Development of 1,2,4-Oxadiazoles as Potent and Selective Inhibitors of the Human Deacetylase Sirtuin 2: Structure–Activity Relationship, X-ray Crystal Structure, and Anticancer Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b01609

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit μM level by using the Sirt2 substrate a-tubulin-acetylLys40 peptide and inactive up to 100 μM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD+, and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed anti proliferative effects at 10 or 25 μM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.

Full text of DOI:10.1021/acs.jmedchem.6b01609

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Article
Development of 1,2,4-Oxadiazoles as Potent and Selective
Inhibitors of the Human Deacetylase Sirtuin 2: Structure-Activity
Relationship, X-Ray Crystal Structure and Anticancer Activity
Sebastien Moniot, Mariantonietta Forgione, Alessia Lucidi, Gebremedhin Solomon Hailu,
Angela Nebbioso, Vincenzo Carafa, Francesca Baratta, Lucia Altucci, Nicola Giacchè,
Daniela Passeri, Roberto Pellicciari, Antonello Mai, Clemens Steegborn, and Dante Rotili
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01609 • Publication Date (Web): 27 Feb 2017
Downloaded from http://pubs.acs.org on February 27, 2017
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Development of 1,2,4-Oxadiazoles as Potent and Selective Inhibitors of the
Human Deacetylase Sirtuin 2: Structure-Activity Relationship, X-Ray Crystal
Structure and Anticancer Activity
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†,¶
§,£,¶
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Sébastien Moniot, Mariantonietta Forgione,
Alessia Lucidi, Gebremedhin S. Hailu, Angela
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#
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Nebbioso, Vincenzo Carafa, Francesca Baratta, Lucia Altucci, Nicola Giacché,° Daniela
,§
,†
,§
Passeri,° Roberto Pellicciari,° Antonello Mai,* Clemens Steegborn,* Dante Rotili*
†
Department of Biochemistry and Research Center for BioꢀMacromolecules, University of
Bayreuth, 95440 Bayreuth, Germany
§
Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5,
00185 Rome, Italy, Laboratory affiliated to Istituto Pasteur Italia – Fondazione Cenci Bolognetti
£
Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena, 291,
0
0161 Rome, Italy
#
Department of Biochemistry, Biophysics and General Pathology, Second University of Naples,
Vico L. De Crecchio 7, 80138 Naples, Italy
°
TES Pharma S.r.l. Via P. Togliatti 20, 06073 Corciano Perugia, Italy
¶
These authors contributed equally to this work.
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ABSTRACT
Sirt2 is a target for the treatment of neurological, metabolic and ageꢀrelated diseases including
cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4ꢀoxadiazole scaffold. These
compounds are potent Sirt2 inhibitors active at singleꢀdigit ꢁM level by using the Sirt2 substrate αꢀ
tubulinꢀacetylLys40 peptide, and inactive up to 100 ꢁM against Sirt1, ꢀ3 and ꢀ5 (deacetylase and
desuccinylase activities). Their mechanism of inhibition is uncompetitive towards both the peptide
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substrate and NAD , and the crystal structure of a 1,2,4ꢀoxadiazole analog in complex with Sirt2
and ADPꢀribose reveals its orientation in a still unexplored sub cavity useful for further inhibitor
development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed
antiproliferative effects at 10 or 25 ꢁM after 48 h. Western blot analyses confirmed the involvement
of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2
inhibitors with a compact scaffold and structural insights for further inhibitor improvement.
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INTRODUCTION
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Sirtuins are a family of NAD ꢀdependent lysine deacylases highly conserved from bacteria to
humans. In mammals there are seven members, Sirt1ꢀ7, that localize in different subcellular
compartments, such as cytoplasm (Sirt1 and Sirt2), nucleus (Sirt1, Sirt2, Sirt6, and Sirt7) and
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mitochondria (Sirt3, Sirt4 and Sirt5). All sirtuins share a conserved NAD ꢀbinding and catalytic
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core domain, but possess distinct Nꢀ or Cꢀterminal extensions. Initially reported to be ADPꢀ
ribosyltransferases and/or deacetylases acting on acetylꢀlysine substrates from both histones and
nonꢀhistone proteins, some of them are now known to remove other acyl (malonyl, glutaryl,
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succinyl, myristoyl) rather than acetyl groups from protein lysines. Due to this multifaceted
activity on various substrate proteins, sirtuins are involved in many biological processes, including
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transcriptional regulation, genome stability, metabolic regulation, and cell survival.
Sirt2 is primarily localized in the cytoplasm and strongly expressed in neuronal tissue, where it
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contributes to protection against oxidative stress and memory function.
Sirt2 is also expressed
in many other tissues and can transiently translocate to the nucleus during G2/M transition. Here it
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deacetylates histone H4K16 thus modulating chromatin condensation during metaphase. Sirt2
regulates mitosis also through deacetylation of αꢀtubulin, and is linked with cancer metabolism by
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interaction with p65, FOXO1, FOXO3, and HIFꢀ1α.
The exact role of Sirt2 in cancer is
controversial, as for other sirtuins (Sirt1, Sirt3, Sirt6). Sirt2 has been suggested to be a tumor
suppressor because Sirt2ꢀknockout mice developed spontaneous tumors in multiple organs,
probably due to a Sirt2 lossꢀmediated deregulation of the anaphaseꢀpromoting complex/cyclosome
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activity, leading to increased levels of the mitotic death factors AuroraꢀA and ꢀB. Such tumor
development may be also due to higher genomic instability and chromosomal aberrations, as a
result of reduction of both the mitotic deposition of H4K20me1 and the levels of the related histone
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methyltransferase PRꢀSet7 after Sirt2 depletion. On the other hand, analyses of Sirt2 expression in
human cancer samples produced contradictory results, and Sirt2 knockdown or Sirt1/Sirt2
pharmacological inhibition repressed cancer cell proliferation, through activation and/or
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accumulation of tumor suppressor proteins such as p53 or FOXO1, or through destabilization of
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oncoproteins such as cꢀMyc and KꢀRAS.
In breast cancer, Sirt2 is the predominant sirtuin and
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appears to act as a tumor suppressor or tumor promoter depending on the breast tumor grade.
Finally, the overexpression of Sirt2 attenuated drug sensitivity and led to multidrug resistance in
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acute myeloid leukemia (AML) through increased expression of phosphorylated ERK1/2, and has
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been recently reported as a novel and unfavorable prognostic biomarker in AML patients.
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A number of Sirt2ꢀselective inhibitors have been described so far: AGK2 (1), AKꢀ7,
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MIND4, SirReal2 (2) and its analogues, the 4ꢀchromanone compound 6f, the benzamide
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analog 17k, the nicotinamide derivatives 33i and 64 and analogues, ICLꢀSIRT078, the
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macrocyclic peptide S2iL5 (3), and the thiomyristoyl peptide TM (Figure 1). However, various
shortcomings such as limited solubility or bioavailability or failures to further improve these mostly
large compounds lead to a requirement of further Sirt2 inhibitor scaffolds. Furthermore, while their
capability to exert neuroprotective effects in cellular models of neurodegenerative diseases has
mostly been reported, the anticancer properties of Sirt2 inhibitors are little studied.
Insert Figure 1
In our previous attempt to identify potent and isoformꢀselective sirtuin inhibitors, we docked a
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library of structurally diverse compounds in the crystal structures of Sirt2, ꢀ3, ꢀ5 and ꢀ6. This
study yielded two potent and Sirt2 selective inhibitors, but their compound scaffolds were not
suitable for cellular applications (see Figure S1 in Supporting Information). We thus used one of the
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less potent but already Sirt2ꢀselective compounds from this screen, the 3,5ꢀdisubstituted 1,2,4ꢀ
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oxadiazole CSC37 (4), as the starting point for development of novel Sirt2 inhibitors. Extensive
structureꢀactivity relationship (SAR) studies have been applied on the 3ꢀ(4ꢀchlorophenyl)ꢀ5ꢀ
(piperidinꢀ1ꢀylmethyl)ꢀ1,2,4ꢀoxadiazole 4 to obtain new hit compounds showing potent and highly
selective inhibition of Sirt2 in vitro (Figure 2). In particular, the influence of the pꢀchloro
substitution at the C3ꢀphenyl ring of 4 has been evaluated by shifting the chloro atom from para to
meta or ortho position, and by removing it or by replacing the phenyl with a 3ꢀpyridyl ring
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(compounds 5ꢀ8). The piperidinyl moiety has been removed or replaced with smaller or bigger
amines (compounds 9ꢀ12), or with morpholine, thiomorpholine, Nꢀmethylꢀ or Nꢀphenylpiperazine
moieties carrying an additional heteroatom in their structures (compounds 13ꢀ16). We also tested
the replacement of the piperidine ring by a 4ꢀhydroxymethylpiperidine, bearing a further
hydrophilic function to improve water solubility, or with a cyclohexylamine moiety, thus keeping
the nitrogen external to the cycle and converting it into a secondary, more basic amine (compounds
17, 18). Some 3ꢀ(4ꢀnitrophenyl)ꢀ1,2,4ꢀoxadiazole analogues bearing N,Nꢀdimethylamino, N,Nꢀ
diethylamino, or pyrrolidinomethyl chain at C5 were also prepared (compounds 19ꢀ21). The
influence of the length of the spacer at the C5 substituent has been evaluated by increasing it from 1
to 3 methylene groups (compare 22 and 23 with 4). Surprisingly, when we tested the intermediate 5ꢀ
(bromomethyl)ꢀ and the 5ꢀ(3ꢀbromopropyl)ꢀ3ꢀ(4ꢀchlorophenyl)ꢀ1,2,4ꢀoxadiazoles 24 and 30 we
noticed that they displayed more potent Sirt2 inhibition than the corresponding 5ꢀ(piperidinꢀ1ꢀ
ylmethyl) analogues 4 and 22. Thus, we selected 30, the most potent among them, as a new hit
compound and we performed on it some structural modifications, such as the shift of the 4ꢀchloro
substituent to meta or ortho position as well as the replacement with other atoms/groups
(compounds 31ꢀ43). Further derivatives carrying the 3ꢀ(4ꢀbromophenyl) moiety at C3 were
prepared to gain more SAR data (compounds 44ꢀ46).
