Synthesis, structure-activity relationships and brain uptake of a novel series of benzopyran inhibitors of insulin-regulated aminopeptidase

Article abstract of DOI:10.1021/jm401540f

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) enhance fear avoidance and spatial memory and accelerate spatial learning in a number of memory paradigms. Using a virtual screening approach, a series of benzopyran compounds was identified th

Full text of DOI:10.1021/jm401540f

Article
pubs.acs.org/jmc
Synthesis, Structure−Activity Relationships and Brain Uptake of a
Novel Series of Benzopyran Inhibitors of Insulin-Regulated
Aminopeptidase
Simon J. Mountford,† Anthony L. Albiston,‡ William N. Charman,§ Leelee Ng,∥ Jessica K. Holien,⊥
Michael W. Parker,⊥,# Joseph A. Nicolazzo,○ Philip E. Thompson,*,† and Siew Yeen Chai*,∥
†Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia
‡College of Health and Biomedicine, Victoria University, St. Albans Campus, St. Albans, Victoria 8001, Australia
§Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052,
Australia
∥Department of Physiology, Monash University, Clayton, Victoria 3800, Australia
⊥ACRF Rational Drug Discovery Centre and Biota Structural Biology Laboratory, St. Vincent’s Institute of Medical Research, Fitzroy,
Victoria 3065, Australia
#Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne,
Parkville, Victoria 3010, Australia
○Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria
3052, Australia
ABSTRACT: Peptide inhibitors of insulin-regulated amino-
peptidase (IRAP) enhance fear avoidance and spatial memory
and accelerate spatial learning in a number of memory paradigms.
Using a virtual screening approach, a series of benzopyran
compounds was identified that inhibited the catalytic activity of
IRAP, ultimately resulting in the identification of potent and
specific inhibitors. The present study describes the medicinal
chemistry campaign that led to the development of the lead
candidate, 3, highlighting the key structural features considered as
critical for binding. Furthermore, the in vivo pharmacokinetics and
brain uptake of compounds (1 and 3) were assessed in rats and were complemented with in vitro human and rat microsomal
stability studies. Following intravenous administration to rodents, 3 exhibits brain exposure, albeit it is rapidly converted to 1, a
compound which also exhibits potent inhibition of IRAP.
sequence.13−18 These angiotensin IV mimetics are proposed
to be metabolically more stable than the native peptide, but
optimal blood−brain barrier (BBB) permeability is still an issue
for the development of these compounds as therapeutically
INTRODUCTION
■
Cognitive and memory impairments afflict one-quarter of the
population over 65 years of age and result from a wide range of
clinical conditions including Alzheimer’s disease, brain trauma,
and stroke. Currently, most Food and Drug Administration
approved treatments belong to the class of cholinesterase
inhibitors and have limited efficacy. The M1 aminopeptidase,
useful cognitive enhancers.
We previously reported on small molecule inhibitors of IRAP
identified by elaboration from a virtual screening campaign.19
The inhibitors are benzopyran-based compounds that are
insulin regulated aminopeptidase (IRAP EC 3.4.11.3), is a
specific, competitive inhibitors of IRAP (Figure 1). The
relatively new target for the development of therapies for
memory impairments.1 Peptide inhibitors of IRAP (angiotensin
inhibitors were efficacious in a hippocampal glucose uptake
assay and were demonstrated to enhance the performance of
IV or LVV-hemorphin 7) have been demonstrated to enhance
performance in both fear avoidance2−4 and spatial memory5,6
rats tested in two distinct memory paradigms, the novel object
recognition and spontaneous alternation tasks.19 The current
tasks. More importantly, these peptides reverse performance
deficits induced by global ischemia,7 bilateral perforant pathway
study describes the medicinal chemistry pathway from in silico
lesion,5 perturbations of central cholinergic systems,8−11 or
screening to the current candidate compounds, the structure−
chronic alcohol exposure.12
activity relationships governing their potency in inhibiting IRAP
Peptidomimetic inhibitors of IRAP have been designed based
on β-homo amino acid substitutions, disulfide cyclizations, and
aromatic scaffold replacement of the angiotensin IV
Received: October 24, 2013
© XXXX American Chemical Society
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Figure 1. Structures of the benzopyran based IRAP inhibitors.
