Matrix metalloproteinase inhibitors based on the 3-mercaptopyrrolidine core

Article abstract of DOI:10.1021/jm400529f

New series of pyrrolidine mercaptosulfide, 2-mercaptocyclopentane arylsulfonamide, and 3-mercapto-4-arylsulfonamidopyrrolidine matrix metalloproteinase inhibitors (MMPIs) were designed, synthesized, and evaluated. Exhibiting unique properties over other MMPIs (e.g., hydroxamates), these newly reported compounds are capable of modulating activities of several MMPs in the low nanomolar range, including MMP-2 (～2 to 50 nM), MMP-13 (～2 to 50 nM), and MMP-14 (～4 to 60 nM). Additionally these compounds are selective to intermediate- and deep-pocket MMPs but not shallow-pocketed MMPs (e.g., MMP-1, ～850 to >50 000 nM; MMP-7, ～4000 to >25 000 nM). Our previous work with the mercaptosulfide functionality attached to both cyclopentane and pyrrolidine frameworks demonstrated that the cis-(3S,4R)-stereochemistry was optimal for all of the MMPs tested. However, in our newest compounds an interesting shift of preference to the trans form of the mercaptosulfonamides was observed with increased oxidative stability and biological compatibility. We also report several kinetic and biological characteristics showing that these compounds may be used to probe the mechanistic activities of MMPs in disease.

Full text of DOI:10.1021/jm400529f

Article
pubs.acs.org/jmc
Matrix Metalloproteinase Inhibitors Based on the
3‑Mercaptopyrrolidine Core
Yonghao Jin,† Mark D. Roycik,† Dale B. Bosco, Qiang Cao, Manuel H. Constantino, Martin A. Schwartz,
and Qing-Xiang Amy Sang*
Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida,
United States
ABSTRACT: New series of pyrrolidine mercaptosulfide, 2-mercaptocyclopentane arylsulfonamide, and 3-mercapto-
4-arylsulfonamidopyrrolidine matrix metalloproteinase inhibitors (MMPIs) were designed, synthesized, and
evaluated. Exhibiting unique properties over other MMPIs (e.g., hydroxamates), these newly reported compounds
are capable of modulating activities of several MMPs in the low nanomolar range, including MMP-2 (∼2 to 50 nM),
MMP-13 (∼2 to 50 nM), and MMP-14 (∼4 to 60 nM). Additionally these compounds are selective to intermediate-
and deep-pocket MMPs but not shallow-pocketed MMPs (e.g., MMP-1, ∼850 to >50 000 nM; MMP-7, ∼4000 to >25 000 nM).
Our previous work with the mercaptosulfide functionality attached to both cyclopentane and pyrrolidine frameworks
demonstrated that the cis-(3S,4R)-stereochemistry was optimal for all of the MMPs tested. However, in our newest compounds
an interesting shift of preference to the trans form of the mercaptosulfonamides was observed with increased oxidative stability
and biological compatibility. We also report several kinetic and biological characteristics showing that these compounds may be
used to probe the mechanistic activities of MMPs in disease.
affinities for each of the MMPs, they were initially proposed
as the ideal inhibitors to block the pathological activities of
MMPs.9 However, TIMPs are essentially broad-spectrum
inhibitors, lacking selectivity for individual MMPs, and
themselves are involved in various non-MMP related
physiological processes (e.g., apoptosis).10−13 This led to a
decade’s endeavor to develop small molecule MMP inhibitors
(MMPIs). Generally MMPIs mimic substrate peptide struc-
tures, incorporating a noncleavable zinc-binding group in place
of the scissile bond, and thus presumably mimic substrate
binding with the enzyme active site. Since the catalytic domains
of all MMPs present a high degree of homology,14 the
specificity and selectivity of inhibition by these early
compounds were minimal, with numerous off-target effects
and binding promiscuity observed.15
INTRODUCTION
■
Matrix metalloproteinases (MMPs) are a family of metzincin
endopeptidases chiefly responsible for remodeling the extrac-
ellular matrix (ECM). Further division of the family on the
basis of domain arrangement and substrate specificity results in
six subgroups: collagenases (MMP-1, -8, -13, and -18);
gelatinases (MMP-2 and -9); stromelysins (MMP-3, -10, and
-11); matrilysins (MMP-7 and -26); membrane-type MMPs
(MMP-14, -15, -16, -17, -24, and -25); and others (MMP-12,
-19, -20, -21, -23, -27, and -28). Members of this family also
process nonmatrix and cell surface signaling proteins to serve
important roles in a number of physiological (e.g., epithelial
morphogenesis, neurogenesis) and pathological processes (e.g.,
cancer, inflammation, cardiovascular disease).1−3 In light of
these pathological roles and in the pursuance of developing
targeted treatments, biological and pharmacological regulation
of MMPs continues to be extensively studied. Targeting
particular MMPs, however, requires careful consideration and
places a premium on selectivity so that side effects are
minimized. For example in ischemic events (e.g., stroke),
proteolytic activities of MMP-2 and -9 result in initial blood−
brain barrier disruption,4 while during later stages MMP-9 may
exhibit neuroprotective effects.5 During atherosclerosis MMP-1
and -8 are suggested to partake in plaque destabilization,6,7
while MMPs -2 and -9 facilitate smooth muscle cell migration
and overall plaque stability.8 Hence, targeting specific MMPs is
disease dependent.
Characterized by our group and others,16,17 the principal
specificity pocket, designated the S1′ pocket, is a hydrophobic
cavity positioned to the right (i.e., prime side) of the catalytic
zinc. Critical to the shape of this pocket is the identity of one
key residue in the catalytic domain’s second helix that
determines the pocket’s relative depth.18 The S1′ pocket thus
may be shallow (MMP-1 and -7), intermediate (MMP-2, -8, -9,
and -26), or deep (MMP-3, -12, and -14) and offers a degree of
individuality to MMPs that can be exploited to ascertain
specificity.16,17
The nature of the zinc-binding group (ZBG) is equally
important to both potency and specificity of the MMPI.
Numerous ZBGs have been utilized, including the widely
incorporated hydroxamates and the lesser used carboxylates,
phosphinyls, and mercaptans.19 Hydroxamate is the most
The activity of MMPs is controlled through several
mechanisms: gene expression; compartmentalization; zymogen
activation; and important to this discussion, enzyme inhibition.
Endogenously, the four tissue inhibitors of metalloproteinases
(TIMP-1, -2, -3, and -4) are primarily responsible for MMP
inhibition. Since TIMPs exhibit picomolar to nanomolar
Received: January 8, 2013
© XXXX American Chemical Society
A
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potent ZBG, coordinating Zn2+ in a bidentate fashion to form a
complex with distorted trigonal bipyramidal geometry.
However, the hydroxamate affinity for Zn2+ seems to
overwhelm specific protease binding contributions by other
groups within the structure. Consequently, the selectivity of
hydroxamate-based MMPIs was reduced and resulted in side
effects such as tendinitis fibromyalgia and musculoskeletal
abnormalities in oncology clinical trials.20,21 These results
stimulated our own interest in developing MMPIs with
alternative ZBGs that offer adequate levels of binding without
sacrificing specificity.
The popularity of thiol-based angiotensin-converting enzyme
inhibitors (e.g., (2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl]-
pyrrolidine-2-carboxylic acid)22 and the effectiveness of thiols
(-SH) as metal binding ligands, despite some limitations,23 led
us to investigate MMPIs containing the mercaptan ZBG. The
earliest of our compounds were peptidomimetic mercaptosul-
fides; however, these MMPIs were readily deactivated by
hydrolysis and by oxidation of the thiol.24−26 A second
generation of mercaptosulfides attached to a cyclopentane or
pyrrolidine core was produced.27−29 Incorporation of the 1,2-
mercaptosulfide moiety into a ring system was designed from
structure-based computer modeling using MacroModel (ver-
sion 7) and chemical intuition to increase binding affinity by
restricting rotation of the C−C bond connected to the two
sulfurs, leading to development of the 1,2-cyclopentanemer-
captosulfide moiety as a new pharmacophore. These novel
inhibitors with a cyclopentane ring showed moderately
improved inhibitory activities and significantly higher oxidation
stability. Further modification of the ring from cyclopentane to
pyrrolidine was carried out to enhance water solubility and
allow for a greater degree of functionality (i.e., so that
additional side chains that interact with the nonprime side of
the enzyme active site may be appended) (Figure 1).28,30 This
characteristics to show how these compounds may be used for
probing the mechanistic activities of MMPs in disease.
RESULTS
■
Structure−Activity Relationship Studies. Mercaptosul-
fide Inhibitors. The synthesis of mercaptosulfide inhibitors with
a pyrrolidine pharmacophore is outlined in Scheme 1. Pyrroline
(1) was Boc protected (2) and oxidized with m-CPBA to give
the pyrrolidine epoxide (3) that was treated with thiolbenzoic
acid over alumina to afford the benzoylthioalcohol intermediate
(4). The benzoyl group of 4 was removed with EtONa in
EtOH and t-Boc protecting group was introduced in one pot,
affording compound 5, which was converted into the
differentially protected cis-dithio compound (6) via the
Mitsunobu reaction.34 The acetyl group of intermediate 6
was selectively removed with MeNH2 in MeOH, and the
resulting thiol was coupled with (S)-2-bromo-4-methylpenta-
noic acid to afford the S-alkylated acid intermediate (7). The
acid (7) was coupled with phenylalanine N-methylamide to
give the key intermediate 8. N-Boc group of 8 was selectively
removed in HCl ethyl acetate solution (9) and coupled with
carboxylic acid to afford intermediate 10 that was treated with
HCl in acetic acid to give the mercaptosulfide MMPIs (11). A
series of pyrrolidine mercaptosulfides with varying N-
substituents was initially synthesized to evaluate the ability of
the P residue (Table 1, Figure 2) to confer potency and
selectivity.33 These compounds were tested against MMP-1, -2,
-3, -7, -9, and -14. As the size of the P residue increased (11b vs
11c−e), potency against the enzymes was generally increased
but no substantial changes in selectivity were observed.
Notably, potency against MMP-3 (stromelysin 1) was
dramatically increased as the chain of the N-phthalimidoacyl
group (11c−11e) was lengthened.
Cyclopentane Mercaptosulfonamide Inhibitors. To en-
hance the overall biological stability of these compounds, the P′
residue of the 3-mercaptopyrrolidine inhibitors was changed
from a peptidomimetic alkylsulfide group to an arylsulfona-
mide.35,36 This modification was intended to enhance enzyme−
inhibitor binding through hydrogen bonding to the enzyme
backbone and guide the hydrophobic substituent into the S1′
pocket.37 On the nonprime side of the compounds, a
cyclopentane scaffold was first employed to determine effective
aryl substituents and the stereochemical requirements for
inhibition in this new class of compounds.
Figure 1. Representation of the pyrrolidine scaffold to functionalize
the MMPIs for interaction with the nonprime side of the enzyme
active site (P).
structural change improved MMPI stability, leading to
enhanced bioavailability and increased potency of inhib-
ition.17,31,32 A final modification of the peptidomimetic part
to a mercaptosulfonamide, again attached to a cyclopentane or
pyrrolidine core, led to further increases in ZBG stability and
inhibitor potency and selectivity.33
An obvious factor determining the effectiveness of these
MMPIs, in addition to the nature of the P and P′ residues, is
the relative and absolute stereochemistry at C-3 and C-4
needed to maximize MMP binding. Initial work with the
mercaptosulfide functionality26 attached to both cyclopentane
and pyrrolidine frameworks demonstrated that the cis-(3S,4R)-
stereochemistry was optimal for all of the MMPs tested.17,31,32
However, in the mercaptosulfonamides the trans stereoisomer
turned out to be the most potent, with little preference between
absolute configurations.
The stereoisomeric 2-mercaptocyclopentane arylsulfona-
mides were synthesized from trans-2-azidocyclopentanol as
outlined in Scheme 2. Kinetic resolution of racemic trans-2-
azidocyclopentanol 12 using lipase Amano AK-20 yielded
(1S,2S) 13 and acetylated (1R,2R)-14 that was treated with
based to give (1R,2R)-15 as described similarly by Ami and
Ohrui.38 Each of these chiral trans-2-azidocyclopentanols was
reduced to amino alcohol, and then the amino group was
protected with Boc to afford 16. Compound 16 was converted
through Mitsunobu reaction34 into compound 17, which was
first treated with HCl in MeOH to remove both protecting
groups and then with arylsulfonyl chloride in DCM to give
chiral cis-mercaptosulfonamide 18. For the synthesis of chiral
trans-mercaptosulfonamide, chiral trans-2-azidocyclopentanol
(12) was converted to chiral cis-2-azidocyclopentanol (20)
through Mitsunobu reaction and hydrolysis. The azide group of
compound 20 was reduced to amine and subsequently treated
with arylsulfonyl chloride to give intermediate 21. The hydroxyl
group of 21 was converted to acetylthio group through
We have produced an array of mercaptosulfonamide MMPIs
capable of modulating the activity of numerous MMPs in the
low nanomolar range and report several kinetic and biological
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a
Scheme 1. Synthesis of Pyrrolidine Mercaptosulfide Inhibitors
a
Reagents and conditions: (a) Boc2O, CH2Cl2, 0 °C; (b) m-CPBA, CH2Cl2, 0 °C to rt; (c) Al2O3, PhCOSH, Et2O, rt; (d) EtONa/EtOH, Boc2O, 0
°C; (e) Ph3P, DEAD, AcSH, THF, 0 °C to rt; (f) (2S)-2-bromo-4-methylpentanoic acid, K2CO3, DMF, rt; (g) L-Phe-NHMe, EDCI, HOBt, Et3N,
CH2Cl2, 0 °C to rt; (h) HCl−EtOAc; (i) R1CO2H, DCC, HOBt, Et3N, CH2Cl2, 0 °C to rt; (j) HCl/AcOH, rt.
Mitsunobu reaction to give compound 22, which was treated
with HCl in MeOH to give the desired chiral trans-
mercaptosulfonamides (23). Compound 23f was obtained
directly from trans-azidocyclopentanol through azide reduction
and amine sulfonation.
