δ-thiolactones as prodrugs of thiol-based glutamate carboxypeptidase II (GCPII) inhibitors

Article abstract of DOI:10.1021/jm401703a

δ-Thiolactones derived from thiol-based glutamate carboxypeptidase II (GCPII) inhibitors were evaluated as prodrugs. In rat liver microsomes, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, 1) was gradually produced from 3-(2-oxotetrahydrothiopyran-3-yl)propionic acid (5), a thiolactone derived from 1. Compound 1 was detected in plasma at concentrations well above its IC ₅₀ for GCPII following oral administration of 5 in rats. Consistent with the oral plasma pharmacokinetics, thiolactone 5 exhibited efficacy in a rat model of neuropathic pain following oral administration.

Full text of DOI:10.1021/jm401703a

Brief Article
pubs.acs.org/jmc
δ‑Thiolactones as Prodrugs of Thiol-Based Glutamate
Carboxypeptidase II (GCPII) Inhibitors
Dana V. Ferraris,†,§ Pavel Majer,§,∥ Chiyou Ni,§ C. Ethan Slusher,† Rana Rais,† Ying Wu,†,§
Krystyna M. Wozniak,†,§ Jesse Alt,†,§ Camilo Rojas,†,§ Barbara S. Slusher,†,‡,§
and Takashi Tsukamoto*,†,‡,§
‡
†Brain Science Institute and Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21205, United States
§Eisai Inc., Baltimore, Maryland 21224, United States
∥Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo N. 2, 166 10, Prague 6,
Czech Republic
ABSTRACT: δ-Thiolactones derived from thiol-based gluta-
mate carboxypeptidase II (GCPII) inhibitors were evaluated as
prodrugs. In rat liver microsomes, 2-(3-mercaptopropyl)-
pentanedioic acid (2-MPPA, 1) was gradually produced from
3-(2-oxotetrahydrothiopyran-3-yl)propionic acid (5), a thio-
lactone derived from 1. Compound 1 was detected in plasma
at concentrations well above its IC50 for GCPII following oral
administration of 5 in rats. Consistent with the oral plasma
pharmacokinetics, thiolactone 5 exhibited efficacy in a rat
model of neuropathic pain following oral administration.
diverse scaffolds presented by the thiol-based compounds than
other series. For instance, rigorous SAR studies of thiol-based
GCPII inhibitors led to the discovery of 3-(2-mercaptoethyl)-
biphenyl-2,3′-dicarboxylic acid 2 (E2072) containing a biphenyl
scaffold distinct from that of 1.6 Compound 2 was found to
inhibit GCPII with much higher potency (IC50 = 2 nM) than 1.
Compound 2 showed significantly improved potency over 1 in
a preclinical model of neuropathic pain following oral
administration, presumably because of its enhanced GCPII
inhibitory potency coupled with the improved oral pharmaco-
kinetic properties.7
From a drug development perspective, however, there has
been a reluctance to pursue thiol-containing compounds as
therapeutic agents. Unlike other zinc-binding groups, the thiol
group is relatively nucleophilic and prone to oxidation. These
chemical properties compromise the metabolic stability and
increase the risk of inducing immune reactions when conjugates
are formed with endogenous proteins. Indeed, some of the
adverse reactions reported for captopril are believed to be due
in large part to its thiol group.8 In addition, a more immediate
concern lies with the complexity involved in the development
of consistent processes to produce thiol compounds of high
quality free from the corresponding homodisulfide impurities.
Furthermore, the instability of thiol-containing compounds
often presents a challenge to identifying a stable formulation
with an acceptable shelf life.
INTRODUCTION
■
Glutamate carboxypeptidase II (GCPII) is a membrane-bound
binuclear zinc metallopeptidase that cleaves N-acetylaspartyl-
glutamate (NAAG) into N-acetylaspartate (NAA) and
glutamate (Glu) in the extracellular space of the nervous
system. Inhibition of GCP II has gained considerable attention
as an alternative therapeutic approach to blocking postsynaptic
glutamate receptors for treating neurodegenerative disorders
associated with glutamate excitotoxicity.
