Synthesis and Biological Evaluation of New Triazolo- and Imidazolopyridine RORγt Inverse Agonists

Article abstract of DOI:10.1002/cmdc.201600500

Retinoic-acid-related orphan receptor γt (RORγt) is a key transcription factor implicated in the production of pro-inflammatory Th17 cytokines, which drive a number of autoimmune diseases. Despite diverse chemical series having been reported, combining high potency with a good physicochemical profile has been a very challenging task in the RORγt inhibitor field. Based on available chemical structures and incorporating in-house knowledge, a new series of triazolo- and imidazopyridine RORγt inverse agonists was designed. In addition, replacement of the terminal cyclopentylamide metabolic soft spot by five-membered heterocycles was investigated. From our efforts, we identified an optimal 6,7,8-substituted imidazo[1,2-a]pyridine core system and a 5-tert-butyl-1,2,4-oxadiazole as cyclopentylamide replacement leading to compounds 10 ((S)-N-(8-((4-(cyclopentanecarbonyl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide) and 33 ((S)-N-(8-((4-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide). Both derivatives showed good pharmacological potencies in biochemical and cell-based assays combined with excellent physicochemical properties, including low to medium plasma protein binding across species. Finally, 10 and 33 were shown to be active in a rodent pharmacokinetic/pharmacodynamic (PK/PD) model after oral gavage at 15 mg kg^?1, lowering IL-17 cytokine production in ex vivo antigen recall assays.

Full text of DOI:10.1002/cmdc.201600500

DOI: 10.1002/cmdc.201600500
Communications
Synthesis and Biological Evaluation of New Triazolo- and
Imidazolopyridine RORgt Inverse Agonists
Samuel Hintermann,[a] Christine Guntermann,[b] Henri Mattes,[a] David A. Carcache,[a]
Juergen Wagner,[a] Anna Vulpetti,[a] Andreas Billich,[b] Janet Dawson,[b] Klemens Kaupmann,[b]
Joerg Kallen,[c] Rowan Stringer,[d] and David Orain*[a]
Retinoic-acid-related orphan receptor gt (RORgt) is a key tran-
scription factor implicated in the production of pro-inflamma-
tory Th17 cytokines, which drive a number of autoimmune dis-
eases. Despite diverse chemical series having been reported,
combining high potency with a good physicochemical profile
has been a very challenging task in the RORgt inhibitor field.
Based on available chemical structures and incorporating in-
house knowledge, a new series of triazolo- and imidazopyri-
dine RORgt inverse agonists was designed. In addition, replace-
ment of the terminal cyclopentylamide metabolic soft spot by
five-membered heterocycles was investigated. From our ef-
forts, we identified an optimal 6,7,8-substituted imidazo[1,2-
a]pyridine core system and a 5-tert-butyl-1,2,4-oxadiazole as
cyclopentylamide replacement leading to compounds 10 ((S)-
N-(8-((4-(cyclopentanecarbonyl)-3-methylpiperazin-1-yl)methyl)-
7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-car-
boxamide) and 33 ((S)-N-(8-((4-(5-(tert-butyl)-1,2,4-oxadiazol-3-
yl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyri-
din-6-yl)-2-methylpyrimidine-5-carboxamide). Both derivatives
showed good pharmacological potencies in biochemical and
cell-based assays combined with excellent physicochemical
properties, including low to medium plasma protein binding
across species. Finally, 10 and 33 were shown to be active in
a rodent pharmacokinetic/pharmacodynamic (PK/PD) model
after oral gavage at 15 mgkgꢀ1, lowering IL-17 cytokine pro-
duction in ex vivo antigen recall assays.
Two isoforms, RORCvar1 (also known as RORg1) and the T-cell-
specific RORCvar2 (RORg2, RORgt) have been identified. RORC-
var2 is considered an attractive drug target based on its in-
volvement in the production of pro-inflammatory Th17 cyto-
kines, such as IL-17, which drive a number of autoimmune dis-
eases.[1–3] Owing to the structural identity of the ligand-binding
domains of the two RORC isoforms, it is highly unlikely that
RORCvar2-specific inhibitors will be identified which do not
also inhibit RORCvar1. A plethora of structurally diverse RORC
inhibitors[4] have been described blocking Th17 differentiation
in vitro, and selected examples have also been reported to in-
hibit inflammation in rodent in vivo models of autoimmune
disease.[2] Successful clinical outcomes with several anti-IL-17
antibodies have validated the IL-17 pathway as a key pathway
for the treatment of inflammation. Recently, Vitae Pharmaceuti-
cals reported a first successful four-week clinical proof-of-con-
cept trial with the RORC inhibitor VTP-43742[5] for the treat-
ment of psoriasis.
In recent years, the vast majority of reported RORgt inverse
agonists use the alkyl aryl sulfone warhead first described by
GlaxoSmithKline (GSK) researchers,[6] including the likely clinical
candidate from Vitae Pharmaceuticals (1, Figure 1). Compound
1 contains an interesting weakly basic center that may confer
some advantageous physicochemical properties. Indeed, com-
bining high potency and good solubility has been a very chal-
lenging task in the RORC inhibitor field. Along the same lines,
in 2014 GSK scientists reported a new class of compounds
Retinoic-acid-related orphan receptor C (RORC) is a nuclear
hormone receptor that plays an important role in immunity.
[a] Dr. S. Hintermann, Dr. H. Mattes, Dr. D. A. Carcache, Dr. J. Wagner,
Dr. A. Vulpetti, Dr. D. Orain
Global Discovery Chemistry, Novartis Institutes for BioMedical Research,
Novartis Campus, 4002 Basel (Switzerland)
E-mail: david.orain@novartis.com
[b] Dr. C. Guntermann, Dr. A. Billich, Dr. J. Dawson, Dr. K. Kaupmann
ATI, Novartis Institutes for BioMedical Research,
Novartis Campus, 4002 Basel (Switzerland)
[c] Dr. J. Kallen
CPC, Novartis Institutes for BioMedical Research,
Novartis Campus, 4002 Basel (Switzerland)
[d] Dr. R. Stringer
MAP, Novartis Institutes for BioMedical Research,
Novartis Campus, 4002 Basel (Switzerland)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600500.
