Original 2-(3-Alkoxy-1 H -pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

Article abstract of DOI:10.1021/acs.jmedchem.5b00606

Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure-activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH.

Full text of DOI:10.1021/acs.jmedchem.5b00606

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Article
Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors
of Human Dihydroorotate Dehydrogenase (DHODH)
Marianne Lucas-Hourani, Hélène Munier-Lehmann, Farah El Mazouni, Nicholas Malmquist,
Jane Harpon, Eloi Paul COUTANT, Sandrine Guillou, Olivier Helynck, Anne Noel, Artur
Scherf, Margaret Anna Phillips, Frederic Tangy, Pierre-Olivier Vidalain, and Yves Louis Janin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00606 • Publication Date (Web): 16 Jun 2015
Downloaded from http://pubs.acs.org on June 22, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate
Dehydrogenase (DHODH)
ab
cd
e
Marianne LucasꢀHourani, Hélène MunierꢀLehmann, Farah El Mazouni, Nicholas A.
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Malmquist, Jane Harpon, Eloi P. Coutant, Sandrine Guillou, Olivier Helynck, Anne
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e
ab
abg
Noel, Artur Scherf, Margaret A. Phillips, Frédéric Tangy, PierreꢀOlivier Vidalain, Yves
cd*
L. Janin
a: Unité de Génomique Virale et Vaccination, Département de Virologie, Institut Pasteur, 28
Rue du Dr. Roux, 75724 Paris Cedex 15, France
b: Unité Mixte de Recherche 3569, Centre National de la Recherche Scientifique, 25 Rue du
Dr. Roux, 75724 Paris Cedex 15, France
c: Unité de Biologie des Interactions HôteꢀParasite, Département de Parasitologie et
Mycologie, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France
d: Unité de Chimie et Biocatalyse, Département de Biologie Structurale et Chimie, Institut
Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France
e: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas,
6
001 Forest Park Blvd, Dallas, Texas 75390ꢀ9041
f: Unité Mixte de Recherche 3523, Centre National de la Recherche Scientifique, 28 Rue du
Dr. Roux, 75724 Paris Cedex 15, France
g: Current address: Laboratoire de Chimie et de Biochimie Pharmacologiques et
Toxicologiques, Team CBNIT, UMR8601 CNRSꢀUniversité Paris Descartes, 45 Rue des
Saint Pères, 75006 Paris, France
*
Eꢀmail: yves.janin@pasteur.fr . Phone: 33 (0)1 40 61 39 92.
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Abstract
Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2ꢀ
3ꢀalkoxy-1H-pyrazolꢀ1ꢀyl)pyrimidine derivatives as well as 2ꢀ(4ꢀbenzylꢀ3ꢀethoxyꢀ5ꢀmethyl-
H-pyrazolꢀ1ꢀyl)ꢀ5ꢀmethylpyridine, we describe here the syntheses and evaluation of an array
(
0
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1
of azineꢀbearing analogues. As in our previous report, the structureꢀactivity study of this
series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus
replication. Among other inhibitors, this round of syntheses and biological evaluation iteration
led to the highly active 5ꢀcyclopropylꢀ2ꢀ(4ꢀ(2,6ꢀdifluorophenoxy)ꢀ3ꢀisopropoxyꢀ5ꢀmethyl-1H-
pyrazolꢀ1ꢀyl)ꢀ3ꢀfluoropyridine. Inhibition of DHODH by this compound was confirmed in an
array of in vitro assays, including enzymatic tests and cellꢀbased assays for viral replication
and cellular growth. This molecule was found to be more active than the known inhibitors of
DHODH brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the
design of an original series of immunosuppressive agent. Moreover, since other series of
inhibitors of human DHODH have been found to also affect Plasmodium falciparum
DHODH, all the compounds were assayed for their effect on P. falciparum growth. However,
the modest in vitro inhibition solely observed for two compounds did not correlate with their
inhibition of P. falciparum DHODH.
1
As explained in more details in our previous report, in the course of a screening campaign of
2ꢀ11
new chemical entities
against infectious agents, compounds 1 and 2 were found active on
12
our whole cell measles virus replication assay. An initial structureꢀactivity study led to
confirm the potential of this original chemotype and to greatly improved antiviral compounds,
including the 2ꢀ(3ꢀisopropyloxy-1H-pyrazolꢀ1ꢀyl)pyrimidine derivative 3, which displayed a
subnanomolar MIC50 on this measles virus replication assay. Moreover, a search for the
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biochemical mechanism of action of this series pointed out that, as for other recently reported
1
3ꢀ18
compounds,
the inhibition of the cellular dihydroorotate dehydrogenase (DHODH) is at
1
the origin of the antiviral effect.
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N
N
N
N
F
N
N N
O
N N
N N
O
O
O
F
1 (MIC50 = 2.7 ꢀM) 2 (MIC50 = 0.65 ꢀM) 3 (MIC50 = 0.66 nM)
Figure 1. Structures of Compounds 1-3.
From these results, in order to increase the chemical diversity possible in this series, we
initially replaced the pyrimidine ring by a pyridine moiety since its additional carbon would
1
provide another position for structureꢀactivity studies. Accordingly, as in our first report, we
are describing here a succession of synthesis campaigns followed by comments on the
antiviral effect obtained. Most of these results are presented in Tables in which the first result
column (%) provides the yield of the reaction depicted and the second column describes the
19
observed antiviral effect expressed as pMIC50 values. This corresponds to the negative log
of the minimum compound concentration required to inhibit viral growth by 50% when using
20
a recombinant measles virus strain expressing a luciferase as a reporter. We first undertook
the preparation of the 2ꢀpyridyl derivatives 6a-t depicted in Table 1. As compound 2, these
1
0
analogues were prepared from the 3ꢀethoxypyrazole 4 and commercially available 2ꢀ
halogenopyridines 5a-p. From 2ꢀhalogenopyridines featuring electronꢀattracting substituents,
the use of cesium carbonate in dry dimethylformamide or acetonitrile and heating at 150 °C
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with a microwave oven for 1 h efficiently gave the Nꢀpyridyl derivatives. On the other hand,
from 2ꢀfluoropyridines featuring electron donating substituents, a temperature of 180 °C
(under pressure) was found necessary and, because of its decomposition into dimethylamine,
7
dimethylformamide was replaced by acetonitrile. For the preparation of compound 6a or 6p,
0
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9
0
we used 2ꢀbromopyridine 5a or 5p and Taillefer and Cristau copperꢀcatalyzed arylation
21
conditions. Moreover, as for compound 1, the regioselectivity of these reactions was
7
unambiguously checked in a couple of instances. The 5ꢀcyclopropyl bearing derivative 6q
was prepared in 48 % yield via a SuzukiꢀMiyaura reaction between cyclopropyl boronic acid
and the 5ꢀbromo derivatives 6j. Alternatively, as described in the Experimental section, the 2ꢀ
fluoropyridines 5qꢀs were prepared via SuzukiꢀMiyaura reactions between cyclopropyl
boronic acid and the corresponding 5ꢀbromo precursors. From them, the analogues 6q-s were
then obtained by Nꢀarylation of 4. The Nꢀarylation of compound 4 with 2ꢀ(6ꢀfluoropyridinꢀ3ꢀ
yl)propanꢀ2ꢀol (5t) gave the hydroxylated analogue 6t and a reduction of its alcohol function
led to the 5ꢀisopropyl derivative 6u. Interestingly, the palladiumꢀcatalyzed hydrogenation at
room temperature of 6t was mostly inefficient. To avoid the use of high hydrogen pressure,
7
which would probably have also resulted in the hydrogenation of the pyridine ring, we used
triethylsilane in the presence of trifluoromethane sulfonic acid and achieved its hydrogenation
into 6u in an unoptimized 24 % yield. Concerning the antiviral effects, as seen in Table 1, the
“methyl scan” leading to compounds 2 and 6bꢀd, pointed out the importance of occupying the
position 5 of the pyridine ring for a tangible antiviral effect (pMIC50 = 6.2 for compound 2).