With this new set of inhibitors (Table 2, Figure 3),
investigations into the effect of P′ aryl group substitution
demonstrated that the 4-phenoxyphenyl (23c) maximized
inhibition of intermediate (MMP-2 and -9) and deep
(MMP-13 and -14) S1′ pocket MMPs when compared to
those incorporating a phenyl (23a) or biphenyl (23b) group. In
contrast to the preferred cis stereochemistry observed with our
Table 1. Effect of N-Substituent on MMP Inhibition by
Pyrrolidine Mercaptosulfides
Kiapp (nM)
a
compd
MMP-1 MMP-2 MMP-3 MMP-7 MMP-9 MMP-14
11a
11b
11c
11d
11e
260
99
200
14
4100
990
300
31
230
91
50
12
11
5.3
5.7
4.9
3.9
0.98
310
6.2
70
110
75
17
8.5
1.7
6.0
7.0
52
1.9
a
Each inhibitor was a 1:1 mixture of cis-(3R,4S) and cis-(3S,4R)
diastereomers.
Figure 2. Structural representation of compounds 11a−e presented in Table 1.
C
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a
Scheme 2. Stereospecific Synthesis of 2-Mercaptocyclopentane Arylsulfonamides
a
Reagents and conditions: (a) Amano AK-20 lipase, isopropenyl acetate, tert-butyl methyl ether, rt; (b) LiOH, THF−MeOH−H2O, rt; (c) (1) PPh3,
THF−H2O, heat; (2) NaHCO3, Boc2O, 0 °C to rt; (d) 4-nitrobenzoic acid, PPh3, DEAD, THF, 0 °C to rt; (e) AcSH, PPh3, DEAD, THF, 0 °C to
rt; (f) (1) 2 N HCl in MeOH; (2) ArSO2Cl, Et3N, CH2Cl2, 0 °C to rt; (g) LiOH, THF−MeOH−H2O, rt; (h) (1) PPh3, THF−H20, heat; (2)
NaHCO3, ArSO2Cl, 0 °C to rt; (i) AcSH, PPh3, DEAD, THF, 0 °C to rt; (j) MeNH2, MeOH, rt.
Table 2. MMP Inhibition by 2-Mercaptocyclopentane Arylsulfonamides
Kiapp (nM)
compd
MMP-1
MMP-2
810
∼2.5 × 104
430
MMP-3
MMP-7
MMP-9
970
MMP-13
200
>1.0 × 105
160
MMP-14
18a
18b
18c
23a
23b
23c
23d
23e
23f
>1.0 × 105
>2.0 × 105
>2.0 × 105
>2.0 × 105
>2.0 × 104
>2.0 × 105
>1.0 × 105
>5.0 × 104
410
∼2.5 × 104
68
>1600
280
>2.0 × 104
∼1.0 × 104
3400
7200
240
>2.0 × 105
∼1.0 × 104
>2.0 × 105
>2.0 × 105
>1.0 × 105
>2.0 × 105
>2.0 × 105
9600
630
3400
3000
230
1200
18
>2.0 × 104
>1.0 × 105
3.6 × 104
>2.0 × 105
5700
25
7
15
18
2100
4.6
38 10
17
31
67
42
3
10
10
>2.0 × 105
>2.0 × 105
∼5.0 × 104
5700
∼2.0 × 105
∼1.0 × 105
>2.0 × 105
>2.5 × 104
23g
4300
2.8 × 104
Figure 3. Structural representation of compounds 18a−c and 23a−g presented in Table 2.
mercaptosulfide compounds,17,26,31,32 the trans stereochemistry
was more potent (23c vs 18a). Moreover, there was limited
enantiomeric stereoselectivity by the trans-mercaptosulfona-
mides (23c vs 23d and 23e), while there was a distinct
D
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preference in the less potent cis-mercaptocyclopentane
sulfonamides for the (1R,2S)-enantiomer (18c). The sulfona-
mide functionality was also evaluated by modifying it to an
amido functionality. This resulted in diminished potency as
seen when the trans-2-(4-phenoxybenzamido)-
cyclopentanethiol (23g) is compared to the corresponding
sulfonamide (23c). Expectedly, the structure lacking an
effective ZBG (23f) showed no significant inhibitory activity
when HO- was substituted for HS-.
Pyrrolidine Mercaptosulfonamide Inhibitors. Findings from
the cyclopentane series of mercaptosulfonamides were applied
to the target pyrrolidine scaffold. The synthetic route to the
( )-trans-3-mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine series of inhibitors is summarized in Scheme 3.
acylation after removing the N-Boc group and reduction of the
disulfide bond with TCEP.
Inhibition values for a selection of mercaptopyrrolidine
compounds (Figure 4) are shown in Table 3. As with the trans-
mercaptosulfonamide cyclopentanes, there was little enantio-
meric stereoselectivity (29a vs 39a and 39b). The N-
carbamoylpyrrolidine derivative (29b) afforded good water
solubility without the deleterious effect of the charged amino
group of the unsubstituted pyrrolidine (29a). Similar to the
earlier mercaptosulfides, attachment of a phthalimidoethylami-
nocarbonyl group to the pyrrolidine nitrogen (29d) as a
nonprimed side (P3) residue greatly improved selectivity for
MMP-3 and -9 (i.e., roughly 10-fold) and moderately for
MMP-1 and -13.
Biological Compatibility. Compound Stability. Although
structurally distinct from the prototypical hydroxamate MMPIs,
the mercaptan-based compounds exhibit comparable potencies,
ranging from submicromolar to single digit nanomolar Ki
values.39−41 However, early generations of these compounds
suffered from rapid oxidation of the thiol group, resulting in a
reduced ability of these compounds to coordinate the active site
Zn2+ due to disulfide formation.32 In order to assess the stability
of these new compounds, studies were performed to evaluate
the thiol’s zinc chelation over time.
Scheme 3. Synthesis of ( )-trans-3-Mercapto-4-
arylsulfonamidopyrrolidines
a
Compared to the mercaptosulfide inhibitors, the mercapto-
sulfonamides showed increased oxidative stability in solution,
moderately inhibiting MMP-9 for periods 5 times longer than
earlier compounds (Figure 5). In addition to increasing
potency, incorporation of either the cyclopentyl or pyrrolidinyl
ring to the nonprimed side of the inhibitor enhanced resistance
of the thiol to air oxidation. However, differences between ring
types had no apparent effect on compound stability, as 23c and
29b (Figure 5, panels A and B) exhibited indistinguishable
stability profiles. Evaluation of longer alkyl additions to the
pyrrolidinyl nitrogen (29f and 29e) revealed direct correlation
with reduced thiol stability (Figure 5, panels C and D).
Methylation of the sulfonamide nitrogen (29c) also resulted in
reduced thiol stability despite increases in potency toward
several MMPs (data not shown). An additional factor affecting
the stability of these compounds is the presence of serum.
Appearing to reduce oxidative degradation, higher percentages
of serum in culture medium prolong inhibitory activity of 23c
and 29f (Figure 5, panels A and C).
Cytotoxicity. Before use in cellular assays, compound toxicity
is assessed in human mesenchymal stem cells (hMSCs). This
cell type was selected, as current research surrounds the
involvement of MMPs in the proliferation and differentiation of
this cell line.42−44 Consistent with its use in other cell lines in
our lab, 23c exhibited the greatest level of toxicity, reducing the
number of cells by 40% at 100 μM (Figure 6, panel A). The
decrease in cell number was attributed to MMP inhibition,
since the noninhibitory analogue (23f) revealed no difference
in cell number. Similar trends were observed with 29b, 29d,
and 29e (Figure 6, panels B, C, and D).
a
Reagents and conditions: (a) NaN3, NH4Cl, MeOH/H2O, 65 °C;
(b) PPh3, THF−H2O; NaHCO3, ArSO2Cl, 0 °C to rt; (c) PPh3,
DEAD, THF, rt; (d) t-BuSH, t-BuOK, MeOH, 0 °C to rt; (e) RX, t-
BuOK, DMF, 0 °C to rt; (f) (1) 1.5 M HCl/EtOAc, rt, (2) R1COCl,
Et3N, CH2Cl2, rt; (g) (1) 2-nitrobenzenesulfenyl chloride, HOAc, rt,
(2) tri(carboxyethyl)phosphine, 1 N NaOH, THF, rt.
Pyrrolidine epoxide (3) was treated with NaN3 to give azido
alcohol (24) that was first reduced to amino alcohol and then
coupled with arylsulfonyl chloride to afford compound 25. N-
Sulfonylaziridine (26), which was obtained from 25 through
Mitsunobu reaction,34 was treated with t-BuSH to give
compound 27. N-Boc deprotection and subsequent N-acylation
followed by removal of t-Butyl protecting group afforded the
inhibitor 29. N-Methylated inhibitor 29c was also made in the
same way except for the methylation step of 27. A different
approach was required to prepare the individual enantiomers of
these compounds, which is outlined in Scheme 4. Kinetic
resolution of the 3,4-hydroxyazidopyrrolidine 24 gave (3S,4S)-
enantiomer 30 and acetylated (3R,4R)-enantiomer 31 that was
treated with base to afford (3R,4R)-enantiomer 32. The
hydroxy group of each enantiomer was activated by mesylation
and then treated with AcOK to give the cis-acetoxyazide (33).
The acetyl group of 33 was removed to give cis-3,4-
hydroxyazidopyrrolidine 34 that was reduced and coupled
with sulfonyl chloride to give compound 35. Compound 36,
obtained from compound 35 through Mitsunobu reaction, was
treated with MeNH2 to remove the acetyl group to give thiol
37, which was reprotected with o-nitrobenzenethio group to
afford disulfide 38. The final compound 39 was obtained by N-
DISCUSSION AND CONCLUSIONS
■
Structure−Activity Relationship Studies. The principle
goal of these studies was to develop potent and selective
MMPIs that incorporate a thiol ZBG. This strategy was initially
selected to circumvent the clinical side effects (e.g., poor plasma
bioavailability, target promiscuity) that generally hampered
early generation hydroxamate MMPIs, albeit recent hydrox-
amates are also avoiding such characteristics.39,45−48 A recent
E
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a
Scheme 4. Stereospecific Synthesis of trans-3-Mercapto-4-arylsulfonamidopyrrolidines
a
Reagents and conditions: (a) Amano AK-20 lipase, tert-butyl methyl ether, isopropenyl acetate, rt; (b) LiOH, THF−MeOH−H2O, rt; (c) (1)
MeSO2Cl, Et3N, CHCl3, rt, (2) AcOK, DMF, 100 °C; (d) LiOH, THF−MeOH−H2O, rt; (e) PPh3, THF−H2O; NaHCO3, ArSO2Cl, 0 °C to rt; (f)
AcSH, PPh3, DEAD, THF, 0 °C to rt; (g) MeNH2, MeOH, rt; (h) 2-nitrobenzenesulfenyl chloride, CH2Cl2, rt; (i) (1) TFA, rt, (2) RCOCl, Et3N,
CH2Cl2, 0 °C to rt; (j) tri(carboxyethyl)phosphine, 1 N NaOH, THF−H2O, rt.
Figure 4. Structural representation of compounds 29a−g, 39a, and 39b presented in Table 3.
report using a molecular dynamics approach to study the
docking of various MMPIs with MMP-9 even indicates that
thiol-based ZBGs are more energetically favorable than their
hydroxamate counterparts for this enzyme.49
The inhibition of MMPs by mercaptosulfonamides is
predicated upon coordination of the thiol with the enzyme’s
catalytic zinc. For inhibitor 29c, where substitution of a methyl
group directly upon the sulfonamide nitrogen did not reduce
inhibitor potency, we speculate the possibility that hydrogen
bonding between the sulfonamide -NH and enzyme is replaced
by the methyl group’s hydrophobic interaction. In fact, potency
was somewhat increased, perhaps by the methyl group
interacting with the S1′* subsite.50 Although our inhibitors
showed somewhat lower binding affinities when compared with
known bidentate ZBGs, the lower dissolution cost and
F
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Table 3. MMP Inhibition by trans-3-Mercapto-4-(4-phenoxybenzenesulfonamido)pyrrolidines
Kiapp (nM)
compd
MMP-1
MMP-2
MMP-3
2500
MMP-7
MMP-9
230
MMP-13
50
MMP-14
29a
29b
29c
29d
29e
29f
∼2.5 × 104
4100
27
>2.5 × 104
>2.5 × 104
>2.5 × 104
>1.2 × 104
3000
3.9
2.3
35
460
15
11
850
350
1.1
28
62
2800
37
2.4
27
21
>6000
3.8
3.1
∼7000
38
810
1.5
1.7
4
>3000
2000
>1.0 × 105
5900
1600
4000
3.6
2.0
>2.0 × 105
6.6
8.5 × 104
29g
39a
39b
>5.0 × 104
5.0 × 104
6300
>1.0 × 105
>2.0 × 105
4100
∼3.0 × 104
210
49
230
Figure 5. Stability profiles of the mercaptosulfonamide MMPIs in cell culture medium (αMEM). For panels A and C, serum concentration was
varied to assess any sequestration of the compounds by albumin. Error bars represent the standard error in the mean (mean SEM).
amenable ionization are more favorable, resulting in only
slightly lower potencies than hydroxamate-based MMPIs.51
Most efforts to establish MMPI selectivity have focused on
modifying the prime side of compounds, with little effort
evaluating nonprime substituents. The nonprime pockets of
MMPs are primarily solvent exposed and display less
segregation between subsites.52 For our purposes, substitution
upon the pyrrolidinyl nitrogen was to enhance future
functionalization of these compounds. However, exploration
of varying N-substituents provided significant changes in
potency. For example, addition of alkyl chains terminating
with more polar head groups generally increased inhibitor
potency (29e and 29f). With particular respect to MMP-3
(stromelysin-1), ring structures attached to long chains had
significant effect on selectivity (11c−e and 29d). Because of the
S2 pocket’s location immediately adjacent to the catalytic zinc,
we hypothesize that the linker and large phthalimido group are
likely interacting with the S3 pocket. As the S3 pocket is more
hydrophobic, it may attract the phthalimido group, which may
then position to participate in a previously noted π−π stacking
interaction with the pocket’s Tyr155.53 Surprisingly, increased
potency of these compounds against MMP-1 (collagenase 1)
was considerably less dramatic (i.e., ∼5-fold increase) than
MMP-3 (i.e., ∼2000-fold increase). This was unexpected, as the
S3 pocket in MMP-1, attributed to Ser155, is larger than those
in MMP-2, -3, -7, and -9, which all contain Tyr155 and are
believed to energetically accommodate larger P residues.54
At the prime side, a noticeable trend was observed with
modulation of the diphenyl ether and sulfonyl group.
