Chiral Mercaptoacetamides Display Enantioselective Inhibition of Histone Deacetylase6 and Exhibit Neuroprotection in Cortical Neuron Models of Oxidative Stress

Article abstract of DOI:10.1002/cmdc.201100522

Mercaptoacetamide-based ligands have been designed as a new class of histone deacetylase (HDAC) inhibitors for possible use in the treatment of neurodegenerative diseases. The thiol group of these compounds provides a key binding element for interaction with the catalytic zinc ion, and thus differs from the more typically employed hydroxamic acid based zinc binding groups. Herein we disclose the chemistry and biology of some substituted mercaptoacetamides with the intention of increasing HDAC6 isoform selectivity while maintaining potency similar to their hydroxamic acid analogues. The introduction of a stereocenter α to the thiol group was found to have a considerable impact on HDAC inhibitor potency. These new compounds were also profiled for their therapeutic potential in an invitro model of stress-induced neuronal injury and were found to act as nontoxic neuroprotective agents.

Full text of DOI:10.1002/cmdc.201100522

DOI: 10.1002/cmdc.201100522
Chiral Mercaptoacetamides Display Enantioselective
Inhibition of Histone Deacetylase 6 and Exhibit
Neuroprotection in Cortical Neuron Models of Oxidative
Stress
Jay H. Kalin,[a] Hankun Zhang,[a] Sophie Gaudrel-Grosay,[a] Giulio Vistoli,[b] and
Alan P. Kozikowski*[a]
Mercaptoacetamide-based ligands have been designed as a
new class of histone deacetylase (HDAC) inhibitors for possible
use in the treatment of neurodegenerative diseases. The thiol
group of these compounds provides a key binding element for
interaction with the catalytic zinc ion, and thus differs from the
more typically employed hydroxamic acid based zinc binding
groups. Herein we disclose the chemistry and biology of some
substituted mercaptoacetamides with the intention of increas-
ing HDAC6 isoform selectivity while maintaining potency simi-
lar to their hydroxamic acid analogues. The introduction of a
stereocenter a to the thiol group was found to have a consid-
erable impact on HDAC inhibitor potency. These new com-
pounds were also profiled for their therapeutic potential in an
in vitro model of stress-induced neuronal injury and were
found to act as nontoxic neuroprotective agents.
Introduction
Histone deacetylases (HDACs) are implicated in the epigenetic
regulation of gene expression via chromatin modification. A
condensed, transcriptionally silent chromatin structure is ob-
tained when the anionic phosphate groups of the DNA back-
bone interact with the cationic lysine residues of histone pro-
teins. Histone deacetylases are part of this process, facilitating
these ionic interactions by removing N-acetyllysine residues
from nucleosomal histone tails. Moreover, HDACs accept a vari-
ety of non-histone substrates such as transcription factors and
regulators, as well as structural and chaperone proteins.[1–4] It is
this latter mechanism by which selective HDAC6 inhibitors are
believed to exert their neuroprotective properties, as HDAC6
inhibition has been linked to upregulation of the transport of
brain-derived neurotrophic factor (BDNF), a protein involved in
neurogenesis, mediation of peroxiredoxins, an important class
of redox regulators, and regulation of Hsp90, an important
chaperone protein involved in a number of cell signaling pro-
cesses.[4–7] Additionally, HDAC6-selective inhibitors have been
suggested to lack some of the major side effects associated
fects.[10,14] Many known HDACIs such as suberoylanilide hy-
droxamic acid (SAHA) and trichostatin A (TSA) contain a hy-
droxamic acid zinc binding group (ZBG); however, in addition
to being metabolically labile (orally administered SAHA has a
half-life of 1.5–2 h in humans), hydroxamates are very potent
metal-chelating agents that could lead to off-target activity at
other zinc-containing enzymes such as matrix metalloproteas-
es, MMP-1, MMP-2, and MMP-3, ultimately resulting in undesir-
able toxicity (Supporting Information figure 1).[10,15–18] In neuro-
nal models of oxidative stress, many hydroxamic acid based
HDACIs were found to exhibit some intrinsic toxicity, whereas
their mercaptoacetamide counterparts were found to be fully
neuroprotective, suggesting that alternative ZBGs may be pre-
ferred depending on the desired therapeutic goal.[15,19–21] How-
ever, examples of neuroprotective hydroxamic acid HDACIs
have also been identified.[15]
Previously we reported on the synthesis of a series of mer-
captoacetamide-based HDACIs that exhibit some HDAC6 selec-
tivity and were found to have better therapeutic profiles in
protecting neurons against oxidative stress-induced cell death
with
broad-spectrum
histone
deacetylase
inhibitors
(HDACIs).[8–10] Thus, our particular interest has focused on
HDAC6, which is highly expressed in the brain and therefore
serves an additional role in controlling gene transcription in
neurons.[11]
[a] J. H. Kalin,+ Dr. H. Zhang,+ Dr. S. Gaudrel-Grosay, Prof. A. P. Kozikowski
Department of Medicinal Chemistry and Pharmacognosy
University of Illinois at Chicago
833 S. Wood Street, Chicago, IL 60612-7211 (USA)
E-mail: kozikowa@uic.edu
The development of selective HDACIs has emerged as a
challenging field in drug discovery, as many of the residues in-
volved in substrate recognition and catalytic activity are con-
served across the HDAC isoforms.[12,13] Only a few HDAC6-selec-
tive inhibitors are known; they are of great interest, not only
as tools for probing the biological functions of the isoform,
but also as possible therapeutic agents with few side ef-
[b] Dr. G. Vistoli
Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”
Universitꢀ degli Studi di Milano, Via Mangiagalli 25, 20133, Milan (Italy)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100522.
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A. P. Kozikowski et al.
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relative to their hydroxamic acid homologues.[15] The mecha-
nism of HDAC inhibition by mercaptoacetamide-based HDACIs
is, theoretically, similar to that of the hydroxamates, in that
both have the potential to chelate zinc in a monodentate or
bidentate fashion depending on the isoform.[13,22] In addition
to our previously reported analogues, numerous examples of
thiol-containing HDACIs have been reported.[10,22,23] However,
one potential drawback of these mercaptoacetamide HDACIs
is their ability to undergo oxidative dimerization to
the disulfide. Although this disulfide bond can be re-
duced within cells to afford the parent monomers, it
can present a problem when profiling the activity of
these inhibitors against purified proteins.[24] Thus, we
synthesized a novel series of modestly potent mer-
captoacetamide-based HDAC6-selective inhibitors
that display increased resistance to dimerization, and
evaluated their ability to protect primary cortical neu-
rons from oxidative stress-induced cell death.
As we were aware of the fact that the hydroxamic acid
group may be associated with neuronal toxicity, we synthe-
sized an analogue to compare it with our mercaptoacetamide-
based ligands both in terms of potency and neuroprotective
properties (Scheme 3). The hydroxamic acid 9 was prepared in
two steps from the isoxazole acid 2 using standard PyBOP cou-
pling protocol and then hydroxyamine to supplant the ob-
tained methyl ester.
Results and Discussion
Chemistry
The synthetic procedures used for the preparation of
our first series of amide-linked mercaptoacetamide
isoxazoles are outlined in Scheme 1. Here, a nitrile
oxide cycloaddition was employed to generate the
trisubstituted isoxazole 1 starting from (E)-benzalde-
hyde chloro-oxime and ethyl-2-butynoate using mi-
Scheme 1. Reagents and conditions: a) N-chlorosuccinimide, Al2O3, microwave, 408C;
b) ethyl-2-butynoate, Et3N, THF, microwave, 708C; c) NaOH, CH3OH/H2O, 608C; d) NH2-
(CH2)5NHBoc, PyBOP, DIEA, CH2Cl2, RT; e) TFA, CH2Cl2, RT; f) 5, PyBOP, DIEA, CH2Cl2, RT;
g) TFA, Et3SiH, CH2Cl2, 08C!RT.
crowave irradiation. This ester was saponified to yield
acid 2 and then allowed to react with a diamine
linker in the presence of the coupling agent PyBOP
to yield 3. The Boc protecting group was removed to
give amine 4, and then a second coupling reaction
was carried out with different trityl- or pivaloyl-pro-
tected mercaptoacetic acids 5a–d. The pivaloyl-pro-
tected prodrug 6d was thus made available for
study. The trityl group was removed from com-
pounds 6a–6c to afford three mercaptoacetamide li-
gands with no (7a), one (7b), or two (7c) methyl
groups a to the thiol of the mercaptoacetamide.
Because it is well known that the absolute stereo-
chemistry of a particular molecule can greatly influ-
ence its activity, we also chose to investigate the indi-
vidual enantiomers of compound 7b. The enantio-
mers of compound 6b were separated by chiral
column chromatography and then deprotected to
afford the single enantiomers, (R)-7b and (S)-7b
(Scheme 2). To determine the absolute stereochemis-
try of the two isolated enantiomers, starting from (S)-
2-aminopropanoic acid and 4, we synthesized the
R isomer of compound 6b and compared the reten-
tion time of the pure enantiomer to that of the race-
mic mixture; we then observed that the R isomer was
the first to elute from the column (Supporting Infor-
mation scheme 1 and figure 2).[25]
Scheme 2. Reagents and conditions: a) TFA, Et3SiH, CH2Cl2, 08C!RT.
Scheme 3. Reagents and conditions: a) NH2(CH2)6CO2Me·HCl, PyBOP, DIEA, CH2Cl2, RT;
b) NH2OH·HCl, KOH, CH3OH, RT.
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Chiral Mercaptoacetamides
As shown in Scheme 4, the synthesis of alkene-linked mer-
captoacetamides 13a–d was performed in 5 or 6 steps starting
from the previously obtained acid 2. Compound 2 was re-
duced to the alcohol and subsequently oxidized to aldehyde
11. A Wittig reaction was then employed with aldehyde 11
Scheme 5. Reagents and conditions: a) I2, PPh3, imidazole, CH2Cl2, 08C!RT;
b) HO(CH2)5NHBoc, NaH, DMF, ꢀ208C!RT; c) TFA, CH2Cl2, RT; d) 5b, PyBOP,
DIEA, CH2Cl2, RT; e) TFA, Et3SiH, CH2Cl2, 08C!RT.
Synthesis of the alkyne- and cinnamoyl-linked mercaptoace-
tamides was performed by starting from commercially avail-
able or readily obtained iodoisoxazoles 17a–b. The Sonoga-
shira and Heck reactions, followed by conditions similar to
those described in Scheme 1, were used with 17a–b to gener-
ate the alkyne- and cinnamoyl-linked compounds 18a–e and
20a–b, respectively (Scheme 6). With the desired compounds
in hand, we next studied their activity in both the isolated
enzyme assays and in cell-based neuroprotection studies.
Scheme 4. Reagents and conditions: a) Borane-THF, THF, 08C!RT; b) MnO2,
CH2Cl2, RT; c) BrPh3P(CH2)6NHBoc, nBuLi, THF, 08C!RT; d) TFA, CH2Cl2, RT;
e) 5, PyBOP, DIEA, CH2Cl2, RT; f) TFA, Et3SiH, CH2Cl2, 08C!RT.
and a prepared phosphonium salt to produce compound 12.
Procedures similar to those described in Scheme 1 were ap-
plied to compound 12 to generate the a-unsubstituted,
mono-, and dimethylmercaptoacetamides. To investigate the
difference in activity between the geometric isomers generat-
ed during the Wittig reaction, the isomers of compounds 13a
and 13d were isolated by chiral column chromatography, and
HDAC isoform inhibition
IC50 values were determined for these new mercaptoacetamide
ligands at selected HDAC isoforms using a fluorescence-based
assay, and the results are listed in Table 1. Mercaptoacetamide
7a showed sub-micromolar HDAC6 activity with some selectiv-
ity over both the class I (>21-fold) and class IIa (>38-fold) iso-
zymes. The related hydroxamic acid 9 exhibited similar activity
and selectivity at the class I and class IIa isoforms (>14- and
1
their molecular configuration was assigned based on H NMR
(Supporting Information scheme 2 and figure 3).
Ligand 16, the compound containing an ether linker, was
synthesized from alcohol 10 by conversion into the iodo pre-
cursor via an Appel-like reaction.
This was subsequently allowed
to react with Boc-aminopentanol
to yield intermediate carbamate
14. The obtained product was
transformed as previously de-
scribed in Scheme 1 into the de-
sired
a-methylmercaptoaceta-
mide (Scheme 5). Again, to in-
vestigate the effect of stereo-
chemistry on compound activity,
the enantiomers of compound
15 were isolated by chiral
column chromatography and
then deprotected to yield (R)-16
and (S)-16 (Supporting Informa-
tion scheme 3). The absolute ste-
reochemistry was assigned in
the same manner as that for
compound 7b (Supporting Infor-
mation scheme 4 and figure 4).
Scheme 6. Reagents and conditions: a) ClCOCH3, ZnCl2, nBuLi, THF, ꢀ788C; b) NH2OMe·HCl, pyridine, Na2SO4,
CH3OH, reflux; c) ICl, CH2Cl2, reflux; d) HCC(CH2)nNHBoc, PdCl2(PPh3)2, CuI, DIEA, DMF, microwave, 1308C; e) TFA,
CH2Cl2, RT; f) 5, PyBOP, DIEA, CH2Cl2, RT; g) TFA, Et3SiH, CH2Cl2, 08C!RT; h) tert-butyl 4-vinylbenzylcarbamate,
Pd(OAc)2, nBu4NCl, Na2CO3, DMF, microwave, 1308C.
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A. P. Kozikowski et al.