Among a variety of GCPII inhibitors reported to date,1−3
thiol-based inhibitors have shown promising pharmacological
profiles in preclinical studies (Figure 1). For example, 2-(3-
mercaptopropyl)pentanedioic acid 1 (2-MPPA) represents the
first orally active GCPII inhibitor with an IC50 of 90 nM.4
Compound 1 showed efficacy in a variety of preclinical animal
models by oral administration.5 Further structural optimization
studies revealed that GCPII is more tolerant of structurally
Received: November 4, 2013
Published: December 19, 2013
Figure 1. Chemical structures of 1−6.
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
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One approach to circumventing some of the issues associated
with thiol-containing drugs is to explore prodrugs in which the
thiol group is protected in the form of a metabolically cleavable
thioester. For instance, M100240 (3) is a thioacetyl derivative
of MDL 100,173 (4), a dual angiotensin-converting enzyme
(ACE)/neutral endopeptidase (NEP) inhibitor (Figure 1). Oral
administration of 3 to healthy subjects resulted in the
substantial plasma exposure to 4 while significantly lower
plasma levels of 3 were detected,9 suggesting rapid in vivo
hydrolysis of the thioester moiety of 3.
A common structural feature shared by nearly all potent
thiol-based GPCII inhibitors is the presence of a 5-
mercaptopentanoic acid backbone. This feature allows us to
explore δ-thiolactones as potential prodrugs of thiol-based
GCPII inhibitors. Such an approach may offer more stable
forms of the drugs by temporally masking a reactive thiol group
and yet rapidly generating the parent compounds in vivo.
Herein we report the synthesis and pharmacological
evaluations of δ-thiolactones 5 and 6 derived from two
structurally distinct thiol-based GCPII inhibitors, 1 and 2
(Figure 1).
mixture by 1H and 13C NMR confirmed the formation of 10 as
a byproduct.
In the enzyme assay using N-acetyl-L-aspartyl[3H]-L-gluta-
mate as a substrate and purified human recombinant GCPII,12
5 and 6 showed substantially weaker inhibitory potency
compared to the parent compounds with IC50 values of 2
and 20 μM, respectively. The reduced potency can be
attributed to the loss of the zinc-binding thiol group and the
key α-carboxylate group interacting with the glutamate
recognition site of GCPII.
To assess the ability of 5 and 6 to serve as prodrugs, release
of the parent compounds was investigated in simulated gastric
fluid (pH 1.2) and rat liver microsomes. Neither 5 nor 6
generated the parent compounds even after an extended period
of incubation (48 h) in simulated gastric fluid at 37 °C. As
shown in Figure 2, however, time-dependent formation of 1
RESULTS
■
As illustrated in Scheme 1, δ-thiolactone 5 was synthesized by
refluxing a solution of 1 in the presence of p-toluenesulfonic
a
Scheme 1. Synthesis of δ-Thiolactones 5 and 6
Figure 2. Formation of 1 from 5 in rat liver microsomes. Values and
error bars represent the mean and standard deviation of triplicate
measurements.
from 5 was observed in rat liver microsomes. Because of the
weak intensity of the parent ion, a reliable bioanalytical method
to quantify 5 was not successfully developed. Consequently, we
could not determine the stoichiometric balance between
prodrug 5 and the parent 1. Thus, the possibility of 5 forming
other metabolites besides 1 cannot be ruled out. Nevertheless,
gradual generation of 1 in rat liver microsomes indicates the
potential of δ-thiolactone 5 to act as a prodrug of 1. In contrast,
δ-thiolactone 6 was completely stable in the rat liver
microsomes over 1.5 h (data not shown). Although speculative,
it is conceivable that a sterically hindered environment around
the thioester moiety of 6 renders it resistant to hepatic
esterases.
a
Reagents and conditions: (a) PTSA, toluene, reflux; (b) BnBr, 4%
In light of the rat liver microsomal stability data, we chose to
conduct a preliminary in vivo pharmacokinetics study (n = 2,
four time points) of δ-thiolactone 5 in rats to determine if it
serves as a prodrug of 1. As shown in Figure 3, following oral
administration of δ-thiolactone 5, 1 was detected in plasma at
concentrations well above its IC50 for GCPII. Among four time
points (t = 0.25, 0.5, 1, and 3 h), plasma levels of 1 were highest
at the earliest time point. Although its levels were nearly 7-fold
lower than plasma levels of 1 at the same time point following
oral administration of 1, the two curves become nearly identical
after 1 h.
