Discovery and process development of a novel TACE inhibitor for the topical treatment of psoriasis

Article abstract of DOI:10.1016/j.bmc.2017.07.054

Targeting the TNFα pathway is a validated approach to the treatment of psoriasis. In this pathway, TACE stands out as a druggable target and has been the focus of in-house research programs. In this article, we present the discovery of clinical candidate

Full text of DOI:10.1016/j.bmc.2017.07.054

Accepted Manuscript
Discovery and process development of a novel TACE inhibitor for the topical
treatment of psoriasis
Jean-Guy Boiteau, Gilles Ouvry, Jean-Marie Arlabosse, Stéphanie Astri,
Audrey Beillard, Yushma Bhurruth-Alcor, Laetitia Bonnary, Claire Bouix-
Peter, Karine Bouquet, Marilyne Bourotte, Isabelle Cardinaud, Catherine
Comino, Benoît Deprez, Denis Duvert, Angélique Féret, Feriel Hacini-
Rachinel, Craig S. Harris, Anne-Pascale Luzy, Arnaud Mathieu, Corinne
Millois, Nicolas Orsini, Jonathan Pascau, Artur Pinto, David Piwnica, Gaëlle
Polge, Arnaud Reitz, Kevin Reversé, Nicolas Rodeville, Patricia Rossio,
Delphine Spiesse, Samuel Tabet, Nathalie Taquet, Loïc Tomas, Emmanuel Vial,
Laurent F. Hennequin
PII:
S0968-0896(17)31064-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.054
BMC 13891
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2017
17 July 2017
28 July 2017
Please cite this article as: Boiteau, J-G., Ouvry, G., Arlabosse, J-M., Astri, S., Beillard, A., Bhurruth-Alcor, Y.,
Bonnary, L., Bouix-Peter, C., Bouquet, K., Bourotte, M., Cardinaud, I., Comino, C., Deprez, B., Duvert, D., Féret,
A., Hacini-Rachinel, F., Harris, C.S., Luzy, A-P., Mathieu, A., Millois, C., Orsini, N., Pascau, J., Pinto, A., Piwnica,
D., Polge, G., Reitz, A., Reversé, K., Rodeville, N., Rossio, P., Spiesse, D., Tabet, S., Taquet, N., Tomas, L., Vial,
E., Hennequin, L.F., Discovery and process development of a novel TACE inhibitor for the topical treatment of
psoriasis, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.054
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Discovery and process development of a
novel TACE inhibitor for the topical
treatment of psoriasis
Leave this area blank for abstract info.
Jean-Guy Boiteaua*, Gilles Ouvrya∗, Jean-Marie Arlabossea, Stéphanie Astria, Audrey Beillarda, Yushma
Bhurruth-Alcora, Laetitia Bonnarya, Claire Bouix-Petera, Karine Bouqueta, Marilyne Bourotteb, Isabelle
Cardinauda, Catherine Cominoa, Benoît Deprezb, Denis Duverta, Angélique Féreta, Feriel Hacini-Rachinela,
Craig S. Harrisa, Anne-Pascale Luzya, Arnaud Mathieub, Corinne Milloisa, Nicolas Orsinia, Jonathan Pascaua,
Artur Pintob, David Piwnicaa, Gaëlle Polgea, Arnaud Reitza, Kevin Reverséa, Nicolas Rodevillea, Patricia
Rossioa, Delphine Spiessea, Samuel Tabeta, Nathalie Taqueta, Loïc Tomasa, Emmanuel Viala and Laurent F.
Hennequina.
aNestlé Skin Health R&D, Les Templiers 2400 Route des Colles, 06410, Biot, France
bUniv. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for living Systems, F-59000 Lille, France

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Discovery and process development of a novel TACE inhibitor for the topical
treatment of psoriasis
Jean-Guy Boiteaua*, Gilles Ouvrya∗, Jean-Marie Arlabossea, Stéphanie Astria, Audrey Beillarda, Yushma
Bhurruth-Alcora, Laetitia Bonnarya, Claire Bouix-Petera, Karine Bouqueta, Marilyne Bourotteb, Isabelle
Cardinauda, Catherine Cominoa, Benoît Deprezb, Denis Duverta, Angélique Féreta, Feriel Hacini-Rachinela,
Craig S. Harrisa, Anne-Pascale Luzya, Arnaud Mathieub, Corinne Milloisa, Nicolas Orsinia, Jonathan
Pascaua, Artur Pintob, David Piwnicaa, Gaëlle Polgea, Arnaud Reitza, Kevin Reverséa, Nicolas Rodevillea,
Patricia Rossioa, Delphine Spiessea, Samuel Tabeta, Nathalie Taqueta, Loïc Tomasa, Emmanuel Viala and
Laurent F. Hennequina.
aNestlé Skin Health R&D, Les Templiers 2400 Route des Colles, 06410, Biot, France
bUniv. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for living Systems, F-59000 Lille, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Targeting the TNFα pathway is a validated approach to the treatment of psoriasis. In this
pathway, TACE stands out as a druggable target and has been the focus of in –house research
programs. In this article, we present the discovery of clinical candidate 26a. Starting from hits
plagued with poor solubility or genotoxicity, 26a was identified through thorough
multiparameter optimisation. Showing robust in vivo activity in an oxazolone-mediated
inflammation model, the compound was selected for development. Following a polymorph
screen, the hydrochloride salt was selected and the synthesis was efficiently developed to yield
the API in 47% overall yield.
Received in revised form
Accepted
Available online
Keywords:
TACE inhibitor
Psoriasis
2009 Elsevier Ltd. All rights reserved.
Solubility
Polymorph
Aminonitrile synthesis
1. Introduction
TACE inhibitors in the treatment of acne broadening the use of
these molecules in dermatology.4
Given the efficacy of TNFα-targeted antibodies in the
treatment of moderate to severe psoriasis, it is tempting to
hypothesize that disrupting the TNFα pathway locally could
potentially be a way to treat mild to moderate psoriasis with a
topically applied drug.1 One key druggable enzyme for this
approach could be the Tumour Necrosis Factor α Converting
Enzyme (TACE or ADAM17),2 Indeed, many potent and
selective TACE inhibitors have been discovered and developed
in both academia and industry, in the most part aimed at the
treatment of rheumatoid arthritis.3 Unfortunately, lack of
efficacy, poor pharmacokinetics and/or toxicity potentially linked
to poor selectivity vs. related ADAMs or MMPs have precluded
these inhibitors from reaching the market. Most of these
limitations would not apply for a topically administered, locally-
acting drug highlighting the attractiveness of this approach.
Furthermore, recent studies have suggested a potential benefit of
With this in mind, researchers at Nestlé Skin Health/Galderma
have strived to identify clinical candidates to validate this
hypothesis. Although a first compound5 has now reached the
clinic, the need to secure this approach led us to embark in a
back-up program aimed at identifying structurally distinct
candidates.
———
∗ Corresponding authors. Tel : +33(0)-492952948 ; email : jean-guy.boiteau@galderma.com; Tel.: +33(0)-492383062; e-mail:
gilles.ouvry@galderma.com