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Table 1. In vitro HDAC isoform inhibitory activity.[a]
Compd
Class I
HDAC2
Class IIa
Class IIb
HDAC6
HDAC1
HDAC3
–
HDAC4
–
10
>30
HDAC5
–
15
>30
>30[e]
>30[e]
>30
6.0
>30
–
6d
7a
7b[b]
(R)-7b
(S)-7b
7c
–
–
–
5.7ꢁ0.5[e]
28
>30
14
>30
0.26ꢁ0.03[e]
1.1ꢁ0.01[e]
0.28ꢁ0.04[e]
>30[e]
>30
>30[e]
>30[e]
>30
>30[e]
>30[e]
>30
11
>30
–
15ꢁ1[e]
>30[e]
>30[e]
>30
9.4
>30
>30[e]
>30[e]
>30
>30
>30
–
>30[e]
>30
2.6
>30
9
3.0ꢁ0.2[e]
0.18ꢁ0.08[e]
2.7ꢁ0.1[e,f]
4.8ꢁ1.8[e,f]
3.5ꢁ0.5[e,f]
9.8
13a[c]
cis-13a
trans-13a
13b[b,c]
13c[c]
13d[c]
cis-13d
trans-13d
16[b]
>30
>30
–
>30[e]
28ꢁ4[e]
–
–
–
>30
>30
>30
>30
>30
>30
>30
>30
–
>30
>30
>30
–
>30
>30
>30
–
>30
>30[e]
>30[e]
–
–
5.5
–
–
>30[e]
9.6
14
>30
–
>30
–
–
8.3
13
>30
>30
–
>30
>30
0.85
(R)-16
(S)-16
18a[b]
18b[b]
18c
1.5ꢁ0.03[e]
–
–
7.5
9.4
3.4ꢁ0.2[e]
0.83ꢁ0.27[e]
>30[e]
>30[e]
1.4
–
>30[e]
2.7
3.7
>30
>30
>30
>30
>30
–
2.0
2.6
2.1
>30
>30
–
>30
>30
–
>30
>30
>30
>30
18d
>30
18e
–
–
20a[b]
20b
>30
>30
>30
>30
>30
>30
>30
>30
TSA[d]
0.006ꢁ0.003
0.017ꢁ0.008
0.008ꢁ0.004
0.047ꢁ0.061
0.013ꢁ0.007
0.0017ꢁ0.0005
[a] These results were determined by CRO Reaction Biology (Malvern, PA, USA); ten-dose IC50 values were determined by using threefold serial dilutions
starting at concentrations of 30 mm. Compounds with IC50 >30 mm were considered to be inactive. A dashed line indicates that the compound was not
tested at that isoform. [b] Racemic mixture. [c] Mixture of geometric isomers. [d] IC50 values for TSA are reported as the average of five experiments ꢁSD
for HDAC1, 3, 4, and 6, and the average of four experiments ꢁSD for HDAC2 and 5. [e] Evaluated in duplicate. [f] p>0.05 (difference not statistically signifi-
cant), determined using the unpaired t-test available in GraphPad Prism 5 (La Jolla, CA, USA).
>33-fold, respectively). The slight difference in potency ob-
served between 7a and 9 may result from the ability of 9 to
form an additional hydrogen bond to the enzyme. It is known
that the NH group of the hydroxamate ZBG is able to form a
hydrogen bond with a histidine residue in the HDAC active
site, whereas the mercaptoacetamide ZBG lacks this functional-
ity.[26] The addition of a methyl group to the mercaptoaceta-
mide ZBG of 7a, compound 7b, resulted in a slight decrease
in potency (7a IC50 =0.26 mm, 7b IC50 =1.1 mm); however, the
activity at all other HDAC isoforms was abolished. Because 7b
was a racemic mixture, we isolated the single enantiomers and
observed that the R isomer displays sub-micromolar activity at
HDAC6, similar to the original unsubstituted compound 7a,
whereas (S)-7b is completely inactive. Compound (R)-7b main-
tained selectivity over all the other HDAC isoforms except
HDAC3, for which the ten-dose IC50 was found to be 15 mm.
Additional modifications were made to the linker region and
cap group of these mercaptoacetamides. Replacing the amide
linker of 7a by an alkene, 13a, resulted in micromolar HDAC6
activity and a good selectivity profile, with the IC50 at all other
tested isoforms being >30 mm. To investigate the effect of
geometric isomerism on compound activity, the isomers of
13a—cis- and trans-13a—were isolated and found to exhibit
similar activity at HDAC6 and a less than twofold difference in
activity at HDAC1, suggesting that the geometric configuration
of the double bond at this position in the linker has little
effect on ligand binding. The pivaloyl prodrug of 13a, com-
pound 13d, along with the individual geometric isomers, cis-
13d and trans-13d, displayed no inhibition at any of the
HDAC isoforms tested. This was expected, as these IC50 values
were measured using purified protein; as such, no metabolic
enzymes were present to release the active form of the
drug.[27] The other pivaloyl prodrugs, 6d and 18e, were not
screened against the isolated HDAC enzymes for this reason.
With an ether linker replacing the amide, as in compound
16, HDAC6 activity was still observed in the sub-micromolar
range, and selectivity over the class I (>6-fold) and class IIa (>
35-fold) isoforms was similar to that of compound 7a. Again,
we investigated the effect of absolute stereochemistry on com-
pound activity and found that the R isomer of 16 exhibits
HDAC6 activity in the sub-micromolar range, whereas (S)-16 is
completely inactive. These results combined with the results
obtained for the enantiomers of compound 7b led us to con-
clude that the R configuration of the monomethyl mercaptoa-
cetamide ZBG is preferred for HDAC6 activity.
Lastly, the compounds containing alkyne linkers and mono-
methyl mercaptoacetamide ZBGs (18a–b) exhibited micromo-
lar activity, with only some selectivity over the HDAC4 isoform.
Compounds containing a cinnamoyl linker (20a–b) showed no
HDAC activity, perhaps due to the rigidity of this linker. The ad-
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Chiral Mercaptoacetamides
dition of two methyl groups to the ZBG, as with 18c and 18d,
completely abolished activity at every tested HDAC isoform
and for all cap group and linker modifications. This loss of ac-
tivity can likely be attributed to steric interactions in the active
site. Although the HDAC proteins are able to accommodate
some steric bulk, the addition of a second methyl group likely
renders the compound unable to adopt a conformation that
does not clash with active site residues.
Molecular modeling
With the intent of rationalizing the stereoselectivity observed
with the reported mercaptoacetamides, a molecular docking
study was carried out considering the two enantiomers of 7b,
(R)-7b and (S)-7b, and three relevant HDAC subtypes. To mini-
mize the number of exploited homology models, the following
structures were studied: a) X-ray structure of the human
HDAC2 isozyme (PDB ID: 3MAX) as representative of the class I
subtypes; b) X-ray structure of the human HDAC4 isozyme
(PDB ID: 2VQM) as representative of the class IIa subtypes; and
c) homology model of the second catalytic domain of human
HDAC6 (class IIb) as recently generated by us.[14] Given the
high degree of sequence homology between HDAC1 and
HDAC2, the results obtained for HDAC2 can be considered
similar to what would be observed with HDAC1. The docking
calculations were carried out considering the anionic form of
the thiol for the two enantiomers. Also, the two His–Asp dyads
that characterize the catalytic core of the HDAC isozymes were
considered in their singly protonated state such that only the
first histidine residue (His145 for HDAC2, His158 for HDAC4,
and His610 for HDAC6-CDII) was protonated, while the second
histidine residue (His146 for HDAC2, His159 for HDAC4, and
His611 for HDAC6-CDII) was considered neutral and in its Ne
tautomeric form to minimize steric hindrance within the cata-
lytic pocket. This choice was justified by considering the pre-
cise planarity that characterizes the first dyad and increases its
basicity, whereas the other dyad does not show similar planari-
ty, resulting in a less basic histidine residue. This is also in line
with other enzymatic studies demonstrating that the first pro-
tonation highly disfavors a second protonation of His146, al-
lowing facile proton transfer between the two dyads.[28]
Figure 1. a) Putative complexes for (R)-7b and (S)-7b with the resolved
HDAC2 crystal structure (PDB ID: 3MAX) illustrating that only the active
R isomer can elicit bidentate chelation, which is reinforced by hydrogen
bonding interactions between the amido group of the ligand and Tyr308.
b) Putative complexes for the two enantiomers (R)-7b and (S)-7b with the
second catalytic subunit of the HDAC6 subtype (previously published),[14] il-
lustrating that only the active R isomer can elicit bidentate chelation rein-
forced by hydrogen bonding between the sulfur atom and Tyr782. This dif-
ference was also reflected by the position of the cap group, as only the
active R isomer was able to stabilize hydrogen bonding interactions with
Asp567 and Ser568 on the cavity rim.
that inhibitors of class I isozymes are characterized by a tight
bidentate chelation with the zinc ion which is reinforced by a
hydrogen bonding interaction with the conserved tyrosine res-
idue and is not dependent on the ionization state of the inhib-
itor.[29] The differing arrangement of the ZBG moieties also re-
flect on the interactions stabilized by the cap group, such that
the R isomer is able to assume a more folded geometry and
can stabilize two relevant hydrogen bonds with His183 and
Tyr209 in addition to a rich set of hydrophobic contacts,
whereas the S isomer assumes a more extended conformation
and does not display these interactions.
The electrostatic energy scores, as determined with VEGA
using a distance-dependent dielectric function, for all mini-
mized complexes between HDAC isozymes and enantiomers
(R)-7b and (S)-7b were lower for the R isomer than for the
S isomer, confirming the observed stereoselectivity (Supporting
Information table 1). The observed difference in binding mode
for the two enantiomers can be explained by considering the
interactions available to the amido group of each ligand (Fig-
ure 1a). Indeed, in both complexes, the sulfur atom ap-
proaches the zinc ion, which also interacts with protonated
His145; however, only the amido group of the R isomer was
able to interact with the zinc ion and stabilize hydrogen bond-
ing interactions with the highly conserved Tyr308 residue. Con-
versely, the amido group of the S isomer was unable to elicit
these significant interactions, and also clashes with Cys156.
This is in line with recent mechanistic studies, which found
The role of the cap group moiety is even more preponder-
ant in HDAC4 (complexes not shown) as the ZBG group realiz-
es a similar interaction pattern for both enantiomers, in that
the sulfur atom contacts the zinc ion and the charged His158
residue in both cases. Conversely, the phenyl isoxazole moiety
of the R isomer is able to stabilize hydrophilic interactions be-
tween two arginine residues (Arg37 and Arg154) that charac-
terize the cavity rim of HDAC4. This finding is in agreement
with the weak monodentate chelation observed with the clas-
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A. P. Kozikowski et al.
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s IIa isoforms due to the lack of a conserved tyrosine residue,
which globally renders the obtained complexes less dependent
on interactions with the metal ion and more dependent on in-
teractions with the cap group and linker.
As depicted in Figure 1b, the binding modes observed be-
tween the two enantiomers, (R)-7b and (S)-7b, and HDAC6 are
similar to what was observed with HDAC2. The sulfur atom of
the ZBG of both enantiomers contacts the zinc ion and proton-
ated His610; however, only the carbonyl group of the R isom-
er’s ZBG is able to interact with the zinc ion, allowing biden-
tate chelation. In addition, the sulfur atom of the R isomer’s
ZBG also contacts Tyr782. These putative complexes suggest
the importance of bidentate chelation to the zinc ion with
regard to activity, and also suggest the plausible role of Tyr782
in further stabilizing the thiol group. The various interactions
stabilized by the cap group moieties also seem to possess a
relevant role in HDAC6 binding, as the phenyl isoxazole group
of the R isomer elicits hydrogen bonding interactions with
Asp567 and Ser568, while the cap group of the S isomer as-
sumes a more lateral arrangement, stabilizing only weak hy-
drophobic contacts.
Figure 2. Qualitative determination of the rate of dimerization for com-
&
^
*
pounds 7a ( ), (R)-7b ( ), and 7c ( ) as measured by LC–MS.
Neuroprotection and glutathione depletion
Treating central nervous system diseases with HDAC inhibitors,
especially HDAC6 inhibitors, is viewed as a rational therapeutic
approach. Therefore, our ligands were tested for their ability to
provide primary cortical neuron protection in an in vitro model
of oxidative stress-induced neurodegeneration.[30,31] In this
model, neurodegeneration is induced by the presence of the
cysteine homologue, homocysteine, at 5 mm, which depletes
the cellular antioxidant glutathione via competitive inhibition
of cysteine uptake at the plasma membrane cysteine/gluta-
mate antiporter. Because cysteine is required for the synthesis
of glutathione, the inhibition of its uptake results in gluta-
thione depletion. Cellular redox homeostasis therefore be-
comes disrupted which leads to accumulation of endogenous-
ly produced and unopposed oxidants, resulting in neuronal de-
generation over the course of approximately 24 hours. Impor-
tantly, primary neurons at this early developmental stage lack
ionotropic and metabotropic receptors and are not susceptible
to excitotoxicity; rather, cell death is induced by the accumula-
tion of unopposed free radicals, and the neurons exhibit a
number of apoptotic features.
Altogether, the docking results confirm the differences in se-
lectivity observed between the considered subtypes which,
based on these calculations, result from the different roles of
the ZBG and cap group moieties in determining the binding
mode. Interestingly, the different binding modes observed be-
tween the ZBG of each enantiomer and HDAC2 are essentially
due to the different residues surrounding the metal ion; how-
ever, they may also be due to the presence of the adjacent
foot cavity, which characterizes the binding pocket of the
class I subtypes and allows for greater diversity amongst the
computed poses, which is reflected by a greater difference in
computed energy scores.
Finally, it was interesting to observe how the various ar-
rangements of the linker, despite its flexibility, have a con-
straining effect on the interactions stabilized by the cap group
(especially with regard to the class II isozymes). This is probably
a result of the rigidity induced by the two amido functionali-
ties.
At 10 mm, the hydroxamic acid 9 shows little neuroprotective
activity, but does exhibit some toxicity, which was not ob-
served with the mercaptoacetamides. Compound 7a, which
displays good HDAC6 activity, is moderately neuroprotective at
7.5 mm and fully neuroprotective at 10 mm, while the prodrug
6d is moderately neuroprotective at 5 mm and fully neuropro-
tective at 7.5 mm (Figure 3a, 3b, and Supporting Information
figure 5). It has been established that thioesters help to im-
prove cell permeability by acting as prodrugs for the active
thiols, which are released once they enter the cell.[24,27,32,33]
Therefore, it is likely that 6d is metabolized to 7a inside the
cell and that 7a is responsible for the observed neuroprotec-
tive properties. Furthermore, the thioester prodrug 6d may ex-
hibit greater cell permeability than the mercaptoacetamide 7a
which would explain the lower concentrations of 6d required
to exhibit neuroprotection.[34] The same trend was also ob-
served for the geometric isomers of 13a, cis-13a and trans-
13a, and their respective prodrugs, cis-13d and trans-13d.
Alkene-linked mercaptoacetamides cis-13a and trans-13a dis-
play moderate neuroprotective activity at 20 mm and full neu-
roprotection at 50–100 mm, whereas their respective prodrugs,
Mercaptoacetamide dimerization
To determine if the addition of a-methyl groups relative to the
thiol in these mercaptoacetamides increases resistance to di-
merization, three ligands, 7a, (R)-7b, and 7c, were assayed by
LC–MS to determine their qualitative rate of dimerization
(Figure 2). We found that compound 7a, which contains no
methyl groups, dimerized at a much greater rate than both
the monomethyl, (R)-7b, and dimethyl, 7c, compounds. In
comparing compounds (R)-7b and 7c, we found that the addi-
tion of a second methyl group adjacent to the thiol further de-
creases the rate of dimerization; however, this decrease was
not nearly as significant as the addition of the first methyl
group.
