Targeting Acidic Mammalian chitinase Is Effective in Animal Model of Asthma

Article abstract of DOI:10.1021/acs.jmedchem.7b01051

This article highlights our work toward the identification of a potent, selective, and efficacious acidic mammalian chitinase (AMCase) inhibitor. Rational design, guided by X-ray analysis of several inhibitors bound to human chitotriosidase (hCHIT1), led

Full text of DOI:10.1021/acs.jmedchem.7b01051

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Article
Targeting Acidic Mammalian Chitinase is Effective in Animal Model of Asthma
Marzena Mazur, Jacek Olczak, Sylwia Olejniczak, Robert Koralewski, Wojciech Czestkowski, Anna
Jedrzejczak, Jakub Golab, Karolina Dzwonek, Barbara Dymek, Piotr L. Sklepkiewicz, Agnieszka Zagozdzon,
Tom Noonan, Keyvan Mahboubi, Bruce Conway, Ryan Sheeler, Paul Beckett, William M. Hungerford,
Alberto D. Podjarny, Andre Mitschler, Alexandra Cousido-Siah, Firas Fadel, and Adam Golebiowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01051 • Publication Date (Web): 28 Dec 2017
Downloaded from http://pubs.acs.org on December 29, 2017
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Targeting Acidic Mammalian Chitinase is Effective in Animal Model of Asthma
Authors:
Marzena Mazur, Jacek Olczak, Sylwia Olejniczak, Robert Koralewski, Wojciech Czestkowski,
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†
†
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†,¶
†,⁂
†
†
Anna Jedrzejczak, Jakub Golab, Karolina Dzwonek, Barbara Dymek, Piotr L. Sklepkiewicz,
†
†
†
†
†
Agnieszka Zagozdzon, Tom Noonan, Keyvan Mahboubi, Bruce Conway, Ryan Sheeler, Paul
‡
,#
‡,
∥
§
§
§
Beckett, William M. Hungerford, Alberto Podjarny, Andre Mitschler, Alexandra CousidoꢀSiah,
§, &
*,†
Firas Fadel, Adam Golebiowski
†
OncoArendi Therapeutics SA, Żwirki i Wigury 101, 02ꢀ089 Warsaw, Poland
⁂
Department of Immunology, Medical University of Warsaw, 1A Banacha Str., 02ꢀ097 Warsaw,
Poland
‡
The Institute for Pharmaceutical Discovery, Business Drive 23, 06ꢀ405, Branford, CT, USA
§
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, 1 rue
Laurent Fries, 67404 Illkirch, France
Abstract. This article highlights our work toward the identification of a potent, selective, and
efficacious acidic mammalian chitinase (AMCase) inhibitor. Rational design, guided by Xꢀray
analysis of several inhibitors bound to human chitotriosidase (hCHIT1) led to the identification of
compound 7f as a highly potent, AMCase inhibitor (IC50 values of 14 and 19 nM against human and
mouse enzyme, respectively) and selective (>150x against mCHIT1) with very good PK properties.
This compound dosed once daily at 30 mg/kg p.o. showed significant antiꢀinflammatory efficacy in
HDMꢀinduced allergic airway inflammation in mice, reducing inflammatory cell influx in the BALF
and total IgE concentration in plasma, which correlated with decrease of chitinolytic activity.
Therapeutic efficacy of compound 7f in the clinically relevant aeroallergenꢀinduced acute asthma
model in mice provides a rationale for developing AMCase inhibitor for the treatment of asthma.
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Introduction:
Allergic asthma, which affects nearly 300 million people, is a chronic inflammatory disease,
dominated by type 2 immunity. Immune responses to inhaled allergens include eosinophilic
inflammation, goblet cell metaplasia, mucus oversecretion, subepithelial fibrosis and airway
hypersensitivity. Nearly 85% of asthmatics are responsive to chitinꢀcontaining house dust mite (HDM)
allergens and animal models of asthma frequently use HDM extract to induce allergic airway
inflammation. Although mammals do not produce chitin, the second most abundant polymer in nature,
they express two enzymes digesting it: acidic mammalian chitinase (AMCase) and chitotriosidase
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(
CHIT1), as well as several chitinꢀbinding proteins, such as YKLꢀ40 (BRPꢀ39 in mice), YM1, YM2,
1
or oviductin. Among true chitinases, AMCase has been shown to be produced during type 2
2
,3
2,4ꢀ
inflammatory responses in several diseases, both in murine asthma models and in allergic patients.
6
2
Early studies have shown that AMCase neutralization with monoclonal antibodies , its genetic
7
ablation with shRNA or inhibition of its enzymatic activity with the pseudotrisaccharide natural
2
,8
products allosamidin or demethylallosamidin results in diminution of ILꢀ13ꢀmediated allergic
inflammation or ovalbumin (OVA)ꢀinduced allergy in the lungs. Several potent natural product
chitinase inhibitors have been identified, including the mentioned above pseudosaccharides
9
10
allosamidin and demethylallosamidin, the cyclic peptides argadin and argifin, and several xanthine
11
derivatives, including theophylline, caffeine, and pentoxifylline (Figure 1).
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Figure 1. Examples of AMCase and CHIT1 inhibitors
The therapeutic potential or use of these compounds as biological tools is limited due to their sizes,
complex chemistries, limited availabilities, and poor pharmacokinetic profiles in addition to
pleiotropic modes of action, especially for the xanthine derivatives. Bisdionin F, a rationally designed
and selective (vs CHIT1) AMCase inhibitor alleviated the primary features of allergic inflammation
1
2
including eosinophilia in the OVA model in mice. Wyeth developed a moderately active, dual
1
3
inhibitor (compound 1) of AMCase and CHIT1 . Graphene oxide was shown to inhibit enzymatic
1
4
activity of AMCase and to attenuate Th2ꢀtype immune responses in a murine model of asthma .
Conversely, some studies have cast doubt as to whether AMCase inhibition would be effective in
asthma treatment. Mice congenitally lacking AMCase (constitutive CHIA, wholeꢀbody knockꢀout
animals) developed normal airway inflammation in HDM or OVAꢀinduced allergy models and
1
5
allosamidin as well as inhibitor 1 failed to inhibit airway inflammation in this study. In other reports,
16
AMCase overexpression resulted in ameliorated type 2 response induced by chitin and transgenic
mice overexpressing enzymatically inactive AMCase were reported to develop increased allergic
1
7
airway inflammation .
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However, congenital defects in AMCase might not represent a suitable model for studying the role of
this enzyme in acute responses induced in adult animals. Moreover, no data on the pharmacokinetic
1
5
properties of the AMCase inhibitors used in this study was provided. Studies in mice overexpressing
AMCase focused on the response induced by artificial chitin polymers covalently attached to
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polystyrene beads.
Finally, enzymatically deficient AMCase was proven to bind a chitin
resembling this protein to BRPꢀ39, which similarly to its human homolog YKLꢀ40 is induced in
1
8
19,20
aeroallergen asthma models and in human asthma patients , respectively. Therefore, the
contrasting observations on the role of AMCase in animal asthma models require development of
more in vitro active compounds with a pharmacokinetic profile suitable for in vivo studies. To this
end, we have developed a new series of compounds with excellent in vitro activity, selectivity versus
CHIT1 and suitable PK properties allowing oral administration once daily. Finally, we demonstrated
strong antiꢀinflammatory effects of these compounds in an HDMꢀinduced airway inflammation model
in mice that reveal AMCase to be an appropriate therapeutic target in asthma.
SYNTHETIC CHEMISTRY
13
Molecule 1 has been reported by Cole and coꢀworkers. The synthesis of an amide analog 4 started by
acylation of 1ꢀ(tertꢀbutoxycarbonyl)piperazine to give intermediate 3 (Scheme 1). Unmasking the
second amine group and installation of 2ꢀaminotriazole ring, using modified previously reported
21
synthesis led to molecule 4.
a
Scheme 1. Preparation of amide 4
a
Reagents and conditions: (a) 1ꢀ(tertꢀButoxycarbonyl)piperazine, TBTU, DIPEA, DCM, rt; (b) HCl,
EtOAc, rt; (c) S,S'ꢀDimethyl NꢀCyanodithioiminocarbonate, K CO , MeCN, 82°C; (d) Hydrazine,
2
3
MeCN, 82°C.