Modifying the lone benzene (23a) to a biphenyl (23b) and
finally a diphenyl ether (23c) dramatically increased inhibitory
potency for MMP-13. Additionally, replacement of the sulfonyl
group with a carbonyl (23g) not only reduced MMP-13
inhibition but potency toward every MMP. These results are
consistent with other reports of thiol-based MMP inhibitors in
G
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Figure 6. Cytotoxicity profiles of the mercaptosulfonamide MMPIs in hMSCs. Error bars represent standard error in the mean (mean SEM).
that the diphenyl ether and sulfonyl groups are critical for
maintaining potency.55
Additional studies are being conducted to further investigate
this effect.
Finally, cytotoxicity analyses revealed that these compounds
exhibit only moderate toxicity (i.e., killing ≤20% of cells), up to
100 μM. This differs from an earlier report, in part, which
indicated that sulfur-containing compounds are generally more
cytotoxic.56 In comparison to a recent study evaluating the
effects of the aminobisphosphate alendronate, the mercapto-
sulfonamides display far less cytotoxicity, as bisphosphonates
were toxic to all cells at concentrations 10-fold lower than those
typically observed for the mercaptosulfonamides.57
Hydroxamate-based inhibitors are quite successful for some
other zinc metalloproteinases, like SAHA (suberoylanilide
hydroxamic acid) for histone deacetylases.47,58−62 Even though
the popularity of MMPIs as putative therapeutics was
diminished because of the notable failures of hydroxamate-
based MMPIs in oncology clinical trials, they are still valuable
investigative tools for MMP functional studies. For example,
preliminary evidence from our lab indicates that several of these
mercaptosulfonamides are capable of reducing the adipogenic
potential of human mesenchymal stem cells, suggesting unique
roles of MMPs in modulating the fate of adult stem cells.
Moreover, selectively targeting individual MMPs in a variety of
disease models will inevitably provide vital information about
the functions of these enzymes, information that may prove to
be critical to identifying possible treatment options.
More surprising was the demonstration that in contrast to
the preferred cis stereochemistry observed in our previous work
with the mercaptosulfides,17,26,31,32 trans-mercaptosulfonamides
were most effective (18a vs 23c). It is possible that replacement
of the prime side (i.e., S1′) peptidomimetic groups with 4-
phenoxybenzene, as seen in 23c, necessitates different stereo-
chemistry to maximize binding energy. Such modifications may
have arranged the MMPIs in such a way that cis enantiomers
are no longer in the correct plain for coordination. We are
currently further evaluating this finding.
Biological Studies. Early applications of thiol ZBGs were
hampered by their metal-binding promiscuity and susceptibility
to metabolic transformations. For example, stability assays with
noncyclic mercaptosulfides demonstrated significant sensitivity
to air oxidation. Yet the incorporation of cyclopentyl and
pyrrolidinyl rings at the P1 position of mercaptosulfonamides
increased inhibitor half-life. When compared to another thiol-
based chelator, N-(methyl)mercaptoacetamide, where 91% of
the thiol was oxidized within the first 8 min,56 our
mercaptosulfonamides appear dramatically more stable. How-
ever, in contrast to a previous report indicating that thiol
stability varied depending on ring structure, i.e., position of
endocyclic heteroatoms, and ring substituents,56 no preference
was discovered during our investigation.
The culture medium utilized with these assays contains
approximately 4 times the amount of serum than other typical
media. Consequently, it was not surprising to observe a
“plateau” toward the later time points in each run. We attribute
this to the MMPIs being sequestered by the serum albumin and
subsequently released in a dynamic equilibrium of some form.
EXPERIMENTAL SECTION
General Information. All chemicals, reagents, and solvents for
syntheses were analytical grade, purchased from commercial sources.
Solvents were purified according to standard procedures, and all air-
and moisture-sensitive reactions were performed under nitrogen. H
spectra were collected on a Bruker ARX 300 NMR spectrometer with
■
1
H
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chemical shifts in parts per million (ppm) downfield from
tetramethylsilane as an internal standard. Mass spectrometry data
were collected on a Jeol JMS-600H, applying ESI. Melting points were
determined on a Buchi melting point apparatus and are uncorrected.
Column chromatography was performed on Merck silica gel 60. The
reactions were monitored by TLC using Merck 60 F254 silica gel
glass-backed plates; zones were detected visually under UV irradiation
(254 nm). Combustion analysis was performed by Atlantic Microlab,
Inc., Norcross, GA, U.S. Purity of key compounds was established by
elemental analysis and/or Ellman’s reagent thiol titration and
determined to be >95%.
was added dropwise and the reaction mixture was stirred at room
temperature overnight. The mixture was concentrated under reduced
pressure and the residue was purified by flash chromatography on silica
gel (20% ethyl acetate in hexane) to give (1R,2S)-cis-2-azidocyclo-
pentyl p-nitrobenzoate (19) as a solid (1.00 g, 92%): mp 51.5−52.0
°C; 1H NMR (300 MHz, CDCl3) δ 8.33 (d, J = 9, 2H), 8.27 (d, J = 9,
2H), 5.42 (dt, J = 6, 5, 1H), 4.00 (dt, J = 5, 6, 1H), 1.88−2.25 (m,
5H), 1.65−1.83 (m, 1H); [α]2D0 −97.3° (c 0.59, CH2Cl2).
To a stirred solution of (1R,2S)-(−)-cis-2-azidocyclopentyl p-
nitrobenzoate (19) (500 mg, 1.81 mmol) in THF−MeOH (3:1, 10
mL) at 0 °C was added 1 N LiOH (3 mL) dropwise. The reaction
mixture was stirred at room temperature until no starting material was
detectable by TLC. The reaction mixture was evaporated under
reduced pressure, and ethyl ether (25 mL) was added to the residue.
The solution was washed with water, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography on silica
gel (50% ethyl acetate in hexane) to give (1R,2S)-cis-2-azidocyclo-
pentanol (20) as an oil (225 mg, 98%): 1H NMR (300 MHz, CDCl3)
δ 4.13 (br p, J = 5, 1H), 3.78 (dt, J = 5, 6, 1H), 1.78−2.00 (m, 4H),
1.53−1.75 (m, 3H); [α]2D0 +66.7° (c 1.44, CH2Cl2).
Pyrrolidine Mercaptosulfides. The inhibitors listed in Table 1
(see Figure 2 for structures) were synthesized as diastereomeric
mixtures as described,17 and as outlined in Scheme 1. Their
characterization data are the following:
11a. 1H NMR (300 MHz, CD3OD) δ 7.25 (m, 5H), 4.66 (m, 1H),
4.05 (m, 0.5H), 3.83−3.00 (m, 7.5H), 2.87 (m, 1H), 2.72 (s, 1.5H),
2.71 (s, 1.5H), 1.57 (m, 1H), 1.26 (m, 1H), 1.16 (m, 1H), 0.76 (m,
6H).
11b. 1H NMR (300 MHz, CD3OD) δ 7.23 (m, 5H), 4.72 (m, 1H),
4.10−3.20 (m, 7H), 3.11 (m, 1H), 2.78 (m, 1H), 2.72 (m, 3H), 2.03
(m, 3H), 1.59 (m, 2H), 1.25 (m, 1H), 0.77 (m, 6H).
By use of the same procedures, (1R,2R)-(−)-trans-2-azidocyclo-
pentanol (15) was converted to (1S,2R)-cis-2-azidocyclopentyl p-
nitrobenzoate (19 enantiomer) (mp 51.5 °C, [α]2D0 +97.9° (c 1.22,
CH2Cl2)) and then to (1S,2R)-cis-2-azidocyclopentanol (20 enan-
1
11c. H NMR (300 MHz, CDCl3) δ 7.87 (m, 2H), 7.74 (m, 2H),
7.40−7.15 (m, 5H), 7.01 (m, 0.5H), 6.93 (m, 0.5H), 6.00−5.78 (m,
1H), 4.67 (m, 1H), 4.40 (s, 1H), 4.36 (s, 1H), 4.00−3.00 (m, 8H),
2.76 (m, 3H), 2.06 (m, 0.5H, SH), 1.95 (m, 0.5H, SH), 1.67 (m, 2H),
1.50 (m, 1H), 0.88 (m, 6H). Anal. Calcd for C30H36N4O5S2·1/4H2O:
C, 59.93; H, 6.12; N, 9.32; S, 10.66. Found: C, 59.94; H, 5.92; N, 9.10;
S, 10.41.
tiomer): H NMR identical to that of the (1R,2S) enantiomer; [α]D20
1
−64.4° (c 1.12, CH2Cl2).
(
)-, (1S,2S)-(−)-, and (1R,2R)-(+)-trans-2-(tert-
Butoxycarbonylamino)cyclopentanol. To a stirred solution of
( )-trans-2-azidocyclopentanol (12) (1.27 g, 10.0 mmol) in THF−
H2O (4:1, 10 mL) was added Ph3P (2.88 g, 12.0 mmol), and the
mixture was stirred at room temperature for 2 h and at 65 °C for 2 h.
The solution was cooled with stirring to 0 °C, and Et3N (1.4 mL, 10.0
mmol) was added. Then a solution of di-tert-butyl dicarbonate (2.18 g,
10.0 mmol) in THF (5 mL) was added dropwise. The reaction
mixture was warmed to room temperature and was stirred for 3 h. The
mixture was concentrated under reduced pressure. Ethyl acetate was
added to the residue, and the organic layer was washed with 10% citric
acid and saturated aqueous NaHCO3. Then it was dried over Na2SO4
and evaporated. The residue was purified by flash chromatography on
silica gel (50% ethyl acetate in hexane) to give ( )-trans-2-(tert-
butoxycarbonylamino)cyclopentanol (( )-16) (1.8 g, 90%): 1H NMR
(300 MHz, CDCl3) δ 4.67 (br s, 1H), 3.98 (m, 2H), 3.62 (m, 1H),
1.93−2.18 (m, 2H), 1.59−1.85 (m, 3H), 1.45 (s, 9H), 1.27−1.41 (m,
1H).
11d. 1H NMR (300 MHz, CD3OD) δ 7.82 (m, 4H), 7.22 (m, 5H),
4.67 (m, 1H), 3.84−3.0 (m, 9H), 3.10 (m, 1H), 2.85 (m, 1H), 2.70 (s,
1.5H), 2.69 (s, 1.5H), 2.36 (m, 2H), 1.98 (m, 2H), 1.59 (m, 1H), 1.24
(m, 2H), 0.77 (m, 6H).
11e. 1H NMR (300 MHz, CD3OD) δ 7.80 (m, 4H), 7.24 (m, 5H),
4.69 (m, 1H), 3.85−2.80 (m, 11H), 2.70 (s, 1.5H), 2.69 (s, 1.5H),
2.28 (m, 2H), 1.65 (m, 5H), 1.32 (m, 4H), 0.78 (m, 6H).
2-Mercaptocyclopentane Arylsulfonamides (Scheme 2 and
Figure 3). (1S,2S)-(+)- and (1R,2R)-(−)-trans-2-Azidocyclopentanol.
A modified version of the procedure of Ami and Ohrui was used.38 To
a stirred solution of ( )-trans-2-azidocyclopentanol (1.90 g, 14.9
mmol) in tert-butyl methyl ether (75 mL) was added isopropenyl
acetate (3.3 g, 33.0 mmol) and lipase Amano AK-20 (4.5 g) (Amano
Enzyme Inc., Japan), and the mixture was stirred at room temperature
for 3 days. The reaction mixture was filtered. The filtrate was
evaporated under reduced pressure, and the residue was subjected to
flash chromatography on silica gel. Elution with 50% ethyl acetate in
hexane gave (1R,2R)-trans-2-azidocyclopentyl acetate (14) as an oil
(1.26 g, 7.45 mmol, 50%): 1H NMR (300 MHz, CDCl3) δ 4.96 (dt, J
= 7, 4, 1H), 3.87 (m, 1H), 2.05 (s, 3H), 1.92−2.18 (m, 2H), 1.57−
1.83 (m, 4H); [α]2D0 −56.7° (c 1.2, CH2Cl2). Continued elution with
50% ethyl acetate in hexane afforded (1S,2S)-trans-2-azidocyclopenta-
nol (13) as an oil (0.93 g, 49%): 1H NMR δ 4.2 (m, 1H), 3.7 (m, 1H)
1.9−2.2 (m, 2H), 1.5−1.9 (m, 5H); [α]2D0 +76.0° (c 1.1, CH2Cl2),
(lit.63 [α]2D0 +84.0°, (c 1.1, CH2Cl2)).
By the same procedure, (1S,2S)-(+)-trans-2-azidocyclopentanol
(13) gave (1S,2S)-trans-2-(tert-butoxycarbonylamino)cyclopentanol
1
(16) as a solid (370 mg, 73%): mp 81 °C; H NMR (300 MHz,
CDCl3) identical to that of ( )-16; [α]2D0 −23.6° (CH2Cl2) (lit.63 mp
87 °C, [α]2D0 −21.0° (c 1.0, CH2Cl2)).
By the same procedure, (1R,2R)-(−)-trans-2-azidocyclopentanol
(15) (160 mg, 1.26 mmol) afforded (1R,2R)-trans-2-(tert-
butoxycarbonylamino)cyclopentanol (16 enantiomer) (180 mg,
1
71%): mp 81 °C; H NMR (300 MHz, CDCl3) identical to that of
( )-16; [α]2D0 +23.4° (CH2Cl2) (lit.63 mp 87 °C, [α]2D0 +21.0° (c 1.0,
CH2Cl2)).