The lack of substituents or the introduction of a small fluorine atom seen in 6a, 6e and 6f also
gave almost inactive compounds although some improvement can be observed when
comparing the antiviral effect of compound 6a (pMIC50 < 5) and the 3ꢀfluorinated derivative
6e (pMIC50 = 5.2). The recourse to a trifluoromethyl (compounds 6g) or a chlorine atom
(compound 6i) on position 5 gave slightly more active analogues (both pMIC50 of 5.5) but the
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bigger bromine present in compound 6j did not improve this further (pMIC = 5.3). On the
5
0
other hand, the combination of the 3ꢀfluoro and 5ꢀbromo substituents seen in compound 6k
appeared to be the cause of slight synergic effect (pMIC50 = 5.9) when considering the
pMIC50 of the parent monoꢀsubstituted compounds 6e and 6j (respectively 5.2 and 5.3). In
contrast to a fluorine group, the 3ꢀmethyl substituent of 5ꢀbrominated derivative 6l led only to
a weak antiviral effect. A loss of effect was also seen for the three derivatives 6m, 6n and 6o
featuring respectively a cyano, an ester or a methyl ketone on position 5. To a lesser extent, a
similar phenomenon was observed with the methoxy of compound 6p (pMIC50 = 5.5). It is
only the introduction of the cyclopropyl group featured by compound 6q that led to an
improved inhibition of the virus replication (pMIC50 = 7.0 nM). Combination of this group
with the fluorine of compound 6r led to an unchanged antiviral effect (pMIC 7.0), whereas
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5
0
the nitrile of compound 6s replacing this fluorine caused a loss (pMIC = 6.6). Interestingly,
50
the branched isopropyl derivative 6u turns out to be an order of magnitude less active
pMIC50 = 5.9) than the cyclopropyl analogue 6q. Finally, the polar hydroxyl moiety of
(
compound 6t (pMIC50 5.3) led to an even bigger loss of effect in comparison with the
hydrogenꢀbearing isopropyl group of compound 6u.
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Table 1. Preparation and Antiviral Effect of
Compounds 6a-u.
R4
R5
R4
R5
R3
R6
H
R3
R6
N
N N
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
N
X
5a-s
N N
O
i
O
4
6a-n, 6p
ii
compd R3 R4
R5
H
H
H
H
H
H
R6
H
H
%iii
pMIC50
< 5
5.4
< 5
< 5
5.2
< 5
5.5
< 5
5.5
5.3
5.9
< 5
< 5
< 5
< 5
5.5
7.0
7.0
6.6
5.3
5.9
6
a
H
H
H
66
6b
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
57
39
6
c
Me
H
H
6d
Me 39
H
F
6e
F
74
62
47
89
40
44
33
62
47
19
15
6f
H
6g
H
CF
H
3
H
CF
H
H
H
H
H
H
H
H
H
H
H
H
H
6h
6i
H
H
3
Cl
Br
Br
Br
CN
6j
H
6k
6l
6m
6n
6o
F
Me
H
H
H
2
CO Me
COMe
OMe
cꢀPr
cꢀPr
cꢀPr
iv
6
6
p
q
H
H
53
48
v
6r
F
17
18
67
6
6
s
t
CN
H
COHMe
iꢀPr
2
vi
6u
H
24
2 3
i: Cs CO , DMF/MeCN, microwave 150ꢀ180 °C.
ii: ꢀlog(MIC50), MIC50 in mol/L, standard deviation < 2%.
7
iii: Using 2ꢀbromopyridine and a copper catalyst.
iv: Using 2ꢀbromoꢀ5ꢀmethoxypyridine and a copper catalyst
see text and Experimental section.
v: From 6j, see text and Experimental section.
vi: From 6t, see text and Experimental section.
1
As for the pyrimidineꢀcontaining series, by arylation of the 4ꢀaryloxypyrazoles 7a-q with the
2
ꢀfluoropyridine 5q, we prepared the 4ꢀaryloxy derivatives 8a-q depicted in Table 2. Later on,
we also prepared the corresponding pyridazine homologues 10b-q from 3ꢀchloroꢀ6ꢀ
cyclopropylpyridazine (9). For comparison purposes, Table 2 provides the pMIC50 for these
two series. With few variations, the pattern of antiviral effect for analogues 8a-q featuring a
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5ꢀcyclopylpyridine is somehow mirroring the one seen for the 5ꢀethylpyrimidyl bearing
1
homologues. In comparison with the phenoxyꢀbearing compound 8a (pMIC = 7.0), orthoꢀ
5
0
substitution with a fluorine, a chlorine of a bromine atom improved the antiviral effect
pMIC50 of respectively 7.7, 7.9 and 7.8). A trifluoromethyl group instead of these halogens
(
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had a lesser effect (compound 8e, pMIC50 = 7.3). Shifting this trifluoromethyl group on the
meta position of the phenoxy ring did not alter much the antiviral effect (pMIC = 7.1 for 8f)
5
0
whereas placing this group on the para position led to a substantial loss (pMIC = 5.3 for 8g).
5
0
A similar trend is observed with the fluorine atom of compounds 8k-m, the orthoꢀfluoro
derivative 8k being the most active (pMIC50 = 7.4) and the para fluorinated analogue 8m far
less effective (pMIC50 = 5.9). Trends in the polyhalogenated derivatives are somehow less
clearꢀcut although orthoꢀsubstituted derivatives are the most active but, as in the pyrimidine
1
series, with a pMIC of 9.0 the 2,6ꢀdiflurophenoxy derivative 8q is the best antiviral of this
5
0
group of analogues. Very similar comments can be made on the antiviral effects of
compounds 10b-q and the differences between any given pair of analogues is always less than
an order of magnitude.
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1
1
1
1
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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
Table 2. Preparation and Antiviral Effect of
Compounds 8a-q and 10b-q.