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Insert Figure 2
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Kinetic studies and a crystal structure of Sirt2 in complex with a 1,2,4ꢀoxadiazole analog
provide mechanistic insights and interaction details for the Sirt2 inhibitory activity of our 1,2,4ꢀ
oxadiazoles. Selected compounds were further tested in leukemia as well as in MDAꢀMBꢀ231
breast cancer cells to determine their effects on cell cycle and apoptosis induction in cellular cancer
models. Functional assays to detect the acetylation levels of α–tubulin in the NB4 leukemia cells
support Sirt2 inhibition as the molecular mechanism for these compounds. Finally, two of the new
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,2,4ꢀoxadiazole hit compounds (35 and 39) were tested in a large panel of leukemia cells
characterized by different genotypes to determine their potential as antiproliferative agents.
RESULTS AND DISCUSSION
Chemistry. The synthetic route for the preparation of the 3ꢀarylꢀ1,2,4ꢀoxadiazole derivatives 4ꢀ
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and the appropriate acyl halides in dry dichloromethane and in the presence of triethylamine at
room temperature gave the crude N'ꢀacyloxyarylimidamide intermediates that without further
purification were heated in toluene or oꢀxylene at reflux to obtain the 1,2,4ꢀoxadiazole compounds
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, 24ꢀ43 (Scheme 1A). Compounds 4-8, 10ꢀ23 were synthesized by treating the selected 5ꢀ
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bromoalkyl)ꢀ3ꢀ(substituted)phenylꢀ1,2,4ꢀoxadiazoles 24 (for 4, 10-18), 25 (for 5), 26 (for 6), 27
for 7), 28 (for 8), 29 (for 19ꢀ21), 30 (for 22) and 33 (for 23) with different commercially available
(
amines, sodium iodide, and potassium carbonate in dry DMF at 60 °C for 1ꢀ2 h (Scheme 1B). The
reaction of the 4ꢀbromoꢀN'ꢀhydroxybenzimidamide 47g with methyl 2ꢀchloroꢀ2ꢀoxoacetate or 2,2,2ꢀ
trifluoroacetic anhydride in a mixture (10:1 v/v) of dry dichloromethane and dry pyridine at room
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temperature or reflux directly furnished the cyclized 5ꢀmethoxycarbonylꢀ and 5ꢀtrifluoromethylꢀ
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,2,4ꢀoxadiazoles 44 and 45, respectively. Further treatment of 44 with a 7M solution of ammonia
in methanol gave the 5ꢀcarboxyamide analog 46 (Scheme 1C).
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Chemical and physical data of compounds 4ꢀ46 are listed in Table S1 in Supporting Information.
Insert Scheme 1
Assessment of Sirt2 inhibiting potency and selectivity. The inhibitory effects of 4ꢀ24, 30ꢀ46
on Sirt2 deacetylase activity were tested in a continuous assay using an αꢀtubulinꢀacetylLys40
peptide as the substrate. The residual Sirt2 activity in presence of 10 and 100 µM of the candidate
inhibitors was evaluated (Figure 3A).
Insert Figure 3
Comparison of the Sirt2 inhibiting activity exerted by 4ꢀ8 suggests that, at the C3ꢀphenyl ring of
the 1,2,4ꢀoxadiazole moiety, the presence as well as the position of the chlorine atom is a
determinant for the inhibitory activity. Indeed, shifting it from para (4) to meta (5) position retains a
certain potency, which is lost with the chlorine atom in ortho (6) or with its complete removal (7) or
by changing the C3ꢀphenyl to a C3ꢀ3ꢀpyridyl ring (8). At the C5ꢀmethylene group of 4 it seems to
be required a cyclic amine moiety for the potency against Sirt2 [see the drop of potency after its
removal (9) or replacement with a N,Nꢀdimethylamino group (10, 19)]. Changes of the piperidine
moiety of 4 with the smaller pyrrolidine (11), or the larger azepane (12) or the heteroatomꢀ
containing morpholine (13), thiomorpholine (14), piperazine (15, 16), or 4ꢀ
hydroxymethylpiperidine (17) led to compounds with similar or lower Sirt2 inhibiting potency, with
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the exception of 11 and 14 that displayed higher inhibition than 4 at 100 ꢁM. Interestingly, also the
replacement of the piperidine with a cyclohexylamine moiety (18), thus changing a tertiary with a
secondary amine function linked to the C5ꢀmethylene group, led to increased inhibition at 100 ꢁM.
Replacing the pꢀchloro with a pꢀnitro substituent at the C3ꢀphenyl ring (19-21) as well as increasing
the length of the spacer between the C5 oxadiazole position and the cyclic amine from 1 to 3
methylene groups (22, 23), strongly decreased or totally abolished the Sirt2 inhibiting activity.
Together with these 5ꢀaminomethylꢀsubstitutedꢀ1,2,4ꢀoxadiazoles, we tested also the
intermediate 5ꢀ(bromomethyl)ꢀ and 5ꢀ(3ꢀbromopropyl)ꢀ3ꢀ(4ꢀchlorophenyl)ꢀ1,2,4ꢀoxadiazoles 24
and 30 against Sirt2 at 10 and 100 ꢁM. Surprisingly, they displayed a substantial improvement in
potency at both tested doses with respect to the corresponding 5ꢀ(piperidinꢀ1ꢀylmethyl) analogues 4
and 22 (Figure 3A). Again, shifting the chlorine atom of 30 from para to meta (31) or ortho (32)
position as well as its total removal (33) decreased the Sirt2 inhibitory potency of the compounds.
Replacement of the pꢀchloro with a pꢀmethyl (34), pꢀmethoxy (35), pꢀtrifluoromethyl (36), pꢀnitro
(37), pꢀfluoro (38), or pꢀbromo (39) substituent in some cases provided more potent (34, 37)
derivatives than 30 only at 100 ꢁM, while with 39 the same inhibition as 30 at 10 ꢁM and improved
inhibition at 100 ꢁM were detected. Thus a further exploration of SAR around 39 was performed,
by preparing the two regioisomers 40 and 41 and by changing the C5ꢀ(3ꢀbromopropyl) chain with a
C5ꢀ(3ꢀchloropropyl) (42) or a C5ꢀ(4ꢀbromobutyl) (43) chain. Whilst the shift of the bromine at the
C3ꢀphenyl ring from para to meta or ortho position abated the Sirt2 inhibition (compare 39 with 40
and 41), the bromo/chloro exchange at the end of the C5ꢀside chain or its enlargement to four
methylene units led to compounds still endowed with high inhibitory activity.
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Finally, replacement of the 3ꢀbromopropyl side chain at C5 of the 3ꢀ(4ꢀbromophenyl)ꢀ1,2,4ꢀ
oxadiazole scaffold with a carbomethoxy (44), trifluoromethyl (45), or carboxamide (46) function
yielded in all cases poorly potent or inactive compounds.
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In order to evaluate and compare more finely the potencies of some potent compounds we
determined the IC50 values against Sirt2 for 30, 35-39, 42 and 43 (Table 1). The 3ꢀ(4ꢀbromophenyl)ꢀ
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the synthesized oxadiazoles, it being slightly more efficient than its 3ꢀ(4ꢀchlorophenyl) counterpart
30. Little changes at the para position of the oxadiazole C3ꢀphenyl ring (see 35ꢀ38) or at the C5ꢀ
side chain (see 42, 43) had only moderate effects on the Sirt2 inhibitory potency of the derivatives,
with only the pꢀfluoro analog 38 being one order of magnitude less potent. We could take in account
that 36, 37, and 43 might be slightly less potent than indicated by their IC50 values due to an
increased background, likely due to solubility limitations.