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Chiral Mercaptoacetamides
Figure 3. Neuroprotection plots for compounds a) 7a, b) 9, c) (R)-7b, and d) (S)-7b. Rat primary cortical neurons were incubated with varying concentrations
of each compound either with (&) or without (&) 5 mm HCA for 24 h, after which cell viability was determined using an MTT assay.
cis-13d and trans-13d, are neuroprotective at 10 mm, but
devoid of any significant HDAC activity (Supporting Informa-
tion figure 5).
The monomethyl mercaptoacetamides 13b, 18a, and 18b
displayed neuroprotective activities that correlate well with
their HDAC6 inhibitory activity. Compounds 18a and 18b dis-
played IC50 values at HDAC6 between 1 and 3 mm and are neu-
roprotective at 10 mm in vitro. Compound 13b, however, is 3–
12-fold less potent then the other monomethyl mercaptoace-
tamides in the enzyme assay. This was reflected by the need to
use concentrations of 20 mm to effect neuroprotection. Com-
pound 7c did not display any neuroprotective activity which is
consistent with the results of the HDAC6 enzyme inhibition
assay (Supporting Information figure 5).
Figure 4. Prevention of glutathione depletion by selected mercaptoaceta-
For some compounds, however, the activity at HDAC6 failed
to track closely with the observed neuroprotective activity.
Compounds (R)-7b and (S)-7b displayed peculiar neuroprotec-
tive profiles such that while compound (R)-7b is neuroprotec-
tive as expected, compound (S)-7b is also neuroprotective, al-
though it does not display HDAC6 activity (Figure 3c and 3d).
The same trend was observed with (R)-16 and (S)-16. Com-
pounds 13c, 18c, 18d, 18e, 20a, and 20b are also neuropro-
tective but show no activity at HDAC6, suggesting that an off-
target mechanism may play a role in the neuroprotective prop-
erties of these compounds (Supporting Information figure 5).
Therefore, the ability of selected compounds to prevent glu-
tathione depletion was measured (Figure 4). We found that (S)-
7b, but not (R)-7b, is able to prevent glutathione depletion,
suggesting this as an additional neuroprotective pathway for
these compounds. Moreover, 7a is also very effective in pre-
venting glutathione depletion which likely contributes to the
mides. Rat primary cortical neurons were incubated with each compound at
10 mm in the absence (&) or presence (&) of the cysteine homologue HCA
(5 mm).
excellent neuroprotective profile observed with this com-
pound. A similar albeit less pronounced effect was observed
with (R)-16 and (S)-16, in that the S isomer is more effective in
preventing glutathione depletion than the R isomer. Many of
the other compounds tested displayed a moderate ability to
prevent glutathione depletion, including cis- and trans-13a as
well as their respective prodrugs, cis- and trans-13d.
Of note, however, is compound 9, which is able to prevent
glutathione depletion without being neuroprotective. This is
likely due to the intrinsic neuronal toxicity that is often ob-
served with hydroxamic acids: although this compound is
active at HDAC6 and is able to prevent glutathione depletion,
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A. P. Kozikowski et al.
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broad. HRMS experiments were performed on a Q-TOF-2TM instru-
ment (Micromass). TLC was performed with Merck 250 mm 60 F254
silica gel plates. Column chromatography was performed with
Merck silica gel (40–60 mesh). Microwave-assisted reactions were
carried out in a sealed tube using a Biotage Initiator. Preparative
HPLC was carried out with an ACE 5AQ column (250ꢁ10 mm) on a
Shimadzu LC8A with SPD-10avp detector (l 254 and 280 nm) and
a flow rate of 3.5 mLminꢀ1 with a gradient (Method A: from 30%
CH3CN in H2O to 100% CH3CN over 30 min; Method B: from 10%
CH3CN in H2O to 100% CH3CN over 28 min). Chiral HPLC was car-
ried out with a Chiralpak AD column (250ꢁ10 mm) on a Shimadzu
LC8A with SPD-10AV detector (l 230 and 240 nm) and a flow rate
of 2.4 mLminꢀ1 with an isocratic mobile phase (80:20 hexane/2-
propanal). The purity of all tested compounds was ꢂ95%, as de-
termined by analytical HPLC (Agilent 1100 with a G1314A detector
(l 254 nm) and a flow rate of 1.4 mLminꢀ1 using a Synergi 4 m
hydro-RP column (150ꢁ4.6 mm) or a Luna 5 m C18 column (150ꢁ
4.6 mm) and a gradient from 60% CH3CN in H2O to 100% CH3CN
over 25 min).
the aforementioned ZBG precludes any substantial neuropro-
tective activity. When dealing with epigenetic regulators (and
small molecules in general), it is unlikely that only one biologi-
cal pathway will be affected and thus the neuroprotective ac-
tivity of these compounds probably results from a combination
of effects. In addition to HDAC6 inhibition and the prevention
of glutathione depletion, other mechanisms that may contrib-
ute to the observed neuroprotective activity of these com-
pounds include the inhibition of MMPs and other zinc-depen-
dent enzymes, interference with HCA itself, and/or the intrinsic
antioxidant activity of the thiols themselves.[35] Therefore, fur-
ther investigation into the potential neuroprotective mecha-
nisms of these compounds is necessary to fully elucidate all of
the biological pathways involved.
Conclusions
Two HDACIs are currently marketed for the treatment of cuta-
neous T-cell lymphoma, and a host of others are the subjects
of ongoing clinical trials.[36] The continued development of
novel HDACIs as therapeutics is warranted; however, the ma-
jority of the reported compounds lack isozyme selectivity, thus
leading to various levels of undesirable toxicity. While pan-ac-
tivity may be acceptable for oncology applications, it is unlike-
ly to be acceptable in other therapeutic areas. As detailed
herein, we have developed a series of selective, stable, low-
molecular-weight HDAC6 inhibitors that exhibit neuroprotec-
tion in neuronal models of oxidative stress for potential use in
the treatment of neurodegenerative diseases, traumatic brain
injury, and other health problems, such as myocardial infarc-
tion, in which oxidative stress is an issue. We demonstrated
that mercaptoacetamide 7a (HDAC1 IC50 =5.7 mm, HDAC6
IC50 =0.26 mm) exhibits potency and selectivity similar to that
of the hydroxamic acid analogue 9 (HDAC1 IC50 =3.0 mm,
HDAC6 IC50 =0.18 mm). Therefore, mercaptoacetamide ZBGs
may find potential use in place of the traditional hydroxamic
acids. In addition, replacement of the hydroxamic acid func-
tionality with a mercaptoacetamide alleviates the toxicity ob-
served in cortical neurons, suggesting this as a suitable ap-
proach toward an improved safety profile. Selective inhibition
of HDAC6 also results in dose-dependent protection against
oxidative stress in cultured cortical neurons, further supporting
the idea that HDAC6 inhibition may be a viable way to treat
certain neurological diseases.[30] Therefore, HDAC6-selective
mercaptoacetamides offer an attractive alternative to the con-
ventional hydroxamic acid based inhibitors, as they afford a
decrease in toxicity and the potential for enhanced cell perme-
ability via prodrug formation.
Ethyl 5-methyl-3-phenylisoxazole-4-carboxylate (1): (E)-benzalde-
hyde oxime (2.4 g, 19.8 mmol) was mixed with neutral Al2O3 (20 g).
N-chlorosuccinimide (3.0 g, 22.4 mmol) was added, and the reac-
tion mixture was irradiated at 408C for 3ꢁ10 min, mixing between
each step. The mixture was extracted with CCl4 (40 mL), and the
solvent was evaporated. The obtained yellow oil was dissolved in
anhydrous THF (30 mL). Ethyl 2-butynoate (1.16 mL, 10.0 mmol)
and Et3N (6.0 mL, 43.0 mmol) were added slowly. The reaction mix-
ture was irradiated at 708C for 1 h, filtered and washed with
EtOAc, and then the filtrate was concentrated in vacuo. Purification
of the crude reaction mixture by column chromatography (SiO2,
0!10% EtOAc/hexanes) afforded the title compound (1.0 g, 43%)
as a pale-yellow oil. 1H NMR (400 MHz, CDCl3): d=7.62–7.60 (m,
2H), 7.43–7.38 (m, 3H), 4.21 (q, J=7.2 Hz, 2H), 2.70 (s, 3H),
1.92 ppm (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=175.6,
162.3, 161.7, 129.6, 129.2, 128.5, 127.9, 108.2, 60.5, 13.7, 13.3 ppm.
5-Methyl-3-phenylisoxazole-4-carboxylic acid (2): NaOH (540 mg,
13.3 mmol) was added to isoxazole 1 (2.68 g, 11.6 mmol) in
CH3OH/H2O 1:1 (80 mL). The reaction mixture was stirred at 608C
for 4 h, and then the CH3OH layer was evaporated. The aqueous
layer was acidified with 1n HCl to pH 3 and then extracted with
EtOAc (3ꢁ50 mL). The combined organic layers were washed with
H2O (100 mL), brine (50 mL), and dried over anhydrous Na2SO4 to
afford the title compound (2.31 g, 98%) as a white powder.
1H NMR (400 MHz, CDCl3): d=7.65–7.62 (m, 2H), 7.49–7.43 (m, 3H),
2.77 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=177.7, 166.8, 162.7,
129.9, 129.4, 128.2, 127.9, 107.5, 13.9 ppm.
tert-Butyl 5-(5-methyl-3-phenylisoxazole-4-carbonylamino)pen-
tylcarbamate (3): N-Boc-1,5-diaminopentane (2.6 mL, 12.6 mmol),
PyBOP (7.12 g, 13.7 mmol), and diisopropylethylamine (DIEA, 6 mL,
34.2 mmol) were added to isoxazole 2 (2.32 g, 11.4 mmol) in CH2Cl2
(100 mL). The reaction mixture was stirred at room temperature for
20 h and quenched with H2O. The aqueous layer was extracted
with CH2Cl2 (3ꢁ50 mL). The combined organic layers were washed
with brine (50 mL) and dried over anhydrous Na2SO4. Purification
of the crude reaction mixture by column chromatography (SiO2,
0!20% EtOAc/hexanes) afforded the title compound (4.35 g,
Experimental Section
Chemistry
1
98%) as a white powder. H NMR (400 MHz, CDCl3): d=7.49–7.47
1H NMR and 13C NMR spectra were recorded on a Bruker spectrom-
eter at 300/400 MHz and 75/100 MHz, respectively, with TMS as an
internal standard. Standard abbreviations indicating multiplicity
were used: s=singlet, d=doublet, t=triplet, q=quartet, m=mul-
tiplet, dd=doublet of doublets, dt=doublet of triplets, and br=
(m, 2H), 7.39–7.36 (m, 3H), 5.82 (br, 1H), 4.71 (br, 1H), 3.13–3.08
(m, 2H), 2.94–2.89 (m, 2H), 2.54 (s, 3H), 1.32 (s, 9H), 1.28–1.22 (m,
4H), 1.08–1.03 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=172.4,
161.2, 159.6, 155.6, 129.9, 128.5, 128.3, 127.8, 111.1, 78.5, 38.8, 29.1,
28.3, 27.9, 25.9, 25.8, 12.2 ppm.
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Chiral Mercaptoacetamides
Methyl-3-phenylisoxazole-4-carboxylic acid (5-aminopentyl)a-
mide TFA (4): Trifluoroacetic acid (TFA, 7.7 mL, 103.7 mmol) was
added to isoxazole 3 (4.02 g, 10.4 mmol) in CH2Cl2 (60 mL). The re-
action mixture was stirred at room temperature for 4 h, and then
the solvent was removed in vacuo to afford the title compound
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-tritylsulfanyl-
propionyl)aminopentyl]amide (6b): Compound 6b (73%) was
prepared from compound 5b according to the methodology de-
scribed for 6a. 1H NMR (400 MHz, CDCl3): d=7.59–7.56 (m, 2H),
7.51–7.47 (m, 3H), 7.44–7.42 (m, 6H), 7.30–7.22 (m, 6H), 7.21–7.19
(m, 3H), 6.01 (br, 1H), 5.75 (br, 1H), 3.21–3.16 (m, 2H), 3.02 (q, J=
7.6 Hz, 1H), 2.91–2.86 (m, 1H), 2.67–2.62 (m, 1H), 2.66 (s, 3H), 1.43
(d, J=7.6 Hz, 2H), 1.33–1.28 (m, 2H), 1.24–1.20 (m, 2H), 1.08–
1.02 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=173.0, 172.4,
161.5, 159.9, 144.1, 130.3, 129.1, 128.8, 128.6, 128.0, 127.9, 126.8,
111.2, 67.9, 44.3, 39.2, 39.0, 28.5, 28.4, 23.7, 19.9, 12.6 ppm.
1
(4.09 g, 98%) as a yellow oil. H NMR (400 MHz, CD3OD): d=7.61–
7.57 (m, 2H), 7.43–7.39 (m, 3H), 3.27–3.23 (m, 2H), 2.84–2.80 (m,
2H), 2.49 (s, 3H), 1.68–1.58 (m, 2H), 1.54–1.47 (m, 2H), 1.34–
1.26 ppm (m, 2H); 13C NMR (100 MHz, CD3OD): d=171.6, 164.6,
161.9, 131.2, 129.8, 129.2, 128.1, 113.8, 40.5, 40.3, 29.6, 28.0, 24.7,
12.0 ppm.
(R)-5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-tritylsulfa-
nylpropionyl)aminopentyl]amide ((R)-6b): Compound (R)-6b was
isolated from the racemic mixture of compound 6b using chiral
preparative HPLC as described in the synthetic protocol above.