δ-Thiolactone 5 was subsequently tested for its antinoci-
ceptive effects following oral administration (10 mg kg−1 day−1)
using the rat chronic constriction injury model of neuropathic
pain.13 As shown in Figure 4, oral administration of 5 resulted
in significant reduction of thermal hyperalgesia relative to the
vehicle-treated control on days 11, 16, and 18.
NaOH, EtOH, rt; (c) TFAA, reflux.
acid using Dean−Stark apparatus. All attempts to synthesize δ-
thiolactone 6 from 2 via acid-catalyzed condensation failed,
with only unreacted starting material recovered. While
searching for alternative methods to form δ-thiolactones, we
came across an unusual route to thiolactones through S-benzyl
sulfide intermediates.10,11 To test this procedure, 2 was
converted into the corresponding S-benzyl sulfide 7. Cycliza-
tion of 7 in refluxing trifluoroacetic anhydride successfully
provided δ-thiolactone 6. This cyclization reaction presumably
proceeds through an initial formation of the corresponding
anhydride 8, followed by its conversion to the sulfinium
intermediate 9. Subsequent rearrangement of 9 through a six-
membered transition state produces δ-thiolactone 6 and benzyl
trifluoroacetate 10. Indeed, careful analysis of the reaction
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Journal of Medicinal Chemistry
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The degree of antinociceptive effect appears less than that
following oral treatment with 1 (10 mg/kg) reported
previously.4 The inferior efficacy of δ-thiolactone 5 could be
attributed to the significantly lower plasma exposure to 1 at the
early time points when 5 is given orally as shown in Figure 3.
This is in a good agreement with the previously reported PK/
PD analysis, which suggests that Cmax rather than AUC is the
key determinant of in vivo efficacy in this particular preclinical
model.14 As mentioned above, the animals might be exposed to
an enantiomerically enriched form of 1 because of enantiose-
lective hydrolysis of δ-thiolactones 5. This, however, should
have little implication for efficacy, since the two enantiomers of
1 were found to show similar degree of in vivo efficacy in this
animal model.15
Although δ-thiolactone 5 did not offer any particular
pharmacological advantages over the parent 1, the δ-
thiolactone-based prodrug approach should represent an
attractive option from chemistry, manufacturing, and controls
(CMC) perspective, as it alleviates issues associated with
handling of thiol-containing drug substances. While the δ-
thiolactone-based prodrug approach appears promising, one
must be aware of the potential safety risk associated with
thiolactones given the well-documented reactivity of homo-
cysteine thiolactone with endogenous proteins.16 The striking
difference in stability between 5 and 6 suggests varying degrees
of reactivity within δ-thiolactones. Selecting the most suitable
thiol-containing parent compound is therefore the crucial step
in this particular prodrug approach. It is worth noting that the
5-mercaptopentanoic acid scaffold has been embedded in a
variety of thiol-based GCPII inhibitors,4,6,17,18 which can be
exploited in the search for δ-thiolactone prodrugs with superior
pharmacological properties.
□
Figure 3. Plasma concentration versus time profiles of 1 ( ) in rats
following oral administration of δ-thiolactone 5 (10 mg/kg). Plasma
△
concentration versus time curve of 1 ( ) following oral administration
of 1 (10 mg/kg) is superimposed for comparison.