Figure 1. Selected examples of RORgt inverse agonists from the patent liter-
ature: Putative clinical candidates from Vitae Pharmaceuticals (1),[5] GSK (2),
and Pfizer (3).
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(e.g., 2, Figure 1) containing a weakly basic center and claim-
ing in vivo activity after oral dosing.[7] A few months later,
a new patent application from Pfizer disclosed a very similar
series of compounds (e.g., 3, Figure 1), but which lack the
basic center.[8] Herein we present our efforts toward triazolo-
and imidazopyridine central core analogues of compound 2 as
novel RORgt inhibitors with some improved properties.
Buchwald coupling of 17 with dimethoxybenzylamine and sub-
sequent acid-catalyzed deprotection to afford intermediate 18.
For the eastern face, the ester group was reduced to the corre-
sponding alcohol after reduction with lithium aluminum hy-
dride and then converted into the benzylic chloride intermedi-
ate 19 with thionyl chloride. Nucleophilic substitution of 19
with the preformed piperazine amide 20[7a] led to aniline 21,
which was finally acetylated to afford final derivative 22 in
moderate yield (Scheme 2).
The first imidazopyridine system was constructed from com-
mercially or synthetically available precursor 4 bearing a nitro
group on its western face as precursor for the amide nitrogen,
and a bromo group on its eastern face allowing introduction
of the benzylpiperazine motif. Direct cyclization with chloroa-
cetylaldehyde led to formation of the corresponding imidazo-
pyridine 5 in good yield. After nitro group reduction and cou-
pling with 2-methylpyrimidine-5-carboxylic acid, the corre-
sponding amide 7 was obtained. Introduction of the key ben-
zylic piperazine motif was realized via a Suzuki-type coupling
using the preformed trifluoroborate methylpiperazine salt 8.[9]
After Boc group deprotection and amide formation, the first
final derivative 10 was obtained. Aromatic amide cleavage
under basic conditions led to the versatile aniline intermediate
11, allowing exploration of the western part. As an illustrative
example, synthesis of the corresponding methylpyridine amide
12 is described (Scheme 1).
A second imidazopyridine ring system was synthesized
using a completely different approach for introduction of the
western and eastern substituents. Starting from commercial
derivative 13, the corresponding N-oxide 14 was rearranged to
the 2-tert-butylamino intermediate, yielding the aminopyridine
15 after tert-butyl group removal.[10] The imidazopyridine ring
system was constructed as before by cyclization with chloroa-
cetylaldehyde under basic conditions. The aniline as precursor
for the nitrogen amide on the western face was introduced via
Next, synthesis of the triazolopyridine ring system was car-
ried out by starting from commercially available precursor 23,
which was reacted with hydrazine hydrate to yield intermedi-
ate 24. Cyclization with trimethyl orthoformate or trimethyl or-
thoacetate gave access to the corresponding triazolopyridine
intermediates in very high yield, bearing at C2 an H atom for
25 or a methyl substituent for 25a. Following a similar se-
quence as described in Scheme 1, final derivatives 29 and 30
were synthesized. Notably, nitro group reduction proved to be
much more challenging for 25 than for 25a. Only reduction
with iron in acetic acid at room temperature for a short period
of time led to the desired aniline 26 in an acceptable yield
(Scheme 3).
Intermediates 31 or 32 were synthesized according to proce-
dures described in Scheme 1, and all N-oxadiazole tert-butyl
isomers were prepared analogously to well-described pub-
lished procedures,[11] leading to final derivatives 34–36. Synthe-
sis of isoxazole derivative 37 was achieved by cyclization of
the intermediate thioamide with hydroxylamine. Finally, N-pyri-
midine analogue 38 was obtained in moderate yield by nucle-
ophilic aromatic substitution (Scheme 4).
Our in vitro screening cascade started with a biochemical
fluorescence resonance energy transfer (FRET) assay that meas-
ures inhibition of RIP140 co-activator peptide binding to the
Scheme 1. Synthesis of imidazopyridine derivatives 10 and 12. Reagents and conditions: a) chloroacetaldehyde 50 wt% in H2O (4 equiv), 908C, 3 h (crude);
b) Fe (2 equiv), NH4Cl (5.3 equiv), EtOH, reflux, 18 h (crude); c) acid (1.25 equiv), TEA (3 equiv), propylphosphonic anhydride (T3P) 50 wt% in EtOAc (1.5 equiv),
CH2Cl2, RT, 18 h (42% yield over three steps); d) compound 8 (1.2 equiv), Pd(OAc)2 (0.05 equiv), X-Phos (0.1 equiv), Cs2CO3 (2.5 equiv), THF/H2O (10:1), 908C,
40 h (79% yield); e) 4m HCl in dioxane, CH2Cl2, RT, 2 h (quant.); f) cyclopentanecarboxylic acid (1 equiv), DIPEA (5 equiv), T3P 50 wt% in EtOAc (1.2 equiv),
CH2Cl2, RT, 18 h (72% yield); g) NaOH 1m (3 equiv), MeOH, 1208C, 6 h; h) 6-methylnicotinic acid (1.1 equiv), T3P 50% in EtOAc (1.3 equiv), TEA (3 equiv),
CH2Cl2, RT, 48 h (28% yield over two steps).
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Scheme 2. Synthesis of imidazopyridine derivative 22. Reagents and conditions: a) mCPBA (1.2 equiv), CH2Cl2, RT, 48 h (83% yield); b) tBuNH2 (7 equiv), Ts2O
(3.3 equiv), CH2Cl2/PhCF3 (4/1), 08C, 1 h (68% yield); c) TFA, CH2Cl2, 708C, 12 h (74% yield); d) chloroacetaldehyde 50 wt% in H2O (5 equiv), NaHCO3 (2 equiv),
MeOH, 708C, 24 h (70% yield); e) 2,4-dimethoxybenzylamine (1.5 equiv), BrettPhos precat (0.2 equiv), Cs2CO3 (3 equiv), dioxane, 708C, 2 h (45% yield); f) 4m
HCl dioxane, RT, 1 h (98% yield); g) LiAlH4 2m in THF (1 equiv), THF, 08C, 10 min (69% yield); h) SOCl2 (10 equiv), dioxane, RT, 12 h (59% yield); i) 20
(1.1 equiv), DIPEA (3.5 equiv), CH3CN, 708C, 16 h (99% yield, 78% purity); j) 6-methylnicotinoyl chloride (1.1 equiv), DIPEA (4 equiv), CH2Cl2, RT, 12 h (8% yield).