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Most of Nꢀalkyl compounds (7) were made using general procedure (Scheme 2). Reductive amination
of respective phenethylamine 5 with NꢀBocꢀ4ꢀpiperidone, followed by the second reductive amination
using appropriate aldehyde led to intermediate 6. Amino group deprotection followed by installation
of 2ꢀaminoꢀtriazole ring system gave final compound 7.
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a
Scheme 2. General synthesis of analogs 7
a
Reagents and conditions: (a) NꢀBocꢀ4ꢀpiperidone, NaBH(OAc)
3
, DCE, rt; (b) R
2
CHO, NaBH(OAc)
3
,
DCE, rt; (c) HCl, EtOAc, rt; (d) S,S'ꢀDimethyl NꢀCyanodithioiminocarbonate, K
(e) Hydrazine, MeCN, 82°C.
2
CO , MeCN, 82°C;
3
An amide 10 was prepared in the straightforward 4ꢀbromobenzylamine acylation, Bocꢀdeprotection
and 2ꢀaminotriazole ring installation sequence. Urea analog 13a as well as an amide 13b was made
using similar synthetic sequence, starting from monoprotected 4ꢀaminopiperidine 11a. The same
starting material as well as its Nꢀmethyl analog 11b was used to make sulfonamide 15a and its Nꢀ
methyl derivative 15b. Piperidine 16 served as a substrate to make amine 18a and an amide 18b
(
Scheme 3).
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a
Scheme 3. Synthesis of compounds: 10, 13a-b, 15a-b, 18a-b
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a
Reagents and conditions: (a) 4ꢀBromobenzylamine, TBTU, DIPEA, CH
2
Cl
2
, rt; (b) HCl, EtOAc, rt;
(
(
c) S,S'ꢀDimethyl NꢀCyanodithioiminocarbonate, K CO , MeCN, 82°C; (d) Hydrazine, MeCN, 82°C;
2
3
e) 4ꢀbromoaniline, DIPEA, COCl
Cl rt; (g) 4ꢀBromobenzylsulfonyl chloride, DIPEA, CH
Bromobenzaldehyde, NaBH(OAc) , DCE, rt; (i) HCHO, NaBH(OAc) , DCE, rt; (j) 4ꢀBromobenzoic
2
, Toluene, 0°C to rt; (f) 4ꢀBromophenylacetic acid, TBTU,
DIPEA, CH
2
2
,
2
2
Cl , rt; (h) 4ꢀ
3
3
2 2
acid, TBTU, DIPEA, CH Cl , rt.
RESULTS AND DISCUSSION
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After reviewing the known chitinase inhibitors we decided to focus on the literature reported
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compound 1. It is a relatively weak and nonꢀselective AMCase inhibitor [human AMCase
hAMCase) IC50 5.5 µM; human CHIT1 (hCHIT1) IC50 6.8 µM], but its good drugꢀlike profile and
(
analysis of crystallographic data (PDB ID: 3RM4) indicated that it could be further optimized.
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a
Table 1. SAR results of early analogs of inhibitor 1
hAMCase
hCHIT1
mAMCase
(IC50 nM)
mCHIT1
(IC50 nM)
Compd #
(
IC50 nM)
(IC50 nM)
1
4
5450±1032
12250±778
>100,000
>100,000
>100,000
3325±304
>100,000
>100,000
>100,000
6755±134
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
23100±4100
2095±559
1355±403
>100,000
>100,000
>100,000
573±126
>100,000
>100,000
>100,000
19800±2970
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
10
13a
13b
15a
15b
18a
18b
a
IC50 data of each compound were determined in 2−3 independent assays, and mean IC50 ± SD values
are shown in the table. Compd, abbreviation of compound; SD, standard deviation.
First attempts to identify key interactions and optimize activity were not very successful.
Modification of the central nitrogen atom, shifting its position, changing its character (e.g. from the
basic tertiary amine to an amide), generally led to inactive compounds with the major exception of 4ꢀ
aminoꢀpiperidine analogs: compounds 7a-s and 15a.
An Xꢀray crystallography approach was used to understand, at the atomic level, the mechanism behind
the increased activity displayed by both compounds 7a and 7b towards hAMCase and hCHIT1
enzymes. The hCHIT1ꢀcompound 7a complex structure was solved to 1.35 Å resolution by molecular
2
2
replacement (Fig. 2A), using the hCHIT1 native structure (PDB ID: 4WJX) as the search model. As
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hAMCase and hCHIT1 have a similar active site, the Xꢀray structure obtained allows us to propose the
binding pattern of compound 7a for both proteins. We next compared the binding patterns of
compounds 1 and 7a by superimposing the new hCHIT1ꢀcompound 7a structure to the previously
reported AMCaseꢀcompound 1 structure (PDB ID: 3RM4) (Fig 2B). Our comparison indicates that by
replacing the compound 1 piperazine core to a 4ꢀaminoꢀpiperidine ring in compounds 7a and 7b, the
accommodation of these two latter within the active site of hCHIT1 is improved and a similar
mechanism could be proposed for hAMCase. Indeed, we noticed that our new lead compound 7a
entered ~1 Å deeper in hCHIT1’s pocket as compared to compound 1. Interestingly, this difference is
crucial for enabling Asp213 to establish a strong Hꢀbond of 2.80 Å with the nitrogen of the central 4ꢀ
aminopiperidine ring. In contrast to compound 1, the Asp213 interacted with its piperazine central
nitrogen through a weak Hꢀbond of 3.7 Å (Fig 2 2B, C). Accordingly, we reasoned that strengthening
the interaction between Asp213 and the core of the screened compounds is a key factor to increase the
potency of compound 7a towards both hAMCase and hCHIT1.
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a
Table 2. SAR of analogs 7
hAMCase
hCHIT1
mAMCase
(IC50 nM)
mCHIT1
(IC50 nM)
Compd #
R1
R2
(
IC50 nM)
(IC50 nM)
3
48 ±21
175±7.1
108±17
245±35
2700±85
7
a
6
60±8
389±12.7
7470±1160
7b
7c
182±18
385±40
95±3
7080±396
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7d
73±4
105±36
945±21
36±12
2715±190
7
e
f
134±8
185±43
15650±636
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0
7
14.2±1.0
8.7±1.3
8.7±1.0
5.4±1.6
232±48
123±47
123±25
163±70
18.8±0.4
8.0±2.5
8.0±2.9
11.2±2.1
2955±50
1269±54
2025±205
1508±2.8
7g
7
7
7
h
i
j
3.5±0.4
149±77
9.7±3.5
6.2±0.4
2585±261
573±60
7k
3.4±1.1
36±9
7l
18450±3040
>100,000
24400±
544±13
>100,000
7
m
1111±182
970±34
1525±304
1011±154
12550±2192
7n
1430±382
1288±738
7121±397
6965±92
7o
4137±1760
1175±205
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4
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5
5
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7
p
1545±177
765±149
4160±56
1735±445
3070±438
0
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8
9
0
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7r
13200±1980
7330±57
333±31
>100,000
7s
2025±163
2255±120
47600±3677
a
IC data of each compound were determined in 2−3 independent assays, and mean IC ± SD values
5
0
50
are shown in the table. Compd, abbreviation of compound; SD, standard deviation.
This observation is in agreement with the IC50 values showed in Table 1. All other placements of
central nitrogen atom as well as its acylation/urea formation as in compounds 4, 10, 13a, 13b, 18a and
18b led to very weak inhibitors or inactive molecules, presumably because of breaking critical
interaction of central nitrogen atom with Asp213 of the enzyme. The only exception in this series was
sulfonamide 15a, capable of forming hydrogen bond between Asp213 and hydrogen atom of the NHꢀ
sulfonamide group. NꢀAlkylation of sulfonamide nitrogen atom (i.e. formation of compound 15b)
gave also an inactive hAMCase/hCHIT1 molecule, further verifying critical interactions with Asp213.