Figure 1. Apratastat (1), DPC-333 (2), representative structure from the first
generation of TACE inhibitors developed at Galderma/Nestlé Skin Health (3)
and representative compound from previously published work on the back-up
series (4).8
•
Cell potency. As mentioned in our previous
publication, the disconnect between enzymatic and
cellular assays8 for TACE inhibitors prompted us
to follow SAR in a cellular setting directly.
Figure 1 highlights two of the most studied TACE inhibitors,
apratastat (1)6 and DPC333 (2)7 alongside a close analog of
Nestlé Skin Health/Galderma’s clinical candidate (3).5 Previously
disclosed work on the back-up program led to the identification
of (4),8 which, although potent in the enzymatic assay, displayed
only micromolar activity in a cellular setting.
•
Solubility in aqueous solution and in PG/EtOH96.
The Propylene Glycol/EtOH(96%) mixture is
believed to be a good surrogate to apprehend the
compound’s solubility in standard topical
formulation. As the solid form is
a major
contributor to solubility,13 the assay was performed
on crystalline material only, as assessed by
polarized light microscopy.14
In order to bypass this unexplained enzyme-cell drop-off, a
cellular assay looking at inhibition of TNFα in human peripheral
blood monocellular cells (PBMC) was selected as the primary
activity assay. Following the approach described previously,8 β-
sulfonamide hydroxamates and α-sulfonamide hydroxamates
containing the key quinoline group found in DPC-333 (2) were
synthesized.9
•
•
Stability in PG/EtOH96 after 1 month at 40°C at
0.01%w/w. Stability in formulation is paramount
for a topical drug to achieve reasonable shelf-life.15
Stability in human hepatocytes/plasma protein
binding. One way to limit systemic exposure is to
ensure a significant amount of liver metabolism.
2. Results and Discussion
2.1 Chemistry
•
•
Stability in human keratinocytes. Keratinocytes are
responsible for metabolism occurring in the skin.16
Compounds presented in this article were synthesized using
the following general approaches (Scheme 1). Starting from the
Phototoxicity. Phototoxicity for topically applied
drugs is considered a major liability as the
concentrations reached by topically applied
compounds in the upper, sun–exposed, layers of
the skin can be very high.17
known
4-((2-methylquinolin-4-yl)methoxy)benzenesulfonyl
chloride 5,10 we could easily access β sulfonamide-esters 7
through straightforward sulfonamide formation from the
corresponding β amino-esters 6. Such esters could also be
synthesized by Michael addition on the corresponding β-
disubstituted, α, β unsaturated ester 9 of sulfonamide 8. The
esters were converted to hydroxamic acids 10 using either
potassium cyanide-catalysed conditions, using THP-protected
hydroxamates as intermediates or DBU-catalysed conditions.8,11
A similar approach was used to synthesize α-sulfonamide-
hydroxamates 13 from the corresponding α-amino-esters 11.12
•
Genotoxicity. As some hydroxamic acids have
been shown to be genotoxic,18 a way to screen for
genotoxicity early on was incorporated in our
screening cascade. In our hands, the Human Green
Screen monitoring Gadd45 gene expression turned
out to be a useful tool for this early screen.19
Using the general approach described above, a series of
sulfonamide-hydroxamates with different linkers were
synthesized. Out of this first round of synthesis, 2 compounds
particularly stood out: 2-(1-acetyl-3-((4-((2-methylquinolin-4-
yl)methoxy)phenyl)sulfonamido)azetidin-3-yl)-N-
hydroxyacetamide (14) and 2-acetyl-N-hydroxy-6-((4-((2-
methylquinolin-4-yl)methoxy)phenyl)sulfonamido)-2-
azaspiro[3.3]heptane-6-carboxamide (15).
Table 1 highlights the different profiles obtained in
comparison to compound 3, a close analog of Nestlé Skin
Health/Galderma’s clinical candidate.
Table 1. Cyclic linker exploration
Scheme 1. General synthetic approach to compounds described in this
manuscript: (i) DIPEA, DCM; (ii) NH3 (7N in MeOH); (iii) DBU,
acetonitrile; (iv) KCN, NaOHaq, MeOH, THF; (v) (a)KOH, MeOH, H2O;
(b)NH2OTHP, Et3N, EDCI.HCl, HOBt, DCM; (c) TFA, MeOH, DCM (vi)
DBU, NH2OH, MeOH; (vii) Et3N, DCM.
3
14
15
TNFα inhibition (hPBMC)
210
<1
8
8
36
84
17
IC50 nMa
Aqueous Solubility (µM)b
<0.5
0.8
Solubility in
PG/EtOH(96%) (3/7) (mM)
Stability in PG/EtOH(96%)
(3/7)
Hu Hep Clint (µL/min/1e6
cells)b
Hu keratinocytes t1/2 (hrs)b
Phototoxic irritation factorb
89
24
98
<5
95
2.2 Hit identification and screening cascade
<5
The following screening cascade was put in place for this
program:
>24
1
>24
1
>24
c
-

Genotoxicity (Human
Green Screen)b
With no clear strategy as to how to work around the
genotoxicity warning, the team prioritized work on derivative 14
with the objective of improving the solubility in PG/EtOH(96%)
and also installing metabolic instability in hepatocytes.
Negative
Negative
Positive
a See supplementary material for assay description. Geomean of at least 2
determinationsi
b See supplementary material for assay description
c Not tested
2.3 Lead optimization
β-sulfonamide hydroxamate 14 turned out to be a significantly
more potent than the first generation of inhibitors. However, the
compound displayed suboptimal solubility and was stable in
hepatocytes. No toxicity flags were found and the compound was
stable in formulation. In contrast, α-sulfonamide hydroxamate 15
displayed improved solubility (both aqueous and non-aqueous).
Displaying a good level of potency and stability in formulation,
this derivative was also stable in hepatocytes. Unfortunately, 15
carried a clear genotoxicity warning.
2.3.1 β-hydroxamates sulfonamides
Solubility in solvents has been shown to be linked to the
strength of the crystal lattice.11,21 With no small molecule X-ray
of compound 14 available, the general strategy was to add steric
hindrance, and minimize hydrogen bond acceptors and hydrogen
bond donors in different regions of the molecule while
monitoring the impact on both potency and PG/EtOH(96%)
solubility.
Table 2. Sulfonamide analogs of β-sulfonamide hydroxamate 14
TNFα inhibition
Solubility
in PG/EtOH(96%)
(3/7) mMb
Cmpd
R1
R2
R3
R4
R5
A
(hPBMC)
IC50 nMa
8
14
16a
16b
16c
16d
16e
16f
16g
17
H
Me
H
H
H
H
H
H
-
H
H
H
H
Me
H
H
H
-
H
H
H
H
H
Me
H
H
-
Me
Me
Me
Me
Me
Me
Me
Et
H
H
H
H
H
H
F
-OCH2-
-OCH2-
-SCH2-
-CH2-
0.8
c
590
-
720
1.0
1.9
0.9
0.4
240
-OCH2-
-OCH2-
-OCH2-
-OCH2-
-
>10000
>10000
61
c
-
H
-
27
0.4
1.8
-
1300
a See supplementary material for assay description. Geomean of at least 2
determinations
and thus de-symmetrising the molecule was tried (18g and 18h).
The eutomer showed good potency with improved solubility (the
solubility of 18g is expected to drop to the level of 18h’s). The
interest in the pyrrolidine series quickly dissipated as they turned
out to be positive in our genotoxicity screen (data not shown).
In parallel, the substitution of the azetidine nitrogen was
explored (Table 4) Exploration of other amide groups
highlighted the isobutyl group (19a) as the best compromise with
no impact on solubility but accelerated clearance in hepatocytes
(Clint=13µL/min/1e6 cells). Switching to tertiary amines (19b)
gave an interesting profile with reasonable potency, improved
solubility in PG/EtOH(96%) and improved aqueous solubility
(70µM). Unfortunately, 19b turned out to be quite unstable in
PG/EtOH(96%) with only 72% of parent remaining after one
month at 40°C. The major impurity detected displayed the same
mass as the parent compound and was hypothesized to be the
protonation-assisted opening of the azetidine by the neighbouring
sulfonamide nitrogen.23 We thought that one way to impact this
would be to reduce the pKa of the tertiary amine. Analog 19c was
synthesized to test this hypothesis. Although the compound was
more stable (88% remaining after 1 month) it did not meet our
>95% stability criteria.
b See supplementary material for assay description.
c Not tested
Methylation of the sulfonamide (16a) led to a significant loss
in potency. Unfortunately, we could not isolate crystalline
material and document possible impacts on solubility. Reported
changes in the phenyl-quinoline linker (16b and 16c)22 were not
well tolerated from a potency perspective in this series and had
no beneficial impact on solubility. A methyl scan on the central
phenyl ring (16d and 16e) generated inactive compounds with
solubility as low as 14’s. Slight modifications of the sterics and
electronics of the quinoline moiety somewhat maintained
potency (16f and 16g) but once again had limited impact on
solubility. Lastly, replacing the whole quinoline moiety by a
methyl alkyne-a known TACE binding motif22(a)- in order to
reduce the overall number of aromaticsbarely improved the
solubility of the series (17).
Focus was then shifted to the optimisation of the azetidine
moiety (Table 3). While deletion led to a drastic drop in
pharmacological activity (18a), dimethyl substitution brought
some of the potency back, albeit with no real impact on solubility
(18b). Cyclobutyl (18d) and oxetane (18e) rings were preferred
when compared to cyclopropyl (18c) but none of these improved
solubility. A hybrid approach between 14 and 15 provided potent
and slightly more soluble analog 18f. Moving to the pyrrolidine
Interestingly, the supposed rearrangement was detected on all
tertiary azetidines in both the β-sulfonamide hydroxamate and α-
sulfonamide hydroxamate series. N-methylation of the