1H NMR (400 MHz, CDCl3): d=7.59–7.56 (m, 2H), 7.51–7.48 (m, 3H),
7.44–7.42 (m, 6H), 7.31–7.27 (m, 6H), 7.24–7.20 (m, 3H), 5.92 (br,
1H), 5.46 (br, 1H), 3.22–3.17 (m, 2H), 3.02 (q, J=7.6 Hz, 1H), 2.92–
2.87 (m, 1H), 2.71 (s, 3H), 2.69–2.61 (m, 1H), 1.43 (d, J=7.6 Hz,
3H), 1.33–1.27 (m, 2H), 1.25–1.20 (m, 2H), 1.07–1.02 ppm (m, 2H);
13C NMR (100 MHz, CDCl3): d=174.0, 172.4, 161.6, 160.2, 144.5,
130.7, 129.5, 129.3, 129.1, 128.5, 128.3, 127.2, 110.9, 68.3, 44.7, 39.6,
39.3, 28.9, 28.8, 24.1, 20.4, 13.1 ppm; [a]D20 = +39.9 (c=1, CHCl3).
Tritylsulfanylacetic acid (5a): CPh3OH (13.0 g, 50 mmol) and TFA
(5.0 mL, 65 mmol) were added to thioglycolic acid (3.5 mL,
50 mmol) in CHCl3 (50 mL). The reaction mixture was stirred at
room temperature for 3 h. The volatiles were removed in vacuo.
The crude product was recrystallized from CH2Cl2/hexanes 1:1
(30 mL) and washed with cold Et2O to afford the title compound
(15.9 g, 95%) as a white powder. 1H NMR (300 MHz, CDCl3): d=
7.43–7.40 (m, 6H), 7.30–7.19 (m, 9H), 3.02 ppm (s, 2H); 13C NMR
(75 MHz, CD3OD): d=171.7, 144.2, 129.3, 127.6, 126.6, 66.7,
34.3 ppm.
2-Tritylsulfanylpropionic acid (5b): Compound 5b (75%) was pre-
pared from 2-mercaptopropanoic acid according to the methodol-
ogy described for 5a. H NMR (400 MHz, CD3OD): d=7.43–7.41 (m,
6H), 7.28–7.19 (m, 9H), 2.90 (q, J=7.2 Hz, 1H), 1.01 ppm (d, J=
7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD): d=177.1, 145.8, 130.8,
128.9, 127.9, 69.3, 44.0, 19.4 ppm.
(S)-5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-tritylsulfa-
nylpropionyl)aminopentyl]amide ((S)-6b): Compound (S)-6b was
isolated from the racemic mixture of compound 6b using chiral
preparative HPLC as described in the synthetic protocol above.
1H NMR (400 MHz, CDCl3): d=7.58–7.56 (m, 2H), 7.53–7.49 (m, 3H),
7.43–7.41 (m, 6H), 7.30–7.28 (m, 6H), 7.26–7.19 (m, 3H), 5.93 (br,
1H), 5.44 (br, 1H), 3.20–3.18 (m, 2H), 3.02 (q, J=7.6 Hz, 1H), 2.93–
2.86 (m, 1H), 2.70 (s, 3H), 2.68–2.62 (m, 1H), 1.43 (d, J=7.6 Hz,
3H), 1.32–1.28 (m, 2H), 1.23–1.19 (m, 2H), 1.04–1.02 ppm (m, 2H);
13C NMR (100 MHz, CDCl3): d=173.5, 172.1, 161.2, 159.6, 143.9,
130.1, 129.0, 128.7, 128.6, 127.9, 127.7, 126.6, 110.7, 67.8, 44.1, 39.1,
38.7, 28.3, 28.2, 23.6, 19.8, 12.5 ppm; [a]D20 =ꢀ36.5 (c=1, CHCl3).
1
2-Tritylsulfanylisobutyric acid (5c): Compound 5c (80%) was pre-
pared from 2-mercaptoisobutyric acid according to the methodolo-
gy described for 5a. 1H NMR (400 MHz, CDCl3): d=7.68–7.66 (m,
6H), 7.38–7.26 (m, 9H), 1.48 ppm (s, 6H); 13C NMR (100 MHz,
CDCl3): d=180.0, 144.2, 129.9, 127.6, 126.7, 68.4, 51.2, 27.1 ppm.
Pivaloylsulfanylacetic acid (5d): Pivaloyl chloride (2.7 mL,
22 mmol) and Et3N (6.1 mL, 44 mmol) were added to thioglycolic
acid (1.39 mL, 50 mmol) in dioxane (10 mL) at 08C. The reaction
mixture was stirred at room temperature overnight, filtered and
acidified with 1n HCl to pH 1. This layer was extracted with CH2Cl2
(3ꢁ100 mL). The combined organic layers were dried over anhy-
drous Na2SO4. Purification of the crude reaction mixture by prepa-
rative HPLC Method B afforded the title compound (0.61 g, 17%)
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-tritylsulfanyli-
sobutyryl)aminopentyl]amide (6c): Compound 6c (98%) was pre-
pared from compound 5c according to the methodology de-
scribed for 6a. 1H NMR (400 MHz, CDCl3): d=7.59–7.56 (m, 2H),
7.47–7.45 (m, 3H), 7.44–7.42 (m, 6H), 7.26–7.22 (m, 6H), 7.20–7.17
(m, 3H), 6.49 (br, 1H), 6.01 (br, 1H), 3.20–3.15 (m, 2H), 3.10–3.05
(m, 2H), 2.62 (s, 3H), 1.35 (s, 6H), 1.33–1.28 (m, 2H), 1.19–1.13 (m,
2H), 1.07–1.02 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=174.1,
172.5, 161.9, 160.2, 144.4, 130.3, 129.1, 129.0, 128.6, 128.0, 127.8,
126.9, 111.7, 67.9, 52.3, 43.3, 39.3, 28.6, 28.5, 28.4, 23.9, 12.6 ppm.
1
as a colorless oil. H NMR (300 MHz, CDCl3): d=9.80 (s, 1H), 3.52 (s,
2H), 1.89 ppm (s, 9H); 13C NMR (75 MHz, CD3OD): d=205.1, 174.7,
46.2, 30.8, 27.0 ppm.
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-tritylsulfany-
lacetyl)aminopentyl]amide (6a): Compound 5a (3.85 g,
11.5 mmol), PyBOP (7.0 g, 13.4 mmol), and DIEA (5 mL, 29.0 mmol)
were added to isoxazole 4 (3.85 g, 9.6 mmol) in CH2Cl2 (100 mL).
The reaction mixture was stirred at room temperature for 20 h and
quenched with H2O. The aqueous layer was extracted with CH2Cl2
(3ꢁ50 mL). The combined organic layers were washed with brine
(50 mL) and dried over anhydrous Na2SO4. Purification of the crude
reaction mixture by column chromatography (SiO2, 0!5% CH3OH/
CH2Cl2) afforded the title compound (5.32 g, 92%) as a white foam.
1H NMR (400 MHz, CDCl3): d=7.58–7.55 (m, 2H), 7.53–7.49 (m, 3H),
7.42–7.38 (m, 6H), 7.31–7.20 (m, 9H), 5.97 (br, 1H), 5.38 (br, 1H),
3.21–3.17 (m, 2H), 3.09 (s, 2H), 2.89–2.85 (m, 2H), 2.71 (s, 3H),
1.32–1.23 (m, 4H), 1.05–1.01 ppm (m, 2H); 13C NMR (100 MHz,
CDCl3): d=173.3, 167.7, 161.4, 159.9, 143.8, 130.3, 129.3, 128.9,
128.7, 128.1, 128.0, 126.9, 111.2, 67.7, 39.2, 38.9, 35.7, 28.6, 28.5,
23.8, 12.6 ppm.
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-pivaloylsulfa-
nylacetyl)aminopentyl]amide (6d): Compound 6d (14%) was pre-
pared from compound 5d according to the methodology de-
scribed for 6a. Purification of the obtained crude mixture was
1
done using preparative HPLC Method A. H NMR (400 MHz, CDCl3):
d=7.59–7.57 (m, 2H), 7.54–7.49 (m, 3H), 6.22 (br, 1H), 5.46 (br,
1H), 3.45 (s, 2H), 3.24–3.19 (m, 2H), 3.17–3.12 (m, 2H), 2.71 (s, 3H),
1.42–1.31 (m, 4H), 1.24 (s, 9H), 1.16–1.11 ppm (m, 2H); 13C NMR
(100 MHz, CDCl3): d=207.26, 173.8, 168.7, 161.6, 160.0, 130.5,
129.1, 129.0, 128.3, 111.1, 46.5, 39.3, 39.1, 32.4, 28.9, 28.7, 27.2, 23.7,
12.9 ppm; ESI-HRMS calcd for [C23H31N3O4S+H]+: 426.21080 m/z,
found: 426.2130 m/z; HPLC purity: 98.0%.
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-mercaptoace-
tyl)aminopentyl]amide (7a): TFA (6.5 mL, 88.0 mmol) and triethyl-
silane (2.8 mL, 17.6 mmol) were added to isoxazole 6a (5.31 g,
8.8 mmol) in CH2Cl2 (50 mL) at 08C. The reaction mixture was
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A. P. Kozikowski et al.
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stirred at room temperature for 3 h. DMF (2 mL) was added, and
volatiles were removed in vacuo. Purification of the crude reaction
mixture by preparative HPLC Method A afforded the title com-
3 mmol) were added to isoxazole 2 (0.2 g, 1.0 mmol) in CH2Cl2
(20 mL). The reaction mixture was stirred at room temperature for
20 h and then quenched with H2O. The aqueous layer was extract-
ed with CH2Cl2 (3ꢁ30 mL). The combined organic layers were
washed with brine (30 mL) and dried over anhydrous Na2SO4. Pu-
rification of the crude reaction mixture by column chromatography
(SiO2, 0!5% CH3OH/CH2Cl2) afforded the title compound (0.29 g,
1
pound (2.45 g, 77%) as a white powder. H NMR (400 MHz, CDCl3):
d=7.59–7.54 (m, 2H), 7.52–7.48 (m, 3H), 6.70 (br, 1H), 5.44 (br,
1H), 3.26–3.20 (m, 6H), 2.71 (s, 3H), 1.85 (t, J=9.0 Hz, 1H), 1.51–
1.44 (m, 2H), 1.41–1.34 (m, 2H), 1.22–1.14 ppm (m, 2H); 13C NMR
(100 MHz, CDCl3): d=173.7, 169.1, 161.5, 160.0, 130.5, 129.1, 128.9,
128.2, 111.1, 39.5, 39.0, 28.9, 28.7, 28.2, 23.8, 12.8 ppm; ESI-HRMS
calcd for [C18H23N3O3S+H]+: 362.15329 m/z, found: 362.1519 m/z;
HPLC purity: 99.7%.
1
86%) as a white solid. H NMR (400 MHz, CDCl3): d=7.43–7.41 (m,
2H), 7.36–7.27 (m, 3H), 5.91 (br, 1H), 3.49 (s, 3H), 3.07–3.02 (m,
2H), 2.46 (s, 3H), 2.13 (t, J=7.2 Hz, 2H), 1.46–1.37 (m, 2H), 1.24–
1.18 (m, 2H), 1.13–1.05 (m, 2H), 1.03–2.97 ppm (m, 2H); 13C NMR
(100 MHz, CDCl3): d=173.6, 172.1, 161.2, 159.7, 129.9, 128.2, 127.8,
126.7, 111.3, 51.0, 38.9, 33.4, 28.5, 28.2, 26.0, 24.3, 12.1 ppm.
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-mercaptopro-
pionyl)aminopentyl]amide (7b): Compound 7b (46%) was pre-
pared from compound 6b according to the methodology de-
scribed for 7a. 1H NMR (400 MHz, CDCl3): d=7.57–7.55 (m, 2H),
7.54–7.47 (m, 3H), 6.54 (br, 1H), 5.50 (br, 1H), 3.40–3.35 (m, 1H),
3.24–3.15 (m, 4H), 2.69 (s, 3H), 1.99 (d, J=8.4 Hz, 1H), 1.50 (d, J=
7.2 Hz, 3H), 1.47–1.42 (m, 2H), 1.40–1.34 (m, 2H), 1.21–1.15 ppm
(m, 2H); 13C NMR (100 MHz, CDCl3): d=173.7, 172.8, 161.5, 160.0,
130.5, 129.1, 128.9, 128.2, 111.1, 39.4, 39.0, 38.1, 28.8, 28.7, 23.8,
22.2, 12.8 ppm; ESI-HRMS calcd for [C19H25N3O3S+H]+: 376.16894
m/z, found: 376.1692 m/z; HPLC purity: 98.9%.
5-Methyl-3-phenylisoxazole-4-carboxylic acid (6-hydroxycarba-
moylhexyl)amide (9): NH2OH [generated from filtration at 08C of a
mixture of NH2OH·HCl (0.4 g, 5.8 mmol) and KOH (325 mg,
5.8 mmol) in CH3OH (2 mL) heated at 408C] and KOH (120 mg,
2.1 mmol) were added to isoxazole 8 (0.1 g, 0.29 mmol) in CH3OH
(1 mL). The reaction mixture was stirred at room temperature for
1.5 h and quenched with H2O. The aqueous layer was extracted
with EtOAc (3ꢁ10 mL). The combined organic layers were dried
over anhydrous Na2SO4. Purification of the crude reaction mixture
by preparative HPLC Method A afforded the title compound
(R)-5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-mercapto-
propionyl)aminopentyl]amide ((R)-7b): Compound (R)-7b (43%)
was prepared from compound (R)-6b according to the methodolo-
gy described for 7a. 1H NMR (400 MHz, CDCl3): d=7.57–7.54 (m,
2H), 7.54–7.50 (m, 3H), 6.56 (br, 1H), 5.53 (br, 1H), 3.40–3.36 (m,
1H), 3.24–3.15 (m, 4H), 2.69 (s, 3H), 1.99 (d, J=8.4 Hz, 1H), 1.50 (d,
J=7.2 Hz, 3H), 1.48–1.42 (m, 2H), 1.40–1.35 (m, 2H), 1.21–
1.15 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=173.3, 172.6,
161.2, 159.7, 130.1, 128.7, 128.5, 127.8, 110.8, 39.1, 38.6, 37.8, 28.5,
28.4, 23.5, 21.9, 12.5 ppm; [a]D20 = +3.9 (c=0.1, CHCl3); ESI-HRMS
calcd for [C19H25N3O3S+H]+: 376.1689 m/z, found: 376.1692 m/z;
HPLC purity: 98.7%.
1
(0.21 g, 21%) as a white solid. H NMR (400 MHz, CD3OD): d=8.20
(br, 1H), 7.66–7.64 (m, 2H), 7.52–7.44 (m, 3H), 3.31–3.27 (m, 2H),
2.54 (s, 3H), 2.08 (t, J=7.2 Hz, 2H), 1.62–1.48 (m, 4H), 1.36–
1.30 ppm (m, 4H); 13C NMR (100 MHz, CD3OD): d=171.5, 164.6,
161.9, 131.3, 129.8, 129.2, 129.1, 113.9, 40.6, 30.0, 29.7, 27.6, 26.6,
11.9 ppm; ESI-HRMS calcd for [C18H23N3O4 +H]+: 346.17613 m/z,
found: 346.1768 m/z; HPLC purity: 96.2%.