It is worth noting the AMCase/CHIT1 selectivity of molecule 15a, although its activity was still
moderate. A second important distinctive point revealed by the hCHIT1ꢀcompound 7a coꢀcrystal
structure refers to the remarkable inversion of Trp99 which plays an important role in binding the
chitoꢀoligosaccharide substrate. Indeed, both hCHIT1 and hAMCase structures in complex with
allosamidin, compound 1 (Fig 1, 2B, C) and other published ligands coꢀstructures show a similar
1
1ꢀ13
position of Trp99 in their native structures.
However, in our hCHIT1ꢀcompound 7a coꢀstructure,
the Trp99 residue shows a 106° movement induced by a πꢀstacking interaction as the distance between
the methyl group at the R1 position of the compound and the aromatic ring is 3.7 Å (Fig 2A, B, C).
Moreover, this new Trp99 position induces a stacking interaction with the 4ꢀaminoꢀpiperidine group
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thus strengthening the binding with the compound. As the compound 1 does not have a R1 group,
the position of Trp99 adopts the native conformation and does not interact with the piperazine group
as the distance between them is 4.9 Å (Fig 2B, C). Thus, we can conclude that substituting the
piperazine ring with the 4ꢀaminoꢀpiperidine, together with the addition of a new hydrophobic function
at the R1 position in compounds 7a and 7b strongly contribute to the new interaction of the
compounds with Trp99. We suggest that the recruitment of Trp99 plays an important role in the
improved potency of these compounds.
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It is important to note that we observe a difference in binding of the aminotriazole group to the highly
conserved catalytic site of CHIT1. Indeed, while Asp138, Tyr27 and Tyr212 directly Hꢀbond this
moiety of compound 7a, the catalytic residue Glu140 interacts indirectly with the N of the
aminotriazole group via a water molecule (Fig. 3). Therefore, this water molecule gets stabilized by
establishing two strong Hꢀbonds of 2.8 Å with Glu140 and with the N of the aminotriazole group.
2
2
Previous studies have shown that Asp138 can adopt a double conformation in the apo enzyme: one
linked to Asp136 and a second one oriented to Glu140. In the presence of compound 7a, Asp138 is
oriented toward Asp136 through a low barrier Hꢀbond (Fig. 3) and a water molecule mediating the
interaction between Glu140 and compound 7a occupies the position of the carbonyl group in Asp138
second conformation thus forcing Asp138 to remain in its first conformation. In contrast, a direct
interaction is established between Glu140 and the N of the aminotriazole group of compound 1 and no
detectable water molecule is found in the pocket. This difference gives a structural mechanistic
explanation that could be one of the factors responsible for the increased potency seen in compound 7a
vs. 1. This structural information provided essential knowledge to further develop our compounds.
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Figure 2 Structure of the hCHITꢀcompound 7a complex and comparison to the hAMCaseꢀcompound
1
complex structure. A) contacts of compound 7a represented as stick (green carbons) in the active site
pocket of hCHIT1 represented as surface and colored according to charges. The 2mFoꢀFc electronꢀ
density map (1 σ cutoff) compound 1 is shown as a mesh colored in grey. B) Superimposition of the
active site residues in hCHIT1 complexed to compound 7a (stick model, carbons and distances
colored in green) and the active site residues in hAMCase complexed to compound 1 (stick model,
carbons and distances colored in orange). C) Zoomed superposition of compounds 7a and 1 and key
residues of hCHIT1 and hAMCase active site. The interaction between Glu140 residue in hCHIT1 and
the N of the aminotriazole group is mediated by a water molecule (shown as a red sphere) while the
interaction is direct in hAMCaseꢀcompound 1 complex. The Trp99 in hCHIT1 is inverted by 106° in
hCHIT1ꢀcompound 7a complex. D) Accommodation of R2 function (bromobenzyl moiety in
compound 7a and 1) within the hydrophobic pocket of hCHIT1 and hAMCase).
To obtain an inhibitor selective for hAMCase, several compounds were generated by modifying
substituents R1 and R2. Our in vitro assays revealed that compounds 7g and 7i display a 15ꢀ and 44ꢀ
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fold selectivity against hAMCase as compared to hCHIT1, respectively. Therefore, we sought to
investigate the structural basis that explains such high selectivity. For this, we have used the soaking
method with the two identified hit compounds and solved their Xꢀray crystal coꢀstructures in complex
with hCHIT1 at 1.27 Å and 1.44 Å resolutions respectively. Both structures show that the main body
of the two compounds binds similarly to compound 7a in hCHIT1 catalytic groove (Fig. 3).
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Figure 3. Superimposition of the active site region of the three complex structures hCHIT1ꢀ
compound 7a, 7i and 7g. Key residues are represented by lines with oxygen atoms in red, nitrogen
atoms in dark blue, sulfur atoms in yellow. A) Active site pocket of hCHIT1 represented as surface
and colored according to charges. In A) and B) compounds 7a, 7i and 7g are represented in stick,
carbons are colored in green, violet and magenta, respectively, chlorine atoms are colored in light
green and bromide atoms in dark red. The water molecule mediating the interaction with the
aminotriazole group is color coded according to the compound it interacts with. In B) the distance
between atoms is colorꢀcoded according to the compound it interacts with.
The R2 which corresponds to bromobenzyl function in 7a and 7g is a chlorobenzyl in 7i. This moiety
in the three compounds occupies in the same way the hydrophobic pocket formed by Leu362, Ala302,
Leu301, Met300, and Tyr267 residues with 1 Å shift to compound 1. The presence of a phenyl
function in R1 in compound 7i shows a stacking contact with Met300 which should take place with
Ile300 in hAMCase. The superposition of the three hCHIT1ꢀligands coꢀstructures shows that the
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position of the Trp99 dictated by the stacking is slightly deviated of (~1.5 Å) depending on the R1
fragment (Fig 3). This deviation of Trp99 seems to be accompanied by slight displacement (of ~1 Å)
of the key Glu140 residue and the water molecule involved in the Glu140ꢀligand interaction (Fig 3).
Consequently, this displacement appears to be crucial to reinforce the contact between Glu140 and the
water molecule. Thus, the distance of this Hꢀbond shifts from 2.75 Å in the hCHIT1ꢀcompound 7a
complex to a low barrier Hꢀbond of 2.50 Å in the complexes hCHIT1ꢀcompound 7g and 7i
respectively (Fig. 3B). As the key residues involved in the interaction with compounds 7g and 7i
except Met300 in hCHIT1 (Ile300 in hAMCase) are conserved in both proteins, we believe that these
two compounds as well as compound 7f bind in a similar way to the AMCase active site. However, the
increased potency seen against hAMCase compared to hCHIT1 could be explained by the fact that
residue 269 is an arginine in hCHIT1 whereas it is a histidine in the case of AMCase. In the case of
hCHIT1 the presence of a long positively charged side chain facing a hydrophobic function, either the
isopropyl or phenyl in compounds 7g and 7i respectively, may have a negative effect on compound
binding (distance with the phenyl group and Arg269 is 3.6 Å). In contrast, this is not the case with the
hAMCase as the Nε2 and Cε1 at the edge of the His269 imidazole ring establish van der Waals
contacts with the isopropyl group in compound 7g and the phenyl group in compound 7i. Taking all
these observations together and extrapolating them on hAMCaseꢀinhibitor interaction, one can say that
the isopropyl group in compounds 7g and 7f as well as the phenyl group in compound 7i enhance the
hydrophobic interaction with Ile300 and His269 in hAMCase leading to an improved stacking
conformation of Trp99 favoring the short Hꢀbond between Glu140 and the water mediated compound
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7i interaction. These contacts together with the strong bonds with Asp213, Tyr212, and Asp138
provide a mechanistic explanation of the higher selectivity of these inhibitors against hAMCase.