Table 1. IC50 Values Against Sirt2 for Selected Compounds
compd structure
IC50, ꢁM
background, %
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7
2.5 ± 0.2
12.0
10.4 ± 0.2
3.9 ± 0.3
6.6 ± 0.9
19.8±1.0
19.3
32.9
27.6
17.1
38
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2
2
2
2
2
2
2
2
2
3
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5
5
5
5
5
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39
42
1.5 ± 0.1
3.6 ± 0. 3
15.5
15.6
25.7
0
1
2
3
4
5
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9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
2.6 ± 0.4
To evaluate the isoform selectivity of the new compounds, the oxadiazoles 30, 39 and 42 were
tested against the sirtuin isoforms Sirt1, ꢀ3, and ꢀ5 (deacetylase and desuccinylase activities) using
their established substrate peptides Acꢀp53 (Sirt1), AcꢀAceCS2 (Sirt3), and AcꢀCPS1 or SucꢀPrx
(Sirt5), respectively (Figure 3B).
The activities of these sirtuin isoforms were not significantly affected by any of the tested 1,2,4ꢀ
oxadiazole Sirt2 inhibitors at concentrations up to 100 µM. These results show that Sirt2 inhibition
is not a promiscuous effect for these compounds, and that this inhibitor class displays outstanding
selectivity toward Sirt2 despite its compact size. The improvement in potency and, in particular, in
selectivity of the new 1,2,4ꢀoxadiazoles compared to many known Sirt2 inhibitors is illustrated by
2
8
comparison with 1, a widely used Sirt2ꢀspecific inhibitor (Figure 3B). While the inhibition of
Sirt2 by 30, 39 or 42 is slightly more pronounced than by 1, no effect of the tested 1,2,4ꢀoxadiazoles
is seen on other Sirt isoforms at 100 ꢁM, while 1 starts to show nonꢀspecific effects at this
concentration.
Mechanism of Sirt2 Inhibition by 1,2,4-Oxadiazoles. To gain first insight into the inhibition
mechanism of 39, we analyzed it via MichaelisꢀMenten kinetic experiments. Both the αꢀtubulin
1
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peptide substrate and the NAD coꢀsubstrate were titrated at different inhibitor concentrations
Figure 4). In both cases, fitting of the individual MichaelisꢀMenten curves showed a decrease of
Vmax and K with increasing 39 concentrations. The complete data sets were thus fitted with an
(
m
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30
31
32
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
uncompetitive inhibition model, which yielded K
m
values of 128.5 ± 13.1 and 123.7 ± 4.2 µM,
values of 2.2 ± 0.1 µM for 39
+
respectively for the peptide substrate and NAD . The resulting K
i
from both competition experiments are in good agreement with the IC50 values (Table 1), as
expected for uncompetitive inhibitors tested under substrate saturation. Comparable data were
obtained from analogous competition experiments with 30 (data not shown), supporting the
conclusion that this compound class inhibits uncompetitively with both substrates.
An uncompetitive mechanism, ie improved inhibition in presence of increasing substrate
concentrations, indicates that the inhibitors bind to a closed conformation of Sirt2 induced by
substrate binding.
Insert Figure 4
Crystal Structure of Sirt2 in Complex with a 1,2,4-Oxadiazole Analog and ADP-ribose. To
characterize further the inhibition mechanism and binding details of our compounds, we tried a
variety of coꢀcrystallization and soaking approaches for solving a complex structure. Finally, we
were able to solve the structure of a Sirt2/1,2,4ꢀoxadiazole complex by soaking the compound 30 in
Sirt2/ADPꢀribose crystals. The resulting highꢀresolution structure was refined to Rcryst/Rfree values
of 17.3/19.4 % (Table 2) and contained well defined electron density for both the ADPꢀribose
molecule and for the 1,2,4ꢀoxadiazole inhibitor (Figure 5A and Figure S2 in Supporting
Information). The electron density located in the hydrophobic pocket at the back of the active site of
Sirt2 can clearly be attributed to the soaked ligand, however the abnormally high Bꢀfactors for the
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2
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2
2
2
2
2
2
3
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3
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5
5
6
Br atom as well as the presence of significant residual negative density in the Fourier difference
map after refinement (Figure S3 in Supporting Information) led to the conclusion that rather than
compound 30, an analogue of this compound with a hydroxyl group instead of the bromine at the
C5 substituent, the 5ꢀ(3ꢀhydroxypropyl)ꢀ3ꢀ(4ꢀchlorophenyl)ꢀ1,2,4ꢀoxadiazole 48, is bound to Sirt2
(Figure S3). This observation is further supported by the absence of any anomalous signal for a Br
0
1
2
3
4
5
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7
8
9
0
1
2
3
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1
2
3
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8
9
0
atom in this region although the data were collected at the bromine K absorption edge. Hydrolysis
47ꢀ48
of bromoalkyl chains in water and/or buffer has been reported before,
and the sonication,
heating, and freezeꢀthawing preꢀtreatment employed for the crystal soaking experiments appear to
have amplified the bromoꢀhydroxy exchange (Figure S4 in Supporting information). Surprisingly,
all attempts to crystallize pure 30 and 39 with Sirt2 and ADPꢀribose were unsuccessful leading to
more than 20 empty crystal structures, and only when the bromoꢀhydroxy conversion of 30 to 48 in
part occurred (Figure S4) we were able to obtain a complex.
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Table 2. Diffraction Data and Refinement Statistics
Sirt2/ADP-ribose/48 complex
Diffraction Data Statistics
Wavelength (Å)
Resolution range (Å)
Space group
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.9184
48.93ꢀ1.89 (1.93ꢀ1.89)
1 1 1
P2 2 2
Unit cell (Å)
76.56 76.71 113.83
399403 (24802)
54438 (3465)
100 (100)
Total reflections
Unique reflections
Completeness (%)
Multiplicity
7.3 (7.2)
Mean I/σ(I)
14.4 (2.5)
Wilson Bꢀfactor
Rꢀmerge (%)
26.3
9.0 (74.1)
Rꢀmeas (%)
10.4 (86.8)
99.9 (56.0)
CC1/2 (%)
Refinement Statistics
Rꢀwork (%)
17.3 (28.1)
19.4 (28.7)
5494
4840
106
Rꢀfree (%)
Non H atoms
Protein
Ligands (Zn, 48, ADPꢀribose)
Solvent
476
RMSD(bonds) (Å)
RMSD(angles) (°)
Ramachandran most favored (%)
Ramachandran outliers (%)
Average Bꢀfactor (Ų)
Protein (Ų)
0.003
0.66
97
0.16
38.2
37.3
Ligands (Ų) (Zn, ADPr, 48)
Solvent (Ų)
34.0
45.3
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5
5
5
5
6
Insert Figure 5
To analyze the capability of 48 to inhibit Sirt2, we synthesized and tested it in our enzyme assay
(see Scheme S1 and Figure S5 in Supporting Information). The obtained IC50 values against Sirt2
for 48 and its acetyl analog 49, intermediate for the synthesis, are: 41.2 ± 11.9 ꢁM for 48, and 21.2
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
±
3.8 ꢁM for 49. These results confirm that 48 is still active against Sirt2, but less potent than 30,
and that the insertion of a highly polar substituent (ie, hydroxyl) at the C5 chain causes a severe
drop of Sirt2 inhibition. However, such loss of potency can be in part counteracted by converting
the hydroxyl into an acetoxy function (48 to 49). Thus, the hydroxyl group of 48 enables inhibition
and crystal structure analysis of the Sirt2 complex with this compound family representative, but it
is not required for highly potent inhibition.
The conversion from 30 to 48 also raises the possibility of a covalent mode of inhibition,
however such a mechanism is very unlikely. First, no covalent connection can be observed in the
crystal structure between 48 and either Sirt2 or ADPꢀribose (Figure S2 in Supporting Information;
see below). In addition, preincubation of Sirt2 with 39 (or 30) did not reveal any influence on the
compound inhibitory potency, which further support the fact that inhibition mechanism of this
compound family does not involve covalent modification of the enzyme (Figure S6 in Supporting
Information).
In the Sirt2/48 complex, the compound occupies the acyl channel at the back of the active site of
49ꢀ50
Sirt2 with its alkyl chain positioned in the extended Cꢀsite (“ECSꢀI” in reff.