Table 4. Azetidine substitution of β-sulfonamide hydroxamate 14
sulfonamide led to increased stability supporting the
rearrangement hypothesis.24
Carbamates (19d) and ureas (19e) were well tolerated but did
not improve the overall profile. One breakthrough came with
basic compounds exemplified by 19f. Indeed, not only did they
generally maintain a high level of potency, but the solubility in
both PG/EtOH(96%) and water (80µM) were often improved.
For quite a while, compound 19f was considered our front-
runner with an overall good balance of properties and no toxicity
flags. However, the clearance of 19f and similar basic analogs in
hepatocytes was too low and more importantly not well predicted
by microsomal clearance. Figure 2 depicts the intrinsic clearance
measured in human hepatocytes (in µL/min/1e6 cells) vs. %parent
product remaining after 15 minutes in human microsomes for all
of the compounds from the back-up project. Basic compounds
(exemplified by 19f and represented by red stars in Figure 2)
clearly stood out of the expected correlation between microsomes
and hepatocytes. It appeared that these compounds showed
almost no metabolism in hepatocytes albeit being sometimes
substantially metabolised by microsomes. One possible
explanation would be a lack of cell permeability. This was not
detected before as it did not impact the cell potency of these
compounds. However, it is not clear if TACE is an
intramolecular or extramolecular target which could explain this
disconnect.8,25
TNFα
Solubility PG/
inhibition
Cmpd
A
EtOH(96%) (3/7)
mMb
(hPBMC) IC50
nMa
14
8
0.8
19a
19b
19c
6
0.9
4.9
3.4
12
10
19d
19e
6
5
1.1
0.5
19f
4.6
11
a See supplementary material for assay description. Geomean of at least 2
determinations
b See supplementary material for assay description.
Table 3. Azetidine replacements of β-sulfonamide hydroxamate 14
TNFα
inhibition
(hPBMC) IC50
nMa
Solubility
PG/EtOH(
96%) (3/7)
mMb
Cmpd
A
14
8
0.8
18a
18b
18c
18d
2300
43
4.1
1.6
1.6
84
c
-
13
18e
10
0.2
3
18f
4
Figure 2. Hepatocyte intrinsic clearance (in µL/min/1e6 cells) vs. %parent
product remaining after 15 minutes incubation with human microsomes.
Basic compounds are represented by red stars.
18gd
18hd
8
17
With these results in hand, the focus shifted away from basic
compounds. At this stage, the team felt that options to tackle the
β-sulfonamide hydroxamate solubility liability issues had been
exhausted and the decision was made to shift towards the α-
sulfonamide hydroxamate.
42
7.9
a See supplementary material for assay description. Geomean of at least 2
determinations
b See supplementary material for assay description.
c Not tested
2.3.2 α-hydroxamates sulfonamides
d
The absolute stereochemistry of enantiomers 18g and 18h was not
determined.
The exploration of this series started with the replacement of
the azaspiro[3.3]heptane moiety (Table 5). Complete deletion of
the group (20a) maintained a certain level activity but had a
detrimental impact on solubility. Dimethyl (20b) or cycloalkyl
substitution (20c, 20d, 20e and 20f) gave interesting results with
cyclobutyl maintaining the potency of the lead (20d). However, it

tested positive in our genotoxicity assay. Cyclohexyl analog (20f)
turned out to be the best compromise with slightly diminished
potency, reasonable solubility and, more importantly, no
genotoxicity warning. Moving to a piperidine (20g) gave a more
promising profile but unfortunately all piperidines tested were
found to be genotoxic. The right balance was struck with
azetidine derivative (20h) with increased potency, good solubility
and no genotoxicity alert.
Table 5. Azaspiro[3.3]heptane 15 analogs
Scheme 2. Synthesis of compounds 24 and 25: (i) Iodomethane, K2CO3,
DMF, r.t. 16hrs; (ii) KCN, NaOHaq, NH2OH.H2O, MeOH;
A quick exploration of N-alkyl groups was undertaken around
this vector (Table 6). We were pleased to find that methylation
of 20h gave a very promising compound (26a) with improved
solubility and potency. Extending to an ethyl group (26b) or a 2-
methoxy ethyl group (26c) was tolerated but did not bring any
clear advantage. The bulkier isopropyl group led to a drastic drop
in potency. Methylation of the sulfonamide of the isobutyl
derivative 26e yielded similar results with a robust impact on
solubility while maintaining potency (26f). An additional benefit
of the N-Me derivatives was increased clearance in human
hepatocytes.
TNFα
Solubility
PG/
EtOH(96%)
(3/7) mMb
inhibition
(hPBMC)
IC50 nMa
Genotoxicity
(HGS)b
Cmpd
A
15
36
17
Positive
c
-
20a
20b
20c
82
0.1
c
-
c
-
140
210
At this stage, the team decided that compounds 26a and 26f
had the potential to move toward preclinical development.
46
Positive
Positive
Table 6. N-alkyl exploration on azetidine series
c
-
20d
20e
30
68
c
-
0.2
11
20f
100
61
Negative
Positive
TNFα
inhibition
Solubility
PG/
(hPBMC) EtOH(96%)
Hu Hep Clint
(µL/min/1e6
cells)b
Cmpd
R1
R2
20g
59
17
IC50 nMa
(3/7) mMb
20h
26a
26b
iPr
iPr
iPr
H
Me
Et
16
9
17
57
44
<5
8
20h
16
Negative
37
11
CH2CH2
OMe
26c
iPr
31
8.9
18
a See supplementary material for assay description. Geomean of at least 2
determinations
26d
26e
26f
iPr
iBu
iBu
iPr
H
790
33
2.2
5
17
6
b See supplementary material for assay description.
c Not tested
As we were exploring the azetidine series, we came upon an
unexpected result. An impurity was isolated during the synthesis
of Boc derivative 22 (Scheme 2). The iodomethane esterification
gave an impurity where the sulfonamide nitrogen had also been
methylated. Hydroxyaminolysis of both compounds gave both
derivatives 24 and 25. When tested, the N-methylated analog
turned out to be equipotent to the N-H derivative (22 vs. 25 nM
in the hPBMC assay). This result was unexpected given the
deleterious impact of N-methylation in the β-sulfonamide
hydroxamate series (Table 2, compound 16a). We do not have a
clear explanation to this observation although we suspect a
conformation preference could explain the difference in
behaviours.
Me
33
45
14
a See supplementary material for assay description. Geomean of at least 2
determinations
b See supplementary material for assay description.
c Not tested
Table 7 summarises key results for both compounds. Cellular
potency measured in hPBMC translated in robust potency in
keratinocytes (KHN). As expected, compounds inhibited robustly
TACE in an enzymatic assay. In a panel of ADAMs and MMPs,
the compounds were selective except for MMP12 and to a lesser
extent MMP3 and ADAM9. The compounds were both soluble in
water and in PG/EtOH(96%). The compounds were stable in PG/
EtOH(96%). Hepatic turnover was detected in human
hepatocytes and the compounds were stable in the presence of
human keratinocytes. No toxicity alerts were found on both
compounds and the human green screen was found to correctly
predict Ames and micronucleus results. Finally the compounds
did not inhibit hERG and displayed micromolar inhibition of
CYP3A4.