Further proof of this interpretation lays in the SAR analysis of compounds 7a-k. An increase of the
size of R1 substituent along with augmenting its hydrophobic character led to the raise of both activity
and selectivity hAMCase vs. hCHIT1.
In parallel to optimization of the R1 substituent we have looked into modification of the
hydrophobic pocket binding fragment R2. The Xꢀray crystallographic data show that the benzyl group
in R2 establishes hydrophobic interaction with Tyr267, Glu297 and Met300 (Ile300 in hAMCase) side
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chains allowing the accommodation of the R2 in this pocket (Fig 2D). The obtained structures reveal
that the introduction of a halogen at the para position of the benzyl ring leads to an interaction between
the C–X (X=Br or Cl) of the R2 function and the residue 295 side chain (Ala in hAMCase and Thr in
hCHIT1) located at the bottom of the pocket. This interaction occurs within a distance of 3.5Å which
increases the ligands potency toward the human chitinases. Removing the halogen group or
substituting it with a bigger hydrophobic function as in compounds 7m to 7r (see Table 2) highly
decreased the compounds activity. This diminution is most likely due to the steric hindrance caused by
these functions which would create clashes with residue 295 side chains perturbing the
accommodation of R2 function in this hydrophobic pocket. Thus, the optimal function selected for R2
is the paraꢀhalogenated phenethyl. Any deviation from pꢀBrꢀphenethyl or pꢀClꢀphenethyl substituents
led to decreased activity, with the small alkyl paraꢀsubstituted phenethyl fragment being the closest in
activity and paraꢀcyclopropyl i.e. inhibitor 7p the best. Compounds 7l-s are samples of variations we
1
1
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0
1
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have tried.
Along with the optimization of the in vitro potency and selectivity, some of the representative
inhibitors were evaluated for their in vitro ADME and in vivo pharmacokinetic properties.
Table 3. In Vivo Pharmacokinetic Profile of Selected Analogues after iv Dose at 3 mg/kg in Mice
Compound
Vss (L/kg) CL (mL/min/kg)
T1/2 (h)
AUC0ꢀinf (mg*h/L)
7
a
12.8
2.33
1.66
3.03
1.16
52.6
9.06
8.53
45.0
11.5
4.40
2.97
3.25
6.78
11.1
0.95
5.52
5.86
1.11
4.34
7
f
7g
7
j
15a
Table 4. In Vivo Pharmacokinetic Profile of Selected Analogues after Oral Dose at 10 mg/kg in
Mice and Microsomal Stability (% remaining after 20 min).
Compound
C
max (mg/L)
0.672
T
max (h)
AUC0ꢀinf (mg*h/L)
3.34
F (%)
99
LM Stb
97.5
7
a
2.0
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6.04
0.25
0.5
1.0
1.0
9.56
27.9
1.75
8.21
52
100
47
76.9
78.3
21
7
g
7j
0.618
3.25
15a
57
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Figure 4. Plasma concentration−time course for compound 7f after 3 mg/kg iv bolus and after 10
mg/kg oral administration (po) into female BALB/c mice.
Microsomal stability showed, in general, a good correlation to in vivo clearance and po exposure in
mice, indicating hepatic first pass is the likely driver of oral bioavailability and phase I metabolism is
the major clearance route for this series. Overall, compounds 7f, 7g and 15a showed good oral
bioavailability, low to moderate clearance, and adequate exposure for efficacy testing following once
daily po dosing in mice. Compounds 7a and 7j had higher clearances and lower systemic absorption
and may require twice daily po dosing. Inhibitor 7f was chosen to be tested in animal models of
asthma.
A common and clinically relevant aeroallergen HDM extract was used to induce acute allergic airway
inflammation in BALB/c mice. HDM administration for three consecutive weeks resulted in increased
leukocytes influx to the lungs (particularly eosinophils) and increased IgE concentration in the plasma
of mice challenged with HDM as compared to control group. These changes were accompanied by a
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significant increase in the chitinolytic activity in bronchoalveolar lavage fluid (BALF) (Fig. 5). Oral
treatment with the compound 7f (30 mg/kg), in a therapeutic scheme starting from day 7 after the start
of HDM administration led to a significant reduction in total leukocyte numbers (Fig. 5A), particularly
the eosinophil numbers (Fig. 5B) in BALF as compared to vehicleꢀtreated controls. Significant antiꢀ
inflammatory effects of compound 7f corresponded with reduced chitinase activity in BALF (Fig. 5C),
as well as a significant reduction of total plasma IgE concentration (Fig. 5D).
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Figure 5. Selective AMCase inhibition with compound 7f exerts significant antiꢀinflammatory effects
in HDMꢀinduced asthma model in mice. Dexamethasone (Dex) was administered ip and 7f was given
po starting from day 7 after the start of HDM administration. (A) Total leukocyte numbers in BAL (n
=
8). (B) Flow cytometry analysis of eosinophils (Siglec F+, CD11cꢀ) represented as total eosinophil
numbers in BAL isolated from mice challenged with PBS (Control) or HDM for 3 consecutive weeks
with or without 7f or dexamethasone treatment. (C) Chitinolytic activity in BAL. (D) Total IgE level
in plasma. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; by oneꢀway ANOVA followed by
Tukey’s multipleꢀcomparison test.
In addition, flow cytometry analysis of BAL leukocyte subpopulations revealed that compound 7f
treatment significantly reduced the total numbers of other subtypes of infiltrating (CD45+) leukocytes
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such as neutrophils, monocytes and B cells in BALF (Fig. 6). T cells (CD3+) show only trend towards
reduction in numbers after 7f treatment without statistical significance. Antiꢀinflammatory efficacy of
compound 7f was observed in three independent acute HDMꢀinduced asthma experimental conditions.
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Figure 6. Selective AMCase inhibition with compound 7f decreases the numbers of different
leukocyte subpopulations in BAL in HDMꢀinduced allergic airway inflammation in mice.
Dexamethasone (Dex) was administered ip and 7f was given po starting from day 7 after the start of
HDM administration. (A) Representative flow cytometry analysis of leukocyte subpopulations such as
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+
alveolar macrophages, eosinophils, neutrophils, monocytes, T and B cells gated from the CD45
BALF leukocytes isolated from mice challenged with PBS or HDM for 3 consecutive weeks with or
without 7f or dexamethasone treatment. (B) Quantitative flow cytometry analysis of leukocyte
numbers such as alveolar macrophages, neutrophils, monocytes, T and B cells in BAL. Data are mean
±
s.e.m. *P < 0.01; **P < 0.01; ***P < 0.001; by oneꢀway ANOVA followed by Tukey’s multipleꢀ
comparison test.
Considering the overall activity profile of compound 7f, it was further characterized. To fully
determine its affinity towards h/mAMCase and h/mCHIT1, inhibitory constants (Ki) for all four
enzymes have been measured and they are in good correlation with earlier established IC50 data
(
hAMCase Ki = 13.5 ± 2.4; hCHIT1 Ki = 312 ± 205; mAMCase Ki = 10.5 ± 1.6; mCHIT1 Ki = 3404
24
±
1749 nM). Compound 7f has passed the test for genotoxicity (AMES test was negative) but
25
showed a potential for cardiotoxicity (hERG binding activity IC50 4 µM). Offꢀtarget in vitro effects
of compound 7f have been evaluated in the CEREP Diversity™ panel consisting of 72 binding and 27
26
enzyme assays and revealed interactions with monoꢀamine transporters for serotonin and dopamine
(
95% inhibition at 10 µM for both).
CONCLUSIONS:
A new series of aminotriazoles with a high inhibitory activity against human and mouse
chitinases (AMCase and CHIT1) has been identified. Structural biology analysis guided medicinal
chemistry efforts, leading to the discovery of several highly potent and selective (AMCase vs CHIT1)
inhibitors. Pharmacokinetic profiles of the top five compounds were further analyzed, resulting in the
nomination of inhibitor 7f for preꢀdevelopment level screening.