) and its paraꢀchloro
phenyl located in a subꢀcavity of the large hydrophobic pocket (see below). The ligand is stabilized
by hydrogen bonds of the 5ꢀhydroxyl of 48 to the mainꢀchain carbonyl atom of Val233 and to the 2'ꢀ
hydroxyl atom of ADPꢀribose. However, these interactions are not essential for potent inhibition,
since 30 with its bromine inhibits Sirt2 much more potently (>10ꢀfold) than 48. All other
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interactions are van der Waals interactions and involve the side chains of Phe96 and 119, of Leu134,
1
38 and 297, of Ile169 and 232 (Figure 5B and Figure S7 in Supporting Information). As already
observed in other Sirt2 crystal structures, the electron density for the loop residues 139ꢀ141 is
relatively weak, and the exact position of the side chain of Leu138, notably, is uncertain indicating
that although 48 binds at the proximity of this flexible region, it does not seem to stabilize its
conformation. The Cꢀsite, which is dedicated to bind the carboxamide group of nicotinamide, is not
covered by 48 and instead occupied by a molecule of DMSO (Figure 5A and 5B).
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17
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42
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47
48
49
50
51
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59
60
The overall Sirt2 conformation in the Sirt2/48 complex is similar to that in the original,
unsoaked crystal structure. Indeed, superposition of the crystal structures of Sirt2 in complex with
ADP ribose to our ligand complex yields low rmsd values [3zgv (0.36 Å) and 5d7o (0.79 Å) on 300
α
C positions] and thus indicate that binding of the ligand does not induce any major conformational
rearrangement (Figure 5C). Most of the differences are located in two regions; the 135ꢀ142 flexible
loop which connects the zincꢀbinding and Rossmannꢀfold domains and constitute part of the ligand
binding site and the αꢀhelix 205ꢀ217 which forms interactions with the former loop.
Comparing the Sirt2/48 complex to other Sirt2 inhibitor structures shows that the overall protein
conformation is very similar to the one observed for complexes with the (S)ꢀ6ꢀchloroꢀ2,3,4,9ꢀ
5
1
tetrahydroꢀ1Hꢀcarbazoleꢀ1ꢀcarboxamide Exꢀ243 (50) (the S enantiomer of the 6ꢀchloroꢀ2,3,4,9ꢀ
5
2
tetrahydroꢀ1Hꢀcarbazoleꢀ1ꢀcarboxamide Exꢀ527 (51) ) and its derivative CHIC35 [(S)ꢀ2ꢀchloroꢀ
51
5
,6,7,8,9,10ꢀhexahydroꢀcyclohept[b]indoleꢀ6ꢀcarboxamide] (rmsd on 301 and 302 C
α
positions of
0
.43 Å and 0.39 Å respectively with PDB: 5d7p and 5d7q). This finding is consistent with the fact
that 48 as well as 50 occupy the extended Cꢀsite in presence of a nucleotide coꢀligand and that both
complex structures were in fact obtained using the same target conformation through soaking of a
31
Sirt2/ADPꢀribose complex. In contrast, superposition of Sirt2/48 with the complex of Sirt2 with 2
+
and NAD (PDBid: 4rmg) yields an rmsd of 2.0 Å. Most conformational differences between these
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2
2
2
2
2
2
2
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5
5
5
6
two structures are due to the fact that 2 blocks Sirt2 in a so called “blockedꢀopen” conformation, in
which the zincꢀbinding domain is not fully closing the active site (Figure 5D). It is interesting to
note that our compound, which is smaller than 2, is able to bind to the closed conformation of Sirt2,
which is also in agreement with the fact that the 1,2,4ꢀoxadiazoles described here are uncompetitive
inhibitors and therefore bind to an “active” conformation of the protein.
0
1
2
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1
2
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4
5
6
7
8
9
0
As illustrated by a superposition of representative Sirt2 inhibitors (Figure 6A), the 1,2,4ꢀ
oxadiazole 48 exhibits an unprecedented orientation in the large, hydrophobic acyl channel pocket
thereby addressing another, Sirt2ꢀspecific sub cavity by intercalating between the side chains of
Leu134 and Leu138. This cavity, uniquely occupied by our compounds, might explain their
exceptional Sirt2ꢀselectivity and can be exploited in further Sirt2 inhibitor development based on
our and also other scaffolds. The superposition of our Sirt2 complex structure with that of the
3
9
inhibitory macrocyclic peptide 3 (Figure 6A; PDBid 4l3o) indicates that the conformation of the
1,2,4ꢀoxadiazole is incompatible with the presence of an acetyl substrate peptide as the two
molecules are clashing. It is, however, conceivable that in the inhibitory relevant quaternary
complex between Sirt2, substrate, coꢀsubstrate, and 1,2,4ꢀoxadiazole, slight conformational changes
allows the 1,2,4ꢀoxadiazole inhibitor to stale catalysis, or that inhibitor binding in fact takes place
49
after intermediate or even product formation, as observed with 50/51.
To compare our complex structure with the SAR data, a set of 1,2,4ꢀoxadiazoles were modelled
inside the active site of Sirt2 based on the crystal structure (Figure 6BꢀE). As exemplified by the
model with 4, the active site of Sirt2 cannot accommodate too bulky groups at the C5 position of
the 1,2,4ꢀoxadiazole ring (Figure 6C). The SAR results about the substitution at the C3 phenyl ring
are also supported by the model of 40 and 41, because the shift of the chlorine atom from para to
either meta or ortho position at the phenyl ring introduce deleterious steric hindrance in the binding
pocket (Figures 6D and 6E).
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Insert Figure 6
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17
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23
24
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30
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
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55
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57
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59
60
1,2,4-Oxadiazoles on Cancer Cells. The 1,2,4ꢀoxadiazoles 4ꢀ24, 30-46 were tested in U937
human leukemia cells at 25 ꢁM for 48 h to determine their effects on cell cycle and induction of
apoptosis (Figure 7). Under the tested conditions, the majority of the compounds did not alter the
percentage of cell cycle phases with respect to the control. The only exceptions were compounds
2
4, 32 and 41, which blocked cell cycle at the G2/M phase, and 35, which gave arrest at the S phase
(Figure 7A). The same compounds, including also 38, displayed the highest apoptosis induction,
determined as fraction of cells in preꢀG1 peak, with percentages in the range 11.1ꢀ37.1%.
Insert Figure 7
Compounds 30, 35, 38 and 39 were chosen according to their potency in Sirt2 inhibition and/or
apoptosis induction in U937 cells, and were tested against a panel of leukemia cells (U937, NB4,
HLꢀ60, and K562) as well as against breast cancer MDAꢀMBꢀ231 cells for 48 h at 5, 10, 25 and 50
ꢁM. The relative doseꢀresponse curves for apoptosis induction (preꢀG1 peak percentage) are
depicted in Figure 8A: among the tested compounds, 39 was highly potent against NB4 cells
already at 10 ꢁM, and displayed >80% of apoptosis in U937 cells at 50 ꢁM. Compound 35 was the
most efficient (>80%) as apoptosis inducer at 25 ꢁM in 3 out of 5 cell lines (NB4, K562 and MDAꢀ
MBꢀ231), while 38 showed 30ꢀ40% apoptosis only at 50 ꢁM against NB4 and MDAꢀMBꢀ231 cells,
and 30 was in general less effective. In the same assays, 1 was totally unable to elicit apoptosis,
giving only 6% apoptosis in HLꢀ60 cells at the highest tested dose (Figure S8 in Supporting
Information).
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2
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4
4
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6
Insert Figure 8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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9
0
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0
1
2
3
4
5
6
7
8
9
0
To confirm that our 1,2,4ꢀoxadiazoles worked in the tested cells by inhibiting Sirt2, the levels of
acetylꢀαꢀtubulin were detected in NB4 and in U937 cells treated with 35 and 39 (5 and 25 ꢁM) for 4
(NB4) or 24 (U937) h (Figure 8B). Compound 1 was used as a reference drug, and the
suberoylanilide hydroxamic acid (SAHA, 52) as a positive control. Western blot analyses in both
cell lines showed no substantial effect on acetylꢀαꢀtubulin levels by 1, while 35 and 39 increased α–
tubulin acetylation.
Compounds 35 and 39 were further tested in a large panel of leukemia cells characterized by
different genotypes (OCIꢀAML3, IMSꢀM2, OCIꢀAML2, MV4ꢀ11, Kasumiꢀ1, Karpass299) in
addition to the already used U937, HLꢀ60 and NB4, to determine their antiproliferative effects. The
relative IC50 values are listed in Table 3. Among the tested cell lines, 35 arrested the growth of
Kasumiꢀ1 and NB4 cells at its biochemical IC50 value (10 ꢁM), and that of MV4ꢀ11 and IMSꢀM2
cells at slightly higher concentration, while with OCIꢀAML3 showed the lowest efficacy. Instead,
3
9 showed a general reduced antiproliferative effect compared to 35, with a preferential efficacy
against Karpass299, MV4ꢀ11 and NB4 cell lines.