Scheme 3. Synthesis of triaziolopyridines 29 and 30. Reagents and conditions: a) hydrazine hydrate (30 equiv), dioxane, 108C, 15 h (90% yield); b) trimethyl or-
thoformate (10 equiv), reflux, 1 h (97% yield); c) trimethyl orthoacetate (9 equiv), reflux, 2 h (90% yield); d) Fe (5 equiv), AcOH, RT, 3 h (50% yield); e) Fe
(2 equiv), NH4Cl (5 equiv), MeOH, reflux, 18 h (42% yield, 75% purity); f) 6-methylnicotinoyl chloride (1.3 equiv), pyridine (3 equiv), CH2Cl2, RT, 12 h (44% yield
for 27 and 64% yield for 27a); g) compound 8 (1.2 equiv), Pd(OAc)2 (0.05 equiv), X-Phos (0.1 equiv), Cs2CO3 (2.5 equiv), THF/H2O (10:1), 908C, 18 h (23% yield
for 28 and 69% yield for 28a); h) 1. 4m HCl in dioxane, CH2Cl2, RT, 16 h (quant.), then 2. cyclopentanecarbonyl chloride (1 equiv), DIPEA (5 equiv), CH2Cl2,
08C, 30 min (11% yield for 29 and 80% yield for 30).
human RORgt ligand binding domain (LBD). To demonstrate
cellular potency against RORgt, a Gal4 reporter gene assay was
established using Jurkat cells stably expressing constructs con-
sisting of the Gal4 DNA binding domain fused to the RORg-
hinge-LBD and Gal4 upstream activator sequences driving tran-
scription of the luciferase firefly gene. Next, the compounds
were assessed for their ability to inhibit IL-17A production by
primary human CD4 Tcells under conditions that favor Th17
differentiation. To determine whether RORgt inverse agonists
are potent in the presence of blood cells and plasma proteins,
20% human whole blood was stimulated with lectin concana-
valin A (ConA) and IL-23, and inhibition of IL-17A secretion was
measured by enzyme-linked immunosorbent assay (ELISA), the
results of which are listed in Table 1.
Compound 2 proved to be a very potent RORgt inverse ago-
nist across all assays tested. From its physicochemical profile, 2
showed moderate solubility at neutral pH and relatively low
stability in human liver microsomes (HLM), whereas the com-
pound was more stable when incubated with rat liver micro-
somes (RLM). In vitro and in vivo metabolism studies identified
the cyclopentyl ring as the main weak spot that was prone to
oxidation. First, however, we turned our attention to modifica-
tions of the central phenyl core in order to improve the physi-
cochemical properties of the scaffold. Based on modeling,
[6,5]-fused heterocyclic systems were assessed. We decided to
keep as one exit vector the basic nitrogen atom of the pipera-
zine motif in contrast to compound 3. The first analogues de-
signed were based on a 6,7,8-substituted imidazo[1,2-a]pyri-
dine core, and derivatives 10 and 12 were synthesized. Com-
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Scheme 4. Synthesis of N-heteroarylpiperazine imidazopyridines 35–38. Reagents and conditions: a) BrCN, DIPEA, CH2Cl2, 08C, 1 h; b) H2NOH, EtOH, 908C,
2.5 h; c) (tBuCO)2O, pyridine, 1208C, 3 h (overall yield 23% for 33 and 45% for 34); d) tBuC(NH)NHOH, ZnCl2, EtOH, 4 days; e) 4m HCl, EtOH, 808C, 24 h (13%
overall yield); f) CuBr2, tBuONO, MeCN, RT, 2 h (81% yield); g) compound 40, K2CO3, DMF, 908C, 16 h (31% yield); h) tBuCOCH2C(=S)SMe, TEA, EtOH, 1008C,
20 h (25% yield); i) H2NOH, EtOH, reflux, 28 h (26% yield); j) 4-bromo-2-methylpyrimidine, DIPEA, butanol, 1208C, 11 h (9% yield).
Table 1. In vitro pharmacological and physicochemical profile of compounds 2, 10, 12, 22, 29, 30, and 33–38.
Compd
IC50 [nm] (inhib. [%])[a,b]
HT-Sol.
cLogP
MDCK Papp A!B
LM CLint [mL(minmg)ꢀ1
Rat Human
]
[g]
FRET[c]
Gal4[d]
hu-Th17[e]
hu-WB[f]
[mgLꢀ1
]
[10ꢀ6 cmsꢀ1
]
2
1 (99)
13 (97)
10 (98)
3 (94)
15 (94)
37 (95)
2.3 (99)
3 (97)
32 (97)
324 (97)
194 (97)
162 (96)
2147 (95)
7271 (86)
164 (97)
100 (97)
303 (97)
1166 (97)
110 (97)
NT
13 (81)
171 (78)
73 (80)
177 (79)
NT
35 (89)
168 (83)
171 (83)
207 (82)
NT
57
127
443
48
367
12
343
119
276
>504
148
>471
3.5
2.0
2.9
2.9
1.9
2.2
2.6
3.6
2.6
1.8
4.0
2.7
11.7
15.7
16.4
12.1
NT
16.6
18.3
4.5
19
4.7
9.4
5.5
33
30
75
97
30
58
168
<25
36
96
171
69
<25
94
61
10
12
22
29
30
33
34
35
36
37
38
453
<25
148
<25
41
<25
<25
49
NT
NT
124 (78)
71 (79)
NT
NT
101 (82)
NT
282 (82)
161 (83)
NT
NT
331 (86)
NT
10 (98)
23 (96)
1 (98)
893 (87)
<25
[a] Values are the mean of at least three independent measurements. [b] In parentheses: maximum inhibition at 10 mm. [c–f] See the Experimental Section
for assay descriptions. [g] Solubility at pH 6.8. NT: Not tested.