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Overall compound 7f is a highly potent, selective (the mouse enzyme) AMCase inhibitor with
very good PK properties. Once daily 30 mg/kg oral doses in mice showed significant antiꢀ
inflammatory efficacy in acute HDMꢀinduced allergic airway inflammation mainly by providing
significant reduction in total IgE levels in plasma, correlating with reduction of AMCase activity and
inflammatory cell influx in the BALF as compared to vehicle controls. Therapeutic efficacy of
compound 7f in the clinically relevant aeroallergenꢀinduced acute asthma model in mice suggests
significant therapeutic potential to treat asthma patients alone or in a combination with first line
existing therapies. Further development of compound 7f was not pursued because of the concerns of
potential cardiovascular safety margin versus hERG and its activity against the dopamine transporter,
but this orally active and highly selective AMCase inhibitor can serve as valuable tool in future studies
on the role of AMCase in various pathologies.
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Samples of compounds 7f will be provided, when possible, to facilitate further preclinical
research on the role of AMCase in asthma and other relevant diseases. Please contact the
corresponding author for details.
EXPERIMENTAL SECTION:
Chemical Methods. All solvents, substrates and reagents that were commercially available were used
without further purification. TLC analysis was performed using preꢀcoated glass plates (0.2 ± 0.03 mm
thickness, GFꢀ254, particle size 0.01–0.04 mm) from Fluorochem Ltd, UK. Column chromatography
was performed using highꢀpurity grade silica gel (pore size 60 Å, 220ꢀ440 mesh particle size, 35ꢀ75
ꢁm particle size) from Fluka. Preparative HPLC was performed on LCꢀ20AP Shimadzu with ELSDꢀ
1
LTII detector equipped with Hypersil GOLD 21.2/250 mm, 5 ꢁm C18 column. H NMR spectra were
recorded on Bruker AVANCE II PLUS NMR spectrometers at 700 MHz. All spectra were recorded in
3 6 2 3
appropriate deuterated solvents (CDCl , DMSOꢀd , D O, CD OD, etc.) that were commercially
available. Resonances are given in parts per million relative to tetramethylsilane. Data are reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, bs = broad
singlet, bd = broad doublet), coupling constants (J in Hz) and integration. ESIꢀMS spectra were
recorded on a Shimadzu LCꢀ20AD LPG separation module with a SPDꢀM20A UV detector and
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LCMSꢀ2020 mass detector equipped with Kinetex 2.1/50 mm, 2.6 ꢁm C18 column eluted with 0.5
mL/min flow of 10ꢀ90% gradient (over 6 min) of acetonitrile in water. HRMSꢀESI spectra were
recorded on Bruker Maxis Impact instrument. Purities of all reported compounds were greater than
95% based on elemental analysis and/or HPLC chromatograms. HPLC analyses were performed on a
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Waters Acquity UPLC system fitted with BEH C18 1.7 µm column (2.1 × 50 mm) and with UV
detection (220 nm) , gradient 3ꢀ97% of acetonitrile in water, flow 0.6mL/min over 3min.
1-(4-(3-Amino-1H-1,2,4-triazol-5-yl)piperazin-1-yl)-2-(4-bromophenoxy)ethanone hydrochloride
(
4).
Step 1: Synthesis of tert-butyl 4-(2-(4-bromophenoxy)acetyl)piperazine-1-carboxylate (3). 4ꢀ
Bromophenoxyacetic acid 2 (250 mg, 1.08 mmol) was dissolved in dichloromethane (5 mL),
diisopropylethylamine (0.23 mL, 1.29 mmol) was added at ambient temperature followed by addition
of Bocꢀpiperazine (201 mg, 1.08 mmol), and when the solution was clear the coupling reagent TBTU
(
347 mg, 1.29 mmol) was added. The reaction mixture was stirred at ambient temperature overnight,
diluted with dichloromethane and washed with 1 M aq NaOH, 2 M aq HCl, brine and dried over
MgSO . The solvent was evaporated and the product was purified by flash chromatography (gradient
elution 0ꢀ1% methanol in dichloromethane) to afford pure amide 3 as offꢀwhite foam (380 mg, 88%).
4
+
1
3
LCꢀMS (ES+): RT 5.5 min, m/z 399.1/401.1 [M+H] . H NMR (700 MHz, CDCl ) δ 7.41 (d, J = 9.1
Hz, 2H), 6.86 (d, J = 9.1 Hz, 2H), 4.70 (s, 2H), 3.62 – 3.53 (m, 4H), 3.46 – 3.30 (m, 4H), 1.49 (s, 9H).
Step 2: Synthesis of 1-(4-(3-amino-1H-1,2,4-triazol-5-yl)piperazin-1-yl)-2-(4-bromophenoxy)ethanone
hydrochloride (4). The NꢀBoc protected amine 3 (350 mg, 0.87 mmol) was dissolved in ethyl acetate
(
2 mL) and treated with a hydrogen chloride (4 M solution in ethyl acetate, 5 mL). The reaction
mixture was stirred at ambient temperature and controlled by TLC (chloroform/methanol 9:1). When
substrate was no longer detected the precipitate was filtered and washed with ether to give amine as
hydrochloride salt (280 mg, 95%), which was sufficiently pure to be used in the following step. The
hydrochloride salt of the amine (137 mg, 0.41 mmol), anhydrous potassium carbonate (143 mg, 1.03
mmol) and S,S'ꢀdimethylꢀNꢀcyanoꢀdithioiminocarbonate (61 mg, 0.41 mmol) were added to
acetonitrile (4 mL) and the reaction mixture was refluxed for 3 h (monitoring by TLC). Hydrazine
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monohydrate (0.06 mL, 1.24 mmol) was then added and the mixture was further refluxed for another 2
h. After this time it was cooled to room temperature and solids were filtered off. The filtrate was
concentrated in vacuo and the crude product was purified by flash chromatography (gradient elution 1ꢀ
5% methanol in dichloromethane), then was converted into hydrochloride salt by hydrogen chloride
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(
4M solution in ethyl acetate), washed with ether and dried in vacuo to give title compound 4 as white
+
1
solid (71 mg, 62%), mp 193ꢀ194°C. LCꢀMS (ES+): RT 3.8 min, m/z 381.0/383.0 [M+H] . H NMR
(
(
700 MHz, DMSOꢀd
6
) δ 7.39 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 4.79 (s, 2H), 3.62 – 3.49
+
m, 4H), 3.29 (bs, 4H). HRMS calculated for C14
H18BrN
6
O
2
[M+H] 381.0669, found 381.0663.
General Procedure for the Synthesis of 1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(R2)-substituted-N-
4-(R1)-substituted-phenethyl)piperidin-4-amine (7a-7s).
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-bromophenethyl)-N-methylpiperidin-4-amine (7a).
(
1
Step 1: Synthesis of tert-butyl 4-((4-chlorophenethyl)(methyl)amino)piperidine-1-carboxylate (6a). To
a stirred solution of NꢀBocꢀ4ꢀpiperidone (200 mg, 1 mmol) and 4ꢀbromophenethylamine (0.18 mL, 1.2
mmol) in 1,2ꢀdichloroethane (3 mL), sodium triacetoxyborohydride (424 mg, 2 mmol) was added. The
reaction mixture was stirred at ambient temperature overnight. Then, 30% aqueous formaldehyde
solution (0.45 mL, 5 mmol) and second portion of sodium triacetoxyborohydride (424 mg, 2 mmol)
were added. The stirring was continued for 1 h. A 5% solution of sodium bicarbonate was added (6
mL) and the biphasic mixture was stirred for 30 minutes. The layers were separated and the aqueous
layer was additionally extracted with dichloromethane (2 × 5 mL). The combined organic extracts
4
were then dried over MgSO , and solvent was removed in vacuo. The crude residue was purified by
flash chromatography (gradient elution 1ꢀ1.5% methanol in dichloromethane) to afford title compound
+
1
6
a as colorless oil (300 mg, 75%). LCꢀMS (ES+): RT 4.3 min, m/z 397.0/399.0 [M+H] . H NMR
700 MHz, CDCl ) δ 7.39 (d, J = 8.3 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 4.17 – 4.11 (m, 2H), 2.72 –
.67 (m, 6H), 2.57 – 2.52 (m, 1H), 2.34 (s, 3H), 1.74 – 1.71 (m, 2H), 1.45 (s, 9H), 1.43 – 1.37 (m, 2H).