H
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
X
N N
N
R3
N
F
O
5q
N N
O
R3
O
O
i
Ar
or
Ar
7
a-q
N
N
X = CH: 8a-q
X = N: 10b-q
9
Cl
ii
ii
pMIC50
8
pMIC50
10b-q
ꢀ
compd
a/10a
b/10b
c/10c
8d/10d
8e/10e
8f/10f
8g/10g
8h/10h
8i/10i
8j/10j
8k/10k
l/10l
m/10m iꢀPr
n/10n
o/10o
p/10p
q/10q
R3
Ar
a-q
8
8
8
Et
Et
Et
Et
Et
Et
Et
Et
Et
C
6
H
5
7.0
7.7
7.9
7.8
7.3
7.1
5.3
7.9
7.1
6.9
7.4
6.8
5.9
7.4
6.5
7.4
9.0
2ꢀFC
6
H
4
7.7
7.1
7.2
6.8
6.4
5.2
7.4
6.9
6.2
7.4
6.4
5.6
7.4
6.4
7.0
8.7
2ꢀClC
6
H
4
4
2ꢀBrC H
6
2ꢀCF
3ꢀCF
4ꢀCF
3
3
3
C
C
C
6
6
6
H
H
H
4
4
4
2,3ꢀCl
2,5ꢀCl
3,5ꢀCl
2
C
C
C
6
6
6
H
H
H
3
2
2
3
3
Et
iꢀPr
iꢀPr
2ꢀFC
3ꢀFC
4ꢀFC
6
6
6
H
H
H
4
4
4
8
8
8
8
8
8
iꢀPr 2,3ꢀF
2
2
2
2
C
C
C
C
6
6
6
6
H
H
H
H
3
iꢀPr 2,4ꢀF
iꢀPr 2,5ꢀF
iꢀPr 2,6ꢀF
3
3
3
2 3
i: Cs CO , MeCN, microwave 180 °C 6h or 2h.
ii: ꢀlog(MIC50), MIC50 in mol/L, standard deviation < 2%.
As depicted in Scheme 1, we also explored the preparation and use of precursors featuring a
5ꢀcyclopropylpyridine moiety to which additional chemistry would allow the introduction of
various group on the carbon 4 of the pyrazole ring. The Nꢀarylation of the 4ꢀiodopyrazole
5
1
1a with 5ꢀcyclopropylꢀ2ꢀfluoropyridine (5q) had to be conducted at 180 °C for up to 12 h
and only led to a rather small amount (13 %) of the 4ꢀiodinated target compound 12a along
with some (9 %) of the reduced derivative 12b. This is quite in contrast with the 55 % yield
we reported for the Nꢀarylation of compound 11a with 2ꢀfluoropyridine at the same
7
temperature for 3 h. Extensive analysis of the reaction mixture pointed out the presence of
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large amount of 3ꢀethoxyꢀ5ꢀmethylpyrazole (11b) resulting from the reduction of the 4ꢀ
iodopyrazole 11a under this long reaction time. Although an explanation for this reduction is
not obvious (a carbene occurrence?), we suggest that the much decreased reactivity of the
electron rich 5ꢀcyclopropylꢀ2ꢀfluoropyridine (5q) toward nucleophilic reaction in comparison
with 2ꢀfluoropyridine is allowing the necessary time for this side reaction to proceed to a large
extent. Somehow related to such behavior is the quick evolution of iodine, or iodochloride,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
upon heating 3ꢀethoxyꢀ4ꢀiodopyrazole in hydrochloric acid. In any case, the iodinated target
compound 12b was treated with butyllithium followed by the addition of benzoylchloride to
give the 4ꢀbenzoyl derivative 13 in 44 % yield. In an attempt to improve these results, we
undertook the Nꢀarylation of the 4ꢀbromopyrazole 11c. Under similar conditions, we could
obtain the 4ꢀbrominated precursor 12c in a much improved 48 % yield. The sodium
borohydride reduction of ketone 13 led to the secondary alcohol 14a whereas the addition of
methyl lithium to ketone 13 provided an access to the tertiary alcohol 14b. From compound
12c, palladiumꢀcatalyzed reactions allowed the preparation of few analogues. A Suzukiꢀ
Miyaura reaction with phenylboronic acid gave 33 % of compound 15 whereas Negishi
reactions with benzylzinc bromide or (1ꢀphenylethyl)zinc chloride gave compounds 6q and 16
in respectively 71 and 42 %. Of note in this part is an optimization of the preparation of 1ꢀ
phenylethylzinc chloride using the lithium chloride, dibromoethane and trimethysilylchlorideꢀ
22ꢀ24
based protocol
as well as the use of CPhos instead of XPhos in an attempt to lessen the
25
extent of βꢀelimination possible with (1ꢀphenylethyl)zinc chloride. Concerning the antiviral
1
effects of these compound, as for the pyrimidineꢀcontaining series, the benzoyl derivative 13
(pMIC50 = 6.9) was slightly less effective than the corresponding methylene analogue 6q
(pMIC50 = 7.0). On the other hand, the alcohol 14a, retained a more sizable antiviral effect
(pMIC50 = 7.4) but the gain is far from what we could observe in the case of the pyrimidineꢀ
1
containing series. Introducing an additional methyl on this carbon led the tertiary alcohol 14b
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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
and to a much reduced antiviral activity (pMIC50 = 6.1). A similar pattern was seen when
adding a methyl to compound 6q (pMIC = 7.0) leading to the much less effective analogue
5
0
16 (pMIC50 = 5.2). Finally, the low antiviral effect of the 4ꢀphenyl derivative 15 (pMIC50 =
6.0) pointed out the importance of a bridge between the pyrazole and the aromatic ring.
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
H
N N
N
i
ii
O
N N
R
O
1
1
1
1a: R = I
1b: R = H
1c: R = Br
R
1
1
1
2a: R = I (pMIC50 < 5)
2b: R = H (pMIC50 < 5)
2c: R = Br (pMIC50 < 5)
N
N
iii or iv
N N
N N
O
O
O
HO
R
14a: R = H (pMIC50 = 7.4)
14b: R = Me (pMIC50 = 6.1)
1
3 (pMIC50 = 6.9)
N
N
N N
v
vi
N N
12c
O
O
R
6
q: R = H (71 %; pMIC50 = 7.0)
15 (pMIC50 = 6.0)
16: R = Me (42 %; pMIC50 = 5.2)
Scheme 1. 5ꢀcyclopropylꢀ2ꢀfluoropyridine (5q), MeCN, microwave 180 °C 12h. ii: a) BuLi,
THF, ꢀ78 °C, b) PhCOCl, ꢀ78 ꢀ>20 °C. iii: NaBH , EtOH. iv: MeLi; THF, 20 °C. v:
4
1
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PhB(OH) , Pddppf, Cs CO , PrOH/H O. vi: PhCH ZnBr, XPhos or PhCHMeZnCl, CPhos,
2
2
3
2
2
Pd(OAc) , THF, 50 °C.