EXPERIMENTAL SECTION
■
General. All solvents were reagent grade or HPLC grade. Unless
otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Compounds 1 and 2
were prepared as previously described.4,6 All reactions were performed
under nitrogen. The analytical HPLC conditions used a gradient of 5%
ACN/95% H2O (both containing 0.1% formic acid) for 0.25 min
followed by an increase to 40% ACN/60% H2O over 1.75 min and
continuation of 40% ACN/60% H2O (containing 0.1% formic acid)
until 4 min (detection at 220 nm) with a Luna C18 column (5 μm,
150 mm × 4.6 mm). All solvents were reagent grade or HPLC grade.
Melting points were obtained on a Mel-Temp apparatus and are
Figure 4. Antinociceptive effects of 5 in the rat chronic constriction
injury (CCI) model of neuropathic pain. Oral administration of 5 at 10
mg kg−1 day−1 significantly attenuated CCI-induced hyperalgesic state
relative to the vehicle-treated control (∗, p < 0.05; ∗∗, p < 0.01).
DISCUSSION AND CONCLUSION
■
1
uncorrected. H NMR spectra were recorded at 400 MHz. 13C NMR
While both δ-thiolactones 5 and 6 were completely stable in
simulated gastric fluid, the two compounds showed different
degrees of microsomal stability, presumably because of the
preference of hepatic esterases for 5 over the sterically hindered
thiolactone 6.
spectra were recorded at 100 MHz. Elemental analyses were obtained
from Atlantic Microlabs, Norcross, GA. The purity of test compounds
was confirmed by elemental analysis (within 0.4% of the calculated
value).
3-(2-Oxotetrahydro-2H-thiopyran-3-yl)propanoic Acid (5). A
solution of 1 (0.530 g, 2.57 mmol) and 10-camphorsulfonic acid
(0.120 g, 0.52 mmol) in toluene (60 mL) was stirred at the refluxing
temperature. The water formed in the reaction was removed by
Dean−Stark apparatus. After 6 h, excess solvent was removed and the
residual oil was purified by silica gel chromatography (hexanes/EtOAc,
4:1) to give 0.187 g of 5 as a white solid (35% yield): mp 80−82 °C;
1H NMR (DMSO-d6) δ 1.48−1.65 (m, 2H), 1.86−2.07 (m, 4H), 2.26
(t, J = 7.5 Hz, 2H), 2.59−2.70 (m, 1H), 3.08−3.26 (m, 2H); 13C NMR
(CD3OD) δ 23.34, 27.47, 29.34, 31.29, 32.32, 50.05, 177.13, 206.5.
Anal. Calcd for C8H12O3S: C, 51.04; H, 6.43; S, 17.03. Found: C,
50.77, H, 6.35; S, 17.25.
Although the lack of plasma data of 5 precludes in-depth
interpretation of the given data, the lower plasma levels of 1 at
the early time points in the rat in vivo pharmacokinetics study
may reflect the gradual conversion of 5 to 1 in liver as indicated
by the microsomal stability test. However, lower plasma levels
of 1 also raise the possibility that δ-thiolactone 5 is poorly
bioavailable and that only a portion hydrolyzed to 1 enters the
circulatory system. After 1 h, there is little difference in plasma
levels, if any, of 1 between oral administration of 5 and 1,
indicative of complete loss of δ-thiolactone in plasma by this
time point. Since δ-thiolactone 5 is a racemic mixture, it is also
possible that only one of the enantiomers is preferentially
hydrolyzed, resulting in lower plasma levels of 1 following oral
administration of 5.
3-(1-Oxoisothiochroman-8-yl)benzoic Acid (6). To a solution
of 2 (200 mg, 0.66 mmol) in ethanol (10 mL) were added a 4%
solution of NaOH (3 mL) and benzyl bromide (120 mg, 0.69 mmol)
at 0 °C. The mixture was stirred at rt for 3h. The solvent was removed
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Journal of Medicinal Chemistry
Brief Article
under reduced pressure, and the residue was partitioned between
EtOAc (20 mL) and 1 N HCl (15 mL). The organic layer was dried
over MgSO4 and concentrated to give 7 as an off-white foam. 1H NMR
(CDCl3) δ 2.73−2.88 (m, 2H) 3.03 (m, 2H), 3.79 (s, 2H) 7.23−7.40
(m, 8H), 7.42−7.51 (m, 1H), 7.52−7.60 (m, 1H), 7.70 (dq, J = 7.7,
1.1 Hz, 1H), 8.00 (dt, J = 7.8, 1.4 Hz, 1H), 8.26−8.33 (brs, 1H). The
resulting foam was dissolved in trifluoroacetic anhydride (4.5 mL) and
refluxed at 60 °C for 2 h. Excess solvent was removed, and the residue
was quenched with saturated NaHCO3 at 0 °C. This was followed by
acidification with 1 N HCl and extraction with EtOAc (20 mL × 2).