pound 10, with the 4-methylpyrimidine amide, showed rela-
tively weak potency in the FRET and Gal4 assays, with IC50
values of 13 and 324 nm, respectively. Surprisingly, almost iden-
tical potencies, around 170 nm, were observed in the human
Th17 assays with or without the addition of blood. Introduc-
tion of this imidazo[1,2-a]pyridine central core decreases the
lipophilicity by 1.5 log units relative to 2 and consequently the
solubility was improved while keeping the high permeation as
measured in the MDCK permeability assay. In addition, the
new core nicely increases the stability in the liver microsome
assays, particularly for the HLM assay. The analogous 4-methyl-
pyridine amide 12 showed a similar profile to that of 10, but
was less stable when incubated with RLM and HLM.
with 12. Despite being more potent in the biochemical read-
out, 22 showed the same pharmacological profile as 12 in the
cellular assays. This regioisomer also showed lower solubility
and much higher instability toward RLM and HLM. Therefore,
we decided to keep the nitrogen architecture of the first imi-
dazopyridine isomer constant. By adding another nitrogen
atom, a corresponding triazolopyridine system was synthesized
with either hydrogen 29 or a methyl substituent 30 attached
to the triazole ring. Despite being nearly equipotent in the bio-
chemical assay, in the 10 nm range, 29 was almost 10 times
less potent in the Gal4 cellular assay than the imidazopyridine
analogue 12. The reason for this discrepancy is not under-
stood, as no clear change in permeation properties were ob-
served. Whilst this core change had a negative impact on cell
potency, it was rather beneficial for microsomal stability, with
clearance in both RLM and HLM below the level of detection.
Next, a second regioisomer of the 5,6,7-substituted imida-
zo[1,2-a]pyridine system was studied. The analogous 4-methyl-
pyridine amide 22 was synthesized as described and compared
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The methyl triazole analogue 30 showed decreased potency in
the biochemical and Gal4 assays pointing toward steric inter-
ference with the binding pocket. In summary, the first round of
optimization led to the discovery of a new heterocyclic central
core with improved solubility and human microsomal stability
whilst keeping good on-target potency.
zoles 33 and 35 showed higher biochemical and cellular po-
tency than the 1,3,4-oxadiazole analogue 36. Notably, com-
pound 36 showed the highest microsomal stability. Between
the two 1,2,4-oxadiazole isomers, oxadiazole 33 with the tert-
butyl group at position 5 was more potent than analogue 35
with the tert-butyl group at position 3. The two isomers have
similar shape but different electronic distribution. The nitrogen
atoms in the oxadiazoles are strong hydrogen bond acceptors,
whereas the oxygen atoms are much weaker.[14] In compound
33 the nitrogen adjacent to the oxygen is believed to overlap
with the carbonyl group of 10, thus better mimicking the key
C=O···H479 hydrogen bond interaction. The extra nitrogen
ortho to the tert-butyl group is not needed for affinity (i.e.,
compare 33 with 37). Assuming a similar overlay, compound
35 has an oxygen atom, a weaker hydrogen bond acceptor, at
the position required to make the hydrogen bond with H479,
thus being less potent. The two corresponding analogues 33
and 34, bearing methyl pyrimidine and methyl pyridine amide,
respectively, were shown to be 5 to 6 times more potent bio-
chemically than their respective cyclopentylamides 10 and 12.
Unfortunately, this higher biochemical potency did not trans-
late into improved cellular potency. Despite increased stability
in RLM, the compounds were less stable in HLM. From 34, the
corresponding isoxazole 37 again showed higher biochemical
potency with respect to 12 and a similar loss in the cellular
assays despite increased permeation. Finally, six-membered
heterocycles to replace the cyclopentylamide were not tolerat-
ed, as illustrated with compound 38.
An X-ray structure of 10 bound to RORgt was obtained at
1.77 ꢁ resolution using the RORgt (264–491) construct, that is,
with the C-terminal helix 12 deleted (Figure 2). The two nitro-
Figure 2. X-ray structure (1.77 ꢁ resolution) of the ligand binding domain of
RORgt (carbon atoms in yellow, nitrogen atoms in blue, oxygen atoms in
red, and sulfur atoms in brown) bound to compound 10 (carbon atoms in
cyan), zoomed in at the ligand binding pocket. Selected water molecules
and hydrogen bond interactions are shown in white, and a selected van der
Waals interaction in yellow. Details of this X-ray structure determination will
be published elsewhere; the coordinates have been deposited in the RCSB
Protein Data Bank (PDB ID: 5M96).
To monitor cellular off-target inhibition and/or cytotoxicity
induced by the compounds, an assay measuring IL-3-induced
proliferation of mouse bone marrow cells was performed as
previously described.[15] Representatives of this compound
series did not show any antiproliferative/cytotoxic effects in
the mouse bone marrow assay when tested at a maximal con-
centration of 10 mm.
gen atoms of the methylpyrimidine group make water-mediat-
ed hydrogen bonds to residues R367, L287, and E379, while
the adjacent amide NH group makes a hydrogen bond with
the carbonyl of the backbone of F377. The imidazopyridine
group is located in a buried pocket formed by M365, V376,
L400, F401, and S404. The methylpiperazine group makes
van der Waals contacts with C320, H323, M365, and F388, and
the adjacent C=O group of 10 accepts a hydrogen bond from
Ne2-H479. The terminal cyclopentyl group would clash with
W317 if helix 12 is in the agonist position. The inverse agonism
of 10 is therefore mediated by a ‘push–pull’ mechanism (‘push’
on W317, ‘pull’ on H479) similar to previous reports.[12]
Compounds 10 and 33 were selected for advanced profiling.
Both compounds showed no significant inhibition in a repre-
sentative panel of 30 targets from different classes (all IC50 >
10 mm) and good selectivity over RORb (IC50: >10 and 2.7 mm
for 10 and 33, respectively) and RORa (IC50 >10 mm) in bio-
chemical assays. Introduction of the imidazopyridine core led
to lower plasma protein binding (PPB). For instance, 10 and 33
showed free fractions in a rat/human PPB assay of 25.6/31.9%
and 10.4/13.8%, respectively, compared with 1.9/3.1% for 2
and <1/<1% for 1.