5-Methyl-3-phenylisoxazol-4-ylmethanol (10): A borane–THF so-
lution (1.5 mL, 1.0m) was added dropwise to a solution of acid 2
(203 mg, 1 mmol) in anhydrous THF (10 mL) at 08C. The mixture
was then stirred at room temperature overnight, after which the
reaction was quenched with H2O (10 mL), and the organic products
were extracted with EtOAc (3ꢁ15 mL). The organic layer was
washed with H2O (10 mL), brine (10 mL) and dried over Na2SO4.
After concentration in vacuo, the crude product was purified by
column chromatography (SiO2, EtOAc/hexanes 70:30!50:50) to
(S)-5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-mercapto-
propionyl)aminopentyl]amide ((S)-7b): Compound (S)-7b (45%)
was prepared from compound (S)-6b according to the methodolo-
gy described for 7a. 1H NMR (400 MHz, CDCl3): d=7.58–7.55 (m,
2H), 7.53–7.48 (m, 3H), 6.51 (br, 1H), 5.48 (br, 1H), 3.41–3.37 (m,
1H), 3.25–3.16 (m, 4H), 2.70 (s, 3H), 1.99 (d, J=8.4 Hz, 1H), 1.50 (d,
J=7.2 Hz, 3H), 1.46–1.43 (m, 2H), 1.38–1.33 (m, 2H), 1.21–
1.15 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=174.0, 173.1,
161.8, 160.2, 130.7, 129.3, 129.2, 128.5, 111.4, 39.7, 39.2, 38.4, 29.1,
29.0, 24.1, 22.5, 13.1 ppm; [a]D20 =ꢀ3.7 (c=0.1, CHCl3); ESI-HRMS
calcd for [C19H25N3O3S+H]+: 376.1689 m/z, found: 376.1691 m/z;
HPLC purity: 99.0%.
1
afford the title product 10 (170 mg, 90%) as a colorless oil. H NMR
(400 MHz, CDCl3): d=7.76–7.73 (m, 2H), 7.42–7.39 (m, 3H), 4.47 (s,
2H), 2.37 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=168.5, 162.3,
129.6, 128.8, 128.7, 128.1, 112.9, 53.2, 10.9 ppm.
5-Methyl-3-phenylisoxazole-4-carbaldehyde (11): The alcohol 10
(190 mg, 1 mmol) and activated MnO2 (870 mg, 10 mmol) were dis-
solved in anhydrous CH2Cl2 (5 mL) under Ar atmosphere. The mix-
ture was stirred at room temperature overnight. After the black
powder was filtered off, the filtrate was dried in vacuo and purified
by column chromatography (SiO2, EtOAc/hexanes 75:25) to afford
the title product 11 (175 mg, 94%) as a white solid. 1H NMR
(400 MHz, CDCl3): d=9.91 (s, 1H), 7.66–7.65 (m, 2H), 7.49–7.47 (m,
3H), 2.74 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=184.3, 176.6,
162.0, 130.3, 128.8, 114.9, 12.8 ppm.
5-Methyl-3-phenylisoxazole-4-carboxylic acid [5-(2-mercaptoiso-
butyryl)aminopentyl]amide (7c): Compound 7c (57%) was pre-
pared from compound 6c according to the methodology de-
scribed for 7a. 1H NMR (400 MHz, CDCl3): d=7.54–7.50 (m, 2H),
7.47–7.43 (m, 3H), 6.96 (br, 1H), 5.63 (br, 1H), 3.21–3.16 (m, 2H),
3.15–3.10 (m, 2H), 2.64 (s, 3H), 2.14 (s, 1H), 1.52 (S, 6H), 1.46–1.38
(m, 2H), 1.36–1.30 (m, 2H), 1.17–1.09 ppm (m, 2H); 13C NMR
(100 MHz, CDCl3): d=175.1, 173.3, 161.5, 159.9, 130.3, 129.0, 128.7,
128.1, 111.1, 47.5, 39.6, 39.0, 30.2, 28.9, 28.6, 23.8, 12.7 ppm; ESI-
HRMS calcd for [C20H27N3O3S+H]+: 390.1846 m/z, found: 390.1861
m/z; HPLC purity: 99.0%.
tert-Butyl
7-(5-methyl-3-phenylisoxazol-4-yl)hept-6-enylcarba-
mate (12): n-Butyllithium (10.8 mL, 17.3 mmol) was added to the
tert-butyl 6-triphenyl-l5-phosphanylhexylcarbamate phosphonium
salt (6.26 g, 11.5 mmol, generated from tert-butyl 6-bromohexylcar-
bamate and PPh3 in toluene at reflux for four days) in THF
(100 mL) at 08C. The reaction mixture was stirred at 08C for
30 min; then the aldehyde 11 (388 mg, 2.07 mmol) in THF (5 mL)
was added. The reaction mixture was stirred at room temperature
Methyl 7-(5-methyl-3-phenylisoxazole-4-carbonylamino)hepta-
noate (8): Methyl 5-aminopentanoate (generated from its acid,
0.193 g, 1.0 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP, 0.612 g, 1.2 mmol), and DIEA (0.5 mL,
434
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ChemMedChem 2012, 7, 425 – 439

Chiral Mercaptoacetamides
for 1.5 h and then quenched with H2O (50 mL). The aqueous layer
was extracted with EtOAc (3ꢁ60 mL). The combined organic layers
were washed with brine (50 mL) and dried over anhydrous Na2SO4.
Purification of the crude reaction mixture by column chromatogra-
phy (SiO2, 0!25% EtOAc/hexanes) afforded the title compound Z/
11.9 ppm; ESI-HRMS calcd for [C20H26N2O2S+H]+: 359.17878 m/z,
found: 359.1785 m/z; HPLC purity: 97.4%.
N-[7-(5-Methyl-3-phenylisoxazol-4-yl)hept-6-enyl]-2-mercaptoiso-
butyramide (13c): Compound 13c Z/E 7:3 (49%) was prepared
from compound 12 in three steps according to the methodology
described for 4, 6c, and 7c. 1H NMR (400 MHz, CDCl3): d=7.74–
7.72 (m, 2H), 7.62–7.60 (m, 2H), 7.44–7.40 (m, 6H), 6.97 (br, 1H),
6.86 (br, 1H), 6.08–6.04 (m, 2H), 5.83–5.77 (m, 2H), 3.24–3.21 (m,
2H), 3.16–3.11 (m, 2H), 2.46 (s, 3H), 2.32 (s, 3H), 2.17–2.13 (m, 2H),
1.92–1.90 (m, 2H), 1.56 (s, 12H), 1.42–1.21 ppm (m, 12H); 13C NMR
(100 MHz, CDCl3): d=175.0, 166.1, 165.5, 161.0, 136.7, 134.8, 129.8,
129.4, 128.6, 127.8, 117.7, 117.1, 112.5, 111.1, 47.7, 39.9, 33.2, 30.3,
29.1, 28.8, 28.5, 26.3, 11.9 ppm; ESI-HRMS calcd for [C21H28N2O2S+
H]+: 371.17987 m/z, found: 371.1785 m/z; HPLC purity: 96.4%.
1
E 8:2 (415 mg, 54%) as a yellow oil. H NMR (400 MHz, CDCl3): d=
7.73–7.70 (m, 2H), 7.62–7.59 (m, 2H), 7.44–7.38 (m, 6H), 6.06–6.02
(m, 2H), 5.82–5.76 (m, 2H), 4.54 (br, 1H), 4.49 (br, 1H), 3.09–2.99
(m, 4H), 2.45 (s, 3H), 2.31 (s, 3H), 2.17–2.12 (m, 2H), 1.91–1.86 (m,
2H), 1.41 (s, 18H), 1.36–1.27 (m, 8H), 1.23–1.15 ppm (m, 4H);
13C NMR (100 MHz, CDCl3): d=166.0, 165.4, 161.0, 155.8, 136.7,
134.9, 129.7, 129.3, 128.4, 127.7, 117.6, 117.0, 112.5, 111.1, 78.9,
40.3, 33.2, 29.7, 28.8, 28.5, 28.3, 26.2, 12.0, 11.7 ppm.
N-[7-(5-Methyl-3-phenylisoxazol-4-yl)hept-6-enyl]-2-mercaptoa-
cetamide (13a): Compound 13a Z/E 7:3 (33%) was prepared from
compound 12 in three steps according to the methodology de-
N-[7-(5-Methyl-3-phenylisoxazol-4-yl)hept-6-enyl]-2-pivaloylsul-
fanylacetamide (13d): Compound 13d Z/E 7:3 (42%) was pre-
pared from compound 12 in two steps according to the methodol-
ogy described for 4 and 6d. Purification of the obtained crude
mixture was done using preparative HPLC Method B. 1H NMR
(400 MHz, CDCl3): d=7.71–7.68 (m, 2H), 7.59–7.57 (m, 2H), 7.41–
7.37 (m, 6H), 6.35 (br, 1H), 6.25 (br, 1H), 6.03–6.00 (m, 2H), 5.79–
5.73 (m, 2H), 3.44 (s, 2H), 3.42 (s, 2H), 3.20–3.14 (m, 2H), 3.11–3.06
(m, 2H), 2.43 (s, 3H), 2.28 (s, 3H), 2.14–2.09 (m, 2H), 1.89–1.83 (m,
2H), 1.44–1.14 ppm (m, 30H); 13C NMR (100 MHz, CDCl3): d=206.8,
168.2, 165.9, 165.4, 160.9, 136.6, 134.8, 129.6, 129.2, 128.4, 127.6,
117.5, 116.9, 112.4, 111.0, 46.3, 39.4, 33.0, 32.3, 29.0, 28.7, 28.4, 27.0,
26.2, 11.7 ppm; ESI-HRMS calcd for [C24H32N2O3S+H]+: 429.22064
m/z, found: 429.2185 m/z; HPLC purity: 98.4%.
1
scribed for 4, 6a, and 7a. H NMR (400 MHz, CDCl3): d=7.75–7.72
(m, 2H), 7.63–7.61 (m, 2H), 7.45–7.41 (m, 6H), 6.97 (br, 1H), 6.60
(br, 1H), 6.09–6.05 (m, 2H), 5.84–5.74 (m, 2H), 3.28–3.15 (m, 4H),
2.47 (s, 3H), 2.33 (s, 3H), 1.94–1.89 (m, 2H), 1.84–1.80 (m, 2H),
1.56–1.20 ppm (m, 12H); 13C NMR (100 MHz, CDCl3): d= 169.1,
166.0, 165.5, 161.0, 136.6, 134.8, 129.6, 129.3, 128.3, 127.6, 117.6,
116.9, 112.5, 111.0, 39.7, 33.1, 29.0, 28.7, 28.4, 28.1, 26.2, 11.7 ppm;
ESI-HRMS calcd for [C19H24N2O2S+H]+: 345.16313 m/z, found:
345.1638 m/z; HPLC purity: 98.1%.
(Z)-2-Mercapto-N-(7-(5-methyl-3-phenylisoxazol-4-yl)hept-6-eny-
l)acetamide (cis-13a): Compound cis-13a (65%) was prepared
from cis-25 according to the procedure described for 7a (see Sup-
porting Information scheme 2 for isolation of cis-25). 1H NMR
(400 MHz, CDCl3): d=7.73 (m, 2H), 7.43 (m, 3H), 6.79 (s, 1H), 6.08
(d, J=10.8 Hz, 1H), 5.77 (dt, J=7.2 Hz, 11.2 Hz, 1H), 3.23–3.15 (m,
4H), 2.33 (s, 3H), 1.91 (q, J=7.2 Hz, 2H), 1.85 (t, J=8.8 Hz, 1H),
1.40 (m, 2H), 1.32 (m, 2H), 1.22 ppm (m, 2H); 13C NMR (100 MHz,
CDCl3): d=170.1, 166.4, 161.3, 136.9, 129.8, 129.7, 128.8, 128.0,
117.3, 111.4, 40.1, 29.2, 29.0, 28.7, 28.3, 26.6, 12.0 ppm; ESI-HRMS
calcd for [C19H24N2O2S+H]+: 345.1631 m/z, found: 345.1628 m/z;
HPLC purity: 98.5%.
(Z)-(S)-2-(7-(5-Methyl-3-phenylisoxazol-4-yl)hept-6-enylamino)-2-
oxoethyl 2,2-dimethylpropanethioate (cis-13d): Compound cis-
13d was isolated from 13d by chiral column chromatography.
1H NMR (400 MHz, CDCl3): d=7.71 (m, 2H), 7.41 (m, 3H), 6.16 (s,
1H), 6.06 (d, J=10.8 Hz, 1H), 5.79 (m, 1H), 3.44 (s, 2H), 3.12 (q, J=
6.8 Hz, 2H), 2.32 (s, 3H), 1.89 (q, J=7.2 Hz, 2H),1.37–1.27 (m, 4H),
1.25–1.15 ppm (m, 11H); 13C NMR (100 MHz, CDCl3): d=207.3,
168.6, 166.3, 161.3, 136.9, 129.9, 129.6, 128.7, 128.0, 117.3, 111.3,
46.7, 39.7, 32.7, 29.3, 29.1, 28.8, 27.4, 26.5, 12.0 ppm; ESI-HRMS
calcd for [C24H32N2O3S+H]+: 429.2206 m/z, found: 429.2217 m/z;
HPLC purity: 99.8%.
(E)-2-Mercapto-N-(7-(5-methyl-3-phenylisoxazol-4-yl)hept-6-eny-
l)acetamide (trans-13a): Compound trans-13a (71%) was pre-
pared from trans-25 according to the procedure described for 7a
(see Supporting Information scheme 2 for isolation of trans-25).
1H NMR (400 MHz, CDCl3): d=7.63 (m, 2H), 7.45 (m, 3H), 6.78 (s,
1H), 6.07 (d, J=16.0 Hz, 1H), 5.77 (dt, J=6.8 Hz, 16.0 Hz, 1H),
3.31–3.06 (m, 4H), 2.48 (s, 3H), 2.17 (q, J=7.2 Hz, 2H), 1.84 (t, J=
9.0 Hz, 1H), 1.57 (m, 2H), 1.48–1.34 ppm (m, 4H); 13C NMR
(100 MHz, CDCl3): d=169.7, 165.8, 161.5, 135.1, 129.8, 129.6, 128.8,
128.7, 118.0, 112.8, 40.2, 33.4, 29.4, 29.1, 28.4, 26.6, 12.3 ppm; ESI-
HRMS calcd for [C19H24N2O2S+H]+: 345.1631 m/z, found: 345.1630
m/z; HPLC purity: 97.5%.