2
As depicted in Table 3, we then retained the 2,6ꢀdifluorophenoxy component of compound 8q
and prepared the 2ꢀpyridyl derivatives 18aꢀs. Analogues 18a-n were obtained by the Nꢀ
arylation of the pyrazole 7q by the corresponding 2ꢀhalogenopyridines 5a, 5g, 5j-o, 5qꢀt and
the 2ꢀfluoropyridines 17l-n (prepared as described in the Experimental section). In the case of
the methoxyꢀbearing analogue 18p, we used again the 2ꢀbromoꢀ5ꢀmethoxypyridine (5p) and
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12
13
14
15
16
17
18
19
20
21
22
23
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35
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41
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43
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47
48
49
50
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52
53
54
55
56
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58
59
60
21
TailleferꢀCristau copperꢀcatalyzed arylation conditions. The volatile 2ꢀbromoꢀ5ꢀ(1,1ꢀ
difluoroethyl)pyridine (17n) was obtained from 2ꢀbromoꢀ5ꢀacetylpyridine (5o). The following
Nꢀarylation of compound 7q with this 2ꢀbromopyridine 17n had to be conducted at a
relatively lower temperature to avoid extensive decomposition and the pure
difluoromethyleneꢀbearing analogue 18n was thus obtained in a modest 12 % yield. A rather
slow catalytic hydrogenation of the cyclopropyl ring of compound 18d enabled an access to
the propyl analogue 18o in a 30 % yield. The preparation of analogues 18pꢀr turned out to be
possible as, under basic conditions, the fluorine atom on the pyridine ring of 18d could be
displaced by methanol, dimethylamine or benzylalcohol using high temperature and a microꢀ
wave oven. In the last case, the catalytic hydrogenation of the resulting 3ꢀbenzyloxyderivative
18r led to the 3ꢀhydroxypyridinyl derivative 18s. Unexpectedly, despite few trials, the direct
hydrolysis of compound 18d into 18s, using sodium hydroxide in THF, was not successful.
As seen in Table 3, with these compounds we could assess the effect of varying the pyridine
substituents while retaining a 2,6ꢀdifluorophenoxy moiety. Going from the hydrogen of the Nꢀ
pyridine derivative 18a (pMIC50 = 4.9) to the 5ꢀmethyl homolog 18b (pMIC50 = 7.6), a 400
fold improvement of the antiviral effect was observed. From then, the 5ꢀcyclopropyl group of
compound 8q (pMIC50 = 9) provided another order of magnitude. On the other hand, the ethyl
1
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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
group of compound 18l (pMIC50 = 7.1) caused a loss of antiviral effect. This is actually
reminiscent of what was observed for the 5ꢀisopropyl bearing compound 6u (pMIC = 5.9) in
5
0
comparison with the 5ꢀcyclopropyl bearing analogue 6q (pMIC50 = 7.0) described above.
Moreover, it is contrasting with what we previously reported for the 5ꢀethyl and 5ꢀ
cyclopropyl pyrimidine analogues which had mostly the same antiviral effect (pMIC50 = 9.2
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
1
and 8.9). Replacement of the methyl group of compound 18b (pMIC = 7.6) by the
5
0
trifluoromethyl of compound 18c (pMIC = 6.5) led to more than a 10 fold loss. If adding a
50
fluorine atom on carbon 3 of the pyridine ring has very little effect when comparing the 5ꢀ
ethyl bearing analogues 18l and 18m (pMIC50 both of 7.1), a two fold loss was seen in
comparison with the 5ꢀcyclopropyl pair 8q and 18d (pMIC50 = 9 and 8.6). On the other hand,
as for the pair of analogues 6j and 6k, a fluorine on the same position is improving the
antiviral effect when comparing the activity of the 5ꢀbromo derivatives 18f and 18g (pMIC50
=
6.3 and 6.9). Attempts to introduce various polar groups on carbon 5 of the pyridine ring
such as the one seen in compound 18i-k did not improve the antiviral effect and the two
fluorine atoms of compound 18n (pMIC50 = 7.1) had no effect in comparison with the 5ꢀethyl
analogue 18l (pMIC = 7.1). Only relatively small losses were observed for the bisꢀsubstitued
50
derivatives 18p-s featuring a cyclopropyl group. Interestingly, the influence of the methoxy
moiety of compound 18p (pMIC50 = 7.5) is comparable to the dimethylaminated derivative
18q (pMIC50 = 7.5) whereas the hydroxylꢀbearing analogue 18o (pMIC50 = 8.1) displayed a
relative stronger activity. Somehow unexpectedly, the larger benzyloxy group of compound
1
8r (pMIC = 7) only caused a relatively small loss of antiviral effect. In any case, none of
50
the analogues described in Table 3 displayed an antiviral effect better than the nanomolar
level of compound 8q.
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Table 3. Preparation and Antiviral Effect of Compounds 18a-
s.
R5
R5
R3
N
R3
N N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
X
H
N
N N
5
a, 5j-t, 17k-m
O
i
O
F
O
F
F
O
7q
18a-m
F
ii
compd
8a
8b
8c
8d
8e
8f
8g
8h
8i
18j
18k
18l
18m
18n
18o
18p
R3
H
H
H
F
CN
H
F
Me
H
H
H
H
F
H
F
MeO
NMe
BnO
HO
R5
H
Me
%
66
34
64
46
96
79
73
84
pMIC50
< 5.0
7.6
1
1
1
1
1
1
1
1
CF
3
6.5
8.6
8.1
6.3
6.9
5.9
7.3
6.6
6.7
7.1
7.1
7.1
6.7
7.5
7.5
7.0
cꢀPr
cꢀPr
Br
Br
Br
OMe
COCH
COHMe
iii
1
64
3
52
68
49
34
12
2
Et
Et
CF
2
CH
3
iv
nꢀPr
cꢀPr
cꢀPr
cꢀPr
cꢀPr
30
70
62
29
v
v
v
18q
2
1
1
8r
8s
vi
72
8.1
2 3
i: Cs CO , DMF/MeCN, microwave, 130ꢀ180 °C.
ii: ꢀlog(MIC50), MIC50 in mol/L, standard deviation < 2%.
iii: Using 2ꢀbromoꢀ5ꢀmethoxypyridine and a copper catalyst, see
text and Experimental section
iv: By catalytic reduction of 18d.
v: From 18d, see text and Experimental section.
vi: Reduction of 18r, see text and Experimental section.
As depicted in Figure 2 and fully described in the Experimental section, from the 2,6ꢀ
difluorophenoxy pyrazole 7q, some other possible azinesꢀbearing compounds such as 3ꢀ
pyridyl, 5ꢀpyrimidyl, the 2ꢀpyrazinyl and the 1,2,4ꢀtriazine derivatives were also prepared.