To a stirred solution of (1R,2R)-(−)-trans-2-azidocyclopentyl
acetate (14) (610 mg, 3.61 mmol) in THF−MeOH (3:1, 15 mL) at
0 °C was added 1 N LiOH (6 mL) dropwise. The reaction mixture
was stirred at room temperature until no starting material was
detectable by TLC. The reaction mixture was evaporated under
reduced pressure, and ethyl ether (50 mL) was added to the residue.
The solution was washed with water, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography on silica
gel (50% ethyl acetate in hexane) to give (1R,2R)-trans-2-
(1R,2S)-(+)-cis-2-(4-Phenoxybenzenesulfonamido)-
cyclopentanethiol (18c). To a stirred solution of Ph3P (393 mg, 1.50
mmol) in THF (5 mL) at 0 °C was added DEAD (236 μL, 1.50
mmol). After 10 min, solutions of (1S,2S)-(−)-trans-2-(tert-
butoxycarbonylamino)cyclopentanol (16) (166 mg, 0.826 mmol)
and thiolacetic acid (107 μL, 1.50 mmol) in THF (2 mL) were added.
The reaction mixture was stirred at room temperature overnight. Then
it was concentrated under reduced pressure. The residue was purified
by flash chromatography on silica gel (10% ethyl acetate in hexane) to
give (1R, 2S)-(+)-cis-2-(tert-butoxycarbonylamino)-
cyclopentanethioacetate (17) (170 mg, 79%) as a solid: mp 72 °C;
1H NMR (300 MHz, CDCl3) δ 4.57 (br s, 1H), 4.18 (br s, 1H), 3.95
1
azidocyclopentanol (15) as an oil (450 mg, 98%): H NMR identical
to that of the (1S,2S) enantiomer; [α]2D0 −84.3° (c 1.1, CH2Cl2).
(1R,2S)-(+)- and (1S,2R)-(−)-cis-2-Azidocyclopentanol. To a
stirred solution of Ph3P (1.55 g, 5.91 mmol) in THF (25 mL) at 0
°C was added DEAD (1.03 mL, 6.54 mmol) dropwise. After 10 min, a
solution of (1S,2S)-(+)-trans-2-azidocyclopentanol (13) (500 mg, 3.93
mmol) and p-nitrobenzoic acid (985 mg, 5.89 mmol) in THF (5 mL)
(q, J = 6, 1H), 2.33 (s, 3H), 2.09−2.21 (m, 1H), 1.93−2.09 (m, 1H),
1.59−1.81 (m, 3H), 1.44 (s, 9H), 1.35−1.53 (m, 1H); [α]2D0 +34.7°
(CH2Cl2).
I
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To a stirred solution of 17 (35 mg, 0.135 mmol) in THF (0.5 mL)
was added 40% aqueous MeNH2 (120 μL, 3.47 mmol). After 20 min
the reaction mixture was concentrated under reduced pressure and the
residue was dissolved in trifluoroacetic acid (0.5 mL). The mixture was
stirred for 2 h. The solvent was removed under reduced pressure, and
the residue was dissolved in CH2Cl2 (2 mL). The solution was cooled
to 0 °C, and Et3N (38 μL, 0.273 mmol) and 4-phenoxybenzene-
sulfonyl chloride (31.5 mg, 0.135 mmol) were added successively. The
reaction mixture was stirred at 0 °C for 3 h. Then it was concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel (20% ethyl acetate in hexane) to give
temperature and was stirred for 3 h. The mixture was concentrated
under reduced pressure. Ethyl acetate was added to the residue, and
the organic layer was washed with 10% citric acid and saturated
aqueous NaHCO3. Then it was dried over Na2SO4 and evaporated.
The residue was purified by flash chromatography on silica gel (50%
ethyl acetate in hexane) to give ( )-trans-S-tert-butyl-2-
(benzenesulfonamido)cyclopentanethiol (90 mg, 100%): 1H NMR
(300 MHz, CDCl3) δ 7.85−7.89 (m, 2H), 7.48−7.61 (m, 3H), 4.75
(br d, J = 3, 1H, NH), 3.01 (dq, J = 4, 8, 1H), 2.81 (q, J = 8, 1H),
2.00−2.22 (m, 2H), 1.44−1.70 (m, 4H), 1.28 (s, 9H).
To a solution of the ( )-trans-S-tert-butyl-2-(benzenesulfonamido)-
cyclopentanethiol (90 mg, 0.30 mmol) in AcOH (1 mL) was added 2-
nitrobenzenesulfenyl chloride (62 mg, 0.33 mmol), and the mixture
was stirred at room temperature for 2 h. The AcOH was evaporated
under vacuum, and to the residue (82 mg, ∼0.2 mmol) were added
THF (2 mL), 1 N NaOH (0.4 mL), and tris(2-carboxyethyl)-
phosphine (TCEP, 63.1 mg, 0.22 mmol). The mixture was stirred at
room temperature under argon for 3 h. Then it was condensed under
vacuum. The residue was dissolved in ethyl acetate, dried over Na2SO4,
and filtered. The solvent was evaporated and the residue was purified
by flash chromatography (25% ethyl acetate in hexane) to give the
1
18c as a solid (41 mg, 87%): mp 112−113 °C; H NMR (300 MHz,
CDCl3) δ 7.83 (d, J= 9, 2H), 7.42 (t, J = 8, 2H), 7.23 (t, J = 8, 1H),
7.06 (m, 4H), 5.02 (d, J = 8, 1H), 3.62 (m, 1H), 3.23 (m, 1H), 2.06
(m, 1H), 1.5−1.8 (m, 5H), 1.33 (d, J = 7, 1H); [α]2D0 +1.30°
(CH2Cl2); HRMS (ESI) calculated for C17H19NO3S2Na+ 372.0699,
found 372.0703.
(1S,2R)-(−)-cis-2-(4-Phenoxybenzenesulfonamido)-
cyclopentanethiol (18b). Following the Ph3P/DEAD procedure
described above, (1R,2R)-(+)-trans-2-(tert-butoxycarbonylamino)-
cyclopentanol (151 mg, 0.75 mmol) was converted to (1S,2R)-cis-2-
(tert-butoxycarbonylamino)cyclopentanethioacetate as a solid (177
mg, 91%): mp 72 °C; 1H NMR (300 MHz, CDCl3) identical to that of
the (1R,2S) enantiomer 17; [α]2D0 −34.8° (CH2Cl2).
1
thiol 23a (45 mg, 58%): H NMR (300 MHz, CDCl3) δ 7.90−7.94
(m, 2H), 7.50−7.63 (m, 3H), 4.67 (br d, J = 6, 1H, NH), 3.25 (p, J =
7.5, 1H), 2.90 (p, J = 7.5, 1H), 1.99−2.19 (m, 2H), 1.63−1.74 (m,
2H), 1.59 (d, J = 7, 1H, SH), 1.33−1.52 (m, 2H); HRMS (ESI)
calculated for C11H15NO2S2Na+ 280.0436, found 280.0442.
To
a stirred solution of 1S, 2R-(−)-cis-2-(tert-
butoxycarbonylamino)cyclopentanethioacetate (50 mg, 0.193 mmol)
in MeOH (0.5 mL) was added 2 N HCl in MeOH (2 mL), and the
mixture was stirred at room temperature for 2 h. The solvent was
removed under reduced pressure, and the residue was dissolved in
CH2Cl2 (2 mL). The solution was cooled to 0 °C, and Et3N (65 μL,
0.466 mmol) and 4-phenoxybenzenesulfonyl chloride (45.0 mg, 0.193
mmol) were added successively. The mixture was stirred at 0 °C for 3
h. Then it was concentrated under reduced pressure. The residue was
purified by flash chromatography on silica gel (20% ethyl acetate in
( )-trans-2-(4-Phenylbenzenesulfonamido)cyclopentanethiol
(23b). A sample of the crude ( )-trans-S-tert-butyl-2-(tert-
butoxycarbonylamino)cyclopentanethiol (59 mg, 0.214 mmol) from
the previous experiment was dissolved in trifluoroacetic acid (1 mL)
and was stirred at room temperature until no starting material
remained by TLC analysis. The trifluoroacetic acid was evaporated
under vacuum, and the residue was dissolved in CH2Cl2 (5 mL). The
solution was cooled to 0 °C, and Et3N (65.6 μL, 0.47 mmol) and 4-
phenylbenzenesulfonyl chloride (46.6 mg, 0.214 mmol) were added.
The reaction mixture was warmed to room temperature and was
stirred for 3 h. The mixture was concentrated under reduced pressure.
Ethyl acetate was added to the residue, and the organic layer was
washed with 10% citric acid and saturated aqueous NaHCO3. Then it
was dried over Na2SO4 and evaporated. The residue was filtered
through a short column of silica gel (50% ethyl acetate in hexane). The
resulting crude product was dissolved in AcOH (1 mL). 2-
Nitrobenzenesulfenyl chloride (41 mg, 0.24 mmol) was added, and
the mixture was stirred at room temperature for 2 h. The AcOH was
evaporated under vacuum to give the crude disulfide that was used
without further purification. To the crude disulfide was added THF (1
mL), 1 N NaOH (0.23 mL), and TCEP (63.1 mg, 0.22 mmol). The
mixture was stirred at room temperature under argon for 3 h. Then it
was condensed under vacuum. To the residue was added ethyl acetate,
and the mixture was dried over Na2SO4 and filtered. The solvent was
evaporated and the residue was purified by flash chromatography (25%
1
hexane) to give 18b as a solid (52 mg, 77%): mp 112−113 °C; H
NMR identical to that of 18c; [α]2D0 −1.30° (CH2Cl2); HRMS (ESI)
calculated for C17H19NO3S2Na+ 372.0699, found 372.0703.
( )-cis-2-(4-Phenoxybenzenesulfonamido)cyclopentanethiol
(18a). By the same procedures, ( )-trans-2-azidocyclopentanol (12)
1
afforded the racemic thiol 18a as a solid: mp 113−114 °C; H NMR
(300 MHz, CDCl3) identical to that of each of the enantiomers.
( )-trans-2-(Benzenesulfonamido)cyclopentanethiol (23a). To a
stirred solution of Ph3P (1.4 g, 5.3 mmol) in THF (25 mL) at 0 °C
was added DEAD (0.87 g, 5.0 mmol) dropwise. After 10 min,
( )-trans-2-(tert-butoxycarbonylamino)cyclopentanol (( )-16) (1.0 g,
5.0 mmol) was added dropwise, and the reaction mixture was stirred at
room temperature overnight. The mixture was concentrated under
reduced pressure and the residue was purified by flash chromatography
on silica gel (10% ethyl acetate in hexane) to give the aziridine N-tert-
butoxycarbonyl-6-azabicyclo[3.1.0]pentane as an oil (0.80 g, 87%): 1H
NMR (300 MHz, CDCl3) δ 2.04−2.16 (m, 2H), 1.53−1.64 (m, 4H),
1.46 (s, 9H), 1.15−1.32 (m, 2H).
1
ethyl acetate in hexane) to give 23b (66 mg, 0.198 mmol, 93%): H
To a stirred solution of the aziridine (550 mg, 3.0 mmol) in MeOH
(20 mL) were added tert-butyl thiol (0.5 mL, 4.4 mmol) and a solution
of sodium methoxide (162 mg, 3.0 mmol) in MeOH (5 mL), and the
reaction mixture was stirred at room temperature overnight. Water was
added to the solution, and the mixture was evaporated under vacuum.
The residue was dissolved in ethyl acetate (50 mL) and the organic
layer was washed with water, dried over Na2SO4, and evaporated to
give the crude ( )-trans-S-tert-butyl-2-(tert-butoxycarbonylamino)-
cyclopentanethiol as an oil (820 mg, 100%): H NMR (300 MHz,
CDCl3) δ 4.60 (broad, 1H), 3.64 (m, 1H). 2.80 (m, 1H), 2.10−2.25
(m, 2H), 1.60−1.75 (m, 4H), 1.45 (s, 9H), 1.36 (s, 9H).
A sample of the oil (82 mg, 0.30 mmol) was dissolved in
trifluoroacetic acid (1 mL), and the solution was stirred at room
temperature until no starting material remained by TLC analysis. The
trifluoroacetic acid was evaporated under vacuum, and the residue was
dissolved in CH2Cl2 (5 mL). The solution was cooled to 0 °C, and
Et3N (124 μL, 0.88 mmol) and benzenesulfonyl chloride (42 μL, 0.33
mmol) were added. The reaction mixture was warmed to room
NMR (300 MHz, CDCl3) δ 7.96 (d, J = 8, 2H), 7.74 (d, J = 8, 2H),
7.62 (d, J = 8, 2H), 7.39−7.52 (m, 3H), 4.69 (br d, J = 5, 1H, NH),
3.29 (p, J = 8, 1H), 2.93 (p, J = 8, 1H), 2.03−2.22 (m, 2H), 1.64−1.75
(m, 2H), 1.62 (d, J = 7, 1H, SH), 1.36−1.54 (m, 2H); HRMS (ESI)
calculated for C17H19NO2S2Na+ 356.0749, found 356.0755.
( 1 R , 2 S ) - ( − ) - a n d ( 1 S , 2 R ) - ( + ) - c i s - 2 - ( 4 -
Phenoxybenzenesulfonamido)cyclopentanol. To a stirred solution
of (1R,2S)-(+)-cis-2-azidocyclopentanol (20) (414 mg, 3.26 mmol) in
THF−H2O (4:1, 5 mL) was added Ph3P (897 mg, 3.42 mmol), and
the mixture was stirred at room temperature for 2 h and at 65 °C for 2
h. The solution was cooled with stirring to 0 °C, and NaHCO3 (310
mg, 3.69 mmol) was added. Then a solution of 4-phenoxybenzene-
sulfonyl chloride (760 mg, 3.26 mmol) in THF (5 mL) was added
dropwise. The reaction mixture was warmed to room temperature and
was stirred for 3 h. The mixture was concentrated under reduced
pressure. Ethyl acetate was added to the residue, and the organic layer
was washed with 10% citric acid and saturated aqueous NaHCO3.