(E)-(S)-2-(7-(5-Methyl-3-phenylisoxazol-4-yl)hept-6-enylamino)-2-
oxoethyl 2,2-dimethylpropanethioate (trans-13d): Compound
trans-13d was isolated from 13d by chiral column chromatogra-
1
phy. H NMR (400 MHz, CDCl3): d=7.62 (m, 2H), 7.45 (m, 3H), 6.20
(s, 1H), 6.06 (d, J=16.0 Hz, 1H), 5.76 (dt, J=7.0 Hz, 16.0 Hz, 1H),
3.47 (s, 2H), 3.21 (q, J=6.8 Hz, 2H), 2.48 (s, 3H), 2.15 (q, J=7.2 Hz,
2H), 1.49 (m, 4H), 1.33 (m, 2H), 1.25 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3): d=207.5, 168.7, 165.8, 161.5, 135.1, 129.9, 129.6,
128.8, 128.7, 118.0, 112.8, 46.8, 39.8, 33.5, 32.7, 29.5, 29.1, 27.5, 26.5,
12.3 ppm; ESI-HRMS calcd for [C24H32N2O3S+H]+: 429.2206 m/z,
found: 429.2212 m/z; HPLC purity: 99.4%.
N-[7-(5-Methyl-3-phenylisoxazol-4-yl)hept-6-enyl]-2-mercapto-
propionamide (13b): Compound 13b Z/E 6:4 (18%) was prepared
from compound 12 in three steps according to the methodology
described for 4, 6b, and 7b. 1H NMR (400 MHz, CDCl3): d=7.75–
7.73 (m, 2H), 7.63–7.62 (m, 2H), 7.46–7.41 (m, 6H), 6.47 (br, 1H),
6.35 (br, 1H), 6.10–6.05 (m, 2H), 5.83–5.79 (m, 2H), 3.43–3.32 (m,
2H), 3.28–3.23 (m, 2H), 3.19–3.13 (m, 2H), 2.48 (s, 3H), 2.34 (s, 3H),
2.19–2.13 (m, 2H), 2.00–1.96 (m, 2H), 1.93–1.91 (m, 2H), 1.55–1.52
(m, 6H), 1.43–1.22 ppm (m, 12H); 13C NMR (100 MHz, CDCl3): d=
172.6, 166.1, 165.6, 161.1, 136.7, 134.8, 129.8, 129.4, 128.6, 127.8,
117.8, 117.2, 112.6, 111.1, 39.7, 38.3, 33.2, 29.2, 28.8, 28.5, 26.3, 22.3,
tert-Butyl 5-(5-methyl-3-phenylisoxazol-4-ylmethoxy)pentylcar-
bamate (14): The alcohol 10 (190 mg, 1 mmol), PPh3 (315 mg,
1.2 mmol), and imidazole (88 mg, 1.3 mmol) were dissolved in an-
hydrous CH2Cl2 (5 mL) under Ar atmosphere. I2 (305 mg, 1.2 mmol)
was added in one portion to the flask through a temporarily
opened neck at 08C. The mixture was stirred at room temperature
for 3 h. After evaporating the solvent, the residue was purified by a
short column (SiO2, EtOAc/hexanes 70:30) to remove baseline im-
purities and to obtain the crude intermediate 4-(iodomethyl)-5-
methyl-3-phenylisoxazole. NaH (60 mg, 1.5 mmol) was added in
one portion to a solution of 5-(Boc-amino)-1-pentanol (305 mg,
ChemMedChem 2012, 7, 425 – 439
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435

A. P. Kozikowski et al.
MED
1.5 mmol) in dry DMF (5 mL) at 08C. The mixture was cooled to
ꢀ208C; then a solution of the crude compound 4-(iodomethyl)-5-
methyl-3-phenylisoxazole in anhydrous DMF (5 mL) was added
slowly. The mixture was warmed to room temperature and stirred
pared from compound (S)-15 according to the methodology de-
scribed for 7a. H NMR (400 MHz, CDCl3): d=7.78 (m, 2H), 7.45 (m,
1
3H), 6.51 (s, 1H), 4.32 (s, 2H), 3.48–3.40 (m, 3H), 3.24 (q, J=6.8 Hz,
2H), 2.49 (s, 3H), 1.99 (d, J=8.0 Hz, 1H), 1.64 (m, 2H), 1.52 (m, 5H),
1.40 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=173.2, 169.1,
163.0, 129.9, 129.4, 129.0, 128.5, 110.8, 70.1, 61.7, 40.0, 38.5, 29.5,
29.4, 23.9, 22.5, 11.5 ppm; [a]D20 =ꢀ13.6 (c=0.08, CHCl3); ESI-HRMS
calcd for [C19H26N2O3S+H]+: 363.1737 m/z, found: 363.1728 m/z;
HPLC purity: 96.4%.
overnight. After quenching with
a saturated NH4Cl solution
(10 mL), the mixture was extracted with EtOAc. The organic layer
was washed with H2O and brine and dried over Na2SO4. The sol-
vent was removed in vacuo, and the residue was purified by
column chromatography (SiO2, EtOAc/hexanes 75:25!50:50) to
1
afford the title product 14 (112 mg, 30%) as a colorless oil. H NMR
5-(3-Fluorophenyl)-4-iodo-3-methylisoxazole (17b): n-Butyllithi-
um (9.4 mL, 15 mmol) was slowly added to 1-ethynyl-3-fluoroben-
zene (1.15 mL, 10 mmol) in THF (50 mL) at ꢀ788C. The reaction
mixture was stirred at 08C for 30 min, then cooled to ꢀ788C. ZnCl2
(2 g, 15 mmol) in THF (15 mL) and then acetyl chloride (1.42 mL)
were added. The reaction mixture was stirred at room temperature
for 1 h and quenched with hexanes (10 mL). The organic layer was
washed with brine (15 mL) and dried over anhydrous Na2SO4. Pu-
rification of the crude reaction mixture by column chromatography
(SiO2, 0!20% EtOAc/hexanes) afforded 4-(3-fluorophenyl)-but-3-
yn-2-one (1.6 g, 98%) as a pale-yellow oil. 1H NMR (400 MHz,
CDCl3): d=7.38–7.16 (m, 4H), 2.47 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=184.2, 163.4, 160.9, 130.3, 128.8, 121.6, 119.7, 119.4,
118.2, 118.0, 88.4, 88.2, 32.6 ppm.
(400 MHz, CDCl3): d=7.78–7.75 (m, 2H), 7.46–7.43 (m, 3H), 4.31 (s,
2H), 3.45 (t, J=6.4 Hz, 2H), 3.09–3.08 (m, 2H), 2.48 (s, 3H), 1.63–
1.58 (m, 2H), 1.49–1.33 ppm (m, 13H); 13C NMR (100 MHz, CDCl3):
d=168.8, 167.2, 162.7, 129.5, 129.2, 128.7, 128.3, 110.5, 70.0, 61.5,
40.2, 29.8, 29.2, 28.4, 24.0, 11.2 ppm.
(R)-N-{5-[(5-Methyl-3-phenylisoxazol-4-yl)methoxy]pentyl}-2-(tri-
tylthio)propanamide ((R)-15): Compound (R)-15 was isolated from
a racemic mixture of compound 15 using chiral column chroma-
tography. Compound 15 was prepared from 14 according to the
methodology described for 4 and 6b. 1H NMR (400 MHz, CDCl3):
d=7.79 (m, 2H), 7.47 (m, 9H), 7.25 (m, 9H), 4.33 (s, 2H), 3.45 (t, J=
6.4 Hz, 2H), 2.97 (m, 2H), 2.69 (m, 1H), 2.50 (s, 3H), 1.57 (m, 2H),
1.46 (d, J=7.5 Hz, 3H), 1.29 ppm (m, 4H); 13C NMR (100 MHz,
CDCl3): d=172.2, 168.9, 162.7, 144.3, 129.6, 129.4, 129.3, 128.7,
128.3, 128.1, 127.0, 110.6, 69.9, 68.1, 61.5, 44.6, 39.6, 29.3, 28.9,
23.6, 20.2, 11.3 ppm; [a]D20 = +36.0 (c=0.2, CHCl3).
NH2OH·HCl (1.67 g, 20 mmol) and pyridine (2.83 mL, 35 mmol)
were added to this ketone in CH3OH (30 mL) with Na2SO4 (2.84 g,
20 mmol). The reaction mixture was stirred at reflux for 17 h and
then the volatiles were evaporated. The crude mixture was dis-
solved in EtOAc, washed with H2O, washed with brine, and dried
over anhydrous Na2SO4 to afford the racemic 4-(3-fluorophenyl)-
(S)-N-{5-[(5-Methyl-3-phenylisoxazol-4-yl)methoxy]pentyl}-2-(tri-
tylthio)propanamide ((S)-15): Compound (S)-15 was isolated from
a racemic mixture of compound 15 using chiral column chroma-
tography. Compound 15 was prepared from 14 according to the
methodology described for 4 and 6b. 1H NMR (400 MHz, CDCl3):
d=7.79 (m, 2H), 7.45 (m, 9H), 7.26 (m, 9H), 4.32 (s, 2H), 3.45 (t, J=
6.3 Hz, 2H), 2.97 (m, 2H), 2.72 (m, 1H), 2.49 (s, 3H), 1.58 (m, 2H),
1.45 (d, J=7.5 Hz, 3H), 1.29 ppm (m, 4H); 13C NMR (100 MHz,
CDCl3): d=172.2, 168.8, 162.7, 144.3, 129.6, 129.4, 129.2, 128.7,
128.3, 128.1, 127.0, 110.6, 69.9, 68.1, 61.5, 44.5, 39.6, 29.3, 28.9,
23.6, 20.2, 11.3 ppm; [a]D20 =ꢀ27.0 (c=0.4, CHCl3).
1
but-3-yn-2-one O-methyloxime (1.6 g, 85%) as a yellow oil. H NMR
(400 MHz, CDCl3): d=7.48–7.03 (m, 8H), 3.96 (s, 3H), 3.95 (s, 3H),
2.10 (s, 3H), 2.06 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=163.4,
161.0, 149.8, 130.0, 127.8, 123.7, 118.7, 118.5, 116.6, 116.3, 97.4,
88.6, 62.5, 62.3, 20.9, 20.4 ppm.
To this oxime in CH2Cl2 (60 mL) was added 1m ICl in CH2Cl2
(10.2 mL, 10.2 mmol). The reaction mixture was stirred at room
temperature overnight, and saturated Na2S2O3 was added. The
aqueous layer was extracted with CH2Cl2 (3ꢁ30 mL). The combined
organic layers were dried over anhydrous Na2SO4. Purification of
the crude reaction mixture by preparative HPLC Method A afforded
the title compound (526 mg, 41%) as a yellow oil. 1H NMR
(400 MHz, CDCl3): d=7.81–7.79 (m, 1H), 7.73–7.71 (m, 1H), 7.45–
7.39 (m, 1H), 7.16–7.11 (m, 1H), 2.31 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=165.6, 163.6, 163.0, 161.2, 130.3, 128.8, 122.7,
117.4, 117.2, 114.1, 113.9, 58.6, 12.4 ppm.
2-Mercapto-N-[5-(5-methyl-3-phenylisoxazol-4-ylmethoxy)pen-
tyl]propionamide (16): Compound 16 (32%) was prepared from
compound 14 in three steps according to the methodologies de-
1
scribed for 4, 6b, and 7b. H NMR (400 MHz, CDCl3): d=7.78–7.76
(m, 2H), 7.47–7.44 (m, 3H), 6.45 (br, 1H), 4.32 (s, 2H), 3.46 (t, J=
6.4 Hz, 2H), 3.43–3.35 (m, 1H), 3.26–3.21 (m, 2H), 2.49 (s, 3H), 1.98
(d, J=8.4 Hz, 1H), 1.65–1.60 (m, 2H), 1.56–1.49 (m, 5H), 1.43–
1.37 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=172.7, 168.8,
162.7, 129.6, 129.3, 128.7, 128.3, 110.6, 69.9, 61.5, 39.8, 38.3, 29.3,
29.2, 23.6, 22.3, 11.3 ppm; ESI-HRMS calcd for [C19H26N2O3S+H]+:
363.17369 m/z, found: 363.1740 m/z; HPLC purity: 99.4%.
2-Mercapto-N-[7-(3-methyl-5-phenylisoxazol-4-yl)hept-6-ynyl]-
propionamide (18a): To the 4-iodo-3-methyl-5-phenylisoxazole
17a (1 g, 3.5 mmol) in DMF (16 mL) was added tert-butyl hept-6-
ynylcarbamate (1.48 g, 7 mmol) [generated from 6-heptynenitrile
by reduction of the nitrile with LiAlH4 in Et2O and Boc protection
of the obtained amine], PdCl2(PPh3)2 (245 mg, 0.35 mmol), CuI
(133 mg, 0.7 mmol) and DIEA (16 mL). The reaction mixture was ir-
radiated at 1308C for 20 min. EtOAc (30 mL) was added. The organ-
ic layer was washed with H2O (3ꢁ50 mL) and dried over anhydrous
Na2SO4. Purification of the crude reaction mixture by column chro-
matography (SiO2, 0!100% EtOAc/hexanes) afforded tert-butyl 7-
(3-methyl-5-phenylisoxazol-4-yl)hept-6-ynylcarbamate (0.87 g, 67%)
as a yellow oil. 1H NMR (400 MHz, CDCl3): d=8.12–8.09 (m, 2H),
7.49–7.43 (m, 3H), 4.53 (s, 1H), 3.14–3.13 (m, 2H), 2.51 (t, J=6.4 Hz,
2H), 2.34 (s, 3H), 1.70–1.63 (m, 2H), 1.53–1.49 (m, 4H), 1.43 ppm (s,
9H); 13C NMR (100 MHz, CDCl3): d=167.6, 162.0, 155.9, 130.2,
(R)-2-Mercapto-N-{5-[(5-methyl-3-phenylisoxazol-4-yl)methoxy]-
pentyl}propanamide ((R)-16): Compound (R)-16 (56%) was pre-
pared from compound (R)-15 according to the methodology de-
1
scribed for 7a. H NMR (400 MHz, CDCl3): d=7.77 (m, 2H), 7.45 (m,
3H), 6.55 (s, 1H), 4.32 (s, 2H), 3.48–3.38 (m, 3H), 3.24 (q, J=6.8 Hz,
2H), 2.48 (s, 3H), 1.99 (d, J=8.0 Hz, 1H), 1.63 (m, 2H), 1.51 (m, 5H),
1.40 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=173.3, 169.1,
162.9, 129.9, 129.4, 129.0, 128.5, 110.8, 70.1, 61.7, 40.0, 38.5, 29.4,
29.3, 23.8, 22.4, 11.5 ppm; [a]D20 = +15.0 (c=0.09, CHCl3); ESI-
HRMS calcd for [C19H26N2O3S+H]+: 363.1737 m/z, found: 363.1729
m/z; HPLC purity: 98.7%.