21
The 3ꢀpyridyl bearing compounds 19a-c were made by the TailleferꢀCristau copper
catalyzed Nꢀarylation of 7q with the commercially available 5ꢀbromoꢀ2ꢀmethyl, 5ꢀbromoꢀ2ꢀ
ethyl and 5ꢀbromoꢀ2ꢀmethoxypyridines. The 5ꢀpyrimidyl derivative 20a was also made with
1
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5
5
5
5
5
5
5
6
the copper catalyzed reaction between 7q and 5ꢀbromoꢀ2ꢀtertꢀbutylpyrimidine. For the
preparation of the 5ꢀcyclopropyl analogue 20b we had recourse to the Nꢀarylation of
compound 7q with a small sample of 2ꢀcyclopropylpyrimidinꢀ5ꢀylboronic acid using Lam and
2
6
Chan reaction conditions. The Nꢀarylation of 2,6ꢀdifluorophenoxy pyrazole 7q with 2,5ꢀ
dibromopyrazine led to the 5ꢀbromopyrazine analogue 21a. Similarly, from 2ꢀchloroꢀ5ꢀ
(trifluoromethyl)pyrazine, the 5ꢀtrifluoromethyl analogue 21b was obtained. The methoxyꢀ
bearing analogue 21c was then made from 21a by displacement of the bromine atom with
methanol under basic conditions. As described in the Experimental section, the preparation of
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
2ꢀbromoꢀ5ꢀcyclopropylpyrazine allowed the synthesis of the cyclopropylꢀbearing analogue
21d by Nꢀarylation. In similar ways, the pyridazineꢀbearing analogues 10q and 22a-b were
prepared from the corresponding chloropyridazines. The methoxy or ethoxyꢀbearing
analogues 22c-d were made by displacement of the chlorine atom of compound 22b with the
corresponding alcohols since an Nꢀarylation trials using 3ꢀchloroꢀ6ꢀmethoxypyridazine led to
extensive side reactions; including, as seen by LC/MS, a methylation of compound 7q. The
1,2,4ꢀtriazineꢀbearing analogue 23 was prepared in a modest 10 % yield by the Nꢀarylation of
compound 7q with 6ꢀethylꢀ3ꢀ(methylthio)ꢀ1,2,4ꢀtriazinꢀ5ꢀol (preparation given) at 200 °C.
Finally, as fully described in the Experimental section we also prepared the Nꢀethyl
imidazoleꢀbearing analogs 24 by the copper catalyzed Nꢀarylation of compound 7q with 1ꢀ
ethylꢀ4ꢀiodo-1H-imidazole. In view of the antiviral effect of the azineꢀbearing analogues 8q,
10q, 19b, 21d, one fact clearly stands out. As for the 2ꢀpyrimidyl analogues previously
studied, an ethyl or a cyclopropyl group para to the pyrazole ring often provides a low
nanomolar antiviral effect. However, this is not true for the 2ꢀcyclopropylpyrimidine
derivative 20b (pMIC50 = 6.8) or the triazine 23 (pMIC50 = 7.6) which is in this case featuring
an additional hydroxyl group. Attempt to replace such alkyl groups by side chains of similar
size but featuring more polar groups mostly failed as seen for the 2ꢀmethoxypyridine
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derivative 19c (pMIC50 = 6.0), the 2ꢀmethoxypyrazine 21c (pMIC50 = 6.1) or the 3ꢀ
methoxypyridazine 22c (pMIC = 7.1). The same trend was observed for the halogenꢀbearing
5
0
analogues 21a-b (pMIC50 = 5.5 and 5.3) or 22b (pMIC50 = 6.8). Finally, the relatively low
antiviral effect of the Nꢀethylimidazole derivative 23 may be another example of the trend
which points at a detrimental effect of polar atoms (a nitrogen in this case) in the vicinity of
this alkyl side chain.
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17
18
19
20
21
22
23
24
25
26
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1
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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
R
N
R
N
N
N N
N N
O
O
F
F
O
F
O
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
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
1
1
1
9a: R = Me (pMIC50 = 7.4)
9b: R = Et (pMIC50 = 8.9)
9c: R = OMe (pMIC50 = 6)
20a: R = t-Bu (pMIC50 = 6.5)
20b: R = c-Pr (pMIC50 = 6.8)
R
N
R
N
N
N
N N
N N
O
O
F
F
O
O
F
F
1
0q: R = c-Pr (pMIC50 = 8.7)
22a: R = Me (pMIC50 = 8.3)
22b: R = Cl (pMIC50 = 6.8)
22c: R = OMe (pMIC50 = 7.1)
22d: R = OEt (pMIC50 = 6.7)
2
2
2
1a: R = Br (pMIC50 = 5.5)
1b: R = CF (pMIC = 5.3)
1c: R = OMe (pMIC50 = 6.1)
3
50
21d: R = c-Pr (pMIC50 = 8.6)
HO
N
N
N
N
N
N N
N N
O
O
F
F
O
O
F
F
2
3 (pMIC50 = 7.6)
24 (pMIC50 = 6.9)
Figure 2. Structure and Antiviral Effect of Compounds 10q and 19ꢀ24.
1
The elucidation of the biochemical target of our series has led us to publish a survey of all
27
the reported inhibitors of DHODH along with their uses. This review pointed out that
teriflunomide (25) depicted in Figure 3, is the only human DHODH inhibitor used in
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medicine against autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
Interestingly, in our cellular assay, teriflunomide (25) displayed an antiviral effect with an
MIC50 of 5 ꢀM which is reflected in the previously reported IC50 of 1 ꢀM on recombinant
2
8,29
human DHODH.
actually triggered the search and the discovery of some offꢀtarget inhibitions in the past.
This value can also be compared to the enzymatic IC of 10 nM reported for brequinar (26),
This relatively modest effect of teriflunomide (25) on the enzyme had
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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50
51
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58
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60
3
0ꢀ32
2
9
5
0
a stronger inhibitor of DHODH which underwent disappointing phase II trials against solid
33ꢀ37
tumors.
HO
O
N
F
F3C
N
H
N
F
teriflunomide (25)
pMIC50 = 5.3
brequinar (26)
pMIC50 = 7.4
HO
O
1
Figure 3. Structure and antiviral effect of teriflunomide (25) and brequinar (26).
In order to assess the potential of our series in comparison with these compounds we
undertook an array of biological assays using compound 18d. In cellulo, as depicted in Figure
4, we could point out that compound 18d is affecting pyrimidine nucleoside biosynthesis.
Indeed, while adding 18d at concentration varying from 4 to 100 nM blocked the measles
virus replication in cells, the addition of the pyrimidineꢀcontaining nucleoside uridine at 10
µg/mL (Figure 4A) restored its replication. On the other hand, the addition of the purineꢀ
containing nucleoside guanosine at 10 µg/mL did not restore this (Figure 4B). Moreover, a
restored virus replication was seen with the addition of orotic acid at 3 mM (Figure 4C) while,
as seen in Figure 4D, dihydroorotic acid at 3 mM had no such effect. These last results thus
narrowed down the biochemical target of compound 18d to DHODH. Accordingly as
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reported, we produced recombinant human DHODH and compound 18d was indeed found to
be an inhibitor of this enzyme with an IC of 25 ± 5 nM.
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Figure 4. Compound 18d is an inhibitor of pyrimidine biosynthesis pathway. HEKꢀ293T cells
were infected with recombinant MV strain expressing luciferase (multiplicity of infection =
0
.1), incubated with DMSO alone or 18d at 4, 20 or 100 nM, and culture medium was
supplemented with uridine (A), guanosine (B), orotate (C) or dihydroorotate (D). After 24 h,
luciferase expression was determined. Experiment was performed in triplicate, and data
represent means ± SD.