Then it was dried over Na2SO4 and evaporated. The residue was
1
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purified by flash chromatography on silica gel (50% ethyl acetate in
hexane) to give (1R,2S)-cis-2-(4-phenoxybenzenesulfonamido)-
cyclopentanol as a solid (420 mg, 75%): mp 110 °C; H NMR (300
2.24 (m, 1H), 1.99 (m, 1H), 1.94 (br s, 1H, OH), 1.73 (m, 2H), 1.61
(m, 2H), 1.36 (s, 9H).
1
To a stirred solution of Ph3P (2.7 g, 10.3 mmol) in THF (25 mL) at
0 °C was added DEAD (1.8 g, 10.3 mmol) dropwise. After 10 min,
( )-trans-2-tert-butylmercaptocyclopentanol (1.64 g, 9.36 mmol) in
THF (5 mL) and hydrazoic acid (1.0 g in 10 mL of benzene, 25
mmol) were added dropwise, and the reaction mixture was stirred at
room temperature overnight. The mixture was concentrated under
reduced pressure and the residue was purified by flash chromatography
on silica gel (20% ethyl acetate in hexane) to give ( )-trans-2-tert-
MHz, CDCl3) δ 7.84 (d, J = 9, 2H), 7.42 (t, J = 8, 2H), 7.23 (t, J = 8,
1H), 7.06 (m, 4H), 4.87 (d, J = 8, 1H), 4.03 (m, 1H), 3.42 (m, 1H),
1.7−1.9 (m, 4H), 1.4−1.7 (m, 3H); [α]2D0 −22.0° (c 1.00, CH2Cl2).
Application of the same procedure to (1S,2R)-(−)-cis-2-azidocyclo-
pentanol (20 enantiomer) afforded (1S,2R)-cis-2-(4-
phenoxybenzenesulfonamido)cyclopentanol: mp 110 °C; 1H NMR
identical to that of the (1R,2S) enantiomer; [α]2D0 +22.3° (c 1.05,
CH2Cl2).
1
butylmercaptocyclopentylazide as an oil (1.3 g, 70%): H NMR (300
(
) -, (1 S, 2S )- (+ ) -, and (1 R, 2R )- (− ) - t r a n s- 2 - ( 4 -
MHz, CDCl3) δ 3.75 (m, 1H), 2.98 (m, 1H), 2.25 (m, 1H), 2.02 (m,
1H), 1.70 (m, 4H), 1.37 (s, 9H).
Phenoxybenzenesulfonamido)cyclopentanethiol (23c, 23d, and
23e). To a stirred solution of Ph3P (262 mg, 1.0 mmol) in THF (5
mL) at 0 °C was added DEAD (157 μL, 1.0 mmol). After 10 min, a
solution of (1R,2S)-(−)-cis-2-(4-phenoxybenzenesulfonamido)-
cyclopentanol (166 mg, 0.50 mmol) and thiobenzoic acid (118 μL,
1.0 mmol) in THF (2 mL) was added. The reaction mixture was
stirred at room temperature overnight. Then it was concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel (20% ethyl acetate in hexane) to give
( 1 S , 2 S ) - t r a n s - 2 - ( 4 - p h e n o x y b e n z e n e s u l f o n a m i d o ) -
cyclopentanethiobenzoate as a solid (180 mg, 79%): mp 132−133 °C;
1H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 8, 2H), 7.72 (d, J = 9, 2H),
To a stirred solution of the azide (470 mg, 2.35 mmol) in THF−
H2O (4:1, 10 mL) was added Ph3P (696 mg, 2.65 mmol), and the
mixture was stirred at room temperature for 2 h and at 65 °C for 2 h.
The solution was cooled with stirring to 0 °C, and Et3N (3.93 μL, 4.79
mmol) was added. Then a solution of 4-phenoxybenzoyl chloride (546
mg, 2.35 mmol) in THF (5 mL) was added dropwise. The reaction
mixture was warmed to room temperature and was stirred for 3 h. The
mixture was concentrated under reduced pressure. Ethyl acetate was
added to the residue, and the organic layer was washed with 10% citric
acid and saturated aqueous NaHCO3. Then it was dried over Na2SO4
and evaporated. The residue was purified by flash chromatography on
silica gel (50% ethyl acetate in hexane) to give ( )-trans-S-tert-butyl-2-
(4-phenoxybenzamido)cyclopentanethiol (0.67 g, 77%): 1H NMR
(300 MHz, CDCl3) δ 7.73 (d, J = 9, 2H), 7.38 (t, J = 8, 2H), 7.17 (t, J
= 7, 1H), 7.04 (d, J = 8, 2H), 7.00 (d, J = 9, 2H), 6.10 (br d, J = 4, 1H,
NH), 4.06 (p, J = 7, 1H), 3.08 (q, J = 7, 1H), 2.20−2.40 (m, 2H),
1.66−1.87 (m, 3H), 1.52−1.66 (m, 1H), 1.38 (s, 9H).
7.59 (t, J = 8, 1H), 7.43 (t, J = 8, 2H), 7.36 (t, J = 8, 2H), 7.19 (t, J = 8,
1H), 6.93 (d, J = 9, 2H), 6.77 (d, J = 9, 2H), 5.54 (br d, 1H), 3.73 (q, J
= 9, 1H), 3.42 (m, 1H), 2.1−2.3 (m, 2H), 1.6−1.9 (m, 4H); [α]D20
−140.6° (c 0.65, CH2Cl2).
To
a stirred solution of (1S,2S)-(−)-trans-2-(4-
phenoxybenzenesulfonamido)cyclopentanethiobenzoate (80 mg,
0.176 mmol) in THF (1 mL) under N2 was added 40% aqueous
MeNH2 (900 μL, 10.5 mmol), and the reaction mixture was stirred for
20 min. The solution was concentrated under reduced pressure and
the residue was purified by flash chromatography on silica gel (20%
ethyl acetate in hexane) to give (1S,2S)-(+)-trans-2-(4-
phenoxybenzenesulfonamido)cyclopentanethiol (23d) as a solid (60
mg, 98%): mp 133−134 °C; 1H NMR (300 MHz, CDCl3) δ 7.84 (d, J
= 9, 2H), 7.42 (t, J = 8, 2H), 7.23 (t, J = 8, 1H), 7.06 (m, 4H), 4.53 (br
d, 1H), 3.23 (pentet, J = 7.5, 1H), 2.91 (p, J = 7.5, 1H), 2.03−2.21 (m,
2H), 1.64−1.76 (m, 2H), 1.60 (d, J = 7, 1H), 1.34−1.53 (m, 2H);
[α]2D0 +16.9° (c 1.00, CH2Cl2).
To a solution of ( )-trans-S-tert-butyl-2-(4-phenoxybenzamido)-
cyclopentanethiol (211 mg, 0.57 mmol) in AcOH (1 mL) was added
2-nitrobenzenesulfenyl chloride (108 mg, 0.57 mmol), and the mixture
was stirred at room temperature for 2 h. The AcOH was evaporated
under vacuum, and to the residue (212 mg, 0.455 mmol) were added
THF (4 mL), 1 N NaOH (0.91 mL), and TCEP (144 mg, 0.50
mmol). The mixture was stirred at room temperature under argon for
3 h. Then it was condensed under vacuum. To the residue was added
ethyl acetate, and it was dried over Na2SO4 and filtered. The solvent
was evaporated and the residue was purified by flash chromatography
1
(25% ethyl acetate in hexane) to give 23g (135 mg, 76%): H NMR
B y t h e s a m e p r o c e d u r e , ( 1 S , 2 R ) - ( + ) - c i s - 2 - ( 4 -
phenoxybenzenesulfonamido)cyclopentanol (180 mg, 0.54 mmol)
was converted to (1R,2R)-trans-2-(4-phenoxybenzenesulfonamido)-
cyclopentanethiobenzoate (170 mg, 69%): mp 132−133 °C; [α]D20
+142.4° (1.05, CH2Cl2). NMR analysis of the product in the presence
of the chiral shift reagents (europium[3] tris-trifluoromethylhydroxy-
methylene-(+)-camphorate and europium[3] tris-heptafluoropropyl-
hydroxymethylene-(+)-camphorate) at both 300 and 400 MHz
indicated it to be in >90% ee.
(300 MHz, CDCl3) δ 7.75 (d, J = 9, 2H), 7.38 (t, J = 8, 2H), 7.17 (t, J
= 7.5, 1H), 7.02 (m, 4H), 6.01 (br d, J = 7, 1H, NH), 4.19 (p, J = 7.5,
1H), 3.10 (dt, J = 7, 8, 1H), 2.30−2.42 (m, 1H), 2.15−2.29 (m, 1H),
1.88 (d, J = 6, 1H, SH), 1.45−1.85 (m, 4H); HRMS (ESI) calculated
for C18H19NO2SNa+ 336.1029, found 336.1034.
( )-trans-2-(4-Phenoxybenzenesulfonamido)cyclopentanol
(23f). To a stirred solution of ( )-trans-2-azidocyclopentanol (553
mg, 4.35 mmol) in THF−H2O (4:1, 10 mL) was added Ph3P (1.26 g,
4.79 mmol), and the mixture was stirred at room temperature for 2 h
and at 65 °C for 2 h. The solution was cooled with stirring to 0 °C,
and Et3N (0.67 mL, 4.79 mmol) was added. Then a solution of 4-
phenoxybenzenesulfonyl chloride (1.01 g, 4.35 mmol) in THF (5 mL)
was added dropwise. The reaction mixture was warmed to room
temperature and was stirred for 3 h. The mixture was concentrated
under reduced pressure, and ethyl acetate was added. The organic
layer was washed with 10% citric acid and saturated aqueous NaHCO3.
Then it was dried over Na2SO4 and evaporated. The residue was
purified by flash chromatography on silica gel (50% ethyl acetate in
hexane) to give 23f as a solid (0.93g, 71%): mp 98 °C; 1H NMR (300
MHz, CDCl3) δ 7.83 (d, J = 9, 2H), 7.42 (t, J = 8, 2H), 7.24 (t, J = 8,
1H), 7.06 (m, 4H), 4.8 (br, 1H, NH), 4.06 (q, J = 6, 1H), 3.24 (m,
1H), 1.88−2.03 (m, 2H), 1.49−1.72 (m, 4H), 1.30−1.43 (m, 1H);
HRMS (ESI) calculated for C17H19NO4SNa+ 356.0927, found
356.0933.
The (1R,2R)-(+)-trans-2-(4-phenoxybenzenesulfonamido)-
cyclopentanethiobenzoate (85 mg, 0.187 mmol) was hydrolyzed as
above to give (1R,2R)-(−)-trans-2-(4-phenoxybenzenesulfonamido)-
1
cyclopentanethiol 23e as a solid (61 mg, 93%): mp 133−134 °C; H
NMR (300 MHz, CDCl3) identical to that of 23d; [α]2D0 −17° (c 1.00,
CH2Cl2).
By the same procedures, ( )-trans-2-azidocyclopentanol afforded
1
the racemic thiol 23c as a solid: mp 156−157 °C; H NMR (300
MHz, CDCl3) identical to that of each of the enantiomers; HRMS
(ESI) calculated for C17H19NO3S2Na+ 372.0699, found 372.0704.
( )-trans-2-(4-Phenoxybenzamido)cyclopentanethiol (23g). To a
stirred solution of cyclopentane oxide (1.57 g, 18.7 mmol) in MeOH
(20 mL) were added t-Bu thiol (2.1 mL, 18.7 mmol) and a solution of
sodium methoxide (1.01 g, 18.7 mmol) in MeOH (10 mL). The
reaction mixture was stirred at room temperature overnight. Water was
added to the solution, and the mixture was evaporated under vacuum.
The residue was dissolved in ethyl acetate (50 mL) and the organic
layer was washed with water, dried over Na2SO4, and evaporated to
give ( )-trans-2-tert-butylmercaptocyclopentanol (3.00 g, 92%): 1H
NMR (300 MHz, CDCl3) δ 3.90 (q, J = 7, 1H), 2.84 (dt, J = 7, 8, 1H),
trans-3-Mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidines (Scheme 3 and Figure 4, Ar = -C6H4-O-C6H5).
( )-N-Boc-trans-3-hydroxy-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (25). A mixture of N-Boc-3-pyrroline oxide (3) (0.925 g,
5.0 mmol), NaN3 (0.65 g, 10 mmol), and NH4Cl (5.0 mmol) in
K
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Journal of Medicinal Chemistry
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MeOH−H2O (6:1, 16 mL) was stirred at 65 °C overnight. The
reaction mixture was concentrated under reduced pressure, and the
residue was extracted thoroughly with ethyl acetate. The combined
ethyl acetate extract was washed with water and saturated brine, dried
over anhydrous Na2SO4, and evaporated. The crude ( )-N-Boc-trans-
3-hydroxy-4-azidopyrrolidine (24) was purified by recrystallization
from ether−hexane: mp 34−36 °C; H NMR (300 MHz, CD3OD) δ
4.16 (m, 1H), 3.95 (m, 1H), 3.62 (m, 1H), 3.51 (m, 1H), 3.35 (m,
2H), 1.47 (s, 9H).
(300 MHz, CD3OD) δ 7.88 (m, 2H), 7.45 (m, 2H), 7.25 (t, J = 7,
1H), 7.10 (m, 4H), 3.74 (m, 1H), 3.55 (m, 2H), 3.17 (m, 2H), 3.00
(m, 1H), 1.44 (s, 9H); MS (ESI) m/e 450.2 (M+); HRMS (ESI)
calculated for C21H26N2O5S2Na+ 473.1172, found 473.1170.