The pharmacokinetic profiles of 10 and 33 were then as-
sessed in rat (Table 2). Both compounds showed similar profiles
with moderate to low volumes of distribution (VSS) and short
MRT and tmax. Compound 33 showed a lower clearance rate
than 10 which may be attributed to the switch from a cyclo-
pentyl to a tert-butyl side chain, which is less prone to oxida-
tive metabolism. In addition, 33 showed a greater oral bioavail-
ability. When free drug exposure was taken into account, 10
and 33 showed similar free AUC and Cmax values. Both com-
pounds were therefore further tested in a rat PK/PD model.
To generate antigen-specific cells in vivo, a delayed-type hy-
persensitivity (DTH) study was performed. Female Lewis rats
In a second round of analogue design, we focused on po-
tential modifications of the cyclopentylamide, which might be
a place for oxidative metabolism as observed for 2. The corre-
sponding N-Boc piperazine analogues showed biochemical po-
tency nearly identical to that of the N-cyclopropylamide piper-
azine (data not shown), but were metabolically less stable. The
tert-butyloxadiazole system is a very well-known stable bioisos-
tere of the Boc group,[13] and the three possible regioisomers
33, 35, and 36 were synthesized on the newly identified 6,7,8-
substituted imidazo[1,2-a]pyridine system. The 1,2,4-oxadia-
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and in vivo. This new imidazopyridine scaffold showed de-
creased plasma protein binding across species leading to
higher free fractions. Compounds 10 and 33 had good oral PK
and decreased IL-17A production at low doses in vivo in a PK/
PD experiment. We believe that such compounds are excellent
tool compounds to further explore the in vivo biology around
RORgt inhibition. Indeed, more data from in vivo pharmacolo-
gy studies using 10 will be published in a separate paper.
Table 2. Rat PK parameters of compounds 10 and 33.
Parameter
10
33
Dose (i.v./p.o.) [mgkgꢀ1
]
1.0/3.0
48ꢁ6
1.0/3.0
26
2.1
1.3
1.4
CL [mLminꢀ1·kgꢀ1
]
V
SS [Lkgꢀ1
]
3.0ꢁ1.5
4.5ꢁ2.6
1.1ꢁ0.7
188ꢁ26
44ꢁ11
23ꢁ4
t1/2 [h]
MRT [h]
AUC i.v. d.n. free [nmh][a]
AUC p.o. d.n. free [nmh][a]
F [%]
132
58 ꢁ12
44ꢁ9
23ꢁ7
0.5ꢁ0.0
89.6/86.2
Cmax d.n. free [nm][a]
tmax [h]
27ꢁ8
Experimental Section
0.3ꢁ0.0
74.4/68.8
General: Chemicals were purchased from commercial sources and
were used without further purification. Reactions were magnetical-
ly and mechanically stirred and monitored by thin-layer chroma-
tography (TLC) on Merck silica gel 60F254 glass plates (visualized by
UV fluorescence at l=254 nm) or analytical Waters Acquity UPLC
instrument equipped with PDA detector, Waters Acquity SQD mass
spectrometer and Waters Acquity HSS T3 1.8 mm 2.1ꢂ50 mm
column. Peak detection is reported at full scan 210–450 nm eluting
with a gradient composed of water/0.05% formic acid/3.75 mm
ammonium formate and acetonitrile/0.05% formic acid. Mass spec-
trometry results are reported as the ratio of mass over charge. The
PPB rat/human [%]
[a] d.n.: dose normalized.
were sensitized with 100 mg methylated bovine serum albumin
(BSA) in complete Freund’s adjuvant (DIFCO). Fourteen days
after sensitization, rats were challenged with 10 mL methylated
BSA into the right ear and with 10 mL 5% glucose into the left
ear. Compounds 10 and 33 were dosed twice daily by oral
gavage starting just before the antigen challenge until the end
of the study (50 h, necropsies were carried out 2 h post-final
dose). Cells were harvested from the draining popliteal lymph
nodes and were used in ex vivo recall assays to assess IL-17A
pathway inhibition by the compounds. Cells were seeded into
96-well microtiter plates (4ꢂ105 cells per well) and antigen-
specific Tcells were re-stimulated with methylated BSA
(50 mgmLꢀ1) or with the lectin ConA and incubated for 72 h.
Supernatants were collected, and IL-17A cytokine concentra-
tions were determined by ELISA (Table 3).
1
purity of new compounds was >95%, as determined by H NMR
and LC–MS after chromatography. Flash column chromatography
was performed with Biotageꢃ SNAP silica gel cartridges, eluting
with distilled technical-grade solvents on an Isolera One apparatus
from Biotage. NMR data were recorded on a Bruker spectrometer
operating at 600 MHz. Chemical shifts (d) are reported in ppm. The
data are reported as: s=singlet, d=doublet, t=triplet, q=quadru-
plet, m=multiplet, and bs=broad singlet.
8-Bromo-7-methyl-6-nitroimidazole[1,2-a]pyridine 5: A mixture
of 3-bromo-4-methyl-5-nitropyridin-2-amine 4 (27.4 g, 118 mmol)
and 2-chloroacetyl aldehyde (50 wt% in water, 74.2 g, 4 equiv) was
stirred at 908C for 3 h. The mixture was cooled to RT and diluted
with 100 mL of MeOH before the volatiles were removed in vacuo.
To the residue, 100 mL of CH2Cl2 were added and the mixture was
stirred for few minutes. The resulting precipitate was filtered off,
washed with CH2Cl2 and after high vacuum drying, the title com-
pound was obtained as a beige solid (33 g, quant., 93% purity)
which was used without further purification. MS (m/z): 256.2/258.2
[M+H]+; 1H NMR (600 MHz, [D6]DMSO): d=9.86 (s, 1H), 8.37 (s,
1H), 8.01 (s, 1H), 2.68 ppm (s, 3H).
Table 3. PK/PD: ex vivo IL-17 inhibition.
10[a]
33[a]
ConA stimulation:
inhibition of IL-17A production [%]
mBSA stimulation:
inhibition of IL-17A production [%]
Blood-free exposure at 2 h [nm]
ꢀ62
ꢀ71
ꢀ77
ꢀ81
481ꢁ103
149ꢁ20
8-Bromo-7-methylimidazo[1,2-a]pyridin-6-amine 6: A mixture of
[a] Tested at 15 mgkgꢀ1 p.o.