Table 7. Full characterisation of 26a and 26f
To evaluate the efficacy of 26a in vivo, a dose response-study
mouse model of oxazolone-induced chronic skin
in
a
inflammation was performed. In this model, daily topical
application of oxazolone induces a skin inflammation that is
triggered by the activation of both innate and adaptive immunity.
Mechanistically, initial oxazolone paintings promote the
activation in the skin draining lymph nodes of oxazolone-specific
T cells.26 Following repeated application with the hapten, these
cells are then recruited into the skin where they exert their
cytotoxic activities against haptenised-keratinocytes, thereby
further amplifying the inflammatory reaction. This ultimately
induces inflammatory lesions characterized by increased
epidermal thickness and trans-epidermal water loss, significant
neutrophil and T cell infiltrations in the skin accompanied by
secretion of IL-1β, IFN-γ and TNF-α cytokines. Thus, this model
is considered as a useful pharmacological model for chronic
dermatitis featuring epidermal hyperplasia in which TNF-α plays
a crucial role, such as in psoriasis. As expected, vehicle only
painting did not induce any inflammation while oxazolone
painting induced a major inflammation (Figure 3A). This could
be significantly reduced by the control corticosteroid
betamethasone valerate (data not shown). Interestingly,
concomitant topical application of 26a significantly and dose-
dependently reduced the severity of skin lesions induced by
oxazolone (Figure 3A). The reduction of the clinical score was
associated with a full inhibition of TNF-α secretion and
epidermis thickening (Figure 3B and 3C). These results
demonstrate that topical inhibitors of TACE are endowed with
potent anti-inflammatory properties, and can actively block T-
cell mediated immune responses within the skin.
26a
26f
TNFα inhibition (NHEK) IC50 nMa
TACE enz IC50 nMb
3
4
20
7
ADAM9/ADAM10 IC50 µMb
MMP1/MMP2/MMP3/
0.09/0.95
0.48/1.3
4.5/0.89/0.09/
0.18/2.6/2.1/
0.013/1.3/6.5
4.6/1.0/0.09/
0.21/3.7/2.2/
0.006/1.2/4.4
MMP7/MMP8/MMP9/
MMP12/MMP13/
MMP14 IC50 µMb
Aqueous solubility (µM)b
84
56
62
45
Solubility PG/EtOH96 (3/7) mMb
Stability PG/EtOH96 (3/7)b
Hu KHN t1/2b
Human fuplasma (%)b
Phototoxic irritation factorb
99
>24h
1.9
1
99
>24h
0.3
1
Genotoxicity (Human Green
Screen)b
Amesb
Micronucleusb
hERG IC50 µMb
CYP 3A4 inhibition µMb
Negative
Negative
Negative
Negative
>100
Negative
Negative
>100
8.7
1.6
a See supplementary material for assay description. Geomean of at least 2
determinations
b See supplementary material for assay description.
Based on its promising overall profile and its superior cellular
potency (Table 7), compound 26a was selected for further in
vivo evaluation.
2.4 In vivo pharmacology
TNF- at D10
150
ꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
120
90
60
30
0
Figure 3. Topical treatment with 26a compound alleviates the oxazolone-induced chronic skin inflammation. Skin inflammation was induced by daily topical
application of oxazolone on the back of female SKH1 mice. The clinical score was determined as described in the Experimental Section (A). At study’s end (day
10), TNF-α protein level in skin lysates was dosed (B) and skin histology sections were stained (H&E) for measurement of epidermis thickness (C). Results are
expressed as mean values+ SD. (p values: *: < 0.05, **: < 0.01, ***: < 0.001; 2way-ANOVA, Dunett's multiple comparison test).
2.5 Process Research and Development
required; the medicinal chemistry route was suitable to produce
milligram quantities but needed some improvement in order to
manufacture kilogram batches. (Scheme 3)
Based on its interesting properties described in the first
sections, compound 26a was selected as the clinical candidate.
To start preclinical evaluation, large quantities of our API were

Scheme 3. Discovery synthesis of 26a: (i) trimethylsilyl diazomethane, DCM/MeOH (83%); (ii) Et3N, DCM (57%), chromatography; (iii) Trifluoroacetic acid,
DCM (100%); (iv) isobutyryl chloride, Et3N, THF (76%), chromatography; (v) Dimethylsulfate, K2CO3, DMF (96%); (vi) DBU, NH2OH, MeOH (61%),
preparative HPLC
of development. Therefore the hydrochloride salt of compound
26a was selected as the clinical candidate.
The protected azetidine amino acid 27 was treated with
trimethylsilyl diazomethane to give amino ester 28 which was
engaged in the coupling reaction with sulfonyl chloride 5 to
afford compound 29. Boc removal with TFA followed by
acylation with isobutyryl chloride of the resulting azetidine
provided ester 31. Methylation of sulfonamide was performed
with methyl sulfate to afford ester 32. The desired hydroxamic
acid
26a
was
synthesized
using
DBU-mediated
hydroxyaminolysis, purified by preparative HPLC and isolated as
a crystalline powder (m.p. 110 °C (Phase I), HPLC purity 99%)
with on overall yield of 21%.
If the overall yield was acceptable; several improvements
were needed according to a process development point of view:
•
Chromatographic steps used for the purification of
all intermediates had to be replaced by
crystallisation to achieve purity specifications on
scale
Figure 4. XRPD pattern of different polymorphic forms of compound 26a.
Green (Phase I, recrystallized from ACN), pink (Phase II, recrystallized from
AcMe), blue (Phase III, recrystallized from EtOH), brown (Phase IV,
recrystallized from IPA)
•
•
On the safety side, hazardous reagents like
trimethylsilyl diazomethane or dimethylsulfate
had to be replaced by safer reagents.
The synthetic route selected for a scalable synthesis was based
on a cyanation of an imine obtained from methylamine and a
protected azetidinone. This approach had two major benefits:
The final preparative HPLC purification in order
to separate the hydroxamic acid from the
carboxylic acid side product had to be replaced by
a crystallization procedure.
•
The utilization of high material cost amino acid
was circumvented
•
The direct introduction of the N-methyl group that
avoided the dimethylsulfate as the methylating
agent. (Scheme 4)
•
And finally the amino acid raw material was found
to be highly expensive and was commercially
available only on gram scale.
Our first experiment on this project was to perform a solid
form characterization of compound 26a. An early screening of
polymorph was realized in order to determine whether the
isolated crystalline solid was the most stable polymorph and if
the solubility determined on early batches was relevant.
Compound 26a was recrystallized from different solvents (EtOH,
iPrOH, AcMe, ACN), and submitted to slurry ripening.27 Among
the different conditions tested, we were able to identify at least 4
polymorphic forms of compound 26a, and one of them (phase
IV, figure 4) seemed to occur frequently in different
recrystallization conditions (m.p. 130 °C). Solubility
measurements of this new crystalline form indicated a 10 fold
reduction in PG/EtOH(96%) (7.8 mM), which jeopardized the
formulation work performed on this compound. To increase
water solubility we decided to investigate the hydrochloride salt
of compound 26a. Compound 26a.HCl was synthesized and in
our hands, only one polymorphic form was observed at this stage
Scheme 4. Retrosynthesis of compound 26a
Boc-azetidinone was chosen as starting material based on its
availability on large quantities (Scheme 5). Treatment with a
30% methylamine solution in ethanol with acetic acid at 60 °C
afforded the intermediate imine, which was reacted with
trimethylsilyl cyanide to afford α-aminonitrile 33 in 90%
yield.28,29 In-process-controls for these non UV-active
compounds were realized by NMR, and unfortunately, α-
aminonitrile 33 was isolated as an oil on 250 g scale. Direct
hydrolysis of 33 to the corresponding acid was possible under
basic conditions but provided a non UV-active compound that
was fairly soluble in water and difficult to isolate or handle. We
decided to engage the α-aminonitrile 33 directly in the coupling
reaction with sulfonyl chloride 5 and perform the cyano group