To a solution of 2.5 M HCl in AcOH (1 mL) was added ( )-N-
Boc-trans-3-mercapto-4-(4-phenoxybenzenesulfonamido)pyrrolidine
(45 mg, 0.10 mmol), and the mixture was stirred at room temperature
for 3 h. Liophylization of the solution gave the product 29a as a white
powder (38.7 mg, 100%): 1H NMR (300 MHz, CD3OD) δ 7.87 (d, J
= 12, 2H), 7.44 (m, 2H), 7.24 (t, J = 7, 1H), 7.12 (m, 4H), 3.72 (m,
1H), 3.62 (m, 2H), 3.37 (m, 1H), 3.19 (m, 2H); MS (ESI) m/e 351.1
1
To a stirred solution of the azido alcohol 24 (1.02 g, 4.45 mmol) in
THF−H2O (10:1, 10 mL) was added Ph3P (1.28 g, 4.88 mmol), and
the mixture was stirred at room temperature for 2 h and at 65 °C for 2
h. The solution was cooled with stirring to 0 °C. Et3N (0.74 mL, 5.34
mmol) was added. Then a solution of 4-phenoxybenzenesulfonyl
chloride (1.04 g, 4.45 mmol) in THF (5 mL) was added dropwise.
The reaction mixture was warmed to room temperature and was
stirred for 3 h. The mixture was concentrated under reduced pressure.
Ethyl acetate was added to the residue, and the organic layer was
washed with 10% citric acid and saturated aqueous NaHCO3. Then it
was dried over Na2SO4 and evaporated. The residue was purified by
flash chromatography (50% ethyl acetate in hexane) to give ( )-N-
Boc-trans-3-hydroxy-4-(4-phenoxybenzenesulfonamido)pyrrolidine
+
(MH+); HRMS (ESI) calculated for C16H19N2O3S2 351.0837, found
351.0837.
(
) - N - C a r b a m o y l - t r a n s - 3 - m e r c a p t o - 4 - ( 4 -
phenoxybenzenesulfonamido)pyrrolidine (29b). To a stirred sol-
ution of 1.5 M HCl in ethyl acetate (5 mL) was added ( )-N-Boc-
trans-3-tert-butylmercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (27) (155 mg, 0.3 mmol), and stirring was continued at
room temperature until no starting material was detected by TLC
(50% ethyl acetate in hexane). The solvent was evaporated under
reduced pressure, and the residue was dissolved in CH2Cl2. The
solution was cooled to 0 °C, and Et3N (46 μL, 33 mmol) and N-
trimethylsilyl isocyanate (TMSNCO) (100 μL, 0.74 mmol) were
added successively. The solution was stirred at room temperature
overnight. The reaction mixture was extracted with water and
saturated brine and was dried over Na2SO4 and filtered. The solvent
was evaporated and the residue was purified by flash chromatography
(5% MeOH in ethyl acetate) to give ( )-N-carbamoyl-trans-3-tert-
butylmercapto-4-(4-phenoxybenzenesulfonamido)pyrrolidine (130
1
(25) as a solid (1.52 g, 79%): mp 98−99 °C; H NMR (300 MHz,
CD3OD) δ 7.85 (d, J = 8, 2H), 7.45 (m, 2H), 7.24 (t, J = 7, 1H), 7.10
(m, 4H), 4.09 (m, 1H), 3.95 (m, 1H), 3.70 (m, 2H), 3.40 (m, 2H),
1.42 (s, 9H); MS, ESI m/e 434.1 (M+); HRMS (ESI) calculated for
C21H26N2O6SNa+ 457.1391, found 457.1391.
(
) - N - B o c - t r a n s - 3 - t e r t - b u t y l m e r c a p t o - 4 - ( 4 -
phenoxybenzenesulfonamido)pyrrolidine (27). To a stirred solution
of Ph3P (0.88 g, 3.35 mmol) and ( )-N-Boc-trans-3-hydroxy-4-(4-
phenoxybenzenesulfonamido)pyrrolidine (25) (1.316 g, 3.04 mmol)
in THF (20 mL) at 0 °C was added DEAD (0.58 mL, 3.33 mmol)
dropwise. The reaction mixture was stirred at room temperature
overnight. Then it was evaporated under reduced pressure. The
residue was purified by flash chromatography (30% ethyl acetate in
hexane) to give the N-(4-phenoxybenzenesulfonyl)aziridine 26 as a
solid (1.23 g, 98%): mp 136−138 °C; 1H NMR (300 MHz, CDCl3) δ
7.88 (m, 2H), 7.43 (m, 2H), 7.22 (m, 1H), 7.10 (m, 4H), 3.72 (m,
2H), 3.59 (m, 1H), 3.47 (m, 1H), 3.38 (m, 2H), 1.42 (s, 9H); MS, ESI
m/e 416.1 (M+); HRMS (ESI) calculated for C21H24N2O5SNa+
439.1290, found 439.1290.
1
mg, 96%): mp 132−133 °C; H NMR (300 MHz, CD3OD) δ 7.85
(d, J = 9, 2H), 7.44 (t, J = 8, 2H), 7.24 (t, J = 8, 1H), 7.10 (m, 2H),
7.09 (d, J = 9, 2H), 3.86 (m, 1H), 3.59 (m, 2H), 3.27 (m, 2H), 3.15
(m,1H), 1.29 (s, 9H); MS (ESI) m/e 449.2 (M+); HRMS (ESI)
calculated for C21H27N3O4S2Na+ 472.1322, found 472.1326.
To a solution of ( )-N-carbamoyl-trans-3-tert-butylmercapto-4-(4-
phenoxybenzenesulfonamido)pyrrolidine (110 mg, 0.245 mmol) in
AcOH (1 mL) was added 2-nitrobenzenesulfenyl chloride (51 mg,
0.269 mmol), and the mixture was stirred at room temperature for 2 h.
The AcOH was evaporated under vacuum, and to the residue were
added THF (4 mL), 1 N NaOH (0.3 mL), and TCEP (77 mg, 0.269
mmol) with stirring under argon. Stirring under argon was continued
for 3 h at room temperature. Then the mixture was concentrated
under vacuum. The residue was dissolved in ethyl acetate, and the
solution was dried over Na2SO4 and filtered. The solvent was
evaporated and the residue was purified by flash chromatography (5%
MeOH in ethyl acetate) to give the mercaptan 29b as a white solid (89
To a stirred solution of the aziridine 26 (416 mg, 1.0 mmol) and
tert-butyl mercaptan (0.15 mL, 1.2 mmol) in anhydrous MeOH (5
mL) at 0 °C was added t-BuOK (0.2 mmol). The mixture was stirred
at room temperature for 4 h. Then it was evaporated under reduced
pressure. Ethyl acetate was added to the residue, and the solution was
extracted with water and saturated brine. Then it was dried over
Na2SO4 and evaporated. The residue was purified by flash
chromatography (20% ethyl acetate in hexane) to give ( )-N-Boc-
trans-3-tert-butylmercapto-4-(4-phenoxybenzenesulfonamido)-
1
mg, 96%): mp 170−171 °C; H NMR (300 MHz, CD3OD) δ 7.87
(m, 2H), 7.44 (m, 2H), 7.24 (m, 1H), 7.10 (m, 4H), 3.78 (m, 1H),
3.68 (m, 1H), 3.59 (m, 1H), 3.20 (m, 2H), 3.08 (m, 1H); MS (ESI)
m/e 393.1 (M+); HRMS (ESI) calculated for C17H19N3O4S2Na+
416.0701, found 416.0699.
1
pyrrolidine (27) as a solid (506 mg, 98%): mp 85−86 °C; H NMR
(300 MHz, CD3OD) δ 7.85 (d, J = 7, 2H), 7.45 (t, J = 7, 2H), 7.25 (t,
J = 7, 1H), 7.09 (d, J = 7, 4H), 3.81 (m, 2H), 3.58 (m, 1H), 3.47 (m,
1H), 3.22 (m,1H), 3.09 (m, 1H), 1.45 (s, 9H), 1.29 (s, 9H); MS, ESI
m/e 506.2 (M+); HRMS (ESI) calculated for C25H34N2O5S2Na+
529.1796, found 529.1795.
(
)-N-Carbamoyl-trans-3-mercapto-4-(N-methyl-4-
phenoxybenzenesulfonamido)pyrrolidine (29c). To a solution of
) - N - B o c - t r a n s - 3 - t e r t - b u t y l m e r c a p t o - 4 - ( 4 -
(
phenoxybenzenesulfonamido)pyrrolidine (27) (236 mg, 0.466 mmol)
in DMF (2 mL) at 0 °C was added t-BuOK (0.69 mmol). The mixture
was stirred at 0 °C for 10 min. Then MeI (0.69 mmol) was added. The
solution was stirred at 0 °C until TLC analysis showed no starting
material remained. Ethyl acetate (20 mL) was added, and the solution
was extracted with water, then was dried over Na2SO4 and filtered.
The solvent was evaporated and the residue was purified by flash
chromatography (ethyl acetate) to give ( )-N-Boc-trans-3-tert-
butylmercapto-4-(N-methyl-4-phenoxybenzenesulfonamido)-
( )-trans-3-Mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine Hydrochloride (29a). To a solution of ( )-N-Boc-trans-3-
tert-butylmercapto-4-(4-phenoxybenzenesulfonamido)pyrrolidine (27)
(117 mg, 0.226 mmol) in AcOH (1 mL) was added 2-nitro-
benzenesulfenyl chloride (46.0 mg, 0.243 mmol), and the mixture was
stirred at room temperature for 2 h. The AcOH was evaporated under
vacuum, and to the residue were added THF (4 mL), 1 N NaOH
(0.25 mL), and TCEP (67 mg, 0.234 mmol). The mixture was stirred
at room temperature under argon for 3 h. Then it was condensed
under vacuum. To the residue was added ethyl acetate, and it was
dried over Na2SO4 and filtered. The solvent was evaporated and the
residue was purified by flash chromatography (ethyl acetate) to give
( )-N-Boc-trans-3-mercapto-4-(4-phenoxybenzenesulfonamido)-
1
pyrrolidine (28, R = CH3) as a white powder (240 mg, 99%): H
NMR (300 MHz, CD3OD) δ 7.84 (d, J = 8, 2H), 7.44 (t, J = 8, 2H),
7.24 (t, J = 8, 1H), 7.10 (m, 2H), 7.09 (d, J = 9, 2H), 4.22 (m, 1H),
3.90 (m, 1H), 3.37 (m, 2H), 3.08 (m, 2H), 2.86 (s, 3H), 1.44 (s, 9H),
1.28 (s, 9H); MS (ESI) m/e 520.2 (M+); HRMS (ESI) calculated for
C26H36N2O5S2Na+ 543.1950, found 543.1950.
1
pyrrolidine as a white solid (82 mg, 81%): mp 78−79 °C; H NMR
L
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To a stirred solution of 1.5 M HCl in ethyl acetate (5 mL) was
residue was purified by flash chromatography (ethyl acetate) to give
the mercaptan 29d as a white solid (48 mg, 87%): mp 140−141.5 °C;
1H NMR (300 MHz, CDCl3) δ 7.93−7.80 (m, 4H), 7.73 (m, 2H),
7.41 (m, 2H), 7.23 (t, J = 8, 1H), 7.15−7.00 (m, 4H), 5.73 (br, 1H),
4.73 (m, 1H), 3.87 (m, 2H), 3.71 (m, 2H), 3.55 (m, 1H), 3.47 (m,
2H), 3.37 (m, 1H), 3.24 (m, 1H), 3.13 (m, 1H), 1.74 (d, J = 7, 1H);
MS (ESI) m/e 566.2 (M+); HRMS (ESI) calculated for
C27H26N4O6S2Na+ 589.1191, found 589.1190.
added
( )-N-Boc-trans-3-tert-butylmercapto-4-(N-methyl-4-
phenoxybenzenesulfonamido)pyrrolidine (28, R = CH3) (130 mg,
0.25 mmol), and stirring was continued at room temperature until no
starting material was detected by TLC. The solvent was evaporated
under reduced pressure, and the residue was dissolved in CH2Cl2. The
solution was cooled to 0 °C, and Et3N (46 μL, 0.33 mmol) and
TMSNCO (100 μL, 0.74 mmol) were added successively. The
solution was stirred at room temperature overnight. The reaction
mixture was extracted with water and saturated brine and was dried
over Na2SO4 and filtered. The solvent was evaporated and the residue
was purified by flash chromatography (5% MeOH in ethyl acetate) to
give ( )-N-carbamoyl-trans-3-tert-butylmercapto-4-(N-methyl-4-
phenoxybenzenesulfonamido)pyrrolidine as a white powder (110
(
)-N-(6-Acetamidohexanoyl)-trans-3-mercapto-4-(4-
phenoxybenzenesulfonamido)pyrrolidine (29e) and ( )-N-(6-Ami-
n o h e x a n o y l ) - t r a n s - 3 - m e r c a p t o - 4 - ( 4 -
phenoxybenzenesulfonamido)pyrrolidine Hydrochloride (29f). To a
stirred solution of 1.5 M HCl in ethyl acetate (1 mL) was added
(
) - N - B o c - t r a n s - 3 - t e r t - b u t y l m e r c a p t o - 4 - ( 4 -
1
phenoxybenzenesulfonamido)pyrrolidine (27) (200 mg, 0.39 mmol),
and stirring was continued at room temperature until no starting
material was detected by TLC (50% ethyl acetate in hexane). The
solvent was evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2. The solution was cooled to 0 °C, and Et3N (60
μL, 0.43 mmol), HOBt·H2O (54 mg, 0.40 mmol), 6-(tert-
butoxycarbonylamino)hexanoic acid (90 mg, 0.39 mmol), and DCC
(80 mg, 0.39 mmol) were added. The mixture was stirred at room
temperature overnight. The solvent was evaporated under reduced
pressure, and the residue was dissolved in ethyl acetate (10 mL). The
organic layer was washed with 10% citric acid and saturated aqueous
NaHCO3. Then it was dried over Na2SO4 and evaporated. The residue
was purified by flash chromatography (50% ethyl acetate in hexane) to
give ( )-N-(6-(tert-butoxycarbonylamino)hexanoyl)-trans-3-tert-butyl-
mercapto-4-(4-phenoxybenzenesulfonamido)pyrrolidine (201 mg,
mg, 95%): H NMR (300 MHz, CD3OD) δ 7.85 (m, 2H), 7.42 (m,
2H), 7.22 (m, 1H), 7.10 (m, 4H), 4.24 (m, 1H), 3.96 (m, 1H), 3.42
(m, 2H), 3.08 (m, 2H), 2.87 (s, 3H), 1.28 (s, 9H); MS (ESI) m/e
463.2 (M+); HRMS (ESI) calculated for C22H29N3O4S2Na+ 486.1479,
found 486.1481.