8-bromo-7-methyl-6-nitroimidazole[1,2-a]pyridine
5
(33 g,
129 mmol), iron powder (14.39 g, 2 equiv) and ammonium chloride
(36.5 g, 5.3 equiv) in 650 mL of EtOH was held at reflux for 18 h.
Additional iron powder was added, and the mixture was held at
reflux until the starting material was fully consumed. Upon com-
pletion, the mixture was cooled down and the volatiles were re-
moved in vacuo. The crude residue was partitioned between 1 L of
CH2Cl2/MeOH (95:5) and 200 mL of a 5% aqueous NaHCO3 solu-
tion. The organic phase was separated and the aqueous phase was
further extracted three times with 300 mL of CH2Cl2/MeOH (95:5).
The organic phases were combined, dried over sodium sulfate and
concentrated in vacuo to afford the crude title compound (18.8 g,
43.1%, 69% purity) which was used without further purification.
Compounds 10 and 33 were found to be potent inhibitors
of IL-17A production by ConA or after methylated BSA stimula-
tion of draining lymph node cells. Notably, free exposure of 10
at 2 h was over-proportional to that expected based on PK
data, whereas 33 was in the expected range. Measured free
drug exposures are in line with the IC50 values from the in vitro
cellular assays for the observed PD effect with a decrease in IL-
17A production.
In conclusion, we have developed a new series of RORgt in-
verse agonists based on an imidazopyridine core showing
good pharmacological potencies, physicochemical profiles, and
excellent selectivity as illustrated by compound 10. In addition,
replacement of the cyclopentylamide by a tert-butyloxadiazole
led to inhibitor 33 with increased rat metabolic stability in vitro
1
MS (m/z): 226.2/228.2 [M+H]+; H NMR (600 MHz, [D6]DMSO): d=
7.91 (s, 1H), 7.86 (s, 1H), 7.49 (s, 1H), 5.83–4.39 (m, 2H), 2.34 ppm
(s, 3H).
N-(8-Bromo-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimi-
dine-5-carboxamide 7: To a mixture of 8-bromo-7-methylimida-
&
ChemMedChem 2016, 11, 1 – 10
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Communications
zo[1,2-a]pyridin-6-amine 6 (8.0 g, 35.4 mmol), 2-methylpyrimidine-
5-carboxylic acid (6.11 g, 1.25 equiv) and triethylamine (10.74 g,
3 equiv) in 250 mL CH2Cl2, propylphosphonic anhydride (T3P;
50 wt% in EtOAc, 33.8 g, 1.5 equiv) was added dropwise. The re-
sulting mixture was stirred at RT for 18 h. To complete the reaction,
2-methylpyrimidine-5-carboxylic acid (3 g), triethylamine (10 mL)
and T3P (50 wt% in EtOAc, 15 mL) were added and the mixture
was stirred for another 18 h at RT. Saturated NaHCO3 aqueous solu-
tion was added to the reaction mixture. The resulting insoluble
material was filtered off, washed with water and after high vacuum
drying, 6.9 g of the title compound were obtained as a beige solid.
The organic phase was dried over sodium sulfate and the solvents
were removed in vacuo, the crude residue was purified by flash
chromatography eluting with CH2Cl2/MeOH (100:0 to 80:20) to
afford 1.08 g of the title compound. Compound 7 was obtained as
a beige solid with a total recovery of 7.98 g (59% yield). MS (m/z):
346.1/348.1 [M+H]+; 1H NMR (600 MHz, [D6]DMSO): d=10.54 (s,
1H), 9.23 (s, 2H), 8.74 (s, 1H), 8.09 (s, 1H), 7.62 (s, 1H), 2.74 (s, 3H),
2.40 ppm (s, 3H).
tylmethyl ether and after high-vacuum drying, the titled com-
pound was isolated as a white powder (3.91 g, 90% yield) and was
used without further purification.
2) (S)-2-Methyl-N-(7-methyl-8-((3-methylpiperazin-1-yl)methyl)i-
midazo[1,2-a]pyridin-6-yl)pyrimidine-5-carboxamide hydrochlo-
ride salt (3.91 g, 7.44 mmol), cyclopentanecarboxylic acid (1.02 g,
1.02 equiv) and triethylamine (7.26 mL, 7 equiv) were dissolved in
100 mL of CH2Cl2 and then, T3P (50 wt% in EtOAc, 5.9 g, 1.2 equiv)
was added dropwise. The resulting mixture was stirred at RT for
18 h and then washed with saturated NaHCO3 aqueous solution
twice. The aqueous phases were combined and extracted with
CH2Cl2. The organic phases were combined, washed with brine,
dried over sodium sulfate and concentrated in vacuo to afford
a light-yellow foam. The crude compound was purified by flash
chromatography eluting with CH2Cl2/MeOH (99:1 to 90:10) to
afford the titled compound as an off-white solid (2.6 g, 72.7%
1
yield). MS (m/z): 476.4 [M+H]+; H NMR (600 MHz, [D6]DMSO): d=
10.29 (s, 1H), 9.22 (s, 2H), 8.64 (s, 1H), 7.92 (s, 1H), 7.53 (s, 1H),
4.53 (bs, 0.5H), 4.17 (m, 1H), 4.0–3.85 (m, 2H), 3.71 (d, 0.5H), 3.10
(t, 0.5H), 2.90 (m, 1H), 2.75–2.55 (m, 5.5H), 2.37 (s, 3H), 2.17 (m,
1H), 1.98 (m, 1H), 1.85–1.45 (m, 8H), 1.17 and 1.05 ppm (d, 3H).