Figure 2. Benzopyran based IRAP inhibitors identified by virtual screening.
Scheme 1. Synthetic Pathway for the Production of 4H-Benzopyran Derivatives
Scheme 2. Synthesis of Compounds Derived from the Precursor Amine
activity, and the evaluation of pharmacokinetic properties of
this new class.
develop more potent inhibitors based on modification of this
template.
Highly functionalized 4H-benzopyrans in general have shown
a range of biological activities, and various methods for their
synthesis have been described. Cai and co-workers have
previously prepared a number of 4H-benzopyran derivatives
based on the general synthetic pathway described in Scheme 1
for use as apoptosis inducers,20 including the use of catalysts
such as DABCO21 and TBAB.22 As well as compounds
synthesized in-house as described, compounds 1−4, 13, 15a−f,
16a,f,g,i−n,s,t, 17a−c,e−h, and 18a−f were obtained from
commercial sources or custom synthesis (Epichem Pty Ltd.).
RESULTS AND DISCUSSION
■
Chemistry. Our starting point for the structure−activity
studies was a range of compounds identified by virtual
screening against a homology model of IRAP followed by
searching of the databases for similar core chemical structure
compounds, as previously reported.19 Compound 1 (HFI-
142),19 along with compounds 5−7 (Figure 2), was identified
and had common elements such as the benzopyran nucleus
with 2-amino-4-aryl substitution and demonstration of high
micromolar range affinity. We therefore pursued a campaign to
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Typically, the 4H-benzopyran derivatives are obtained in two
steps, with aromatic aldehydes 8 and nitriles 9 subject to
Knoevenagel condensation in the presence of piperidine to give
the corresponding arylidene-nitrile intermediate 10, followed
by reaction with a substituted phenol 11 to give the desired
products 12 (Scheme 1). Alternatively, a number of the 4H-
benzopyran derivatives obtained were prepared by a one-pot
reaction with the reaction proceeding via the intermediate 10 in
situ. It is worth noting that, while straightforward, the yields of
these syntheses depended heavily on the choice of reaction
conditions, with neither the stepwise nor one-pot approach
offering a generally preferred method. In terms of reliability, it
is suggested that the two-step pathway was preferred to reduce
the complexity of reaction mixtures and ease of isolation of
products.
For example, compound 17d was prepared in a one-pot
reaction from 3-pyridinecarboxaldehyde, t-butyl cyanoacetate,
and resorcinol in 37% yield. In general however, the one-pot
conditions gave yields below 20%. The two-step procedure was
particularly useful when diversifying at the level of the phenol.
The efficient isolation of intermediates such as ethyl 2-cyano-3-
(pyridin-3-yl)acrylate, prepared in 90% yield by the reaction of
3-pyridinecarboxaldehyde and ethyl cyanoacetate in refluxing
toluene, made the subsequent condensation reaction cleaner
and as seen in the case of 1- and 2-naphthol could be
condensed in ethanol at ambient temperature to give 18g and
18h in 65% and 74% yield, respectively.
Table 1. Substitutions at the 3- and 4-Position of the 4H-
Benzopyrans
a
compd
R
Ar
Ki (μM)
1
CO2Et
CO2Et
CN
pyridin-3-yl
1.8
2
quinolin-3-yl
0.36
>100
>100
50
15a
15b
15c
15d
15e
15f
15g
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16q
16r
16s
16t
4-methoxyphenyl
3-methoxyphenyl
3,4-dimethoxyphenyl
3,5-dimethoxyphenyl
3,4,5-trimethoxyphenyl
pyridin-3-yl
CN
CN
CN
>100
>100
>100
>100
>100
>100
2.9
CN
CN
CN
4-N,N-dimethylaminophenyl
phenyl
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
2-cyanophenyl
pyridin-2-yl
quinolin-2-yl
3.0
2-nitrophenyl
42
3-chlorophenyl
3-cyanophenyl
35
3.2
2,4-dichloropyridine-3-yl
4-methylphenyl
4-bromophenyl
4-chlorophenyl
4-cyanophenyl
14
>100
>100
>100
11
Finally, for the synthesis of compounds 13 and 3 (HFI-
419)19 derived from the precursor amine, treatment of 1 with
acetic anhydride selectively yielded the acetate, but extended
treatment with a large excess allowed triacetylation to occur.