hydrolysis in a second step keeping in mind the potential
instability of the sulfonamide linker. In the original route, the
sulfonamide formation step (28 to 29, Scheme 3) gave poor
results due to substantial hydrolysis of the sulfonyl chloride 5.
Replacement of triethylamine by the pyridine/DMAP couple in
dichloromethane at 35 °C, had a dramatic impact on the purity
profile. With these conditions in hands, hydrolysis of sulfonyl
chloride was minimized to less than 10% and direct
crystallization of sulfonamide 34 from the crude mixture was
possible after swapping pyridine for n-heptane. This reaction was
performed on a 500g scale and afforded the targeted compound
in 87% yield with a 99% HPLC purity.
structure-related compounds was described in literature;30
however, on our substrate a complex mixture of partially
deprotected compounds along with degradation of the
sulfonamide linker was obtained. Basic hydrolyses gave a much
nicer reaction profile, and the selective hydrolysis could be
achieved with sodium hydroxide in methanol at 60 °C. After
completion, concentrated hydrochloric acid was added then acid
35 was extracted with DCM and submitted to a solvent exchange
from DCM to THF. Treating the crude mixture with a 4N
solution of hydrochloric acid in dioxane completed the Boc
removal. Compound 36 was isolated as a dihydrochloride salt
with a quantitative yield but contaminated with 1% of the
corresponding amide 37 which resulted from a partial hydrolysis
of the cyano group and which was difficult to remove at this step.
The selective hydrolysis of the cyano group proved to be
challenging with regards to the stability of both the Boc
protecting group and the sulfonamide linker. Acidic hydrolysis of
Scheme 5. Scalable route to compound 26a.HCl: (i) Trimethylsilylcyanide, methylamine (30% EtOH), AcOH, 60 °C (90%); (ii) Pyridine, DMAP, DCM, (87%);
(iii) NaOH (1M)/MeOH, 60 °C, HCl(37%); (iv) DCM, THF, HCl (4N in dioxane), 40 °C (quantitative over 2 steps); (v) isobutyryl chloride, Et3N, DCM (77%);
(vi) SOCl2, DMF, DCM, 32 °C; NH2OH (50% in EtOH),5-15 °C, AcOEt, HCl (5-6 N in iPrOH) (79%)
Acylation of the free azetidine was performed with isobutyryl
chloride in dichloromethane with triethylamine and provided acid
38. The basic treatment during work-up of this reaction allowed
us to keep acid 38 in the aqueous phase and the amide by-product
39 was extracted from dichloromethane. Acidic treatment of the
aqueous phase induced direct crystallization of 38 in a 77% yield
with a 98% HPLC purity. Formation of the hydroxamic acid 26a
was envisioned by an amide coupling directly from acid 38 and
hydroxylamine but this reaction proved to be much more
complicated than anticipated. The common coupling agents
tested (EDC/HOBT, EDC/HOPO, CDI, T3P) gave complex
mixtures where the expected product was only present at around
40%. Encouraging improvements were achieved when activation
of acid 38 was realized using an acyl chloride. Treatment with
oxalyl chloride gave uncompleted reaction but thionyl chloride in
DCM with a catalytic amount of DMF afforded the acyl chloride
in a clean quantitative manner. To obtain a nice purity profile, the
reaction was performed at 30 °C, but all attempts to get rid of
thionyl chloride excess or to concentrate the reaction mixture
resulted in a partial hydrolysis and acid 38 was always present in
appreciable quantities in the isolated product. Finally we decided
to activate acid 38 with only 0.9 eq. of thionyl chloride at 32 °C,
and this solution was dosed into a cold solution of hydroxylamine
in ethanol. It is interesting to note that in these conditions we
were not able to observe the ethyl ester impurity resulting from
reaction of acyl chloride on ethanol.
induced the crystallization of 26a.HCl in 79% yield with 96%
purity. This batch was in accordance with our internal
specifications for preclinical studies. Development of the
synthetic route is currently underway to realize telescoping of
first steps and avoid isolation of oily amide 33.
3. Conclusion
Having rapidly identified promising lead molecules, the
general strategy put forth by the team for the lead optimisation
was two-pronged: (1) maintaining the excellent potency of β-
sulfonamide hydroxamate 14 while focusing on improving
solubility in PG/EtOH(96%); (2) maintaining the good
potency/solubility balance of α-sulfonamide hydroxamate 15
while identifying core-replacements with no genotoxicity
liability. These efforts led to the identification of compound 26a,
a potent and soluble TACE inhibitor with robust activity in an
oxazolone-induced chronic skin inflammation model. Once
selected for development, a polymorph screen was undertaken
and resulted in the selection of compound 26a.HCl. The key
point of the process development was based on the cyanation of
an imine, followed by a selective hydrolysis of the cyano group
of the resulting α-aminonitrile in the presence of a sulfonamide
linker. The final hydroxamic acid formation was realized by
coupling hydroxylamine with an acyl chloride followed by the
crystallization of the hydrochloride salt of our API. Compound
26.HCl was obtained in 47 % overall yield over 6 steps in 96%
HPLC purity, with a scalable process that resulted in a drastic
lowering of material cost.
In this case, in-process-control of the crude mixture indicated
the formation of a 85/15 mixture of compound 26a and residual
acid 38. Washing with sodium hydrogen carbonate solution
allowed the solubilisation of the residual acid 38 in the aqueous
phase and a final switch from DCM to AcOEt followed by salt
formation with 5-6N solution of hydrochloric acid in isopropanol
4. Experimental section
4.1 Development route for compound 26a.HCl