The combined extracts were dried over MgSO4 and concentrated. The
residual material was purified by silica gel chromatography (EtOAc/
hexanes/AcOH, 1/1/0.02) to give 55 mg of 6 as a white solid (30%
yield): mp 210−212 °C; 1H NMR (DMSO-d6) δ 3.21−3.29 (m, 2H),
3.37−3.44 (m, 2H), 7.30 (d, J = 7.6 Hz, 1H), 7.44−7.54 (m, 3H), 7.60
(t, J = 7.6 Hz, 1H), 7.76 (s, 1H), 7.89 (d, J = 8.3 Hz, 1H), 13.01 (brs,
1H); 13C NMR (DMSO-d6) δ 28.75, 31.55, 127.66, 128.39, 128.83,
129.02, 130.51, 130.73, 130.82, 132.27, 132.74, 140.25, 142.03, 143.03,
167.24, 190.16. Anal. Calcd for C16H12O3S·0.25H2O: C, 66.53; H,
4.36; S, 11.10. Found: C, 66.51; H, 4.40; S, 10.97.
ABBREVIATIONS USED
■
GCPII, glutamate carboxypeptidase II; AUC, area under the
curve
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Chemical Stability Studies in Simulated Gastric Fluid.
Solutions of 5 (4.7 mg/mL in H2O) and 6 (1.4 mg/mL in DMSO)
were prepared. A 50 μL aliquot of each of these solutions was added to
1 mL of simulated gastric fluid (pH 1.2, Ricca Chemical 7108-16).
These mixtures were incubated at 37 °C, and 50 μL aliquots were
taken at 30 min, 1 h, 4 h, and 48 h. The samples were analyzed by
HPLC to quantify the remaining thiolactones and the hydrolyzed
compounds.
Metabolic Stability Studies in the Rat Liver Microsomes.
Test compound (10 μM) was incubated in 100 mM potassium
phosphate buffer, pH 7.4, containing rat liver microsomes (0.5 mg/
mL). The reaction was run in triplicate, and at predetermined times (0,
30, 60, and 90 min) aliquots of the mixture were removed and the
reaction quenched by addition of 2 times the volume of ice cold
acetonitrile spiked with the internal standard (losartan). Compound
appearance/disappearance over time was monitored using a liquid
chromatography and tandem mass spectrometry (LC/MS/MS)
method. Chromatographic separation was performed on an Agilent
Eclipse Plus C18 column (1.8 μm, 100 mm × 2.1 mm) at a flow rate of
0.4 mL/min. The solvent system consisted of distilled water with 0.1%
formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent
B). 10% B was used from 0 to 0.2 min followed by a linear gradient of
10% to 70% B over 1.6 min. Detection was achieved under negative
ion electrospray using the [M − H]− peaks. The multiple reaction
monitoring transitions used for quantification of 1 were m/z 205.501
→ 187.432 and m/z 205.501 → 171.492. For quantification of the
internal standard (losartan), m/z 421.71 →179.414 and m/z 421.71 →
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conducted as previously described.14 The chronic constrictive injury
model of neuropathic pain was performed following the procedure
reported by Bennett’s group.13 A more detailed procedure is described
in the recent article.14
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 410-614-0982. E-mail: ttsukamoto@jhmi.edu.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was in part supported by National Institutes of
Health (Grant R01CA161056 to B.S.S.) and the Johns Hopkins
Brain Science Institute NeuroTranslational Drug Discovery
program.
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