Subsequent hydrolysis yielded the monoacetamide (Scheme 2).
The same approach was applied to the synthesis of 4 (HFI-
437).19 Further attempts to modify the amino group of
compound 1, including reductive alkylation, failed to yield any
products.
pyridin-4-yl
3.7
quinolin-4-yl
0.9
4-nitrophenyl
7.7
4-(pyridin-2-yl)phenyl
4-N,N-dimethylaminophenyl
3,4-dimethoxyphenyl
2-thiophene
>100
5.3
6.2
(50)
(50)
2-thiazole
Structure−Activity Relationships. This investigation of
the SAR has been categorized into specific molecular classes.
The results relating to derivatives of 2-amino-4H-chromen-7-ol
with various substituents at the 3- and 4-positions are shown in
Table 1. Of the 3-cyano compounds, the first identified
compound 15c with a 3,4-dimethoxyphenyl substituent was the
only compound in the series to elicit inhibitory activity albeit at
a modest Ki value of 49.7 μM. The Ki values for monomethoxy
substitutions at either the 4- or 3-position (15a,b) could not be
determined under the assay conditions used, likewise with the
3,5-dimethoxy and 3,4,5-trimethoxy analogues (15d,e).
Where an ethyl ester substituent was included in the 3-
position, the inhibitory potencies were much stronger, and
many of the compounds prepared in this series had Ki values of
less than 20 μM. Notably, with the dimethoxyphenyl
substituted compound (15c), the change to an ethyl ester
(16r) yielded an 8-fold increase in potency. The unsubstituted
phenyl derivative (16a) had a Ki value of >50 μM; however, the
nature and placement of substituents on the ring greatly altered
potency. The 3-cyano derivative (16g) had a Ki of 3.2 μM,
while the 4-cyano derivative (16l) had a Ki of 11.4 μM, and the
2-cyano phenyl derivative (16b) was inactive. This trend in
potency was also observed for the 3-chloro derivative (16f)
over the inactive 4-chloro derivative (16k), and likewise, the 4-
nitro derivative (16o) was 5-fold more potent than the 2-nitro
derivative (16e). Compounds that failed to produce a Ki value
in the assay range were the unsubstituted phenyl derivative
a
The numbers in parentheses represent % inhibition at 100 μM.
(16a), and the mono substituted phenyl derivatives 2-cyano
(16b), 4-methyl (16i), 4-bromo (16j), and 4-chloro (16k)
having Ki values >50 μM.
The greatest gains in potency derived from heteroaryl
substitution at the 4-position of the 4H-benzopyrans were with
either pyridine or quinoline groups. The quinoline derivatives,
in general, were more potent than the pyridine derivatives. The
3-quinoline derivative (2) (HFI-435)19 was the most potent of
the series (Ki 0.36 μM) followed by the 4-quinoline derivative
(16n) (Ki 0.9 μM). The most active pyridyl derivative, again
like the quinoline, had the ring nitrogen in the 3-position (1);
however, it was 5-fold less potent than 2. The 2-pyridyl and 2-
quinoline derivatives (16c and 16d, respectively) had very
similar activity (Ki 2.9 and 3.0 μM, respectively) while the 4-
pyridyl derivative (16m) was the least potent of the
unsubstituted heteroaryls examined. The incorporation of the
2,4-dichloro substitution on the 3-pyridyl ring (16h) sup-
pressed activity and was almost 8-fold less potent than 1.