All solvents and reagents were purchased from the suppliers
1
129.6, 128.8, 127.5, 126.0, 123.8, 123.7, 120.2, 117.3, 115.3,
80.2, 66.8, 58.8, 49.6, 37.7, 27.9, 24.9. ESI-HRMS(m/z) calcd
for C27H31N4O5S+ [M + H]+ 523.2010 found 523.2010
and used without further purification. H NMR and 13C NMR
were recorded on a Bruker Avance 400 MHz, 100 MHz.
Chemical shifts are expressed in parts per million (ppm)
downfield from residual solvent peaks, and coupling constants
are reported in hertz (Hz). Splitting patterns are indicated as
follows: br, broad; s, singlet; d, doublet; dd, doublet of doublet; t,
triplet; q, quartet; m, multiplet. X-ray powder diffraction (XRPD)
analyses were performed by Holodiag using Panalytical X’Pert
Pro MPD X-ray powder diffractometer.
4.1.3. Compound 36: 3-((N-methyl-4-((2-methylquinolin-4-
yl)methoxy)phenyl)sulfonamido)azetidine-3-carboxylic
dihydrochloride.
acid
In a 10L double jacketed reactor tert-butyl 3-cyano-3-((N-
methyl-4-((2-methylquinolin-4-yl) methoxy)phenyl)
4.1.1.
(methylamino)azetidine-1-carboxylate.
Compound
33:
tert-butyl
3-cyano-3-
sulfonamido) azetidine-1-carboxylate (450 g; 0.9 mol; 1.0 eq.)
was solubilized in methanol (4 L) then a 1N solution of NaOH
(2 L) was added and the reaction mixture was stirred 30 hours at
60 °C. The methanol was distilled off until the final volume
reached 2.5 L, then the reaction mixture was shilled to 25 °C, and
37% hydrochloric acid was added (160 mL; 1.9 mol; 2.2 eq.)
dropwise in 30 min at around 25 °C. The organic phase was
separated and the aqueous phase was extracted two times with
dichloromethane (5 L). All the organic phases were combined
and the solvents were swapped with THF and concentrated until
the final volume reached 4 L. Hydrogen chloride (4N in dioxane)
was added (800 mL; 3.2 mol; 3.7 eq.) dropwise to this solution
and the reaction mixture was stirred 3 hours at 55 °C. The
reaction mixture was chilled to 25 °C and the precipitate was
filtered off, washed with THF (3 L) and dried under vacuum at
45 °C to afford 3-((N-methyl-4-((2-methylquinolin-4-
yl)methoxy)phenyl)sulfonamido) azetidine-3-carboxylic acid
dihydrochloride (465 g; 100%). The isolated solid had a water
content of 5%.
In a 10 L double jacketed reactor, tertbutyl-3-oxoazetidine-1-
carboxylate (225 g; 1.3 mol; 1.00 eq.) in MeTHF (1500 mL) are
dissolved, then a 30% w/w methylamine solution in ethanol (430
mL; 3.1 mol; 2.4 eq.) are added dropwise. Acetic acid (113 mL; 2
mol; 1.5 eq.) is added dropwise, the temperature reached 37 °C.
The reaction mixture was stirred 15 min at 40 °C. pH was
controlled in order to be at around pH = 7-8. Trimethylsilyl
cyanide (20 mL; 1.6 mol; 1.20 eq.) was added dropwise and the
resulting mixture was stirred 2 hours at 60 °C. A sample (2 mL)
was taken treated with 1N NaOH solution and extracted with
AcOEt. NMR control showed disappearance of the raw material.
The reaction mixture was chilled to 20 °C, then diluted with THF
(750 mL), washed two times with a 1N NaOH solution (2500
mL), and with water (1500 mL), the organic phase was
concentrated in vacuo to afford tert-butyl 3-cyano-3-
(methylamino)azetidine-1-carboxylate (262 g; 90%) as
yellowish oil.
a
1
NMR H (400 MHz, DMSO-d6): 10.19 (br s, 1H); 9.48 (br s,
1
NMR H (400 MHz, DMSO-d6): 4.1 (br d, J = 8.8 Hz, 2H);
3.75 (d, J = 8.8 Hz, 2H); 3.20 (q, J = 6.0 Hz, 1H); 2.24 (d, J = 6.0
Hz, 3H); 1.37 (s, 9H). NMR 13C (100 MHz, DMSO-d6): 155.5,
119.7, 79.5, 58.7, 49.3, 30.3, 28.0. ESI-HRMS(m/z) calcd for
C10H18N3O2+ [M + H]+ 212.1394 found 212.1395
1H); 8.52 (d, J = 8 Hz, 1H); 8.46 (d, J = 8.0 Hz, 1H); 8.11 (m,
2H); 7.93 (t, J = 7.6 Hz, 1H); 8.52 (d, J = 8.0 Hz, 1H); 7.90 (d, J
= 8.5 Hz, 2H); 7.46 (d, J = 8.5 Hz, 2H); 6.00 (s, 2H); 4.33 (m,
2H); 4.24 (m, 2H); 3.02 (s, 3H); 2.77 (s, 3H); 2.51 (s, 9H). NMR
13C (100 MHz, DMSO-d6): 170.6, 161.2, 158.0, 133.4, 131.1,
129.8, 128.7, 124.9, 123.8, 121.8, 120.5, 115.7, 66.5, 60.5, 51.9,
32.2, 21.1. ESI-HRMS(m/z) calcd for C22H24N3O5S+ [M + H]+
442.1431 found 442.1432
4.1.2. Compound 34: tert-butyl 3-cyano-3-((N-methyl-4-((2-
methylquinolin-4-yl)methoxy) phenyl) sulfonamido)azetidine-1-
carboxylate.
To a 15 L double jacketed reactor tert-butyl 3-cyano-3-
(methylamino)azetidine-1-carboxylate (257 g; 1.2 mol; 1.00 eq.)
was solubilized in dichloromethane (4.00 L) at 25 °C. 4-
dimethylaminopyridine (10 g; 0.08 mol; 0.07 eq.) and pyridine
(492 mL; 6.1 mol; 5.0 eq.) were added. 4-((2-methylquinolin-4-
yl) methoxy) benzenesulfonyl chloride hydrochloride (607 g; 1.6
mol; 1.30 eq.) are added to the reaction mixture and was stirred 6
hours at 30 °C. After completion, a 1N solution of NaOH (5L)
was added followed by NaOH pellets (100 g). The organic phase
was washed with a 1N solution of NaOH (3L) then with a 2N
solution of NH4Cl (5L) and finally with water (5L). The organic
phase was concentrated under vacuum, 3L of dichloromethane
were removed and the solvents were swapped with n-heptane
(9L). The reaction mixture was concentrated until the final
volume reached 2L then diisopropyl ether (3L) was added and
the suspension was stirred overnight. The solid was filtered off,
washed with diisopropyl ether (2L) and dried under vacuum at
45 °C to afford tert-butyl 3-cyano-3-((N-methyl-4-((2-
methylquinolin-4-yl)methoxy)phenyl)sulfonamido)azetidine-1-
carboxylate (555 g; 87%) as a yellow solid.31
4.1.4. Compound 38: 1-isobutyryl-3-((N-methyl-4-((2-
methylquinolin-4-yl)methoxy)phenyl)sulfonamido) azetidine-3-
carboxylic acid.
In a 4L double jacketed reactor, triethylamine (176 mL; 1.3
mol; 3.5 eq.) was added to a suspension of 3-((N-methyl-4-((2-
methylquinolin-4-yl)methoxy)phenyl)sulfonamido) azetidine-3-
carboxylic acid dihydrochloride (200 g; 0.4 mol; 1.0 eq.) in
dichloromethane (1.5 L) at 25 °C. The reaction mixture was
chilled to 8 °C then isobutyryl chloride (45.5 mL; 0.4 mol; 1.2
eq.) was added dropwise while maintaining temperature below
12 °C and stirred 15 min at this temperature. 2 L of a 1N solution
of sodium hydroxide was added. The organic phase was
discarded and the aqueous phase was treated with 37% hydrogen
chloride solution (60 mL), the precipitate was filtered off, washed
with a 9/1 water/isopropanol mixture (1 L) and dried under
vacuum at 45 °C to afford 1-isobutyryl-3-((N-methyl-4-((2-
methylquinolin-4-yl)methoxy) phenyl)sulfonamido) azetidine-3-
carboxylic acid, hydrate (159 g; 77%). The water content of the
solid was measured at 3.6%.
1
NMR H (400 MHz, DMSO-d6): 8.12 (d, J = 8.0 Hz, 1H);
1
NMR H (400 MHz, DMSO-d6): 8.12 (d, J = 7.7 Hz, 1H);
7.99 (d, J = 8.0 Hz, 1H); 7.82 (d, J = 8.5 Hz, 2H); 7.76 (t, J = 7.6
Hz, 1H); 7.60 (t, J = 7.6 Hz, 1H); 7.58 (s, 1H); 7.35 (d, J = 8.5
Hz, 2H); 5.73 (s, 2H); 4.45 (s, 2H); 4.10 (m, 2H); 2.84 (s, 3H);
2.68 (s, 3H); 2.46 (m, 1H); 0.97 (d, J = 7.0 Hz, 3H); 0.94 (d, J =
7.0 Hz, 3H). NMR 13C (100 MHz, DMSO-d6): 175.9, 172.2,
7.99 (d, J = 8.1 Hz, 1H); 7.92 (d, J = 8.5 Hz, 2H); 7.76 (t, J = 7.3
Hz, 1H); 7.60 (t, J = 7.3 Hz, 1H); 7.56 (s, 1H); 7.41 (d, J = 8.5
Hz, 2H); 5.78 (s, 2H); 4.41 (d, J = 9.0 Hz, 2H); 4.23 (d, J = 9.0
Hz, 2H); 2.69 (s, 3H); 2.67 (s, 3H); 1.41 (s, 9H). NMR 13C (100
MHz, DMSO-d6): 162.5, 158.7, 155.6, 147.2, 141.6, 130.9,