3
8
By using a metabolite analysis protocol, the HEKꢀ293 T cells content in adenosine
triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine
triphosphate (UTP) treated for 24 h with various concentration of compound 18d could be
determined. As seen in Table 4, intracellular concentrations of uridine and cytidine collapsed
in cells treated with 18d, whereas purine nucleotides concentrations were slightly increased
likely as a consequence of the control loops connecting purine and pyrimidine metabolic
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pathways. This strongly demonstrates in cell cultures the inhibition of de novo pyrimidine
biosynthesis by 18d.
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Table 4. Normalized Cellular Nucleotides Content
(%) in the Presence of Compound 18d at 0.016, 0.8, 4,
20 and 100 nM.
DMSO 0.016
0.8
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213
100
110
± 9
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± 5
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ATP
GTP
CTP
UTP
± 20 ± 13 ± 18
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± 3
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±
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± 1
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±
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± 7
± 2
± 2
± 1
± 1
We recently reported that the inhibition of pyrimidine biosynthesis amplifies cellular response
17
to pathogenꢀassociated molecular patterns such as exogenous RNA molecules. When
transfecting cells with small synthetic RNA molecules (ssRNA) that mimic viral RNA
genomes or transcripts, activation of the interferonꢀstimulated response element (ISRE) that
drives innate immunity genes was enhanced by DHODH inhibition. As depicted in Figure 5,
we thus monitored that compound 18d increased the expression of an ISREꢀluciferase
reporter gene when transfecting cells with ssRNA molecules. This adds to the panel of
cellular assays that support the inhibition of pyrimidine biosynthesis by compound 18d.
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Figure 5. Compound 18d amplifies cellular response to ssRNA molecules. (Top) HEKꢀ293 T
cells with the ISREꢀluciferase reporter gene (STINGꢀ37 cells) were transfected with
increasing doses of synthetic 5’ꢀtriphosphate RNA molecules (ssRNA), and incubated in the
presence of compound 18d or DMSO alone in 96ꢀwell cultures plates. After 24 h, luciferase
expression was determined. (Bottom) Same experiment was performed in the presence of
uridine at 30 µg/mL. Both experiments were performed in duplicate, and the data represents
means ± SD.
Aside from their antiviral effect, pyrimidine biosynthesis inhibitors are also well known for
blocking the proliferation of lymphocytes, and this probably accounts for their
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immunosuppressive property in vivo. Accordingly, we also determined the effect of
compound 18d on the growth of Jurkat T lymphocyte cell line, which has been demonstrated
40,41
to be sensitive to DHODH inhibitors.
As shown in Figure 6, compound 18d strongly
inhibited Jurkat cells proliferation (IC = 0.02 ꢀM), and its level of inhibition compared
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favorably with brequinar (IC50 = 0.2 ꢀM) or teriflunomide (IC50 close to 60 ꢀM, data not
shown).
Figure 6. Inhibition (%) of Jurkat cells proliferation by compound 18d and brequinar (26).
Jurkat cells were incubated with increasing doses of 18d or brequinar. As a control, cells were
treated with DMSO alone. At t = 0 and after 72 h of culture, the number of living cells was
determined using the CellTiterꢀGlo reagent. The inhibition of cellular proliferation is
expressed as a percentage relative to DMSOꢀtreated control wells. The results presented
correspond to the mean ± SD of two independent experiments.
Also of much interest is the recent demonstration that Plasmodium falciparum DHODH is a
42ꢀ46
valid target for the treatment of Malaria
and that a dual inhibition of human and P.
47ꢀ50
falciparum DHODH was noticed for some series.
Accordingly, we screened all these
antiviral compounds for a potential inhibition of P. falciparum growth. No growth inhibition
was seen in a cellular assay at the concentration of 8.66 µg/mL (data not shown) for all the
compounds aside from a modest effect for the pyridazineꢀbearing analogues 10q and 22a
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(IC of 0.33 and 0.47 µM). Of course, these values led us to prepare many more pyridazineꢀ
5
0
bearing analogues, including compound 10b-p, but all of them turned out to be at least an
order of magnitude less effective on the parasite growth. Just in case, compound 10q and 22a
were also assayed for their eventual inhibition of recombinant P. falciparum DHODH but
they turned out to be completely devoid of effect on this biochemical assay.
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In conclusion, along with our previous report, this study made good use of the
2
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alkoxypyrazole chemistry we previously reported,
which somehow cleared a sort of
“
chemical blind spot” existing in pyrazole chemistry. The ensuing screenings of the resulting
new chemical entities led us to extensively explore the structureꢀactivity relationship of a new
series of human DHODH inhibitors. Of note is a cellular antiviral assay which greatly
simplified the evaluation of the compounds prepared and probably filtered out analogues of
low cell membrane permeability. Concerning the potential of this series of inhibitors against
51,52
immune diseases, the current success of teriflunomide (25)
has led to renewed interest in
the search for better inhibitors of human DHODH. Quite a few strong acidꢀbearing human
DHODH inhibitors have thus emerged and are currently at various stage of clinical
41,53ꢀ61
development for the treatments of autoimmune diseases or graft rejection.
Since all
these compounds, as well as brequinar (26), are featuring a carboxylic acid function, we are
currently working on the selection of compounds displaying optimal preclinical properties in
order to secure a proof of effect on an animal model of autoimmune disease. This endeavor is
based on the reasonable assumption that our carboxylic acidꢀfree series of inhibitors could
display a different and beneficial pharmacological profile in comparison with the inhibitors
currently in preclinical or clinical studies. However, so far our best inhibitors can be only
considered as tools as a too short microsomal stability has been a recurrent feature for this
series. Indeed, low half life values were observed for compounds such as 8k (t1/2 = 4 mn) or
8q (t1/2 = 6 mn) on human microsomes and only modest improvements were seen for the
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“fluorineꢀprotected” compound 18d (t1/2 = 27ꢀ41 mn), the “hydroxyꢀprotected” compound 18s
(t1/2 = 38 mn) or the less active analogues 22c (t1/2 = 22 mn) and 18g (t1/2 = 60 mn). Related to
this aspect is a report mentioning similar difficulties for another type of Nꢀarylated
62
pyrazoles. Accordingly, additional work will be required to improve this and the Xꢀray
based determination of the binding mode of this series of inhibitors to human DHODH could
be very useful in this regard. Finally, these results are probably an illustration of the interest
of designing whole cell/phenotypic assays of sufficient sensitivity (a bioluminescent virus in
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the present case). This led us to detect an unexpected biological effect and provided a simple
biological mean not only to undertake further structureꢀactivity study but also to find the
mode of action of this series of compounds.
Experimental section
Measles Virus Inhibition Assay. HEKꢀ293T cells (ATCC) were maintained in Dulbecco's
modified Eagle's medium (DMEM; GibcoꢀInvitrogen) containing 10% fetal calf serum (FCS),
2
penicillin, and streptomycin at 37°C and 5% CO . Antiviral activity of compounds was
determined using a recombinant vaccine strain of measles virus expressing firefly luciferase
20
(
rMV2/Luc) from an additional transcription unit. To determine the MIC , HEKꢀ293T cells
50
+4
were infected with rMV2/Luc (MOI = 0.1), and incubated in 96ꢀwell culture plates at 3x10
cells/well with increasing concentrations of compounds or DMSO alone. After 24 h,
luciferase expression was determined. The MIC50 corresponds to the concentration of a
compound inhibiting luciferase activity by 50%.