(
3
2
Step 2: Synthesis of 1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-bromophenethyl)-N-methylpiperidin-4-
amine (7a). The NꢀBoc protected amine 3 (300 mg, 0.75 mmol) was dissolved in ethyl acetate (2 mL)
and treated with a hydrogen chloride (4 M solution in ethyl acetate, 5 mL). The reaction mixture was
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stirred at ambient temperature and controlled by TLC (chloroform/methanol 9:1). When substrate was
no longer detected the precipitate was filtered and washed with ether to give amine as hydrochloride
salt (270 mg, 97%), which was sufficiently pure to be used in the following step. The hydrochloride
salt of the amine (270 mg, 0.73 mmol), anhydrous potassium carbonate (251 mg, 1.82 mmol ) and
S,S'ꢀdimethylꢀNꢀcyanoꢀdithioiminocarbonate (107 mg, 0.73 mmol) were added to acetonitrile (4 mL)
and the reaction mixture was refluxed for 3 h (monitoring by TLC). Hydrazine monohydrate (0.11 mL,
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2.19 mmol) was then added and the mixture was further refluxed for another 2 h. After this time it was
cooled to room temperature and solids were filtered off. The filtrate was concentrated in vacuo and the
crude product was purified by flash chromatography (gradient elution 1ꢀ5% methanol in
dichloromethane), then was crystallized from acetonitrile to give title compound 7a (170 mg, 62%),
+
1
mp 152ꢀ153°C. LCꢀMS (ES+): RT 1.0 min, m/z 379.2/ 381.2 [M+H] . H NMR (700 MHz, DMSOꢀd
O) δ 7.40 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H), 3.73 (d, J = 12.9 Hz, 2H), 2.62 (dd, J =
.2, 5.9 Hz, 2H), 2.61 – 2.54 (m, 4H ), 2.48 – 2.40 (m, 1H), 2.17 (s, 3H), 1.62 (d, J = 14.9 Hz, 2H),
6
+
D
2
9
1
2
1
6
.31 (qd, J = 12.4, 4.1 Hz, 2H). Elemental analysis calculated (%) for C16H23BrN : C 50.67, H 6.11, N
2.16. Found: C 50.56, H 6.04, N 22.08.
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-chlorophenethyl)-N-methylpiperidin-4-amine
trihydrochloride (7b). The title compound was prepared by a similar procedure as that used for 7a
and purified by preparative HPLC to give 7b as hydrochloride salt (40 mg, 76%), mp 162ꢀ164°C. LCꢀ
+
1
MS (ES+): RT 0.9 min, m/z 335.1/ 337.1 [M+H] . H NMR (700 MHz, DMSOꢀd
6
2
+ D O) δ 7.37 –
7
3
.34 (m, 2H), 7.33 – 7.30 (m, 2H), 3.82 (d, J = 13.0 Hz, 2H), 3.50 (tt, J = 12.2, 3.6 Hz, 1H), 3.36 –
.27 (m, 1H), 3.24 – 3.15 (m, 1H), 3.07 – 2.89 (m, 4H), 2.75 (s, 3H), 2.12 – 1.91 (m, 2H), 1.77 – 1.59
+
(
m, 2H). HRMS calculated for C16
H24ClN
6
[M+H] 335.1745, found 335.1736.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-chlorophenethyl)-N-ethylpiperidin-4-amine (7c). The title
compound was prepared by a similar procedure as that used for 7a and crystallized from acetonitrile to
+
1
give 7c (296 mg, 62%), mp 141ꢀ142°C. LCꢀMS (ES+): RT 1.1min, m/z 349.3/ 351.3 [M+H] . H
NMR (700 MHz, DMSOꢀd + D O) δ 7.28 – 7.24 (m, 2H), 7.22 – 7.17 (m, 2H), 3.77ꢀ3.74 (m, 2H),
6
2
2.62 – 2.58 (m, 3H), 2.58ꢀ2.54 (m, 3H), 1.60 (d, J = 11.4 Hz, 2H), 1.31 (qd, J = 12.3, 4.0 Hz, 2H),
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.90 (t, J = 7.1 Hz, 3H), three protons overlapping with water and DMSO signals. Elemental analysis
calculated (%) for C17
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-bromophenethyl)-N-ethylpiperidin-4-amine (7d). The title
compound was prepared by a similar procedure as that used for 7a and crystallized from acetonitrile to
6
H25ClN : C 58.53, H 7.22, N 24.09. Found: C 58.66, H 7.32, N 24.10.
1
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0
1
2
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0
1
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0
1
2
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7
8
9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
give 7d (150 mg, 52%), mp 137ꢀ138°C. LCꢀMS (ES+): RT 1.7 min, m/z 393.2/ 395.2 [M+H] . H
NMR (700 MHz, DMSOꢀd + D O) δ 7.39 – 7.36 (m, 2H), 7.15 – 7.11 (m, 2H), 3.77 – 3.74 (m, 2H),
6
2
2
2
2
1
.63 – 2.59 (m, 7H), 2.51 (q, J = 7.1 Hz, 2H), 1.60 (d, J = 10.5 Hz, 2H), 1.34 (qd, J = 12.3, 4.3 Hz,
6
H), 0.91 (t, J = 7.1 Hz, 3H). Elemental analysis calculated (%) for C17H25BrN : C 51.91, H 6.41, N
1.37. Found: C 51.89, H 3.37, N 21.33.
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-chlorophenethyl)-N-isopropylpiperidin-4-amine
trihydrochloride (7e). The title compound was prepared by a similar procedure as that used for 7a
and purified by preparative HPLC to give 7e as hydrochloride salt (50 mg, 44%), mp 88ꢀ89°C. LCꢀMS
+
1
(
(
ES+): RT 2.0 min, m/z 363.0/ 365.0 [M+H] . H NMR (700 MHz, DMSOꢀd
6 2
+ D O) δ 7.38 – 7.35
m, 2H), 7.35 – 7.32 (m, 2H), 3.85 – 3.79 (m, 3H), 3.55 (tt, J = 11.9, 3.4 Hz, 1H), 3.30 – 3.18 (m, 2H),
3
.02 – 2.91 (m, 4H), 2.13 – 2.04 (m, 2H), 1.85 – 1.72 (m, 2H), 1.32 (d, J = 6.5 Hz, 3H), 1.24 (d, J =
.5 Hz, 3H). Elemental analysis calculated (%) for C18 *3HCl*H O: C 44.10, H 6.58, N 17.14.
6
H27ClN
6
2
Found: C 44.18, H 6.91, N 16.76.