To a solution of ( )-N-carbamoyl-trans-3-tert-butylmercapto-4-(N-
methyl-4-phenoxybenzenesulfonamido)pyrrolidine (90 mg, 0.194
mmol) in AcOH (1 mL) was added 2-nitrobenzenesulfenyl chloride
(38.6 mg, 0.204 mmol), and the mixture was stirred at room
temperature for 2 h. The AcOH was evaporated under vacuum, and to
the residue were added THF (4 mL), 1 N NaOH (0.25 mL), and
TCEP (67 mg, 0.234 mmol). The mixture was stirred at room
temperature under argon for 3 h. Then it was condensed under
vacuum. To the residue was added ethyl acetate, and it was dried over
Na2SO4. The solvent was evaporated and the residue was purified by
flash chromatography (5% MeOH in ethyl acetate) to give the
1
84%): H NMR (300 MHz, CDCl3) δ 7.82 (dd, J = 9, 3, 2H), 7.43
1
(t, J = 8, 2H), 7.24 (t, J = 7, 1H), 7.08 (m, 4H), 5.13 (br m, 1H, NH),
4.57 (br s, 1H, NH), 3.72−4.10 (m, 2H), 3.00−3.45 (m, 6H), 2.21 (t,
J = 7, 2H), 1.64 (m, 2H), 1.48 (m, 2H), 1.44 (s, 9H), 1.36 and 1.30 (s,
9H), 1.26 (m, 2H); HRMS (ESI) calculated for C31H45N3O6S2Na+
642.2642, found 642.2654.
mercaptan 29c as a white powder (75.1 mg, 95%): H NMR (300
MHz, CD3OD) δ 7.87 (m, 2H), 7.44 (m, 2H), 7.24 (m, 1H), 7.10 (m,
4H), 4.32 (q, J = 9, 1H), 3.83 (dd, J = 10, 8 Hz, 1H), 3.42 (m, 2H),
3.13 (q, J = 10, 2H), 2.83 (s, 3H); MS (ESI) m/e 407.2 (M+); HRMS
(ESI) calculated for C22H29N3O4S2Na+ 430.0864, found 430.0867.
( )-N-(2-Phthalimidoethylaminocarbonyl)-trans-3-mercapto-4-
(4-phenoxybenzenesulfonamido)pyrrolidine (29d). To a stirred
solution of 1.5 M HCl in ethyl acetate (3 mL) was added ( )-N-
Boc-trans-3-tert-butylmercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (27) (77.5 mg, 0.15 mmol), and stirring was continued at
room temperature until no starting material was detected by TLC
(50% ethyl acetate in hexane). The solvent was evaporated under
reduced pressure, and the residue was dissolved in CH2Cl2 (3 mL).
The solution was cooled to −10 °C, and diisopropylethylamine (63
μL, 0.36 mmol) and triphosgene (14.9 mg, 0.05 mmol) in CH2Cl2 (3
mL) were added. The mixture was stirred at −10 °C for 20 min. Then
diisopropylethylamine (63 μL, 0.36 mmol) and 2-phthalimidoethyl-
amine hydrochloride (34 mg, 0.15 mmol) were added successively.
The solution was warmed to room temperature and was stirred for 4 h.
The reaction mixture was extracted with water, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by flash
chromatography (5% MeOH in ethyl acetate) to give ( )-N-(2-
phthalimidoethylaminocarbonyl)-trans-3-tert-butylmercapto-4-(4-
phenoxybenzenesulfonamido)pyrrolidine as a white solid (79 mg,
84%): mp 105−106 °C; 1H NMR (300 MHz, CDCl3) δ 7.92 (m, 2H),
7.85 (m, 2H), 7.72 (m, 2H), 7.42 (m, 2H), 7.24 (m, 1H), 7.10 (m,
4H), 5.62 (d, J = 9, 1H), 4.62 (m, 1H), 3.98−3.70 (m, 3H), 3.62 (m,
1H), 3.48 (m, 3H), 3.24 (m, 2H), 3.14 (m, 1H), 1.32 (s, 9H); MS
(ESI) m/e 622.2 (M+); HRMS (ESI) calculated for C31H34N4O6S2Na+
645.1812, found 645.1814.
To a solution of ( )-N-(6-(tert-butoxycarbonylamino)hexanoyl)-
trans-3-tert-butylmercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (130 mg, 0.21 mmol) in AcOH−CH2Cl2 (1:1, 1 mL) was
added 2-nitrobenzenesulfenyl chloride (42 mg, 0.22 mmol), and the
mixture was stirred at room temperature for 2 h. The solvents were
evaporated under vacuum and the residue was purified by flash
chromatography (50% ethyl acetate in hexane) to give ( )-N-(6-(tert-
butoxycarbonylamino)hexanoyl)-trans-3-(2-nitrobenzenedisulfido)-4-
(4-phenoxybenzenesulfonamido)pyrrolidine: 1H NMR (300 MHz,
CDCl3) δ 8.28 (m, 1H), 8.22 and 8.11 (d, J = 8, 1H), 7.75 (m, 3H),
7.42 (m, 3H), 7.24 (m, 1H), 7.10 (m, 2H), 7.02 (m, 2H), 5.40 and
5.12 (br s, 1H, NH), 4.57 (br s, 1H, NH), 3.75−4.00 (m, 3H), 3.21−
3.53 (m, 3H), 2.95−3.14 (m, 2H), 2.19 (m, 2H), 1.61 (m, 2H), 1.47
(m, 2H), 1.43 (s, 9H), 1.33 (m, 2H).
A solution of ( )-N-(6-(tert-butoxycarbonylamino)hexanoyl)-trans-
3-(2-nitrobenzenedisulfido)-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (78 mg, 0.11 mmol) in trifluoroacetic acid (1 mL) was
stirred at room temperature for 1 h. The trifluoroacetic acid was
evaporated under vacuum, and the residue was dissolved in CH2Cl2 (1
mL). The solution was cooled to 0 °C, and Et3N (34 μL, 0.244 mmol)
and acetyl chloride (8.6 μL, 0.12 mmol) were added. The mixture was
stirred for 2 h. The solution was evaporated under vacuum. The
residue was dissolved in ethyl acetate (5 mL), washed with water, dried
over Na2SO4, and the solvent was evaporated. The residue was purified
by flash chromatography (100% ethyl acetate) to give ( )-N-(6-
acetamidohexanoyl)-trans-3-(2-nitrobenzenedisulfido)-4-(4-
phenoxybenzenesulfonamido)pyrrolidine (68 mg, 94%): 1H NMR
(300 MHz, CD3OD) δ 8.20−8.33 (m, 2H), 7.76 (m, 3H), 7.47 (m,
3H), 7.25 (t, J = 8, 1H), 7.10 (d, J = 9, 2H), 6.99 (m, 2H), 3.92 (m,
2H), 3.68−3.86 (m, 2H), 3.52 (m, 1H), 3.39 (m, 1H), 3.14 (m, 2H),
2.24 (m, 2H), 1.93 and 1.92 (s, 3H), 1.54 (m, 4H), 1.34 (m, 2H). To a
solution of the 2-nitrobenzene disulfide (10 mg, 0.015 mmol) in
THF−H2O (4:1, 1 mL) were added TCEP (5 mg, 0.017 mmol) and 1
N NaOH (20 μL). The mixture was stirred until no starting material
was detected by TLC (100% ethyl acetate). The solvent was
To a solution of ( )-N-(2-phthalimidoethylaminocarbonyl)-trans-3-
tert-butylmercapto-4-(4-phenoxybenzenesulfonamido)pyrrolidine
(62.3 mg, 0.10 mmol) in AcOH (1 mL) was added 2-nitro-
benzenesulfenyl chloride (21 mg, 0.11 mmol), and the mixture was
stirred at room temperature for 2 h. The AcOH was evaporated under
vacuum, and to the residue were added THF (2 mL), 1 N NaOH (0.2
mL), and TCEP (34 mg, 0.12 mmol). The mixture was stirred at room
temperature under argon for 3 h. Then it was concentrated under
vacuum. To the residue was added ethyl acetate, and the solution was
dried over Na2SO4 and filtered. The solvent was evaporated and the
M
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evaporated under reduced pressure and the residue was purified by
short flash column chromatography (100% ethyl acetate) to give the
mercaptan 29e (7 mg, 91%): H NMR (300 MHz, CD3OD) δ 7.88
reduced pressure. Ethyl acetate (20 mL) was added to the residue, and
the solution was washed with brine, dried over Na2SO4, and
evaporated. The residue was purified by flash column chromatography
(50% ethyl acetate in hexane) to give (3R,4R)-trans-N-Boc-3-hydroxy-
4-azidopyrrolidine (32): 1H NMR (300 MHz, CDCl3) identical to that
of the (3S,4S) isomer; [α]2D0 −26.8° (c 1.0, CHCl3).
A sample of the (3S,4S)-trans-N-Boc-3-hydroxy-4-azidopyrrolidine
(30) (80 mg, 0.35 mmol) was dissolved in trifluoroacetic acid (1 mL)
and was stirred until no starting material was detected on TLC (25%
ethyl acetate in hexane). The solution was evaporated under reduced
pressure, and the residue was dissolved in CH2Cl2 (2 mL). The
solution was cooled to 0 °C. Et3N (117 μL, 0.84 mmol) and Cbz-Cl
(60 mg, 0.35 mmol) were added, and the resulting solution was stirred
for 2 h at room temperature. The reaction mixture was washed with
water, dried over Na2SO4, and evaporated. The residue was purified by
flash chromatography (25% ethyl acetate in hexane) to give (3S,4S)-
trans-N-Cbz-3-hydroxy-4-azidopyrrolidine (89 mg, 97%): [α]2D0 +16.1°
(c 1.00, CHCl3) (lit.:64 [α]D25 +14.3° (c 1.06, CHCl3)).
1
(m, 2H), 7.45 (t, J = 8, 2H), 7.25 (t, J = 8, 1H), 7.10 (m, 4H), 3.81−
3.98 (m, 2H), 3.53−3.72 (m, 2H), 3.05−3.40 (m, 4H), 2.26 (m, 2H),
1.92 (s, 3H), 1.46−1.65 (m, 4H), 1.30−1.41 (m, 2H); HRMS (ESI)
+
calculated for C24H32N3O5S2 506.1778, found 506.1786.
To a solution of ( )-N-(6-(tert-butoxycarbonylamino)hexanoyl)-
trans-3-(2-nitrobenzenedisulfido)-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (50 mg, 0.7 mmol) in THF−H2O (4:1, 1 mL) were added
TCEP (23 mg, 0.8 mmol) and 1 N NaOH (100 μL). The mixture was
stirred until no starting material was detected by TLC (50% ethyl
acetate in hexane). The solution was evaporated under reduced
pressure, and the residue was purified by short flash column
chromatography (50% ethyl acetate in hexane) to give the thiol.
The latter was dissolved in 2 M HCl in acetic acid (1 mL), and the
mixture was stirred at room temperature for 1 h. Liophilization of the
solution gave the mercaptan 29f as a white hygroscopic solid (33 mg,
1
(3S,4S)-trans-3-Mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine Hydrochloride (39a). A solution of (3S,4S)-trans-N-Boc-3-
hydroxy-4-azidopyrrolidine (30) (850 mg, 3.72 mmol) and Et3N (622
μL, 4.46 mmol) in CH2Cl2 (10 mL) was cooled with stirring to 0 °C,
and methanesulfonyl chloride (331 μL, 4.28 mmol) was added
dropwise. The solution was stirred at room temperature until no
starting material was detected by TLC (25% ethyl acetate in hexane).
The reaction mixture was washed with 10% aqueous citric acid and
water, then was dried over Na2SO4 and evaporated. The residual crude
methanesulfonate was dissolved in anhydrous DMF (20 mL).
Potassium acetate (1.10 g, 11.2 mmol) was added, and the mixture
was stirred at 105 °C under N2 for 6 h. The reaction mixture was
poured into ice−water and was extracted with ethyl acetate. The
organic layer was washed with brine, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography (25%
ethyl acetate in hexane) to give (3R,4S)-cis-N-Boc-3-acetoxy-4-
94%): H NMR (300 MHz, CD3OD) δ 7.88 (m, 2H), 7.45 (t, J = 8,
2H), 7.25 (t, J = 8, 1H), 7.10 (m, 4H), 3.84−3.97 (m, 2H), 3.71 (dd, J
= 12, 7, 1H), 3.52−3.64 (m, 2H), 3.06−3.18 (m, 1H), 2.93 (br m,
2H), 2.31 (q, J = 7, 2H), 1.66 (br m, 4H), 1.42 (br m, 2H); HRMS
+
(ESI) calculated for C22H30N3O4S2 464.1672, found 464.1672.
(
) - N - ( 6 - A mi n o h e xa no y l ) - t r a n s - 3 - h y d r o x y - 4 - ( 4 -
phenoxybenzenesulfonamido)pyrrolidine Hydrochloride (29g). To
a stirred solution of 1.5 M HCl in ethyl acetate (3 mL) was added
( )-N-Boc-trans-3-hydroxy-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (25) (430 mg, 0.99 mmol), and stirring was continued
until no starting material was detected by TLC (50% ethyl acetate in
hexane). The solvent was evaporated under reduced pressure, and the
residue was dissolved in CH2Cl2. The solution was cooled to 0 °C, and
Et3N (153 μL, 1.1 mmol), HOBt·H2O (135 mg, 1.0 mmol), 6-(tert-
butoxycarbonylamino)hexanoic acid (231 mg, 1.0 mmol), and DCC
(206 mg, 1.0 mmol) were added. The mixture was stirred at room
temperature overnight. The solvent was evaporated under reduced
pressure, and the residue was dissolved in ethyl acetate (10 mL). The
organic layer was washed with 10% citric acid and saturated aqueous
NaHCO3. Then it was dried over Na2SO4 and evaporated. The residue
was purified by flash chromatography (50% ethyl acetate in hexane) to
give ( )-N-(6-(tert-butoxycarbonylamino)hexanoyl)-trans-3-hydroxy-
4-(4-phenoxybenzenesulfonamido)pyrrolidine (400 mg, 74%). The
latter was dissolved in 2 M HCl in acetic acid (3 mL), and the mixture
was stirred at room temperature for 1 h. Liophylization of the solution
1
azidopyrrolidine (33) (860 mg, 86%): H NMR (300 MHz, CDCl3)
δ 5.31 (br m, 1H), 4.07 (m, 1H), 3.60−3.71 (m, 2H), 3.36−3.50 (m,
2H), 2.16 (s, 3H), 1.46 (s, 9H); [α]2D0 −34.5° (c 1.00, CHCl3).