P. falciparum Growth Assay. Compounds were screened against P. falciparum 3D7 strain
parasites with a starting parasitemia of 0.8% at 2% hematocrit in 96ꢀwell plates. Compound
concentrations were 8.66 µg/mL final in the assay. Parasite growth and proliferation was
63
measured using SYBR Green I.
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P. falciparum DHODHs. This enzyme was expressed as recombinant proteins in E. coli and
44,64
purified as previously described.
Steadyꢀstate kinetic analysis was performed using the
44
2
,5ꢀdichloroindophenol (DCIP)ꢀbased spectrophotometric method as described. Enzyme and
substrate concentrations were: DHODH (E = 5ꢀ10nM), substrates (0.2mM Lꢀdihydroorotate
and 0.02mM CoQd). The 100 x compound stock solutions were made in DMSO covering a 3ꢀ
fold dilution series such that final concentrations in the assay for inhibition ranged from 0.001
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–
100 ꢁM.
6
Metabolite Analyses. HEKꢀ293T cells were plated in 6ꢀwell plates (10 cells per well). One
day later, culture medium was supplemented with increasing doses of compound 18d or
DMSO alone. After an additional 24 h of culture, cells were harvested, carefully counted and
monitored for viability by trypan blue exclusion. Cellular nucleotides were quantified as
17
previously described.
ISRE-Luciferase Reporter Assay. STINGꢀ37 cell line was previously described, and
corresponds to HEKꢀ293 T cells that express luciferase under control of five interferonꢀ
17
stimulated response elements (ISRE). Cells were transfected with increasing amounts of
ssRNA molecules using JetPrime PEI reagents (Polyplus transfection), and dispensed in 96ꢀ
well plates at 35,000 cells/well in 100 ꢀl of Dulbecco's modified Eagle's medium (DMEM;
GibcoꢀInvitrogen) containing 10% fetal calf serum (FCS), penicillin, and streptomycin.
DMSO or compound 18d was added to culture medium and after mixing, cells were cultured
for 24 h at 37°C and 5% CO
BrightꢀGlo reagent following manufacturer’s recommendations (Promega).
Inhibition of Jurkat Cells Proliferation. Jurkat cells were cultured in at 5x10 cells per well
in flatꢀbottom 96ꢀwell culture dishes. Cells were maintained at 37°C and 5% CO in RPMI
GibcoꢀInvitrogen) containing 10% fetal calf serum (FCS), pyruvate sodium, nonꢀessential
amino acids, penicillin, and streptomycin. Number of living cells was determined by
2
. Finally, firefly luciferase activity was determined using the
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quantification of adenosine triphosphate (ATP) in culture wells using the CellTiterꢀGlo Assay
(Promega) following manufacturer’s recommendations. This luciferaseꢀbased assay evaluates
by ATP quantification the number of metabolically active cells in culture wells.
Human microsome stability. The microsomal stability assessments were subcontracted to
Oroxcell, Romainville, France.
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Chemistry. A Biotage initiator 2 microwave oven was used for reactions mentioning such
1
13
heating method. H NMR and C NMR spectra were recorded on a Bruker Avance 400
spectrometer at 400 MHz and 100 MHz, respectively. Shifts ( ) are given in ppm with respect
δ
to the TMS signal and coupling constants (J) are given in Hertz. Column chromatography
were performed either on Merck silica gel 60 (0.035 ꢀ 0.070 mm) or neutral alumina
containing 1.5 % of added water using a solvent pump and an automated collecting system
driven by a UV detector set to 254 nm unless required otherwise. Sample deposition was
carried out by adsorption of the mixture to be purified on a small amount of the solid phase
followed by its deposition of the top of the column. The low resolution mass spectra were
obtained on an Agilent 1100 series LC/MSD system using an atmospheric electrospray
ionization system and the high resolution mass spectra (HRMS) were obtained using a Waters
Micromass QꢀTof with an electrospray ion source. Unless stated otherwise, a purity of at least
95 % was obtained for all the compounds by means of chromatography, recrystallisation or
distillation and this level of purity was established by TLC, LC/MS and NMR spectroscopy
Preparations of all the commercially unavailable halogenoazines used in this work.
5-Cyclopropyl-2-fluoropyridine (5q). Under an inert atmosphere, 5ꢀbromoꢀ2ꢀfluoropyridine
(5j) (17 g, 0.096 mol) and cesium carbonate (114 g, 0.35 mol) were dispersed in a 95/5 vv
mixture of toluene and water (500 mL). This was degassed by gently bubbling argon in the
reaction and cyclopropyl boronic acid (10 g, 0.11 mol) and [1,1’ꢀbis(diphenyl
phosphino)ferrocene] dichloropalladium complexed with dichloromethane (1.45 g, 0.0018
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mol) was added. The flask was heated to reflux for 40 minutes, upon cooling the suspension
was diluted in ethyl acetate, the filtered organic layer was washed with water, brine, dried
over magnesium sulfate and concentrated to dryness. The residue was purified by a
distillation at ambient pressure (E760 mm = 206 °C) to give a volatile oil (8.06 g, 62 %). A
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lesser pure fraction (about 7 %) was also collected. H (CDCl
3
): 0.68 (m, 2H); 1.02 (m, 2H);
1
3
1.91 (m, 1H); 6.81 (m, 1H); 7.42 (m, 1H); 8.02 (m, 1H). C (CDCl ): 8.6; 12.2; 108.9 (38
3
Hz); 136.7 (4 Hz); 138.1 (7 Hz); 145.5 (14 Hz); 162.2 (236 Hz). HRMS: too volatile for
analysis.
5
-Cyclopropyl-2,3-difluoropyridine (5r). By using the procedure described for the
preparation of 5ꢀcyclopropylꢀ2ꢀfluoropyridine (5q), this compound was obtained from 5ꢀ
bromoꢀ2,3ꢀdifluoropyrimidine (5k) in 63 % yield, as a volatile oil, after a chromatography
1
over silica gel (cyclohexane – dichloromethane 2/1). H (CDCl ): 0.71 (m, 2H); 1.06 (m, 2H);
3
13
1
.94 (m, 1H); 7.18 (m, 1H); 7.78 (m, 1H). C (CDCl
3
): 9.0; 12.2; 123.5 (3 and 15 Hz); 139.3;
1
39.4 (5 and 13 Hz); 145.4 (29 and 260 Hz); 150.4 (15 and 236 Hz). HRMS: too volatile for
analysis.