(S)-2-Mercapto-N-{5-[(5-methyl-3-phenylisoxazol-4-yl)methoxy]-
pentyl}propanamide ((S)-16): Compound (S)-16 (56%) was pre-
436
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 425 – 439

Chiral Mercaptoacetamides
128.7, 127.4, 125.9, 99.5, 97.2, 79.1, 70.0, 40.4, 29.6, 28.4, 28.2, 26.0,
19.6, 10.5 ppm.
N-[6-(3-Methyl-5-phenylisoxazol-4-yl)hex-5-ynyl]-2-pivaloylsulfa-
nylacetamide (18e): Compound 18e (16%) was prepared with
tert-butyl hex-5-ynylcarbamate and compound 5d in three steps
according to the methodology described for 18a. 1H NMR
(400 MHz, CDCl3): d=8.10–8.08 (m, 2H), 7.49–7.44 (m, 3H), 6.42 (br,
1H), 3.51 (s, 2H), 3.33–3.28 (m, 2H), 2.52 (t, J=6.8 Hz, 2H), 2.33 (s,
3H), 1.70–1.62 (m, 4H), 1.22 ppm (s, 9H); 13C NMR (100 MHz,
CDCl3): d=207.4, 169.4, 167.7, 162.0, 130.3, 128.7, 127.3, 125.9,
99.4, 96.6, 70.3, 46.5, 39.2, 32.3, 28.5, 27.2, 25.7, 19.3, 10.5 ppm; ESI-
HRMS calcd for [C23H28N2O3S+H]+: 413.18934 m/z, found:
413.1898 m/z; HPLC purity: 99.6%.
The Boc protecting group was removed according to the method-
ology described for 4 to afford 7-(3-methyl-5-phenylisoxazol-4-
1
yl)hept-6-ynylamine TFA salt. H NMR (400 MHz, CD3OD): d=8.09–
8.07 (m, 2H), 7.50–7.48 (m, 3H), 2.95–2.89 (m, 2H), 2.57 (t, J=
6.4 Hz, 2H), 2.30 (s, 3H), 1.75–1.68 (m, 4H), 1.62–1.55 ppm (m, 2H);
13C NMR (100 MHz, CD3OD): d=168.9, 163.3, 161.0, 131.7, 130.0,
128.5, 126.9, 100.6, 98.6, 70.7, 40.6, 29.2, 28.1, 26.8, 20.0, 10.4 ppm.
N-[7-(3-Methyl-5-phenylisoxazol-4-yl)hept-6-ynyl]-2-tritylsulfa-
nylpropionamide (43%) was prepared from the obtained amine
according to the methodology described for 6b. 1H NMR
(400 MHz, CDCl3): d=8.12–8.10 (m, 2H), 7.49–7.43 (m, 9H), 7.29–
7.26 (m, 6H), 7.22–7.18 (m, 3H), 5.99 (br, 1H), 3.05–2.97 (m, 2H),
2.76–2.71 (m, 1H), 2.50–2.46 (m, 2H), 2.33 (s, 3H), 1.63–1.58 (m,
2H), 1.44 (d, J=7.2 Hz, 3H), 1.39–1.33 ppm (m, 4H); 13C NMR
(100 MHz, CDCl3): d=172.0, 167.4, 161.8, 144.1, 130.1, 129.2, 128.5,
127.9, 127.3, 126.8, 125.7, 99.3, 97.0, 69.9, 67.9, 44.3, 39.4, 28.4,
28.0, 25.9, 20.0, 19.4, 10.4 ppm.
tert-Butyl 4-[trans-2-(3-methyl-5-phenylisoxazol-4-yl)vinyl]ben-
zylcarbamate (19): tert-Butyl 4-vinylbenzylcarbamate (327 mg,
1.4 mmol) [generated from Boc-protection of 4-vinylbenzylamine],
Pd(OAc)2 (16 mg, 0.07 mmol), nBu4NCl (194 mg, 0.7 mmol), and
Na2CO3 (148 mg, 1.4 mmol) were added to 4-iodo-3-methyl-5-phe-
nylisoxazole (0.2 g, 0.7 mmol) in DMF (3 mL). The reaction mixture
was irradiated at 1308C for 1 h. EtOAc was added. The organic
layer was washed with H2O (3ꢁ20 mL) and dried over anhydrous
Na2SO4 to afford the title compound (180 mg, 66%) as a brown oil.
1H NMR (400 MHz, CDCl3): d=7.71–7.68 (m, 2H), 7.48–7.43 (m, 3H),
7.40–7.38 (m, 2H), 7.30–7.26 (m, 2H), 6.95 (d, J=16.4 Hz, 1H), 6.85
(d, J=16.4 Hz, 1H), 5.28 (s, 1H), 4.31–4.29 (m, 2H), 2.46 (s, 3H),
1.48 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=165.2, 158.6, 155.8,
138.8, 135.7, 131.3, 129.6, 128.6, 127.8, 127.5, 127.2, 126.2, 116.4,
112.6, 79.1, 44.0, 28.1, 11.8 ppm.
Compound 18a (79%) was prepared from the obtained propiona-
mide according to the methodology described for 7a. 1H NMR
(400 MHz, CDCl3): d=8.07–8.05 (m, 2H), 7.45–7.39 (m, 3H), 6.70 (br,
1H), 3.40–3.33 (m, 1H), 3.26–3.21 (m, 2H), 2.47 (t, J=6.8 Hz, 2H),
2.29 (s, 3H), 1.99 (d, J=8.4 Hz, 1H), 1.67–1.60 (m, 2H), 1.58–
1.47 ppm (m, 7H); 13C NMR (100 MHz, CDCl3): d=172.9, 167.4,
161.8, 130.2, 128.5, 127.1, 125.7, 99.3, 97.1, 69.8, 39.5, 37.9, 28.8,
28.0, 25.9, 22.1, 19.4, 10.3 ppm; ESI-HRMS calcd for [C20H24N2O2S+
H]+: 357.16313 m/z, found: 357.1638 m/z; HPLC purity: 98.9%.
2-Mercapto-N-(4-[trans-2-(3-methyl-5-phenylisoxazol-4-yl)vinyl]-
benzyl)propionamide (20a): Compound 20a (10%) was prepared
from compound 19 in three steps according to the methodologies
described for 4, 6b, and 7b. 1H NMR (400 MHz, CDCl3): d=7.74–
7.72 (m, 2H), 7.52–7.46 (m, 3H), 7.44–7.42 (m, 2H), 7.30–7.26 (m,
2H), 6.99 (d, J=16.8 Hz, 1H), 6.89 (d, J=16.8 Hz, 1H), 6.80 (br, 1H),
4.47–4.45 (m, 2H), 3.51–3.47 (m, 1H), 2.50 (s, 3H), 2.05 (d, J=
8.4 Hz, 1H), 1.60 ppm (d, J=8.4 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=172.7, 165.8, 158.9, 135.7, 136.4, 131.3, 130.0, 128.9, 128.1,
127.5, 126.2, 117.2, 112.8, 43.6, 38.2, 22.2, 12.1 ppm; ESI-HRMS
calcd for [C22H22N2O2S+H]+: 379.14748 m/z, found: 379.1477 m/z;
HPLC purity: 96.9%.
7-[5-(3-Fluorophenyl)-3-methylisoxazol-4-yl]hept-6-ynyl-2-mer-
captopropionamide (18b): Compound 18b (20%) was prepared
from compound 17b in four steps according to the methodology
1
described for 18a. H NMR (400 MHz, CDCl3): d=7.83–7.79 (m, 2H),
7.42–7.36 (m, 1H), 7.11–7.06 (m, 1H), 6.67 (br, 1H), 3.39–3.34 (m,
1H), 3.27–3.22 (m, 2H), 2.48 (t, J=6.8 Hz, 2H), 2.29 (s, 3H), 1.99 (d,
J=8.0 Hz, 1H), 1.68–1.61 (m, 2H), 1.58–1.47 ppm (m, 7H); 13C NMR
(100 MHz, CDCl3): d=172.8, 166.0, 163.7, 161.9, 161.3, 130.3, 129.0,
121.4, 117.1, 116.9, 112.7, 112.5, 100.2, 98.0, 69.6, 39.5, 37.9, 28.8,
28.0, 26.0, 22.1, 19.4, 10.3 ppm; ESI-HRMS calcd for [C20H23FN2O2S+
H]+: 375.15370 m/z, found: 375.1540 m/z; HPLC purity: 99.4%.
2-Mercapto-2-methyl-N-(4-[trans-2-(3-methyl-5-phenylisoxazol-4-
yl)vinyl]benzyl)propionamide (20b): Compound 20b (55%) was
prepared from compound 19 in three steps according to the meth-
odologies described for 4, 6c, and 7c. 1H NMR (400 MHz, CDCl3):
d=7.72–7.69 (m, 2H), 7.50–7.45 (m, 3H), 7.43–7.41 (m, 2H), 7.35
(br, 1H), 7.28–7.26 (m, 2H), 6.97 (d, J=16.4 Hz, 1H), 6.87 (d, J=
16.4 Hz, 1H), 4.45–4.43 (m, 2H), 2.47 (s, 3H), 2.26 (s, 1H), 1.64 ppm
(s, 6H); 13C NMR (100 MHz, CDCl3): d=174.9, 165.4, 158.7, 138.0,
136.0, 131.3, 129.8, 127.8, 127.3, 126.0, 116.8, 112.7, 47.3, 43.5, 30.2,
11.9 ppm; ESI-HRMS calcd for [C23H24N2O2S+H]+: 393.16313 m/z,
found: 393.1624 m/z; HPLC purity: 99.0%.
2-Mercapto-2-methyl-N-[7-(3-methyl-5-phenylisoxazol-4-yl)hept-
6-ynyl]propionamide (18c): Compound 18c (24%) was prepared
using compound 5c in four steps according to the methodology
1
described for 18a. H NMR (400 MHz, CDCl3): d=8.12–8.10 (m, 2H),
7.49–7.43 (m, 3H), 6.96 (br, 1H), 3.29–3.24 (m, 2H), 2.52 (t, J=
6.8 Hz, 2H), 2.34 (s, 3H), 2.11 (s, 1H), 1.71–1.66 (m, 2H), 1.62–
1.51 ppm (m, 10H); 13C NMR (100 MHz, CDCl3): d=175.0, 167.6,
162.0, 130.3, 128.7, 127.4, 125.9, 99.5, 97.1, 70.1, 47.8, 39.9, 30.4,
29.0, 28.2, 26.1, 19.6, 10.5 ppm; ESI-HRMS calcd for [C21H26N2O2S+
H]+: 371.17878 m/z, found: 371.1784 m/z; HPLC purity: 96.2%.
HDAC isoform inhibition
2-Mercapto-2-methyl-N-[6-(3-methyl-5-phenylisoxazol-4-yl)hex-
5-ynyl]propionamide (18d): Compound 18d (30%) was prepared
with tert-butyl hex-5-ynylcarbamate and compound 5c in four
steps according to the methodology described for 18a. 1H NMR
(400 MHz, CDCl3): d=8.07–8.05 (m, 2H), 7.45–7.37 (m, 3H), 7.02 (br,
1H), 3.31–3.26 (m, 2H), 2.52 (t, J=6.8 Hz, 2H), 2.30 (s, 3H), 2.15 (s,
1H), 1.70–1.64 (m, 4H), 1.55 ppm (s, 6H); 13C NMR (100 MHz,
CDCl3): d=175.0, 167.5, 161.8, 130.2, 128.6, 127.2, 125.8, 99.3, 96.7,
70.2, 47.5, 39.3, 30.2, 28.6, 25.8, 19.2, 10.4 ppm; ESI-HRMS calcd for
[C20H24N2O2S+H]+: 357.16313 m/z, found: 357.1636 m/z; HPLC
purity: 98.3%.
HDAC inhibition assays were performed by the Reaction Biology
Corporation (Malvern, PA) using human full-length recombinant
HDAC1, 2, 5, and 6 isolated from a baculovirus expression system
in Sf9 cells. A complex of human full-length recombinant HDAC3
co-expressed with human NCOR2 was used in the HDAC3 assays,
and residues 627–1085 of human recombinant HDAC4 were used
for the HDAC4 assays. An acetylated, fluorogenic peptide derived
from residues 379–382 of p53 (RHKKAc) was used as the substrate
in the assays. The reaction buffer contained 50 mm Tris·HCl pH 8.0,
137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, 1 mgmLꢀ1 BSA, and a final
ChemMedChem 2012, 7, 425 – 439
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437

A. P. Kozikowski et al.
MED
concentration of 1% DMSO. The enzyme was delivered into wells
of the reaction plate and compounds were delivered in 100%
DMSO into the enzyme mixture by Acoustic technology (Echo550
instrument; nanoliter range). The plates were spun down and pre-
incubated for 5–10 min. The substrate was then delivered to all re-
action wells to initiate the reaction, which was incubated for 2 h at
308C. After incubation, developer and TSA were added to quench
the reaction and generate fluorescence. Kinetic measurements
were then taken for 1.5 h at 15-min intervals to ensure that devel-
opment was complete. Endpoint readings were taken for analysis
after the development reached a plateau. Dose–response curves
were generated, and the IC50 value for each compound was ex-
trapolated from the generated plots (ten-dose IC50 curves were
generated using a threefold serial dilution pattern starting at
30 mm).
ion and sulfur atom. The best complexes were minimized keeping
all atoms outside of a 15 ꢂ radius around the bound substrate
fixed to favor the mutual adaptability between the ligand and
enzyme. The optimized complexes were then used to re-calculate
the AutoDock docking scores and VEGA energy scores.