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-chlorophenethyl)-N-isobutylpiperidin-4-amine (7f). The
1
title compound was prepared by a similar procedure as that used for 7a and purified by crystallization
from acetonitrile to give 7f (220 mg, 42%), mp 127ꢀ128°C. LCꢀMS (ES+): RT 3.1 min, m/z 377.3/
+
1
3
2
1
6 2
79.3 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.25 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz,
H), 3.77 – 3.64 (m, 2H, overlapping with water), 2.64 – 2.53 (m, 7H), 2.09 (d, J = 7.1 Hz, 2H), 1.61 –
.40 (m, 3H), 1.32 – 1.24 (m, 2H), 0.71 (d, J = 6.5 Hz, 6H). Elemental analysis calculated (%) for
C
19
6
H29ClN : C 60.54, H 7.76, N 22.30. Found: C 60.61, H 7.72, N 22.17.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-bromophenethyl)-N-isobutylpiperidin-4-amine (7g). The
title compound was prepared by a similar procedure as that used for 7a and purified by crystallization
from acetonitrile to give 7g (305 mg, 62%), mp 127ꢀ128°C. LCꢀMS (ES+): RT 3.4 min, m/z 421.25/
+
1
6 2
423.25 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.38 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz,
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H), 3.77 – 3.71 (m, 2H, overlapping with water), 2.60 – 2.51 (m, 7H), 2.09 (d, J = 6.6 Hz, 2H), 1.56 –
.49 (m, 2H), 1.46 – 1.43 (m, 1H), 1.33 – 1.24 (m, 2H), 0.71 (d, J = 6.4 Hz, 6H). Elemental analysis
1
calculated (%) for C H BrN : C 54.16, H 6.94, N 19.94. Found: C 54.20, H 6.84, N 19.95.
19
29
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1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-chlorophenethyl)-N-(cyclohexylmethyl)piperidin-4-amine
0
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trihydrochloride (7h). The title compound was prepared by a similar procedure as that used for 7a
and purified by preparative HPLC to give 7h as hydrochloride salt (32 mg, 53%), mp 154ꢀ155°C. LCꢀ
+
1
MS (ES+): RT 3.9 min, m/z 417.5/419.5 [M+H] . H NMR (700 MHz, DMSOꢀd
8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 3.83 (bd, J = 12.9 Hz, 2H), 3.56 – 3.52 (m, 1H), 3.29 – 3.20
m, 2H), 3.07 (dd, J = 13.7, 7.7 Hz, 1H), 3.00 (t, J = 8.5 Hz, 2H), 2.96 – 2.89 (m, 3H), 2.04 (bd, J =
0.3 Hz, 1H), 1.97 (bd, J = 12.1 Hz, 1H), 1.81 (bd, J = 11.6 Hz, 1H), 1.75 – 1.64 (m, 6H), 1.61 – 1.58
m, 1H), 1.25 – 1.18 (m, 2H), 1.11 (tt, J = 12.8, 3.2 Hz, 1H), 0.99 – 0.91 (m, 2H). Elemental analysis
calculated (%) for C22 *3HCl*H O: C 48.54, H 7.04, N 15.44. Found: C 48.74, H 6.79, N
6 2
+ D O) δ 7.37 (d, J
=
(
1
(
H33ClN
6
2
15.81.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-benzyl-N-(4-chlorophenethyl)piperidin-4-amine (7i). The
title compound was prepared by a similar procedure as that used for 7a and purified by crystallization
from acetonitrile to give 7i (77 mg, 51%), mp 51ꢀ52°C. LCꢀMS (ES+): RT 3.6 min, m/z 411.3/413.3
+
1
[
M+H] . H NMR (700 MHz, DMSOꢀd
6 2
+ D O) δ 7.25 – 7.18 (m, 6H), 7.18 – 7.13 (m, 1H), 7.08 (d, J
=
8.0 Hz, 2H), 3.78ꢀ 3.68 (m, 2H), 3.60 (bs, 2H), 2.66 – 2.52 (m, 6H), 1.62 (bd, J = 12.2 Hz, 2H), 1.38
(
qd, J = 12.0, 3.9 Hz, 2H), one proton overlapping with DMSO signals. HRMS calculated for
+
C H ClN [M+H] 411.2058, found 411.2064.
22
28
6
1
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(2-chlorobenzyl)-N-(4-chlorophenethyl)piperidin-4-amine
7j). The title compound was prepared by a similar procedure as that used for 7a and purified by
crystallization from acetonitrile to give 7j (28 mg, 64%), mp 88ꢀ89°C. LCꢀMS (ES+): RT 3.7 min, m/z
(
+
1
4
1
3
1
1
6 2
45.3/447.3 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.35 – 7.33 (m, 1H), 7.33 – 7.30 (m,
H), 7.22 (d, J = 8.3 Hz, 2H), 7.20 – 7.17 (m, 2H), 7.09 (d, J = 8.3 Hz, 2H), 3.81ꢀ3.79 (m, 2H), 3.76ꢀ
.74 (m, 1H), 3.68 (s, 2H), 2.68 – 2.62 (m, 3H), 2.57 (t, J = 7.2 Hz, 3H), 1.63 (d, J = 12.0 Hz, 2H),
2 6
.38 (qd, J = 13.4, 4.6 Hz, 2H). Elemental analysis calculated (%) for C22H26Cl N : C 59.33, H 5.88, N
8.87. Found: C 59.35, H 5.85, N 18.67.
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1
-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-chlorophenethyl)-N-(naphthalen-1-ylmethyl)piperidin-4-
amine trihydrochloride (7k). The title compound was prepared by a similar procedure as that used
for 7a and purified by preparative HPLC to give 7k as hydrochloride salt (140 mg, 64%), mp 145ꢀ
+
1
1
8
7
2
2
6 2
46°C. LCꢀMS (ES+): RT 4.1 min, m/z 461.4/463.4 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.18 (d, J = 9.0 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.1 Hz, 1H),
.67 – 7.65 (m, 1H), 7.62 – 7.57 (m, 2H), 7.24 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 4.84 (bs,
H), 3.92 (bd, J = 13.4 Hz, 2H), 3.67 – 3.63 (m, 1H), 3.27 – 3.24 (m, 2H), 2.97 – 2.93 (m, 2H), 2.82 –
.78 (m, 2H), 2.22 (d, J = 12.4 Hz, 2H), 1.97 (qd, J = 12.1, 4.2 Hz, 2H). Elemental analysis calculated
(
%) for C H ClN *3HCl*H O: C 54.75, H 5.66, N 14.73. Found: C 54.82, H 5.48, N 14.90.
26 29 6 2
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-methyl-N-phenethylpiperidin-4-amine trihydrochloride (7l).
The title compound was prepared by a similar procedure as that used for 7a and purified by
preparative HPLC to give 7l as hydrochloride salt (33 mg, 38%), mp 158ꢀ160°C. LCꢀMS (ES+): RT
+
1
0
.4 min, m/z 301.2 4 [M+H] . H NMR (700 MHz, DMSOꢀd
7.1 Hz, 1H), 3.84 (bd, J = 12.6 Hz, 2H), 3.54 – 3.49 (m, 1H), 3.37 – 3.33 (m, 1H), 3.25 – 3.21 (m,
H), 3.06 – 3.01 (m, 1H), 2.99 – 2.92 (m, 3H), 2.77 (s, 3H), 2.04 – 2.01 (m, 2H), 1.75 – 1.64 (m, 2H).
Elemental analysis calculated (%) for C16 *3HCl: C 46.90, H 6.64, N 20.51. Found: C 46.61, H
6 2
+ D O) δ 7.34 – 7.29 (m, 4H), 7.25 (t, J
=
1
24 6
H N
6.86, N 20.37.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-methyl-N-(4-methylphenethyl)piperidin-4-amine (7m). The
title compound was prepared by a similar procedure as that used for 7a and crystalized from
+
acetonitrile to give 7m (27 mg, 37%), mp 150ꢀ152°C. LCꢀMS (ES+): RT 0.7 min, m/z 315.2 [M+H] .
1
H NMR (700 MHz, DMSOꢀd
6 2
+ D O) δ 7.19 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 3.85 (d, J
=
13.6 Hz, 2H), 3.52 (tt, J = 11.8, 3.4 Hz, 1H), 3.33 (td, J = 11.6, 5.2 Hz, 1H), 3.20 (td, J = 11.9, 5.5
Hz, 1H), 3.02 – 2.89 (m, 4H), 2.78 (s, 3H), 2.26 (s, 3H), 2.07 – 2.00 (m, 2H), 1.77 – 1.64 (m, 2H).