To a stirred solution of the (3R,4S)-azidoacetate 33 (560 mg, 2.07
mmol) in THF−MeOH (3:1, 8 mL) at 0 °C was added 1 N LiOH (3
mL) dropwise. The reaction mixture was stirred at room temperature
overnight. Then it was evaporated under reduced pressure. Ethyl
acetate (20 mL) was added to the residue, and the solution was
washed with brine, dried over Na2SO4, and evaporated. The residue
was purified by flash chromatography (50% ethyl acetate in hexane) to
give (3R,4S)-cis-N-Boc-3-hydroxy-4-azidopyrrolidine (34) (460 mg,
gave
( )-N-(6-aminohexanoyl)-trans-3-hydroxy-4-(4-
phenoxybenzenesulfonamido)pyrrolidine hydrochloride (29g) as a
white hygroscopic solid (353 mg, 100%): 1H NMR (300 MHz,
CD3OD) δ 7.86 (dd, J = 9, 3, 2H), 7.46 (t, J = 8, 2H), 7.25 (t, J = 8,
1H), 7.10 (m, 4H), 4.24 and 4.12 (m, 1H), 3.70−3.76 (m, 1H), 3.47−
3.63 (m, 3H), 3.34−3.42 (m, 1H), 2.93 (br t, J = 7, 2H), 2.35 and 2.31
(t, J = 7, 2H), 1.67 (m, 4H), 1.44 (m, 2H); HRMS (ESI) calculated for
C22H30N3O5S+ 448.1906, found 448.1904.
1
97%): H NMR (300 MHz, CDCl3) δ 4.35 (br m, 1H), 4.02 (br m,
1H), 3.25−3.72 (m, 4H), 2.16 (br s, 1H), 1.46 (s, 9H); [α]2D0 +32.3° (c
0.69, CHCl3).
To a solution of the (3R,4S)-azido alcohol 34 (470 mg, 2.06 mmol)
in THF−H2O (10:1, 10 mL) was added Ph3P (540 mg, 2.06 mmol),
and the mixture was stirred at room temperature for 2 h and at 65 °C
until no starting material was detected on TLC (25% ethyl acetate in
hexane). The solution was cooled with stirring to 0 °C, and Et3N (345
μL, 2.48 mmol) was added. Then a solution of 4-phenoxybenzene-
sulfonyl chloride (529 mg, 2.26 mmol) in THF (2 mL) was added
dropwise. The reaction mixture was warmed to room temperature and
was stirred for 3 h. The mixture was concentrated under reduced
pressure. Ethyl acetate was added to the residue, and the organic layer
was washed with 10% aqueous citric acid and saturated aqueous
NaHCO3. Then it was dried over Na2SO4 and evaporated. The residue
was purified by flash chromatography (50% ethyl acetate in hexane) to
give (3R,4S)-cis-N-Boc-3-hydroxy-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (35) (670 mg, 75%): 1H NMR (300 MHz, CDCl3) δ 7.83
(d, J = 7, 2H), 7.42 (t, J = 8, 2H), 7.24 (t, J = 8, 1H), 7.06 (m, 4H),
5.14 (br s, 1H), 4.25 and 4.11 (br s, 1H), 3.76 (m, 1H), 3.55 (m, 1H),
3.43 (m, 2H), 3.07 (m, 1H), 2.36 and 2.23 (br s, 1H), 1.43 (s, 9H);
[α]2D0 +15.6° (c 0.80, CHCl3).
Enzymatic Resolution of ( )-N-Boc-trans-3-hydroxy-4-azidopyr-
rolidine (Scheme 4). To a solution of ( )-N-Boc-trans-3-hydroxy-4-
azidopyrrolidine (24) (2.28 g, 10 mmol) in tert-butyl methyl ether (50
mL) were added isopropenyl acetate (3 g, 3 mmol) and lipase Amano
AK-20 (3 g), and the mixture was stirred at room temperature for 72
h. The mixture was filtered. The filtrate was evaporated under reduced
pressure, and the product was separated by flash chromatography
(25% ethyl acetate in hexane). First eluted was (3R,4R)-trans-N-Boc-3-
acetoxy-4-azidopyrrolidine (31) (1.30 g): 1H NMR (300 MHz,
CDCl3) δ 5.09 (br s, 1H), 4.04 (m, 1H), 3.57−3.74 (m, 2H), 3.34−
3.55 (m, 2H), 2.09 (s, 3H), 1.47 (s, 9H); [α]2D0 −30.4° (c 1.0, CHCl3).
Second to be eluted was (3S,4S)-trans-N-Boc-3-hydroxy-4-azido-
1
pyrrolidine (30) (1.07 g): H NMR (300 MHz, CDCl3) δ 4.25 (br s,
1H), 3.93 (br s, 1H), 3.55−3.75 (m, 2H), 3.30−3.50 (m, 2H), 2.05 (br
s, 1H), 1.46 (s, 9H); [α]2D0 +26.8° (c 0.5, CHCl3).
To a stirred solution of (3R,4R)-trans-N-Boc-3-acetoxy-4-azidopyr-
rolidine (31) (1.0 g, 3.4 mmol) in THF−MeOH (3:1, 8 mL) at 0 °C
was added 1 N LiOH (4 mL) dropwise. The reaction mixture was
stirred at room temperature overnight. Then it was evaporated under
To a stirred solution of Ph3P (525 mg, 2.0 mmol) in THF (3 mL)
at 0 °C was added DEAD (315 μL, 2.0 mmol) dropwise. After 10 min,
a solution of alcohol 35 (435 mg, 1.0 mmol) and thiolacetic acid (228
N
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Journal of Medicinal Chemistry
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μL, 2.29 mmol) in THF (3 mL) was added. The resulting reaction
mixture was stirred at room temperature for 6 h. Then it was
concentrated under reduced pressure. The residue was purified by
flash chromatography (15% ethyl acetate in hexane) to give (3S,4S)-
trans-N-Boc-3-acetylthio-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (36) (400 mg, 81%): 1H NMR (300 MHz, CDCl3) δ 7.80
(d, J = 9, 2H), 7.42 (m, 2H), 7.23 (t, J = 8, 1H), 7.06 (m, 4H), 5.21
and 5.00 (br s, 1H, NH), 3.75 (m, 4H), 3.16 (m, 2H), 2.31 and 2.25
(br s, 3H), 1.44 (s, 9H); [α]2D0 +22.8° (c 0.62, CHCl3).
To a stirred solution of the acetylthiopyrrolidine 36 (60 mg, 0.122
mmol) in MeOH (1 mL) under nitrogen was added 40% aqueous
MeNH2 (100 μL, 1.16 mmol), and the mixture was stirred for 10 min.
The solution was concentrated under vacuum and the residue was
purified by flash chromatography (50% ethyl acetate in hexane) to give
(3S,4S)-trans-N-Boc-3-mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (37) (54 mg, 98%) as an oil: 1H NMR (300 MHz,
CD3OD) identical to that of the ( )-modification described earlier;
[α]2D0 +39.2° (c 0.54, MeOH).
a confluency of 75% was reached. At this point varying dosages of
inhibitor (maintaining a constant final DMSO concentration) were
used to supplement fresh medium and added to the cells for a period
of 24 h. The logarithmic panel of inhibitor concentrations ranged from
100 to 1 μM. At the end of the treatment period the inhibitor-
conditioned medium was removed and the cells were trypsinized
(0.25% trypsin, Hyclone) after being washed with phosphate-buffered
saline (pH 7.4).
To quantify the levels of toxicity these compounds elicited on
hMSC growth, cells were pelleted by centrifugation at 450g and
resuspended in 1 mL of medium. An aliquot of the suspension was
then diluted 1:1 with a 0.4% (w/v) trypan blue solution and counted
with a hemocytometer. Determined by normalization of the number of
cells counted for each treatment against its respective control, the
relative groups were plotted with error bars representing the standard
error in the mean (mean
SEM). Each of the treatments and
hemocytometer counts were performed in triplicate, and compounds
at a particular concentration killing more than 20% of the cells were
deemed cytotoxic.
To a solution of 2.0 M HCl in AcOH (1 mL) was added (3S,4S)-
trans-N-Boc-3-mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine (37) (45 mg, 0.10 mmol), and the mixture was stirred at
room temperature for 1 h. Liophilization of the reaction mixture gave
the amine hydrochloride 39a as a white hygroscopic solid (37 mg,
100%): 1H NMR (300 MHz, CD3OD) identical to that of the racemic
inhibitor 29a; [α]2D0 +21.9° (c 0.7, MeOH); HRMS (ESI) calculated
AUTHOR INFORMATION
■
Corresponding Author
* Phone: +1 (850) 644-8683. E-mail: qxsang@chem.fsu.edu.
Author Contributions
+
for C16H19N2O3S2 351.0832, found 351.0824.
†Y.J. and M.D.R. are shared first authors.
(3R,4R)-3-Mercapto-4-(4-phenoxybenzenesulfonamido)-
pyrrolidine Hydrochloride (39b). By the procedures used for the
3S,4S enantiomer, (3R,4R)-trans-N-Boc-3-hydroxy-4-azidopyrrolidine
was converted to the title inhibitor 39b as a white hygroscopic solid:
1H NMR (300 MHz, CD3OD) identical to those of 29a and 39a; [α]2D0
Y.J. performed compound synthesis and characterization.
M.D.R. was responsible for cell culture, cytotoxicity, and
stability results. D.B.B., Q.C., and M.H.C. determined
inhibition constants and IC50 values. M.A.S. and Q.-X.A.S.
conceived the study and experimental designs, and Y.J., M.D.R.,
M.A.S., and Q.-X.A.S. wrote the manuscript.
+
−21.0° (c 1.0, MeOH); HRMS (ESI) calculated for C16H19N2O3S2
351.0832, found 351.0820.
Enzyme Kinetics. Enzymatic assays to characterize inhibitor
potency were performed as previously described.17,65 Assays were
consistently carried out at 25 °C in 50 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer, pH 7.5, with 10 mM
CaCl2, 200 mM NaCl, and 0.01% (w/v) Brij-35. The reducing agent
TCEP was also included at 5 μM to maintain the fully reduced form of
the MMPIs. Briefly, 10 μL of varying concentrations of MMPIs
dissolved in DMSO, 176 μL of HEPES buffer, and 10 μL of enzyme
stock solution were mixed and incubated for 30 min. The assay was
started by addition of 4 μL of a 1 μM solution of the synthetic
substrate Mca-PLGLDpa-AR-NH2 (M-1895, Bachem)66 in 50%
DMSO, and the release of product was monitored by fluorescence
(λex = 328 nm and λem = 393 nm) with a PerkinElmer luminescence
spectrophotometer LS50B (FL WinLab, version 3.0) connected to a
temperature-controlled water bath. Enzyme concentrations ranged
from 0.2 to 7 nM during the assays.
Stability Assays. To assess the relative stability of the thiol
inhibitors over time, the inhibitory potencies of these compounds were
assessed at varying time points. The compounds were initially
dissolved in DMSO and diluted in cell culture medium (containing
either 20%, 10%, or 0% fetal bovine serum) to a concentration
approximately 10 000 times their IC50 value. They were then incubated
in a humidified environment at 37 °C and 5% CO2 for varying periods
up to 72 h. After the initial incubation period, 10 μL of the inhibitor
solution was further incubated with 176 μL of HEPES buffer and 10
μL of MMP-9 enzyme (final concentration of ∼1 nM) for 30 min
prior to initiation of the assay. The relative rates (vi/vo) for each
compound at its various time points were obtained in triplicate as
previously described and plotted versus time to indicate their
corresponding stability.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by grants from the
Department of Defense (DoD) Prostate Cancer Research
Program (Grants DAMD17-02-1-0238 and W81XWH-07-1-
0225), Florida Department of Health James and Esther King
Biomedical Research Program (Grant 1KF04), and Florida
State University (to Q.-X.A.S.), by Grants 1 R21 NS066418
and 2 R01 NS045847-05A2 from the National Institutes of
Health (NIH) (to Gary A. Rosenberg and Q.-X.A.S.), and by
the Molecular Design and Synthesis (MDS) Foundation (to
M.A.S.). The authors gratefully acknowledge Dr. Umesh Goli,
Director of the MASS/BASS Facilities at Florida State
University, for his assistance in mass spectrometry character-
ization of the compounds.
ABBREVIATIONS USED
■
DEAD, diethyl azodicarboxylate; ECM, extracellular matrix;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
hMSC, human mesenchymal stem cell; MMPI, matrix metal-
loproteinase inhibitor; TCEP, tris(2-carboxyethyl)phosphine;
TIMP, tissue inhibitor of metalloproteinase; TMSNCO,
trimethylsilyl isocyanate; ZBG, zinc-binding group
Cell Culture and Compound Toxicity. Low passage hMSCs
were routintely cultured in α-modified minimum essential medium
(αMEM) supplemented with L-glutamine, penicillin, streptomycin,
and 20% fetal bovine serum. Prior to the initial seeding of the cells at
low density (∼60 cells/cm2), cells were plated for 24 h of recovery and
maintained at 37 °C in a humidified atmosphere containing 5% CO2.
The addition of fresh culture medium was provided every 3 days until
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