-Chloro-5-cyclopropylnicotinonitrile (5s). By using the procedure described for the
2
preparation of 5ꢀcyclopropylꢀ2ꢀfluoropyridine (5q), this compound was obtained from 5ꢀ
bromoꢀ2ꢀchloronicotinonitrile in 53 % yield, as a solid, after a chromatography over silica gel
1
(cyclohexane – dichloromethane 1/1). H (CDCl
3
): 0.79 (m, 2H); 1.17 (m, 2H); 1.97 (m, 1H);
): 9.8; 12.3; 110.2; 114.8; 139.1; 139.3;
49.4. 151.3. HRMS calcd for C H BrN + H: 222.9871. Found: 222.9840.
13
7
1
2
.59 (d, 1H, J = 2.5); 8.38 (d, 1H, J = 2.5). C (CDCl
3
9
7
2
-(6-Fluoropyridin-3-yl)propan-2-ol (5t). Under an inert atmosphere, 5ꢀbromoꢀ2ꢀ
fluoropyridine (5j) (3.12 g, 17.7 mmol) was dissolved in dry ether (100 mL) and the solution
cooled to ꢀ78 °C. Butyl lithium (9.3 mL, 18.6 mmol, 2N in cyclohexane) was added. The
resulting precipitate was stirred at ꢀ78 °C for 15 mn, acetone (0.4 mL, 90 mmol, dried over 4
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Å molecular sieves) was added and the reaction was allowed to warm back to room
temperature for 30 mn. This was diluted with a saturated solution of ammonium chloride and
extracted with ethyl acetate. The organic layer was washed with a saturated solution of
ammonium chloride, brine, dried over magnesium sulfate and concentrated to dryness. The
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residue was further purified by a chromatography over silica gel (dichloromethane – ethanol
1
98.5/1.5) to yield compound 5t as an oil (0.57 g, 20 %). H (CDCl , slight differences with the
3
6
5
reported one ): 1.63 (s, 6H); 1.89 (s, 1H); 6.91 (dd, 1H, J = 2.8 and 8.4); 7.94 (m, 1H); 8.33
13
(
m, 1H). C (CDCl
3
): 31.8; 71.1; 108.8 (37 Hz); 138.0 (8 Hz); 142.0 (4 Hz); 144.0 (14 Hz);
10FNO + H: 156.0825. Found: 156.0791.
2-Fluoro-5-ethylpyridine (17l). Under an inert atmosphere, 5ꢀbromoꢀ2ꢀfluoropyridine (5j)
5.35 g, 30.39 mmol), potassium carbonate (16.8 g, 121.59 mmol) were dissolved in dry
8
162.6 (238 Hz). HRMS calcd for C H
(
dimethylformamide (75 mL, dried over 4 Å molecular sieves). Oxygen was removed from
this solution by a slow stream of argon and [1,1’ꢀbis(diphenylphosphino)ferrocene]
dichloropalladium complexed with dichloromethane (0.62 g, 1.52 mmol) was added, followed
by a 1M solution of triethylborane (40 mL, 40.4 mmol) which was slowly added with a
syringe. This darkening suspension was heated at 85 °C for 4 h using an oil bath. The
resulting black suspension was diluted in diethyl ether and water, the organic layer was
washed with water 5 times, brine, dried over magnesium sulfate and cautiously concentrated
to dryness to take into account the volatility of the reaction product. The residue was purified
by a chromatography over silica gel (cyclohexane – dichloromethane from 2/3 to 1/4) to yield
1
the 5ꢀethyl derivative 17l as a volatile oil (1.52 g, 40 %). H (CDCl ): 1.25 (t, 3H, J = 7.6);
3
13
2
.65 (q, 2H, J = 7.6); 6.84 (dd, 1H, J = 3.0 and 8.4); 7.61 (m, 1H); 8.03 (m, 1H). C (CDCl ):
3
15.3; 25.1; 108.9 (37 Hz); 136.7 (4 Hz); 140.5 (8 Hz); 145.6 (14 Hz); 162.2 (236 Hz). HRMS:
too volatile for analysis.
2
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Page 29 of 82
Journal of Medicinal Chemistry
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2,3-Difluoro-5-ethylpyridine (17k). By using the protocol described for the synthesis of 2ꢀ
fluoroꢀ5ꢀethylpyridine (17l), compound 17k was obtained in 17 % yield from 5ꢀbromoꢀ2,3ꢀ
difluoropyridine (5q) as a volatile oil after a chromatography over silica gel (cyclohexane –
1
dichloromethane from 1/1 to 1/4). H (CDCl
3
): 1.27 (t, 3H, J = 7.6); 2.68 (q, 2H, J = 7.6);
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3
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.40 (m, 1H); 7.80 (m, 1H). C (CDCl
3
): 15.1; 25.0; 125.9 (3 and 14 Hz); 138.9 (4 Hz); 140.3
(5 and 12 Hz); 145.2 (28 and 260 Hz); 150.4 (14 and 235 Hz). HRMS: too volatile for
analysis. Another fraction of this chromatography led, after a recrystallisation in cyclohexane,
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to 5,5',6,6'ꢀtetrafluoroꢀ3,3'ꢀbipyridine in a 25 % yield as fine needles. H (CDCl
3
): 7.75 (m,
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2
3
H); 8.18 (m, 2H). C (CDCl ): 125.2 (4 and 16 Hz); 131.1 (5 Hz); 139.7 (6 and 13 Hz);
145.7 (29 and 264 Hz); 152.2 (14 and 242 Hz). HRMS: does not ionizes. Anal. Calcd for
C H F N : C, 52.64; H, 1.77; N, 12.28. Found: C, 52.44; H, 1.84; N, 12.26.
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-Bromo-5-(1,1-difluoroethyl)pyridine (17n). In a closed teflon flask, 1ꢀ(6ꢀbromopyridinꢀ3ꢀ
yl)ethanone (5o) (0.95 g, 4.75 mmol) and bis(2ꢀmethoxyethyl)aminosulfur trifluoride (3.6 g,
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1.4 mmol) were stirred for seven days (as only a 78 % conversion was monitored by
1
HNMR, a longer reaction time may lead to an even better yield). This was diluted in water,
solid calcium chloride was added and the solution was extracted with ethyl acetate. The
organic layer was washed with water, brine, dried over magnesium sulfate and concentrated to
dryness (not for too long, this compound is volatile). The residue was purified by a
chromatography over silica gel (cyclohexane –dichloromethane 1/1) to yield the difluorinated
1
pyridine 17n as a colorless and volatile oil (0.57 g, 54 %). H (CDCl
3
): 1.96 (t, 3H, J = 18);
13
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.57 (d, 1H, J = 8.3); 7.69 (dd, 1H, J = 2.3 and 8.3); 8.20 (m, 1H). C (CDCl ): 25.7 (29 Hz);
3
1
20.5 (240 Hz); 128.0; 133.2 (27 Hz); 135.0 (5 Hz); 143.7 (2 Hz); 146.9 (6 Hz). HRMS calcd
BrF N + H: 221.9730. Found: 221.9667.
2-Bromo-5-cyclopropylpyrazine: Under an inert atmosphere, 2,5ꢀdibromopyrazine (0.21 g,
.88 mmol) and cesium carbonate (1.12 g, 3.43 mmol) were dispersed in a 95/5 vv mixture of
for C
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H
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