Table 3. Antiproliferative Effect (IC values) Displayed by 35 and 39 in a Panel of Leukemia
5
0
Cell Lines
IC , ꢁM
5
0
cell line
subꢀtype
alterations
3
5
39
a
+
OCIꢀAML3
AML
NPM1c , DNMT3A (R882C)
62 ± 6
66 ± 2
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IMSꢀM2
OCIꢀAML2
MV4ꢀ11
Kasumiꢀ1
U937
AML
AML
AML
AML
AML
NPM1c , no other informations
18.4 ± 0.4 100 ± 1
DNMT3A (R635W)
37 ± 2
16 ± 2
85 ± 5
38 ± 8
56 ± 3
>100
10
11
12
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21
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23
24
25
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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58
59
60
FLT3ꢀITD, [t(4;11)(q21;q23)],
MLL/MLLT2(AF4)
AML1ꢀETO [t(8:21)]
10.3 ± 0.3
25 ± 5
+
/ꢀ
p53
b
NB4
APL
PMLꢀRARα, [t(15;17)(q22;q12)]
12 ± 2
49 ± 1
100 ± 2
25 ± 6
ꢀ/ꢀ
HLꢀ60
APL
p53
30 ± 3
c
Karpass299
ALCL
NPM1/ALK, [t(2;5)(p23;q35)]
36 ± 3
a
b
c
AML, Acute Myeloid Leukemia. APL, Acute Promyelocytic Leukemia. ALCL, Anaplastic Large
Cell Lymphoma.
CONCLUSION
Among the epigenetic players (writers, readers and erasers), the class III deacetylase Sirt2 is widely
11
recognized as an attractive target for treatment of neurodegenerative disorders, metabolic
53
10
9, 54
dysfunctions, ageꢀrelated diseases, and cancer,
although its role in the last disease is still
25, 55
23, 28ꢀ29, 31ꢀ39
debated.
A number of Sirt2 inhibitors have been reported to date,
but they suffer
from scarce Sirt2 isoform selectivity and/or they have been studied in CNS rather than in cancer
contexts. Here we describe the identification of a new series of Sirt2 inhibitors, based on the 1,2,4ꢀ
oxadiazole scaffold, starting from the hit compound 3ꢀ(4ꢀchlorophenyl)ꢀ5ꢀ(piperidinꢀ1ꢀylmethyl)ꢀ
40
1
,2,4ꢀoxadiazole 4 previously disclosed by us through virtual screening procedures. Extensive
SAR studies applied on this compound by using the αꢀtubulinꢀacetylLys40 peptide as the Sirt2
substrate highlighted the presence of a para-substituted phenyl ring at the C3 position and of a
cyclic aminomethyl or a ω–haloalkyl chain at the C5 position of the 1,2,4ꢀoxadiazole scaffold as
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crucial determinants to gain Sirt2 inhibition. Selected compounds showed IC50 values in the low,
singleꢀdigit ꢁM range, and were inactive up to 100 ꢁM against Sirt1, Sirt3 and Sirt5 (deacetylase
and desuccinylase activities). Mechanistic studies performed on the C3ꢀparaꢀbromophenyl 39
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showed uncompetitive inhibition with respect to both the α–tubulin peptide substrate and the NAD
coꢀsubstrate, and the crystal structure of Sirt2 in complex with the 3ꢀ(4ꢀchlorophenyl)ꢀ5ꢀ(3ꢀ
hydroxypropyl)ꢀ1,2,4ꢀoxadiazole 48 and ADPꢀribose was determined. The superposition of the
complex structure between Sirt2 and 48 with that of some representative Sirt2 inhibitor complexes
revealed that 48 exhibits an unprecedented orientation in the large hydrophobic pocket addressing
another sub cavity by intercalating between the side chains of Leu134 and Leu138, a cavity never
exploited before that may constitute a rationale for further Sirt2 inhibitor development, also starting
from other scaffolds. Development of more potent inhibitors based on our 1,2,4ꢀoxadiazole moiety
could be pursued by further extension of the 3ꢀ(4ꢀchlorophenyl) substituent to address either the
Sirt2 ‘selectivity pocket’ occupied by the SirReal inhibitors or by introducing interactions within the
Cꢀsite, taking over the position of the DMSO molecule bound in our Sirt2/48 complex structure.
When tested in human leukemia (U937, NB4, HLꢀ60 and K562) cell lines as well as against the
breast cancer MDAꢀMBꢀ231 cell lines, 35 gave >80% of apoptosis at 25 ꢁM in NB4, K562 and
MDAꢀMBꢀ231 cell lines, and 39 was able to induce the same effect in NB4 cells already at 10 ꢁM.
These compounds, when analyzed in Western blot assay to detect their effects on the acetylꢀαꢀ
tubulin levels in NB4 cells, displayed an increase of αꢀtubulin acetylation confirming the
involvement of Sirt2 inhibition in their activity. Compound 1, tested at the same condition, was
practically inactive in both apoptosis induction assays and Western blot analysis.
AML is a disease that groups heterogeneous hematopoietic stem cell disorders. Several recurrent
chromosomal alterations are involved in AML pathogenesis, such as aberrant fusion proteins
+
AML1ꢀETO, PMLꢀRARα or mutant proteins as NPM1c , FLT3ꢀITD or DNMT3A. Thus, we tested
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5 and 39 against a panel of leukemia cell lines characterized by different alterations. The IC50
values reported in Table 3 show that 35 was able to reduce 50% cell growth in Kasumiꢀ1 and NB4
cell lines at its IC biochemical value as Sirt2 inhibitor (10 ꢁM), and in MV4ꢀ11 and IMSꢀM2 cell
50
10
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lines at concentration slightly higher, while 39 showed reduced potency and a preferential efficacy
against Karpass299, MV4ꢀ11 and NB4 cell lines. Further studies will be performed on this 1,2,4ꢀ
oxadiazole series of Sirt2 inhibitors to improve their potency and their anticancer potential.
EXPERIMENTAL SECTION
1
Chemistry. Melting points were determined on a Stuart melting point apparatus SMP10. HꢀNMR
spectra were recorded at 400 MHz using a Bruker AC 400 spectrometer; chemical shifts are
4
reported in δ (ppm) units relative to the internal reference tetramethylsilane (Me Si). Mass spectra
were recorded on an APIꢀTOF Mariner by Perspective Biosystem (Stratford, TX, USA), and
samples were injected by an Harvard pump using a flow rate of 5ꢀ10 ꢁL/min, infused in the
1
Electrospray system. All compounds were routinely checked by TLC and HꢀNMR. TLC was
performed on aluminumꢀbacked silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with
4
spots visualized by UV light or using a KMnO alkaline solution. All solvents were reagent grade
and, when necessary, were purified and dried by standard methods. Concentration of solutions after
reactions and extractions involved the use of a rotary evaporator operating at reduced pressure of ~
2
0 Torr. Organic solutions were dried over anhydrous sodium sulfate. Elemental analysis has been
used to determine purity of the described compounds, that is >95%. Analytical results are within ±
.40% of the theoretical values (Table S2 in Supporting Information). All chemicals were
purchased from Sigma Aldrich s.r.l., Milan (Italy) or from TCI Europe N.V., Zwijndrecht
Belgium), and were of the highest purity. As a rule, samples prepared for physical and biological
0
(
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studies were dried in high vacuum over P
depending on the sample melting point.
2 5
O for 20 h at temperatures ranging from 25 to 40 °C,
General procedures for the syntheses of the 1,2,4ꢀoxadiazoles 9, 24-43 (General Procedure A) and
of 4-8, 10-23 (General Procedure B), spectral data for the final compounds 4-24, 30-43, and
syntheses and spectral data of compounds 44ꢀ46 are described below. Syntheses and spectral data of
compounds 48 and 49 as well as chemical and physical data of compounds 4ꢀ46, 48 and 49 are
reported in Supporting Information.
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General Procedure for the Preparation of the 1,2,4-Oxadiazoles 9, 24-43 (General
Procedure A). A solution of the appropriate (ω–halo)acyl chloride (1.32 mmol) in dry
dichloromethane (2 mL) was added dropwise, while cooling at 0 °C, to a solution of the proper (Z)ꢀ
N'ꢀhydroxybenzimidamide 47 (0.88 mmol) in dry dichloromethane (3 mL) in the presence of
triethylamine (1.58 mmol). The reaction mixture was stirred at 0 °C for 10 min and then at room
temperature for 2 h. After the completion of the reaction, the mixture was evaporated at reduced
pressure and the resulting crude product was triturated with water (10 mL) for 1 h and then filtered
to afford the related (Z)ꢀN'ꢀ[(ωꢀhaloacyl)oxy]benzimidamide as a pink solid that was used in the
next step without further purification.