161.5, 158.7, 147.4, 141.6, 132.2; 129.6, 129.3, 128.9, 126.0,
123.9, 123.8, 120.3, 115.5, 66.8, 59.0, 57.6, 55.3, 32.8, 29.0,
25.0, 18.8, 18.6. ESI-HRMS(m/z) calcd for C26H30N3O6S+ [M +
H]+ 512.1850 found 512.1849
are generated and stained (H&E) to measure the epidermis
thickness. In parallel, to measure the levels of TNF-α, frozen
samples (8-mm punch biopsy previously stored at -80 °C) are
homogenized using a Tissue Ruptor in ice-cold lysis buffer
supplemented with proteases and TACE inhibitor (Apratastat at
10µM in DMSO). After centrifugation and filtration,
supernatants are used to quantify the selected cytokines with
CBA (Cell Based Assay; BD Biosciences). Cytokine
concentrations are expressed in pg/mg of proteins.
4.1.5. Compound 26a.HCl : N-hydroxy-1-isobutyryl-3-((N-
methyl-4-((2-methylquinolin-4-yl)methoxy)phenyl)sulfonamido)
azetidine-3-carboxamide hydrochloride.
In a 4L double jacketed reactor, a suspension of 1-isobutyryl-
3-((N-methyl-4-((2-methylquinolin-4-yl)methoxy)
phenyl)
sulfonamido) azetidine-3-carboxylic acid hydrate (157 g; 0.3
mol; 1.0 eq.) in dichloromethane (1.6 L) and with N,N-
dimethylformamide (1.1 mL; 0.01 mol; 0.05 eq.) was heated to
32 °C then thionyl chloride (65 mL; 0.9 mol; 3.0 eq.) was added
dropwise. The reaction mixture was stirred 6 hours at 32 °C, and
then the reaction mixture was chilled to 20 °C. In another 4L
double jacketed reactor a 50% hydroxylamine solution in ethanol
(1.1 L) was charged and chilled to 5 °C. To this solution the acyl
chloride solution prepared above was dosed while maintaining
the temperature below 15 °C. The reaction mixture was stirred 16
hours at room temperature and treated with water (1 L). The
aqueous phase was extracted with dichloromethane (1 L). The
organic phases were combined and the solvent was swapped with
AcOEt until the final volume reached 1.5 L. a saturated solution
of NaHCO3 (750 mL) and water (500 mL) were added. The
aqueous phase was extracted five times with AcOEt (1 L). The
organic phases were combined concentrated until the final
volume reached 1L. Ethanol (1L) was added followed by a 5-6 N
hydrogen chloride solution in isopropanol (81 mL; 0.5 mol; 1.5
eq.) was added dropwise. The solid was filtered off, washed with
ethanol (500 mL) and diisopropyl ether (500 mL) and finally
dried under vacuum at 45 °C to afford N-hydroxy-1-isobutyryl-3-
((N-methyl-4-((2-methylquinolin-4-
References and notes
1.
(a)
Belge,
K.;
Brück,
J.;
Ghoreschi,
K.;
http://f1000.com/prime/reports/m/6/4; (b) Mease,P. J.; Goffe, B.
S.; Metz, J.; VanderStoep, A.; Finck, B.; Burge, D. G. Lancet,
2000, 356, 685; (c) Gottlieb, A. B.; Masud, S.; Ramamurthi, R.;
Abdulghani, A.; Romano, P.; Chaudhari, U.; Dooley, L. T.;
Fasanmade, A. A.; Wagner, C. L. J. Am. Acad. Dermatol. 2003,
48, 68; (d) Semble, A. L.; Davis, S. A.; Feldman, S. R. Am. J.
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3.
(a) Horiuchi, K. Keio J. Med. 2013, 62, 29; (b) Vani, T.;
Suchitra, M.; Lakshamana, M. G.; Krantikumar, P. Inter. J.
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(a) DasGupta, S.; Murumkar, P. R.; Giridhar, R.; Yadav, M. R.
Bioorg. Med. Chem. 2009, 17, 444; (b) Murumbar, P. R.;
DasGupta, S.; Chandani, S. R.; Giridhar, R.; Yadav, M. R. Expert
Opin. Ther. Patents 2010, 20, 31; (c) Li, N.-G.; Shi, Z.-H.; Tang,
Y.-P.; Li, W.; Yin, L. Duan, J.-A. Curr. Med. Chem. 2012, 19,
2924; (d) Murumkar, P. R.; Giridhar, R.; Yadav, M. R. Expert
Opin. Drug Discov. 2013, 8, 157.
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5.
Aubert, J.; Carlavan, I.; Voegel, J. WO2009/004247, 08 Jan,
2009.
Clary L, Chambon S, Chantalat L, Mauvais P, Roye O, Schuppli
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Chantalat L, et al. WO2011/001089; 06 Jan, 2011.
6.
7.
Levin, J. I.; Chen, J. M.; Laasko, L. M.; Du, M.; Schmid, J.; Xu,
W.; Cummons, T.; Xu, J.; Jin, G.; Barone, D.; Skotnicki, J. S.
Bioorg. Med. Chem. Lett. 2006, 16, 1605.
yl)methoxy)phenyl)sulfonamido)azetidine-3-carboxamide
(a) Duan, J. J.-W.; Chen, L.; Wasserman, Z. R.; Lu, Z.; Liu, R.-
Q.; Covington, M. B.; Qian, M.; Hardman, K. D.; Magolda, R.
L.; Newton, R. C.; Christ, D.D., Wexler, R. R.; Decicco, C. P. J.
Med. Chem. 2002, 45, 4954; (b) Grootveld, M.; McDermott, M.
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A.; Brogdon, B.; Wu, J.-T.; Liu, R.-Q.; Covington, M. B.; Vaddi,
K.; Newton, R. C.; Fossler, M. J.; Garner, C.E.; Deng, Y.;
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hydrochloride (133 g; 79%) as a white crystalline powder.
Compound 26a was isolated as an ethanol solvate with a content
of 7%. HPLC purity 96%, Acquity column BEH C18 1.7 µm
2.1*150 mm; Flow rate= 0.8 mL/min, MeCN+0.1% formic acid.
1
NMR H (400 MHz, DMSO-d6): 11.00 (s, 1H); 8.44 (d, J =
8.0 Hz, 1H); 8.40 (m, 1H); 8.11 (t, J = 7.6 Hz, 1H) 8.07 (s, 1H);
7.93 (t, J = 7.7 Hz, 1H); 7.84 (d, J = 8.0 Hz, 2H); 7.43 (d, J = 8.0
Hz, 2H), 5.99 (s, 2H), 4.46 (m, 2H); 4.09 (m, 2H); 2.98 (s, 3H);
2.84 (s, 3H); 2.45 (m, 1H); 0.97 (d, J = 7.0 Hz, 3H), 0.94 (d, J =
7.0 Hz, 3H). NMR 13C (100 MHz, DMSO-d6) : 176.1, 165.9,
160.9, 157.9, 151.6, 138.1, 133.6, 131.4, 129.8, 128.8, 124.9,
123.7, 121.4, 120.4, 115.6, 66.4, 58.6, 57.2, 55.1, 33.2, 29.1,
20.8, 18.9, 18.6. ESI-HRMS(m/z) calcd for C26H31N4O6S+ [M +
H]+ 527.1959 found 527.1957
8.
9.
Ouvry, G.; Berton, Y.; Bhurruth-Alcor, Y.; Bonnary, L.; Bouix-
Peter, C.; Bouquet, K.; Bourotte, M.; Chambon, S.; Comino, C.;
Deprez, B.; Duvert, D.; Duvert, G.; Hacini-Rachinel, F.; Harris,
C. S.; Luzy, A.-P.; Mathieu, A.; Millois, C.; Pascau, J.; Pinto, A.;
Polge, G.; Reitz, A.; Revers, K.; Rosignoli, C.; Taquet, N.;
Hennequin, L.F. Bioorg. Med. Chem. Lett. 2017, 27, 1848.
The sulfonamide–quinoline moiety can also be found in WAY-
281418, a preclinical candidate from Wyeth/Pfizer: Levin, J.; Li,
Z.; Diamantidis, G.; Lovering, F. E.; Wang, W.; Condon, J. S.;
Lin, Y.; Skotnicki, J. S.; Park, K. US 2006/0211730; 21 Sept.
2006.
10. Wang, Y.; Papamichelakis, M.; Chew, W.; Sellstedt, J.;
Noureldin, R.; Tadayon, S.; Daigneault, S. Org. Process Res. &
Dev. 2008, 12, 1253-1260.
4.2 Evaluation of 26a in an oxazolone-induced chronic
skin inflammation model
11. Beillard, A.; Bhurruth-Alcor, Y.; Bouix-Peter, C.; Bouquet, K.;
Chambon, S.; Clary, L.; Harris, C. S.; Millois, C.; Mouis, G.;
Ouvry, G.; Pierre, R.; Reitz, A.; Tomas, L. Tetrahedron Lett.,
2016, 57, 2165.
Female SKH1 mice (hairless) receive for 10 days daily topical
applications on the back (~6cm², n=6 per group) of 50 µl of
12. Synthesis and full characterization of all new compounds are
described in the Supplementary Material.
vehicle (Acetone/Citrate buffer), 0.2
% oxazolone alone
dissolved in acetone/citrate buffer or in combination with
increasing concentrations of 26a (0.8-2 %). The clinical score is a
global grading corresponding to the sum of erythema, oedema
and desquamation scores inspired from the Lattice system
physician’s global assessment known as the LS PGA.32 Each
individual score uses a scale from 0 to 4 and is daily assessed
starting from the second day of application up to the last day of
application (day 10). In addition at day 10, histological sections
13. (a) Jain, N.; Yalkowsky, S. H. J Pharm. Sci. 2001, 90, 234-252.
(b) Ran, Y.; Yalkowsky, S. H. J. Chem. Inf. Comput. Sci. 2001,
41, 354-357.
14. We were not able to determine melting points with hydroxamate
compounds as they tended to decompose before melting. In
general, DSC traces were also hard to interpret on these
compounds. This was also the case for another program with
hydroxamic acid molecules.