The collected data for compounds 16, give no obvious clue
to the structural basis for activity, with electron-rich and
-deficient substituents in para- or meta-positions capable of
exhibiting inhibitory potency. The series of compounds 16 may
also be able to adopt more than one binding pose to block
enzyme activity,23 and it should be noted that these are all
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racemates. We have previously attempted to rationalize the
binding of compounds 1 (HFI142) and 2 (HFI435) by
molecular docking.23 Overall, the docking suggests the S-
enantiomer will be more active than the R-enantiomer, which is
unable to dock in a reasonable orientation for any active
compound. The S-enantiomer of compound 1 was predicted to
have a direct ring−stack interaction between the benzopyran
rings and Phe544 of IRAP, while the benzopyran oxygen
interacts with the Zn2+ ion. This was supported by mutagenesis
studies that showed sensitivity to mutation at Phe544.23 In that
model, the ester at the 3-position protrudes out of the binding
pocket such that its polar groups are orientated toward the
solvent. This may explain its significant potency advantages
over the cyano compounds (15a−g). The poorly active cyano
compound (15f) was unable to dock in the same pose as 1. The
quinolone derivative 2 cannot be accommodated in this pose
either, but the S-enantiomer readily adopts a flipped pose
where the quinolone nitrogen interacts with the Zn2+ ion. This
demands different interactions with Phe544, but the inhibitor is
still sensitive to mutation.
Table 3. Substitutions at the 7-Position of the 4H-
Benzopyrans
a
compd
R
Ki (μM)
1
7-OH
1.8
13
7-OAc
7-NH2
7-NMe2
7-OMe
(25)
(50)
(25)
(25)
5.6
18a
18b
18c
18d
18e
18f
18g
18h
6-Cl, 7-OH
6-Br, 7-OH
(50)
9.8
8-OH
7−8, fused phenyl
5−6, fused phenyl
>100
>100
a
The numbers in parentheses represent % inhibition at 100 μM.
Given the activity of compound 1, we then examined
variation to the ester functionality (Table 2), while retaining the
the 6-chloro for a 6-bromo (18e) caused complete loss of
activity. Interestingly, shifting the hydroxyl group from the 7-
position to the 8-position (18f) resulted in just a 5-fold loss of
potency.
Table 2. Variation of the Ester at the 3-Position of the 4H-
Benzopyrans
In our model of 1 described above, the 7-OH moiety is
predicted to make a hydrogen bond with Glu295. Substitutions
to this group (13, 18a−c,f−h) results in a significant loss of
potency. The docked solution of 18f suggests that it retains a
hydrogen bond to Glu295, the core shifting slightly in the
binding pocket relative to 1. The distance of the benzopyran
oxygen to the Zn ion is increased which may explain the
modest loss in potency (Figure 3). The requirement for a
phenolic substituent, in contrast to the amino group of 18a or
the ether 18c, suggests that Glu295 has quite specific
requirements for binding ligands. Consistent with that, 18c is
unable to dock into the IRAP binding pocket.
a
compd
R
Ki (μM)
17a
17b
17c
17d
17e
17f
methoxy
propoxy
4.9
1.6
n-butoxy
2.6
t-butoxy
11.9
4.0
2-methoxyethoxy
cyclohexylmethoxy
benzyloxy
(50)
1.7
Finally, variations at the 2-amino group were limited to
acetylation, and this proved to be effective for inhibitory
potency with both the 3-pyridinyl (3) and 3-quinolinyl (4)
substituted compounds (Table 4).
17g
17h
phenyl
(50)
a
The numbers in parentheses represent % inhibition at 100 μM.
The two acetamide compounds (3 and 4) dock into the
IRAP model structure analogously to their parent compounds 1
and 2, resepctively. As previously described, the acetyl group
allows for additional binding contacts in the binding site, albeit
in different positions. In compound 3, the acetamide makes a
second contact with the catalytic zinc ion and in 4 with
aromatic residues Phe550 and Tyr495. Mutagenesis studies give
support to the hypothesis that the pyridinyl and quinolinyl
inhibitors bind in distinct poses. While strikingly different, the
poses predicted by docking seem plausible based upon the
collected SAR data (Figure 4). The solution of cocrystal
structures with IRAP remains an important goal to progress the
development of IRAP inhibitors.
Biopharmaceutical Properties. Four compounds were
selected for further study with respect to selectivity and
biopharmaceutical properties, including solubility, and octa-
nol−water partition coefficients.