The crude (Z)ꢀN'ꢀ[(ωꢀhaloacyl)oxy]benzimidamide (0.852 mmol) was dissolved in dry toluene
(10 mL) and stirred at reflux for 5 h. Toluene was then evaporated under reduced pressure at 60 °C
to achieve a brown/orange crude product that was purified by a silica gel column eluting with a
mixture ethyl acetate:nꢀhexane 1:30 to give the final product.
General Procedure for the Preparation of the 1,2,4-Oxadiazoles 4-8, 10-23 (General
Procedure B). A mixture of 5ꢀ(bromoalkyl)ꢀ3ꢀaryl)ꢀ1,2,4ꢀoxadiazole (among 24-30 or 33) (0.365
mmol), piperidine (1.82 mmol), sodium iodide (0.402 mmol) and dry potassium carbonate (0.548
mmol) in dry DMF (1.5 mL) was heated while stirring at 60 °C for 1 h. The reaction was then
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allowed to cool to room temperature, quenched with water (18 mL) and extracted with ethyl acetate
(4 × 25 mL). The combined organic phases were washed with brine, dried over sodium sulfate,
filtered and evaporated under vacuum to afford a residue that was purified by a silica gel column
10
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60
eluting with a mixture ethyl acetate:nꢀhexane 1:5 to give the pure final product.
1
3-(4-Chlorophenyl)-5-(piperidin-1-ylmethyl)-1,2,4-oxadiazole (4) (General Procedure B). Hꢀ
NMR (400 MHz; CDCl
3
) δ 1.44ꢀ1.50 (m, 2H, CH
2
piperidine ring), 1.64ꢀ1.69 (m, 4H, 2 × CH
2
piperidine ring), 2.59ꢀ2.61 (m, 4H, 2 × CH
2
piperidine ring), 3.90 (s, 2H, oxadiazoleꢀCH
2
N), 7.47ꢀ
7
.49 (d, J = 8.8 Hz, 2H, CH benzene ring), 8.05ꢀ8.08 (d, J = 8.8 Hz, 2H, CH benzene ring). MS
+
(ESI), m/z: 278 [M + H] .
1
3
-(3-Chlorophenyl)-5-(piperidin-1-ylmethyl)-1,2,4-oxadiazole (5) (General Procedure B). Hꢀ
NMR (400 MHz; CDCl ) δ 1.45ꢀ1.50 (m, 2H, CH piperidine ring), 1.62ꢀ1.70 (m, 4H, 2 × CH
piperidine ring), 2.58ꢀ2.61 (t, 4H, 2 × CH piperidine ring), 3.91 (s, 2H, oxadiazoleꢀCH N), 7.42ꢀ
3
2
2
2
2
7.51 (m, 2H, CH benzene ring), 8.00ꢀ8.02 (m, 1H, CH benzene ring), 8.14 (s, 1H, CH benzene
+
ring). MS (ESI), m/z: 278 [M + H] .
1
3
-(2-Chlorophenyl)-5-(piperidin-1-ylmethyl)-1,2,4-oxadiazole (6) (General Procedure B). Hꢀ
NMR (400 MHz; CDCl
3
) δ 1.45ꢀ1.49 (m, 2H, CH
2
piperidine ring), 1.63ꢀ1.69 (m, 4H, 2 × CH
2
piperidine ring), 2.58ꢀ2.61 (m, 4H, 2 × CH
2
piperidine ring), 3.95 (s, 2H, oxadiazoleꢀCH
2
N), 7.38ꢀ
7.47 (m, 2H, CH benzene ring), 7.55ꢀ7.57 (m, 1H, CH benzene ring), 7.95ꢀ7.97 (m, 1H, CH
+
benzene ring). MS (ESI), m/z: 278 [M + H] .
1
3
-Phenyl-5-(piperidin-1-ylmethyl)-1,2,4-oxadiazole (7) (General Procedure B). HꢀNMR (400
MHz; CDCl
3
2 2
) δ 1.46ꢀ1.48 (m, 2H, CH piperidine ring), 1.65ꢀ1.68 (m, 4H, 2 × CH piperidine ring),
2.60 (m, 4H, 2 × CH
2
piperidine ring), 3.91 (s, 2H, oxadiazoleꢀCH
2
N), 7.50ꢀ7.51 (m, 3H, CH
+
benzene ring), 8.11ꢀ8.14 (m, 2H, CH benzene ring). MS (ESI), m/z: 244 [M + H] .
1
5
-(Piperidin-1-ylmethyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (8) (General Procedure B). Hꢀ
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NMR (400 MHz; CDCl
3
) δ 1.43ꢀ1.48 (m, 2H, CH
2
piperidine ring), 1.54ꢀ1.68 (m, 4H, 2 × CH
2
piperidine ring), 2.60 (t, 4H, 2 × CH
2
piperidine ring), 3.93 (s, 2H, oxadiazoleꢀCH
2
N), 7.43ꢀ7.46
(m, 1H, CH pyridine ring), 8.39ꢀ8.41 (m, 1H, CH pyridine ring), 8.76ꢀ8.77 (m, 1H, CH pyridine
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ring), 9.35 (s, 1H, CH pyridine ring). MS (ESI), m/z: 245 [M + H] .
1
3-(4-Chlorophenyl)-5-methyl-1,2,4-oxadiazole (9) (General Procedure A). HꢀNMR (400
MHz; CDCl
3
) δ 2.68 (s, 3H, CH
3
), 7.47ꢀ7.49 (d, J = 8.4 Hz, 2H, CH benzene ring), 8.01ꢀ8.03 (d, J =
+
8
.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 195 [M + H] .
1
-(3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl)-N,N-dimethylmethanamine
(10)
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl
3
) δ 2.45 (s, 6H, 2 × CH
3
), 3.88 (s, 2H, oxadiazoleꢀ
CH
ring). MS (ESI), m/z: 238 [M + H] .
-(4-Chlorophenyl)-5-(pyrrolidin-1-ylmethyl)-1,2,4-oxadiazole (11) (General Procedure B).
2
N), 7.47ꢀ7.49 (d, J = 8.4 Hz, 2H, CH benzene ring), 8.06ꢀ8.08 (d, J = 8.4 Hz, 2H, CH benzene
+
3
1
HꢀNMR (400 MHz; CDCl ) δ 1.95ꢀ1.99 (m, 4H, 2 × CH pyrrolidine ring), 2.83ꢀ2.92 (m, 4H, 2 ×
3
2
CH
2
pyrrolidine ring), 4.03 (s, 2H, oxadiazoleꢀCH
2
N), 7.46ꢀ7.48 (d, J = 8.4 Hz, 2H, CH benzene
+
ring), 8.06ꢀ8.08 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 263 [M + H] .
1
5
-(Azepan-1-ylmethyl)-3-(4-chlorophenyl)-1,2,4-oxadiazole (12) (General Procedure B). Hꢀ
NMR (400 MHz; CDCl ) δ 1.62ꢀ1.63 (m, 4H, 2 × CH azepane ring), 1.71 (m, 4H, 2 × CH azepane
ring), 2.82ꢀ2.85 (m, 4H, 2 × CH azepane ring), 4.07 (s, 2H, oxadiazoleꢀCH N), 7.46ꢀ7.49 (d, J =
3
2
2
2
2
8
.4 Hz, 2H, CH benzene ring), 8.05ꢀ8.07 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 292
+
[
M + H] .
4
-((3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl)methyl)morpholine (13) (General Procedure B).
1
3 2
HꢀNMR (400 MHz; CDCl ) δ 2.68ꢀ2.70 (m, 4H, 2 × CH morpholine ring), 3.78ꢀ3.80 (t, 4H, 2 ×
CH morpholine ring), 3.93 (s, 2H, oxadiazoleꢀCH N), 7.47ꢀ7.49 (d, J = 8.4 Hz, 2H, CH benzene
2
2
+
ring), 8.05ꢀ8.07 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 280 [M + H] .
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-(4-Chlorophenyl)-5-(thiomorpholinomethyl)-1,2,4-oxadiazole (14) (General Procedure B).
1
HꢀNMR (400 MHz; CDCl
3 2
) δ 2.74ꢀ2.77 (t, 4H, 2 × CH thiomorpholine ring), 2.92ꢀ2.95 (m, 4H, 2
×
CH thiomorpholine ring), 3.95 (s, 2H, oxadiazoleꢀCH N), 7.47ꢀ7.49 (d, J = 8.4 Hz, 2H, CH
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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32
33
34
35
36
37
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44
45
46
47
48
49
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51
52
53
54
55
56
57
58
59
60
+
benzene ring), 8.05ꢀ8.07 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 296 [M + H] .