15. Generally, a minimum of 2 year stability is required for a
formulated product at room temperature.
D.; Trzaskos, J. M.; Newton, R. C.; Decicco, C. P.; Duan, J. J.
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E.; Oesch-Bartlomowicz, B.; Werner, C.; Landsiedel, R. Drug
Metab. Rev. 2007, 39, 659.
23. Tentative
structure
for
the
rearranged
product:
17. Based
on
https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecdtg43
2-508.pdf , a test substance with a PIF < 2 predicts: "no
phototoxicity".
A
PIF >2 and<
5
predicts: "probable
phototoxicity" and a PIF > 5: "phototoxicity”. See Supplementary
Material for more details.
24. 98% of compound 40 was remaining after 1 month at 40° C in
PG/EtOH(96%) whereas only 58% of compound 41 was found
18. (a) Wang, C. Y.; Mutat. Res.1977, 56, 7 ; (b) Rossberger, S. ;
Andrae, U. ; Mutat. Res.1985, 145, 201 ; (c) Ziegler-Skylakakis,
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under the same conditions.
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2015, 19, 646.
20. Full description of the assays can be found in the Supplementary
Material.
21. Schultes, S.; de Graaf, C.; Berger, H.; Mayer, M.; Steffen, A.;
Haaksma, E. E. J.; de Esch, I. J. P.; Leurs, R.; Krämer, O. Med.
Chem. Commun. 2012, 3, 584.
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Santos, E. D.; Du, X.; Khafizova, G.; MacEwan, G.; Niu, C.;
Salaski, E. J.; Zask, A.; Cummons, T.; Sung, A.; Xu, J.; Zhang,
Y.; Xu, W.; Ayral-Kaloustian, S.; Jin, G.; Cowling, R.; Barone,
D.; Mohler, K. M.; Black, R. A.; Skotnicki, J. S. Bioorg. Med.
Chem. Lett. 2003, 13, 2799; (b) Chen, X. T.; Ghavimi, B.;
Corbett, R. L.; Xue, C. B.; Liu, R. Q.; Covington, M. B.; Qian,
M.; Vaddi, K. G.; Christ, D. D.; Hartman, K. D.; Ribadeneira, M.
28. Esaki, Tohru WO2010/126030; 04 Nov, 2010.
29. TMSCN had a toxicity concern, this compound was handled in a
dedicated laboratory with an adapted safety suit and pH of the
reaction mixture was controlled in order to keep a basic pH.
30. Mueller, Urs WO2005/021490; 10 Mar, 2005.
31. Diisopropyl ether is not a preferred solvent but gave good results
for precipitation of compound 34 and 26a. Replacement of this
solvent is anticipated in future development phases.
32. Langley, R. G.; Ellis, C. N. J. Am. Acad. Dermatol.2004, 51, 563.