Although the substitution of the pyridinyl moiety for a
quinolinyl at position 4 of the benzopyran resulted in an
approximate order of magnitude increase in affinity for IRAP
(i.e., 2 and 4), the aqueous solubility of the inhibitors was
2-amino, 4-(3-pyridyl), and 7-hydroxyl groups. The resultant
compounds showed a reasonable tolerance to changes in this
position, with the methyl ester (17a), propyl ester (17b), n-
butyl ester (17c), methoxyethyl ester (17e), and benzyl ester
(17g) only marginally altering potency in comparison to that of
1. The introduction of more sterically demanding side chains,
such as the t-butyl ester (17d), did cause a decrease in potency
(Ki 11.9 μM), and the cyclohexyl derivative (17f) was found to
be inactive. Replacement of the ester altogether by phenyl
ketone resulted in the inactive derivative (17h).
Modification of the 7-position of the 4H-benzopyrans was
examined, while the 2-amino, 3-ethyl ester and 4-(3-pyridyl)
groups of 1 were retained (Table 3). It was found that the
phenolic group had no effective counterparts. Replacing the 7-
hydroxyl group with 7-amino (18a), 7-dimethylamino (18b), 7-
methoxy (18c), 7-acetyloxy (13), and a benzo ring fused at the
7−8-positions (18g) and the 5−6-positions (18h) all yielded
inactive analogues. Addition of a chloro group at the 6-position
of the 4H-benzopyran ring while retaining the 7-hydroxyl group
(18d) caused a 3-fold decrease in potency, while substituting
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Figure 3. Overlay of docking solutions for 1 (cyan carbons) and compound 18f (purple carbons) in the IRAP homology model (white carbons).
The movement of the hydroxyl group around the ring to the 8-position causes the compound to shift relative to 1. However, the ability to retain a
hydrogen bond to Glu295 may explain the small 5-fold drop in potency. The figure is presented in cross-eyed stereo with the zinc highlighted as a
sphere and residues Phe544 and Glu295 highlighted as sticks. Potential hydrogen bonds are shown as yellow dashed lines.
both intravenous and intraperitoneal injections suggesting that
urinary excretion is not a major pathway for elimination of this
compound.
Table 4. Acetylation of the 2-Amino Group of the 4H-
Benzopyrans
Given the rapid in vivo clearance of 3, in vitro hepatic
microsomal incubations were undertaken to identify potential
pathways involved in the metabolism of both 3 and 1. In
addition to the formation of glucuronide metabolites, P+16
metabolites (suggestive of oxygenated products of 3 and 1)
were detected in vitro. Furthermore, in microsomal matrices
lacking NADPH and UDPGA cofactors, 3 was converted to 1,
suggesting that nonenzymatic (such as amide hydrolysis)
processes were contributing to the rapid in vitro (and likely in
vivo) disappearance of 3. The human and rat microsome-
predicted hepatic extraction ratios for 1 were 0.71 and 0.86,
respectively, and for 3, the corresponding human and rat
microsome-predicted hepatic extraction ratios were 0.71 and
0.61. These values are suggestive of intermediate-to-high
extraction ratios, which would be expected to result in a
relatively high clearance in vivo, concordant with that observed
for 3 in rat pharmacokinetic studies.