3
-(4-chlorophenyl)-5-((4-methylpiperazin-1-yl)methyl)-1,2,4-oxadiazole
(15)
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl
3
) δ 2.32 (s, 3H, CH
3
), 2.45ꢀ2.62 (m, 4H, 2 × CH
2
piperazine ring), 2.64ꢀ2.80 (m, 4H, 2 × CH
2
piperazine ring), 3.94 (s, 2H, oxadiazoleꢀCH
2
N), 7.47ꢀ
7
.49 (d, J = 8.0 Hz, 2H, CH benzene ring), 8.04ꢀ8.06 (d, J = 8.0 Hz, 2H, CH benzene ring). MS
+
(ESI), m/z: 293 [M + H] .
3
-(4-Chlorophenyl)-5-((4-phenylpiperazin-1-yl)methyl)-1,2,4-oxadiazole
(16)
piperazine ring), 3.28ꢀ
2
N), 6.87ꢀ6.88 (m, 1H, CH
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl
3
) δ 2.84ꢀ2.87 (m, 4H, 2 × CH
2
3
.30 (m, 4H, 2 × CH
2
piperazine ring), 4.01 (s, 2H, oxadiazoleꢀCH
benzene ring), 6.91ꢀ6.96 (d, 2H, CH benzene ring), 7.28ꢀ7.31 (m, 2H, CH benzene ring), 7.48ꢀ7.50
(d, J = 8.4 Hz, 2H, CH benzene ring), 8.06ꢀ8.08 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI),
+
m/z: 355 [M + H] .
(
1-((3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl)methyl)piperidin-4-yl)methanol (17) (General
1
Procedure B). HꢀNMR (400 MHz; CDCl
3
) δ 1.29ꢀ1.44 (m, 3H, CHCH
2
OH and 2 × CHH
piperidine ring), 1.78ꢀ1.81 (d, 2H, 2 × CHH piperidine ring), 2.24ꢀ2.29 (t, 2H, 2 × CHH piperidine
ring), 3.04ꢀ3.07 (d, 2H, 2 × CHH piperidine ring), 3.52ꢀ3.54 (m, 2H, CH OH), 3.93 (s, 2H,
oxadiazoleꢀCH N), 7.47ꢀ7.49 (d, J = 8.4 Hz, 2H, CH benzene ring), 8.05ꢀ8.07 (d, J = 8.4 Hz, 2H,
2
2
+
CH benzene ring). MS (ESI), m/z: 308 [M + H] .
N-((3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl)methyl)cyclohexanamine
(18)
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl ) δ 1.14ꢀ1.29 (m, 4H, 2 × CH cyclohexane ring), 1.63ꢀ
3
2
1
2
.78 (m, 4H, 2 × CH cyclohexane ring), 1.92ꢀ1.95 (d, 2H, 2 × CHH cyclohexane ring), 2.52ꢀ2.56
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5
5
5
5
5
5
6
(
m, 1H, CH
2
CHCH
2
2
cyclohexane ring), 4.16 (s, 2H, oxadiazoleꢀCH NH), 7.47ꢀ7.49 (d, J = 8.8 Hz,
2
H, CH benzene ring), 8.04ꢀ8.06 (d, J = 8.8 Hz, 2H, CH benzene ring). MS (ESI), m/z: 292 [M +
+
H] .
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5
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7
8
9
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2
3
4
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8
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N,N-Dimethyl-1-(3-(4-nitrophenyl)-1,2,4-oxadiazol-5-yl)methanamine
(19)
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl
3
) δ 2.46 (s, 6H, 2 × CH
3
), 3.91 (s, 2H, oxadiazoleꢀ
+
CH
2
N), 8.32ꢀ8.38 (m, 4H, CH benzene ring). MS (ESI), m/z: 249 [M + H] .
N-Ethyl-N-((3-(4-nitrophenyl)-1,2,4-oxadiazol-5-yl)methyl)ethanamine
(20)
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl ) δ 1.15ꢀ1.18 (t, J = 7.2 Hz, 6H, 2 × CH CH ), 2.68ꢀ2.74
3
2
3
(q, J = 7.2 Hz, 4H, 2 × CH
2
CH
3
2
), 4.07 (s, 2H, oxadiazoleꢀCH N), 8.31ꢀ8.38 (m, 4H, CH benzene
+
ring). MS (ESI), m/z: 277 [M + H] .
3
-(4-Nitrophenyl)-5-(pyrrolidin-1-ylmethyl)-1,2,4-oxadiazole (21) (General Procedure B).
HꢀNMR (400 MHz; CDCl ) δ 1.90ꢀ1.94 (m, 4H, 2 × CH pyrrolidine ring), 2.74ꢀ2.77 (m, 4H, 2 ×
CH pyrrolidine ring), 4.07 (s, 2H, oxadiazoleꢀCH N), 8.32ꢀ8.38 (m, 4H, CH benzene ring). MS
1
3
2
2
2
+
(ESI), m/z: 275 [M + H] .
3
-(4-Chlorophenyl)-5-(3-(piperidin-1-yl)propyl)-1,2,4-oxadiazole (22) (General Procedure
1
B). HꢀNMR (400 MHz; CDCl
3
) δ 1.42ꢀ1.43 (m, 2H, CH
CH CH N), 2.38ꢀ2.44 (m, 6H, 2 × CH
CH2CH2CH N), 2.98ꢀ3.01 (t, J = 7.2 Hz, 2H, CH CH CH N), 7.46ꢀ7.48 (d, J = 8.4 Hz, 2H, CH
2
piperidine ring), 1.52ꢀ1.57 (m, 4H, CH
2
piperidine ring), 2.03ꢀ2.10 (m, 2H, CH
2
2
2
2
piperidine ring and
2
2
2
2
+
benzene ring), 8.02ꢀ8.04 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 306 [M + H] .
2
-(4-(3-(3-Phenyl-1,2,4-oxadiazol-5-yl)propyl)piperazin-1-yl)ethan-1-ol
(23)
N ), 2.04ꢀ2.11 (m, 2H,
CH N), 3.00ꢀ3.04 (t, J =
.2 Hz, 2H, CH CH OH), 3.59ꢀ3.62 (t, J = 5.2 Hz, 2H, CH CH OH), 7.49ꢀ7.51 (m, 3H, CH
(General
1
Procedure B). HꢀNMR (400 MHz; CDCl
3
) δ 1.75 (m, 2H, CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
N), 3.28ꢀ3.30 (m, 10H, 4 × CH
2
piperazine ring and CH
2
CH
2
2
7
2
2
2
2
+
benzene ring), 8.07ꢀ8.10 (m, 2H, CH benzene ring). MS (ESI), m/z: 317 [M + H] .
2
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-(Bromomethyl)-3-(4-chlorophenyl)-1,2,4-oxadiazole (24) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl
3
) δ 4.57 (s, 2H, CH
2
Br), 7.48ꢀ7.50 (d, J = 8.4 Hz, 2H, CH benzene ring),
+
8
.03ꢀ8.05 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 273 [M + H] .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
5-(3-Bromopropyl)-3-(4-chlorophenyl)-1,2,4-oxadiazole (30) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.42ꢀ2.49 (m, 2H, CH CH CH Br), 3.15ꢀ3.19 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.57ꢀ3.61 (t, J = 6.4 Hz, 2H, CH CH CH
3
2
2
2
2
2
2
2
2
2
Br), 7.47ꢀ7.49 (d, J = 8.4 Hz, 2H, CH
+
benzene ring), 8.02ꢀ8.04 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 301 [M + H] .
1
5
-(3-Bromopropyl)-3-(3-chlorophenyl)-1,2,4-oxadiazole (31) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.38ꢀ2.49 (m, 2H, CH CH CH Br), 3.15ꢀ3.19 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.57ꢀ3.60 (t, J = 6.4 Hz, 2H, CH CH CH Br), 7.41ꢀ7.50 (m, 2H, CH benzene
3
2
2
2
2
2
2
2
2
2
ring), 7.96ꢀ7.98 (m, 1H, CH benzene ring), 8.08 (s, 1H, CH benzene ring). MS (ESI), m/z: 301 [M
+
+
H]
1
5-(3-Bromopropyl)-3-(2-chlorophenyl)-1,2,4-oxadiazole (32) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.43ꢀ2.50 (m, 2H, CH CH CH Br), 3.18ꢀ3.21 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.57ꢀ3.60 (t, J = 6.4 Hz, 2H, CH CH CH Br), 7.38ꢀ7.47 (m, 2H, CH benzene
3
2
2
2
2
2
2
2
2
2
ring), 7.54ꢀ7.56 (m, 1H, CH benzene ring), 7.91ꢀ7.93 (m, 1H, CH benzene ring). MS (ESI), m/z:
+
3
01 [M + H] .