Mercaptoacetamide dimerization
These experiments were performed on a Shimadzu LC–MS 2010EV
Liquid Chromatograph Mass Spectrometer. Electrospray ionization
was used in positive mode with a scan range of 300–800 m/z with
scans occurring every 0.5 s. A Halo C8 3.0ꢁ30 mm column with
2.7 mm particle size was used at a flow rate of 0.2 mLminꢀ1; gradi-
ent: 25% CH3CN/H2O to 100% CH3CN, 8 min; 100% CH3CN, 2 min;
100% CH3CN to 25% CH3CN/H2O, 0.1 min; 25% CH3CN/H2O,
49.9 min. LC–MS solvents were purchased from commercial sour-
ces and each contained 0.1% formic acid. Each compound
(100 mg) was dissolved in 1 mL CH3OH, and 1 mL aliquots were ana-
lyzed every hour for 24 h. Normalized peaks corresponding to the
[M+H]+ and [M+Na]+ ions for both the monomer and dimer
were extracted from the total ion chromatogram (TIC), and the
area under each respective peak in the extracted ion chromato-
grams (XIC) was calculated using the automatic integration feature
available in Shimadzu’s LCMS Solutions software (Columbia, MD,
USA). The cutoff for peak width was set at 20 s, and the rest of the
parameters were left at default settings. The ratio of total dimer to
total monomer was calculated by adding the areas of the [M+H]+
and [M+Na]+ peaks for each respectively and calculating the quo-
tient. The ratio was then plotted against time for each compound.
Molecular modeling
The two enantiomers (R)-7b and (S)-7b were simulated consider-
ing the thiol function in its anionic form. The conformational be-
havior of these compounds was investigated using a Monte Carlo
procedure (as implemented in the VEGA suite of programs, http://
www.vegazz.net/), which generated 1000 conformers by randomly
rotating the rotors. All geometries so obtained were stored and
optimized to avoid high-energy rotamers. The 1000 conformers
were clustered according to their similarity in order to discard re-
dundancies; in this analysis, two geometries were considered non-
redundant when they differed by >608 in at least one torsion
angle. For each ligand, the so obtained lowest-energy structure
was then exploited in the following docking simulations.
As mentioned above, the resolved structures of HDAC2 and
HDAC4 were retrieved from the RCSB PDB, while the homology
model of the HDAC6 subtype had been recently generated by
us.[14] The experimental structures were completed by adding hy-
drogen atoms; the side chains of Arg, Lys, Glu, and Asp were ion-
ized to remain compatible with physiological pH values, while His
residues were considered neutral by default apart from those be-
longing to cited dyads, which are reported in Supporting Informa-
tion table 1. In particular, in all docking simulations, only the first
histidine residue (His145, His158, and His610 for HDAC2, 4, and 6,
respectively) was protonated. In all simulated HDAC structures, the
second neutral histidine (His146, His159, and His611 for HDAC2, 4,
and 6, respectively) was considered in its Ne tautomeric form in
order to minimize steric hindrance within the catalytic pocket. The
structures so obtained were minimized while keeping the back-
bone fixed to preserve the experimental folding for HDAC2 and 4
and the predicted folding of HDAC6.
Neuroprotection and glutathione depletion
For the neuroprotection studies, cells were rinsed with warm PBS
and then placed in minimum essential medium (Invitrogen) con-
taining 5.5 gLꢀ1 glucose, 10% fetal calf serum, 2 mm l-glutamine,
and 100 mm cysteine. Oxidative stress was induced by the addition
of the cysteine homologue, homocysteine (HCA; 5 mm), to the
media. HCA was diluted from 100-fold concentrated solutions that
were adjusted to pH 7.5. In combination with HCA, the novel
HDAC inhibitors (10 mm) were added. Viability was assessed after
48 h by calcein-acetoxymethyl ester (AM)/ethidium homodimer-1
staining (live/dead assay; Molecular Probes, Eugene, OR, USA)
using fluorescence microscopy and the MTT assay (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method.
Total intracellular glutathione (GSH+GSSG) measurements of pri-
mary neuron cultures were determined with the GSH-Glo Gluta-
thione Assay kit (Promega) according to the manufacturer’s proto-
col. Primary cortical neurons were plated at 1ꢁ105 cells per well in
a poly-d-lysine-coated 96-well plate. After incubation for 24 h, neu-
rons were exposed to HCA in the presence or absence of drug for
8 h. Neurons were lysed with DTT containing lysis buffer to convert
any GSSG to GSH, allowing the determination of total GSH. The ly-
sates were incubated with a reaction buffer containing the luciferin
precursor luciferin-NT and the enzyme GST. In the presence of GSH,
GST converts luciferin-NT into luciferin. A subsequent reaction
buffer containing the enzyme luciferase, which produces light as a
by-product of luciferin metabolism, was added to the lysates, and
the light intensity (which correlates with GSH levels) was measured
with a luminometer (LMax II 384, Molecular Devices). The values
were normalized to protein concentration as determined by a
Bradford protein assay. GSH standards were used to calculate GSH
concentrations.
Docking simulations were performed by GriDock, a parallel tool
based on the AutoDock 4.0 engine.[37] In detail, the grid box was
set to include all residues within a 15 ꢂ radius around the catalytic
metal ion, thus comprising the entire catalytic cavity. The resolu-
tion of the grid was 60ꢁ60ꢁ60 points with a grid spacing of
0.450 ꢂ. Each inhibitor was docked into this grid with the Lamarck-
ian algorithm as implemented in AutoDock. For the docking simu-
lations, the flexible bonds of the ligand were automatically recog-
nized by GriDock and left free to rotate. The genetic-based algo-
rithm ran 20 simulations per substrate with 2ꢁ106 energy evalua-
tions and a maximum number of generations set to 27000. The
crossover rate was increased to 0.8, and the number of individuals
in each population to 150. All other parameters were left at the
AutoDock default settings. The docking results were ranked con-
sidering both AutoDock scores and the distance between the zinc
438
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 425 – 439

Chiral Mercaptoacetamides
[19] D. Kim, C. L. Frank, M. M. Dobbin, R. K. Tsunemoto, W. Tu, P. L. Peng, J. S.
Guan, B. H. Lee, L. Y. Moy, P. Giusti, N. Broodie, R. Mazitschek, I. Delalle,
S. J. Haggarty, R. L. Neve, Y. Lu, L. H. Tsai, Neuron 2008, 60, 803–817.
[20] M. A. Rivieccio, C. Brochier, D. E. Willis, B. A. Walker, M. A. D’Annibale, K.
McLaughlin, A. Siddiq, A. P. Kozikowski, S. R. Jaffrey, J. L. Twiss, R. R.
Ratan, B. Langley, Proc. Natl. Acad. Sci. USA 2009, 106, 19599–19604.
[21] N. Suzuki, T. Suzuki, Y. Ota, T. Nakano, M. Kurihara, H. Okuda, T. Yamori,
H. Tsumoto, H. Nakagawa, N. Miyata, J. Med. Chem. 2009, 52, 2909–
2922.
Acknowledgements
The authors thank Dr. Brett Langley and Kathryn McLaughlin at
Burke Medical Research Institute for performing the neuroprotec-
tion and glutathione depletion studies. The authors also thank
the Alzheimer’s Drug Discovery Foundation (grant no. 271210)
for supporting this work.
[22] S. K. Anandan, J. S. Ward, R. D. Brokx, M. R. Bray, D. V. Patel, X. X. Xiao,
Bioorg. Med. Chem. Lett. 2005, 15, 1969–1972.
[23] J. E. Payne, C. Bonnefous, C. A. Hassig, K. T. Symons, X. Guo, P. M.
Nguyen, T. Annable, P. L. Wash, T. Z. Hoffman, T. S. Rao, A. K. Shiau, J. W.
Malecha, S. A. Noble, J. H. Hager, N. D. Smith, Bioorg. Med. Chem. Lett.
2008, 18, 6093–6096.
[24] R. Furumai, A. Matsuyama, N. Kobashi, K. H. Lee, M. Nishiyama, H. Naka-
jima, A. Tanaka, Y. Komatsu, N. Nishino, M. Yoshida, S. Horinouchi,
Cancer Res. 2002, 62, 4916–4921.
[25] Y. M. Cui, J. Y. Li, L. L. Chen, J. Li, Q. Z. Ye, F. J. Nan, Bioorg. Med. Chem.
2004, 12, 2853–2861.
[26] C. Yan, Z. Xiu, X. Li, S. Li, C. Hao, H. Teng, Proteins 2008, 73, 134–149.
[27] C. A. Hassig, K. T. Symons, X. Guo, P. M. Nguyen, T. Annable, P. L. Wash,
J. E. Payne, D. A. Jenkins, C. Bonnefous, C. Trotter, Y. Wang, J. V. Anzola,
E. L. Milkova, T. Z. Hoffman, S. J. Dozier, B. M. Wiley, A. Saven, J. W. Male-
cha, R. L. Davis, J. Muhammad, A. K. Shiau, S. A. Noble, T. S. Rao, N. D.
Smith, J. H. Hager, Mol. Cancer Ther. 2008, 7, 1054–1065.
[28] P. Bertrand, Eur. J. Med. Chem. 2010, 45, 2095–2116.
[29] R. Wu, Z. Lu, Z. Cao, Y. Zhang, J. Am. Chem. Soc. 2011, 133, 6110–6113.
[30] C. d’Ydewalle, J. Krishnan, D. M. Chiheb, P. Van Damme, J. Irobi, A. P. Ko-
zikowski, P. V. Berghe, V. Timmerman, W. Robberecht, L. Van Den Bosch,
Nat. Med. 2011, 17, 968–974.
Keywords: dimerization
deacetylases · mercaptoacetamides · neurological agents
·
enantioselectivity
·
histone
[1] M. Dokmanovic, C. Clarke, P. A. Marks, Mol. Cancer Res. 2007, 5, 981–
989.
[2] J. Wu, M. Grunstein, Trends Biochem. Sci. 2000, 25, 619–623.
[3] E. Verdin, F. Dequiedt, H. G. Kasler, Trends Genet. 2003, 19, 286–293.
[4] R. B. Parmigiani, W. S. Xu, G. Venta-Perez, H. Erdjument-Bromage, M.
Yaneva, P. Tempst, P. A. Marks, Proc. Natl. Acad. Sci. USA 2008, 105,
9633–9638.
[5] J. J. Kovacs, P. J. Murphy, S. Gaillard, X. Zhao, J. T. Wu, C. V. Nicchitta, M.
Yoshida, D. O. Toft, W. B. Pratt, T. P. Yao, Mol. Cell 2005, 18, 601–607.
[6] X. Wu, P. S. Chen, S. Dallas, B. Wilson, M. L. Block, C. C. Wang, H. Kinya-
mu, N. Lu, X. Gao, Y. Leng, D. M. Chuang, W. Zhang, R. B. Lu, J. S. Hong,
Int. J. Neuropsychopharmacol. 2008, 11, 1123–1134.
[7] J. P. Dompierre, J. D. Godin, B. C. Charrin, F. P. Cordelieres, S. J. King, S.
Humbert, F. Saudou, J. Neurosci. 2007, 27, 3571–3583.
[8] K. Ververis, T. C. Karagiannis, Am. J. Transl. Res. 2011, 3, 454–467.
[9] Y. Zhang, S. Kwon, T. Yamaguchi, F. Cubizolles, S. Rousseaux, M. Kneissel,
C. Cao, N. Li, H. L. Cheng, K. Chua, D. Lombard, A. Mizeracki, G. Matthias,
F. W. Alt, S. Khochbin, P. Matthias, Mol. Cell Biol. 2008, 28, 1688–1701.
[10] T. Suzuki, N. Miyata, Curr. Med. Chem. 2005, 12, 2867–2880.
[11] Y. Kawaguchi, J. J. Kovacs, A. McLaurin, J. M. Vance, A. Ito, T. P. Yao, Cell
2003, 115, 727–738.
[31] A. G. Kazantsev, L. M. Thompson, Nat. Rev. Drug Discovery 2008, 7, 854–
868.
[32] Y. Ying, K. Taori, H. Kim, J. Hong, H. Luesch, J. Am. Chem. Soc. 2008, 130,
8455–8459.
[33] Y. Liu, L. A. Salvador, S. Byeon, Y. Ying, J. C. Kwan, B. K. Law, J. Hong, H.
Luesch, J. Pharmacol. Exp. Ther. 2010, 335, 351–361.
[34] A. Bowers, N. West, J. Taunton, S. L. Schreiber, J. E. Bradner, R. M. Wil-
liams, J. Am. Chem. Soc. 2008, 130, 11219–11222.
[35] W. Chen, R. Hartman, R. Ayer, S. Marcantonio, J. Kamper, J. Tang, J. H.
Zhang, J. Neurochem. 2009, 111, 726–736.
[36] W. K. Kelly, P. A. Marks, Nat. Clin. Pract. Oncol. 2005, 2, 150–157.
[37] G. Vistoli, A. Pedretti, A. Mazzolari, B. Testa, J. Comput. Aided Mol. Des.
2010, 24, 771–787.
[12] K. V. Butler, A. P. Kozikowski, Curr. Pharm. Des. 2008, 14, 505–528.
[13] J. E. Bradner, N. West, M. L. Grachan, E. F. Greenberg, S. J. Haggarty, T.
Warnow, R. Mazitschek, Nat. Chem. Biol. 2010, 6, 238–243.
[14] K. V. Butler, J. Kalin, C. Brochier, G. Vistoli, B. Langley, A. P. Kozikowski, J.
Am. Chem. Soc. 2010, 132, 10842–10846.
[15] A. P. Kozikowski, Y. Chen, A. Gaysin, B. Chen, M. A. D’Annibale, C. M.
Suto, B. C. Langley, J. Med. Chem. 2007, 50, 3054–3061.
[16] R. Konsoula, M. Jung, Int. J. Pharm. 2008, 361, 19–25.
[17] M. Whittaker, C. D. Floyd, P. Brown, A. J. Gearing, Chem. Rev. 1999, 99,
2735–2776.
[18] W. K. Kelly, O. A. O’Connor, L. M. Krug, J. H. Chiao, M. Heaney, T. Curley,
B. MacGregore-Cortelli, W. Tong, J. P. Secrist, L. Schwartz, S. Richardson,
E. Chu, S. Olgac, P. A. Marks, H. Scher, V. M. Richon, J. Clin. Oncol. 2005,
23, 3923–3931.
Received: November 11, 2011
Revised: December 9, 2011
Published online on January 10, 2012
ChemMedChem 2012, 7, 425 – 439
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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