Potassium (S)-((4-(tert-butoxycarbonyl-3-methylpiperazin-1-yl)-
methyl)trifluoroborate 8: A mixture of potassium (bromomethyl)-
trifluoroborate (30 g, 139 mmol) and (S)-1-N-Boc-2-methylpipera-
zine (31.4 g, 1.05 equiv) in 150 mL of THF was held at reflux under
nitrogen atmosphere for 18 h. The mixture was cooled to RT and
the solvent was removed in vacuo. The residue was taken up in
900 mL of acetone and potassium carbonate (41.3 g, 2 equiv) was
added. The resulting mixture was stirred at RT for 18 h. The precipi-
tate was filtered off, washed with acetone and after high vacuum
drying, the title compound was obtained as a white powder
(42.05 g, 88% yield) and was used without further purification. MS
(m/z): 280.2/281.2/282.2 [MꢀH]ꢀ; 1H NMR (600 MHz, [D6]DMSO,
1008C): d=4.14 (m, 1H), 3.72 (m, 1H), 3.17–2.96 (m, 3H), 2.47–2.16
(bm, 2H), 1.65 (bs, 2H), 1.42 (s, 9H), 1.20 (d, 3H).
(S)-N-(8-((4-(5-(tert-Butyl)-1,2,4-oxadiazol-3-yl)-3-methylpipera-
zin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methyl-
pyrimidine-5-carboxamide 33: 1) (S)-N-(8-((4-cyano-3-methylpi-
perazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-meth-
ylpyrimidine-5-carboxamide. At 08C, to a solution of (S)-2-methyl-
N-(7-methyl-8-((3-methylpiperazin-1-yl)methyl)imidazo[1,2-a]pyri-
din-6-yl)pyrimidine-5-carboxamide hydrochloride salt (2.84 g,
5.05 mmol) in 50 mL of CH2Cl2, DIPEA (3.26 g, 5 equiv) was added
followed by BrCN (3m in CH2Cl2, 1.68 mL, 1 equiv). The mixture
was stirred at RT for 1 h and then quenched by addition of brine.
The organic phase was separated, dried over sodium sulfate and
concentrated in vacuo to afford the titled compound as a crude
beige oil (2.05 g, quant.) which was used without further purifica-
tion.
(S)-tert-Butyl-2-methyl-4-((7-methyl-6-(2-methylpyrimidine-5-car-
boxamido)imidazo[1,2-a]pyridin-8-yl)methyl)piperazine-1-car-
boxylate 9: To a degassed solution of N-(8-bromo-7-methylimida-
zo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide 7 (3.63 g,
10.49 mmol), potassium (S)-((4-(tert-butoxycarbonyl-3-methylpiper-
azin-1-yl)methyl)trifluoroborate 8 (6.72 g, 2 equiv), X-Phos (750 mg,
0.15 equiv) and cesium carbonate (10.25 g, 3 equiv) in 60 mL THF/
water (9:1), palladium acetate (177 mg, 0.075 equiv) was added
and the mixture was then stirred under argon atmosphere at 808C
for 40 h. The mixture was diluted with EtOAc and washed with sa-
turated NaHCO3. The organic phase was separated, dried over
sodium sulfate and the resulting crude residue was purified by
flash chromatography eluting with CH2Cl2/MeOH (99:1 to 90:10) to
afford the titled compound as a light-yellow solid (3.98 g, 79%
2) (S)-N-(8-((4-(N-Hydroxycarbamimidoyl)-3-methylpiperazin-1-
yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimi-
dine-5-carboxamide. A mixture of S)-N-(8-((4-cyano-3-methylpiper-
azin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyri-
midine-5-carboxamide (2.05 g, 5.05 mmol) and hydroxylamine
(50 wt% in water, 1.33 g, 4 equiv) in 50 mL of EtOH was held at
reflux for 2 h. The mixture was concentrated to dryness in vacuo to
afford a crude brown oil (2.2 g) which was used without further
purification in the next step.
3) (S)-N-(8-((4-(5-(tert-Butyl)-1,2,4-oxadiazol-3-yl)-3-methylpiper-
azin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methyl-
pyrimidine-5-carboxamide 33. A mixture of the crude (S)-N-(8-((4-
(N-hydroxycarbamimidoyl)-3-methylpiperazin-1-yl)methyl)-7-meth-
ylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide
(2.2 g, 5.05 mmol), pivalic anhydride (3.76 g, 4 equiv) and trimethyl-
amine (3.26 g, 5 equiv) in 100 mL of CH2Cl2 was stirred at RT for 2 h
and then held at reflux for 16 h. The mixture was washed with sa-
turated aqueous NaHCO3 solution twice. The aqueous phases were
combined and extracted with CH2Cl2. The organic phases were
combined, washed with brine, dried over sodium sulfate and con-
centrated in vacuo to afford the crude compound which was puri-
fied by flash chromatography eluting with CH2Cl2/MeOH (99:1 to
90:10) to afford the title compound as an off-white solid (1.04 g,
40.9% yield). MS (m/z): 504.4 [M+H]+; 1H NMR (600 MHz,
[D6]DMSO): d=10.26 (s, 1H), 9.20 (s, 2H), 8.63 (s, 1H), 7.91 (s, 1H),
7.52 (s, 1H), 3.94 (q, 3H), 3.48 (d, 1H), 3.15 (d, 1H), 3.10–2.96 (m,
1
yield). MS (m/z): 480.4 [M+H]+; H NMR (600 MHz, [D6]DMSO): d=
10.29 (s, 1H), 9.22 (s, 2H), 8.63 (s, 1H), 7.91 (s, 1H), 7.53 (s, 1H),
4.07 (bs, 1H), 3.91 (m, 2H), 3.64 (m, 1H), 2.86 (m, 1H), 2.73 (s, 3H),
2.62 (m, 2H), 2.36 (s, 3H), 2.20 (m, 1H), 2.00 (m, 1H), 1.39 (s, 9H),
1.08 ppm (2 s, 3H).
(S)-N-(8-((4-(Cyclopentanecarbonyl)-3-methylpiperazin-1-yl)-
methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-
5-carboxamide 10: 1) (S)-2-methyl-N-(7-methyl-8-((3-methylpi-
perazin-1-yl)methyl)imidazo[1,2-a]pyridin-6-yl)pyrimidine-5-car-
boxamide hydrochloride salt. To a solution of (S)-tert-butyl-2-
methyl-4-((7-methyl-6-(2-methylpyrimidine-5-carboxamido)imida-
zo[1,2-a]pyridin-8-yl)methyl)piperazine-1-carboxylate
9
(3.97 g,
8.28 mmol) in 70 mL of CH2Cl2, 4m HCl in dioxane (20.7 mL,
10 equiv) was added. The resulting mixture was stirred at RT for
2 h. The resulting precipitate was filtered off, washed with tert-bu-
ChemMedChem 2016, 11, 1 – 10
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
1H), 2.71 (s, 4H), 2.64 (d, 1H), 2.37 (s, 3H), 2.19 (dt, 1H), 1.30 (s,
9H), 1.11 ppm (d, 3H).
perimentation guidelines and laws laid down by the Swiss Federal
and Cantonal Authorities, and specifically as described in Basel-
Stadt Experimental License No. 1244.