1
5-(3-Bromopropyl)-3-phenyl-1,2,4-oxadiazole (33) (General Procedure A). HꢀNMR (400
MHz; CDCl ) δ 2.44ꢀ2.50 (m, 2H, CH CH CH Br), 3.14ꢀ3.19 (t, J = 7.2 Hz, 2H, CH CH CH Br),
.58ꢀ3.61 (t, J = 6.4 Hz, 2H, CH CH CH
3
2
2
2
2
2
2
3
2
2
2
2
Br), 7.49ꢀ7.51 (m, 3H, CH benzene ring), 8.08ꢀ8.10 (m,
+
H, CH benzene ring). MS (ESI), m/z: 267 [M + H] .
1
5-(3-Bromopropyl)-3-(4-tolyl)-1,2,4-oxadiazole (34) (General Procedure A). HꢀNMR (400
MHz; CDCl ) δ 2.43ꢀ2.49 (m, 5H, CH and CH CH CH Br), 3.14ꢀ3.17 (t, J = 7.2 Hz, 2H,
3
3
2
2
2
CH
2
CH
2
CH
2
Br), 3.57ꢀ3.60 (t, J = 6.4 Hz, 2H, CH
2
CH
2
CH
2
Br), 7.28ꢀ7.31 (d, J = 8.0 Hz, 2H, CH
2
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2
2
2
2
2
2
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3
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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
+
benzene ring), 7.96ꢀ7.98 (d, J = 8.0 Hz, 2H, CH benzene ring). MS (ESI), m/z: 281 [M + H] .
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5
-(3-Bromopropyl)-3-(4-methoxyphenyl)-1,2,4-oxadiazole (35) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.42ꢀ2.49 (m, 2H, CH CH CH Br), 3.13ꢀ3.17 (t, J = 7.2 Hz, 2H,
3
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH
2
CH
2
CH
2
Br), 3.57ꢀ3.60 (t, J = 6.4 Hz, 2H, CH
2
CH
2
CH
2
3
Br), 3.89 (s, 3H, OCH ), 6.99ꢀ7.02 (d, J
= 8.8 Hz, 2H, CH benzene ring), 8.01ꢀ8.04 (d, J = 8.8 Hz, 2H, CH benzene ring). MS (ESI), m/z:
+
2
97 [M + H] .
5
-(3-Bromopropyl)-3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (36) (General Procedure
1
A). HꢀNMR (400 MHz; CDCl ) δ 2.40ꢀ2.51 (m, 2H, CH CH CH Br), 3.17ꢀ3.22 (t, J = 7.2 Hz, 2H,
3
2
2
2
CH
2
CH
2
CH
2
Br), 3.58ꢀ3.62 (t, J = 6.4 Hz, 2H, CH
2
CH
2
CH
2
Br), 7.76ꢀ7.78 (d, J = 8.0 Hz, 2H, CH
+
benzene ring), 8.21ꢀ8.23 (d, J = 8.0 Hz, 2H, CH benzene ring). MS (ESI), m/z: 335 [M + H] .
1
5
-(3-Bromopropyl)-3-(4-nitrophenyl)-1,2,4-oxadiazole (37) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.45ꢀ2.52 (m, 2H, CH CH CH Br), 3.19ꢀ3.23 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.59ꢀ3.62 (t, J = 6.4 Hz, 2H, CH CH CH Br), 8.28ꢀ8.30 (d, J = 7.2 Hz, 2H, CH
3
2
2
2
2
2
2
2
2
2
+
benzene ring), 8.35ꢀ8.38 (d, J = 7.2 Hz, 2H, CH benzene ring). MS (ESI), m/z: 312 [M + H] .
-(3-Bromopropyl)-3-(4-fluorophenyl)-1,2,4-oxadiazole (38) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.42ꢀ2.49 (m, 2H, CH CH CH Br), 3.15ꢀ3.18 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.57ꢀ3.61 (t, J = 6.4 Hz, 2H, CH CH CH
1
5
3
2
2
2
2
2
2
2
2
2
Br), 7.16ꢀ7.21 (m, 2H, CH benzene
+
ring), 8.07ꢀ8.11 (m, 2H, CH benzene ring). MS (ESI), m/z: 285 [M + H] .
1
3
-(4-Bromophenyl)-5-(3-bromopropyl)-1,2,4-oxadiazole (39) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.43ꢀ2.49 (m, 2H, CH CH CH Br), 3.15ꢀ3.19 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.57ꢀ3.61 (t, J = 6.4 Hz, 2H, CH CH CH
3
2
2
2
2
2
2
2
2
2
Br), 7.63ꢀ7.65 (d, J = 8.8 Hz, 2H, CH
+
benzene ring), 7.95ꢀ7.97 (d, J = 8.8 Hz, 2H, CH benzene ring). MS (ESI), m/z: 347 [M + H] .
1
3
-(3-Bromophenyl)-5-(3-bromopropyl)-1,2,4-oxadiazole (40) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.45ꢀ2.48 (m, 2H, CH CH CH Br), 3.16ꢀ3.20 (t, J = 7.2 Hz, 2H,
3
2
2
2
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2 2 2 2 2 2
CH CH CH Br), 3.58ꢀ3.61 (t, J = 6.4 Hz, 2H, CH CH CH Br), 7.36ꢀ7.40 (t, 1H, CH benzene ring),
7
.64ꢀ7.66 (m, 1H, CH benzene ring), 8.02ꢀ8.04 (m, 1H, CH benzene ring), 8.25 (m, 1H, CH
+
benzene ring). MS (ESI), m/z: 347 [M+H] .
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14
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18
19
20
21
22
23
24
25
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29
30
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32
33
34
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36
37
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39
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3-(2-Bromophenyl)-5-(3-bromopropyl)-1,2,4-oxadiazole (41) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl ) δ 2.38ꢀ2.56 (m, 2H CH CH CH Br), 3.19ꢀ3.22 (t, J = 7.2 Hz, 2H,
CH CH CH Br), 3.56ꢀ3.68 (t, J = 6.4 Hz, 2H, CH CH CH Br), 7.35ꢀ7.39 (m, 1H, CH benzene
3
2
2
2
2
2
2
2
2
2
ring), 7.43ꢀ7.47 (m, 1H, CH benzene ring), 7.75ꢀ7.76 (m, 1H, CH benzene ring), 7.82ꢀ7.84 (m, 1H,
+
CH benzene ring). MS (ESI), m/z: 347 [M + H] .
1
3-(4-Bromophenyl)-5-(3-chloropropyl)-1,2,4-oxadiazole (42) (General Procedure A). Hꢀ
NMR (400 MHz; CDCl
3
) δ 2.36ꢀ2.41 (m, 2H, CH
2
CH
2
CH
2
Cl), 3.15ꢀ3.19 (t, J = 7.2 Hz, 2H,
CH CH CH Cl), 3.71ꢀ3.75 (t, J = 6.4 Hz, 2H, CH
2
2
2
2
CH
2
CH
2
Cl), 7.63ꢀ7.65 (d, J = 8.4 Hz, 2H, CH
+
benzene ring), 7.95ꢀ7.97 (d, J = 8.4 Hz, 2H, CH benzene ring). MS (ESI), m/z: 301 [M + H] .
1
5-(4-Bromobutyl)-3-(4-bromophenyl)-1,2,4-oxadiazole (43) (General Procedure A). HꢀNMR
(400 MHz; CDCl
3
) δ 2.02ꢀ2.09 (m, 4H, CH
2
CH
2
CH
2
CH
2
Br), 2.99ꢀ3.03 (t, J = 7.2 Hz, 2H,
CH CH CH CH Br), 3.47ꢀ3.62 (t, J = 6.4 Hz, 2H, CH
2
2
2
2
2
CH
2
CH
2
CH Br), 7.63ꢀ7.65 (d, J = 8.8 Hz,
2
2
H, CH benzene ring), 7.95ꢀ7.97 (d, J = 8.8 Hz, 2H, CH benzene ring). MS (ESI), m/z: 361 [M +
+
H] .
Preparation of Methyl 3-(4-Bromophenyl)-1,2,4-oxadiazole-5-carboxylate (44). Methyl 2ꢀ
chloroꢀ2ꢀoxoacetate (0.26 mL, 341.8 mg, 2.79 mmol) was added dropwise at 0 °C to a solution of
Z)ꢀ4ꢀbromoꢀN'ꢀhydroxybenzimidamide 47g (500 mg, 2.32 mmol) in a mixture of dry
(
dichloromethane (10 mL) and dry pyridine (1 mL). After 20 h of stirring at room temperature, the
solvent was evaporated under vacuum, and the crude was triturated with water (10 mL), filtered,
dried under vacuum, and finally purified by a silica gel flash chromatography (SNAP 50 KPꢀSil,
Biotage Isolera One) using a linear gradient of ethyl acetate (1% to 7%) in petroleum ether to give
2
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