+
HRMS calculated for C17
H
27
N
6
[M+H] 315.2292, found 315.2289.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-ethylphenethyl)-N-methylpiperidin-4-amine
dihydrochloride (7n). The title compound was prepared by a similar procedure as that used for 7a
and purified by preparative HPLC to give 7n as hydrochloride salt (88 mg, 45%), mp 163ꢀ164°C. LCꢀ
+
1
6 2
MS (ES+): RT 1.7 min, m/z 329.4 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.22 (d, J = 8.1
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 3.85 (bd, J = 13.0 Hz, 2H), 3.53 – 3.49 (m, 1H), 3.36 – 3.31 (m,
1
H), 3.23 – 3.19 (m, 1H), 3.02 – 2.98 (m, 1H), 2.96 – 2.90 (m, 3H), 2.78 (s, 3H), 2.56 (q, J = 7.6 Hz,
H), 2.02 (bd, J = 12.4 Hz, 2H), 1.74 – 1.64 (m, 2H), 1.14 (t, J = 7.6 Hz, 3H). Elemental analysis
2
28 6 2
calculated (%) for C18H N *2HCl*0.5H O: C 52.68, H 7.61, N 20.48. Found: C 52.63, H 7.22, N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20.11.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-isopropylphenethyl)-N-methylpiperidin-4-amine
trihydrochloride (7o). The title compound was prepared by a similar procedure as that used for 7a
and purified by preparative HPLC to give 7o as hydrochloride salt (147 mg, 52%), mp 157ꢀ158°C.
+
1
LCꢀMS (ES+): RT 3.3 min, m/z 343.1 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.20 (d, J =
6
2
8
.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 3.82 (d, J = 12.9 Hz, 2H), 3.50 (tt, J = 11.9, 3.3 Hz, 1H), 3.30
td, J = 12.3, 5.3 Hz, 1H), 3.18 (td, J = 12.2, 5.2 Hz, 1H), 3.02 – 2.87 (m, 4H), 2.82 (sept, J = 6.9 Hz,
H), 2.75 (s, 3H), 2.06 – 1.97 (m, 2H), 1.75 – 1.63 (m, 2H), 1.14 (d, J = 6.9 Hz, 6H). Elemental
analysis calculated (%) for C19 *3HCl: C 50.50, H 7.36, N 18.60. Found: C 50.19, H 7.76, N
(
1
30 6
H N
18.44.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-cyclopropylphenethyl)-N-methylpiperidin-4-amine (7p).
The title compound was prepared by a similar procedure as that used for 7a and purified by
crystallization from acetonitrile to give 7p (195 mg, 62%), mp 108ꢀ110°C. LCꢀMS (ES+): RT 2.0 min,
+
1
m/z 341.35 [M+H] . H NMR (700 MHz, DMSOꢀd
Hz, 2H), 3.75 (d, J = 11.8 Hz, 2H), 2.63 – 2.53 (m, 7H), 2.21 (s, 3H), 1.81 (ddd, J = 13.4, 8.4, 4.9 Hz,
H), 1.66 (d, J = 12.2 Hz, 2H), 1.39 – 1.28 (m, 2H), 0.90 – 0.82 (m, 2H), 0.60 – 0.50 (m, 2H).
Elemental analysis calculated (%) for C19 : C 67.03, H 8.29, N 24.68. Found: C 66.98, H 8.23, N
6 2
+ D O) δ 7.04 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.0
1
28 6
H N
24.41.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-(tert-butyl)phenethyl)-N-methylpiperidin-4-amine (7r).
The title compound was prepared by a similar procedure as that used for 7a and purified by
crystallization from acetonitrile to give 7r (160 mg, 63%), mp 147ꢀ148°C. LCꢀMS (ES+): RT 3.6 min,
+
1
m/z 357.4 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.32 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4
6
2
Hz, 2H), 3.82 (d, J = 12.6 Hz, 2H), 3.56 – 3.40 (m, 1H), 3.30 (td, J = 12.1, 5.0 Hz, 1H), 3.18 (td, J =
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2.1, 5.3 Hz, 1H), 3.09 – 2.80 (m, 4H), 2.75 (s, 3H), 2.04ꢀ1.98 (m, 2H), 1.68 (dtd, J = 32.9, 12.1, 8.4
+
Hz, 2H), 1.22 (s, 9H). HRMS calculated for C20
H
33
N
6
[M+H] 357.2761, found 357.2744.
1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(2,4-dichlorophenethyl)-N-methylpiperidin-4-amine
(7s).
The title compound was prepared by a similar procedure as that used for 7a and purified by
crystallization from acetonitrile to give 7s (33 mg, 38%), mp 234ꢀ236°C. LCꢀMS (ES+): RT 2.3 min,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
m/z 369.2/371.2 [M+H] . H NMR (700 MHz, DMSOꢀd + D O) δ 7.54 (d, J = 2.2 Hz, 1H), 7.45 (d, J
6
2
=
8.3 Hz, 1H), 7.37 (dd, J = 8.3, 2.2 Hz, 1H), 3.91 – 3.83 (m, 2H), 3.55 – 3.49 (m, 1H), 3.26 (bs, 2H),
3.17 – 3.11 (m, 2H), 2.97 – 2.90 (m, 2H), 2.81 (s, 3H), 2.09 – 2.03 (m, 2H), 1.78 – 1.66 (m, 2H).
+
HRMS calculated for C H Cl N [M+H] 369.1356, found 369.1362.
1
6
23
2
6
Synthesis of 1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-bromobenzyl)piperidine-4-carboxamide
dihydrochloride (10).
Step 1: Synthesis of tert-Butyl 4-((4-bromobenzyl)carbamoyl)piperidine-1-carboxylate (9). The title
compound was prepared by a similar procedure as that used for 3 to give 9 (150 mg, 42%). LCꢀMS
+
1
(
ES+): RT 5.5 min, m/z 397.1/399.1 [M+H] . H NMR (700 MHz, CDCl
3
) δ 7.49 – 7.45 (m, 2H), 7.17
7.13 (m, 2H), 5.80 (bs, 1H), 4.41 (d, J = 5.8 Hz, 2H), 4.16 (bs, 2H), 2.76 (bs, 2H), 2.27 (tt, J = 11.6,
.8 Hz, 1H), 1.87 – 1.80 (m, 2H), 1.67 (qd, J = 12.2, 4.4 Hz, 2H), 1.47 (s, 9H).
–
3
Step 2: Synthesis of 1-(3-Amino-1H-1,2,4-triazol-5-yl)-N-(4-bromobenzyl)piperidine-4-carboxamide
hydrochloride (10). The title compound was prepared by a similar procedure as that used for 4 (step 2)
+
1
to give 10 (53 mg, 37%), mp 224ꢀ226°C. LCꢀMS (ES+): RT 3.7 min, m/z 379.1/381.1 [M+H] . H
NMR (700 MHz, DMSOꢀd ) δ 8.43 (t, J = 6.0 Hz, 1H), 7.57 – 7.39 (m, 2H), 7.29 – 7.11 (m, 2H), 4.21
6
(
d, J = 6.0 Hz, 2H), 3.77 (d, J = 12.8 Hz, 2H), 2.95 (t, J = 12.8 Hz, 2H), 2.45 – 2.37 (m, 1H), 1.80 –
+
1
3
1
.72 (m, 2H), 1.65 – 1.52 (m, 2H). HRMS calculated for C15
H20BrN
6
O [M+H] 379.0876, found
79.0868.
-(1-(3-Amino-1H-1,2,4-triazol-5-yl)piperidin-4-yl)-3-(4-bromophenyl)urea (13a).
Step 1: Synthesis of tert-Butyl 4-(3-(4-bromophenyl)ureido)piperidine-1-carboxylate (12a). To a
solution of 4ꢀbromoaniline (0.94 g, 5.46 mmol) in toluene was added diisopropylethylamine (1 mL,
2
5.46 mmol) and the mixture was cooled in iceꢀbath. A 20% solution of COCl (3.2 mL, 6.58 mmol) in
toluene was added in one portion. Bath was removed and after 40 min at rt TLC chloroform/methanol
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