TR-FRET assay: The activity of test compounds to displace
a RIP140 co-activator derived biotinylated peptide from the RORg
ligand binding domain was measured. The assay mixture (16 mL)
included 5 nm recombinant His6-tagged human RORg ligand bind-
ing domain comprising amino acids 264–518, 90 nm biotinylated
RIP140 co-activator peptide (Biotin-NH-Ahx-NSH QKV TLL QLL LGH
KNE EN-CONH2, 0.45 nm Cy5-labelled streptavidin, and 1.5 nm euro-
pium-labeled anti-His6 antibody. After incubation of assay plates
for 1 h at room temperature, donor (615 nm) and acceptor
(665 nm) emission values were recorded using an Envision 2102
multilabel reader. Concentration–response curves for IC50 determi-
nations were constructed from the 665/615 nm emission ratio
using the GraphPad Prism software package.
Acknowledgements
We thank Engin Tasdelen, Alex Bussenault, Ralf Boesch, Richard
Felber, Celine Dubois, Florence Ecoeur, Jessica Weiss, Julia Riker,
Bernhard Jost, Yves Henriquez, Elodie Arduin, Aude Izaac, Celine
Be, and Elke Koch for their excellent assistance.
Keywords: imidazopyridines · inverse agonists · oxadiazoles ·
plasma protein binding · RORgt
Gal4-RORgt-hinge-LBD luciferase reporter gene assay. A Jurkat
cell line stably expressing a pGL4.35 reporter plasmid (Promega)
was generated; this plasmid contains nine repeats of the Gal4 up-
stream activator sequence driving the transcription of the lucifer-
ase gene. A vector containing the human RORgt-hinge-LBD fused
to the DNA-binding domain (DBD) of the Gal4 gene (Promega) was
cloned. Jurkat cells containing the reporter plasmid were transfect-
ed with the RORgt-hinge-LBD fusion plasmid and clones stably ex-
pressing the two constructs were generated. Cells were resuspend-
ed in RPMI 1640 medium containing supplements and 100 UmLꢀ1
penicillin and 100 mgmLꢀ1 streptomycin, and were seeded in mi-
crotiter plates (5ꢂ104 cells per well). The RORgt inhibitors or DMSO
as a control were added to the cells, after 24 h of incubation, cells
were lysed (Steady lite plus, PerkinElmer) and luciferase activity
was measured using an EnVision Multilabel Plate Reader (Perki-
nElmer).
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Human Th17 cell assays. Peripheral blood mononuclear cells were
isolated from human buffy coats by density gradient centrifugation
using Ficoll-Paque. CD4+ Tcells were obtained by immunomagnet-
ic isolation using a CD4+ T-Cell Enrichment Kit according to the
manufacturer’s instructions (Stem Cell). CD4+ Tcells were seeded
at 5ꢂ104 cells per well in CD3 and CD28 pre-coated microtiter
plates and were cultured with a cytokine cocktail containing IL-6
(20 ngmLꢀ1), TGF-b1 (5 ngmLꢀ1), IL-1b (10 ngmLꢀ1), and IL-23
(10 ngmLꢀ1). Compounds were added at the beginning of the cell
cultures, and supernatants were collected after 72 h of incubation.
Supernatants were collected, and IL-17A cytokine concentration
was quantified by ELISA.
Human whole-blood assay. Heparinzed whole blood was obtained
from healthy volunteers and was diluted with high-glucose DMEM
containing supplements, 100 UmLꢀ1 penicillin, and 100 mgmLꢀ1
streptomycin. Diluted blood (20% final concentration) was incubat-
ed with compounds and was stimulated with 10 mgmLꢀ1 ConA
and 10 ngmLꢀ1 recombinant human IL-23. After 72 h incubation
supernatants were collected for IL-17A cytokine quantification by
ELISA according to the manufacturer’s instructions (Bender Med-
systems).
Approval for the use of humans and animals in research: Blood
from healthy volunteers was provided under informed consent and
collected through the Novartis Tissue Donor Program (TRI0128) in
accordance with the Swiss Human Research Act and approval of
the responsible ethic committee (Ethikkommission Nordwest- und
Zentralschweiz number: 329/13). Buffy coats from healthy volun-
teers were provided under informed consent and collected
through the InterRegionale Blutspende of the Swiss Red Cross. The
rat experiments were performed in accordance with the animal ex-
&
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Communications
[12] a) R. I. Olsson, Y. Xue, S. von Berg, A. Aagaard, J. McPheat, E. L. Hansson,
J. Bernstrçm, P. Hansson, J. Jirholt, H. Grindebacke, A. Leffler, R. Chen, Y.
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Manuscript received: October 3, 2016
Revised: November 9, 2016
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Final Article published: && &&, 0000
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COMMUNICATIONS
S. Hintermann, C. Guntermann, H. Mattes,
D. A. Carcache, J. Wagner, A. Vulpetti,
A. Billich, J. Dawson, K. Kaupmann,
J. Kallen, R. Stringer, D. Orain*
Autoimmune challenge: A new series
of RORgt inverse agonists containing tri-
azolo- and imidazopyridine cores has
been identified. Compounds based on
the 6,7,8-substituted imidazo[1,2-a]pyri-
dine core retained high potencies on
the target plus an advantageous physi-
cochemical profile including medium to
high free fraction across species. Deriva-
tives 10 and 33 showed in vivo efficacy
in a rat PK/PD model and inhibited IL-
17A production in an ex vivo challenge.
&& – &&
Synthesis and Biological Evaluation of
New Triazolo- and Imidazolopyridine
RORgt Inverse Agonists
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ChemMedChem 2016, 11, 1 – 10
www.chemmedchem.org
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!