In separate studies assessing BBB penetration in rats, plasma
and brain concentrations of 3 were detected at 5 and 30 min
following an intravenous dose (2 mg/kg) but not at 240 min
postdose in line with a rapid systemic elimination of 3. The
concentrations of 3 in the brain ranged between a mean value
of 87.6 ng/g (5 min postdose) to 10.9 ng/g (30 min postdose),
resulting in B/P ratios varying between 0.23 and 0.35. Similarly,
10 min following intraperitoneal administration of 3 (10 mg/
kg), a B/P ratio of 0.20 was detected (Figure 6). These values
are similar to those obtained following intravenous admin-
istration of 3 to mice, where a 5 min postdose B/P ratio of 0.39
was reported.19 Following intravenous administration of 3,
plasma concentrations of 1 were higher than those obtained for
3 (as observed in the pharmacokinetic studies), and these were
detectable for up to 240 min postdose. Similarly, 1 was detected
in the brain at all time points, resulting in mean B/P ratios
ranging between 0.13 and 0.38. It is unknown whether the 1
detected within the brain was a product of 3 amide hydrolysis
within the brain or whether systemically formed 1 penetrated
the BBB. Given that the physicochemical properties of 1 are
compd
Ar
Ki (μM)
3
4
pyridine-3-yl
quinolin-3-yl
0.48
0.03
compromised. Although all inhibitors demonstrated good
aqueous solubility under acidic conditions, at pH 6.5, 1 and 3
displayed only moderate solubility values, ranging from 25 to
50 μg/mL and 50−100 μg/mL, respectively, and solubility
values for 2 and 4 were approximately 10-fold lower.19 The
partition coefficient, effective LogD7.4 (eLogD7.4) values, for all
four compounds exhibited acceptable values for good oral
absorption (Table 5): however, based on lipophilicity alone, 1
and 3 appear better candidates for BBB penetration, as a logD7.4
of 1−3 is recommended for optimum BBB penetration.24
Pharmacokinetic Properties of 3. Given that 3 exhibited
the most appropriate biochemical activity and pharmaceutical
properties (Table 5),19 the pharmacokinetics and brain uptake
of this analogue were determined following intravenous
administration to rats. Concentrations of 3 were only detectable
in plasma for up to 65 min following intravenous admin-
istration of 2 mg/kg, with this analogue exhibiting a short
apparent elimination half-life (11 min), a high plasma clearance
(172.8 mL/min/kg), and a large volume of distribution (2.8 L/
kg). This rapid disappearance of 3 was associated with the
immediate appearance of the active metabolite 1 in the plasma,
indicative of a rapid in vivo hydrolytic process (Figure 5). While
3 plasma concentrations declined rapidly following intravenous
administration, the plasma concentrations of 1 were detected at
all postdose time points. Following intraperitoneal injection of
10 mg/kg 3, the apparent half-life was extended to 4.6 h,
although there was still rapid deacetylation to 1. A very small
proportion of 3 was recovered in urine (less than 1%) with
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Figure 4. Surface representation of the IRAP homology model (gray) displaying the alternate binding positions of 3 (magenta sticks) and 4 (cyan
sticks). Theses alternate modes are supported in part by the SAR surrounding the 3 series and by mutagenesis studies of Phe544 (yellow sticks),
which is predicted to ring-stack with the benzopyran of 3 but alternatively be an edge-face hydrophobic packing point with the benzopyran of 4. Also
displayed is the Zn ion (purple sphere, positioned behind the benzopyran of 4) and Gln295 (yellow sticks, positioned behind the pyridine ring of 3).
Table 5. Summary of the Pharmacological/Physicochemical
Properties of 4 Key IRAP Inhibitors
Ki
molecular mass
(Da)
aqueous solubility (μg/mL)
a
compd (μM)
eLogD7.4
at pH 6.5
1
2
3
4
1.8
312.33
362.39
354.37
404.43
2.99
3.88
3.16
4.05
25−50
3−6
50−100
6−12
0.36
0.48
0.02
a
For the acetylated analogues, 3 and 4, 0.17 units were added to the
partition coefficient values of the base molecules resulting in calculated
LogD7.4 (cLogD7.4) values of 3.16 and 4.05, respectively. eLogD7.4 is
the experimentally determined effective LogD7.4
favorable for BBB penetration, it is likely that a substantial
amount of the 1 detected within the brain was derived from the
plasma, in line with that observed in mice.19
●
○
Figure 5. Plasma concentrations of 3 ( ) and 1 ( ) following
intravenous administration of 3 (2 mg/kg) to male Sprague−Dawley
rats. Data are presented as the mean of n = 2 replicates.
CONCLUSIONS
■
We have identified and developed the first generation small
molecular weight inhibitors of IRAP based on the structure of 1
identified through virtual screening on a homology model of
the catalytic site of IRAP. This study presents the structure−
activity relationship of analogues of 1 and the physicochemical
and pharmacokinetic properties of 4 of the most active
analogues of this series. These small molecule IRAP inhibitors
will be useful pharmacological tools to probe the physiological
roles of IRAP.
LTA4H25 and is described elsewhere.19,23 The model of IRAP was
then used for structure-based drug design. An initial in-house virtual
screen was conducted and is described elsewhere.19 Further docking
analysis has been conducted on the synthesized analogues.
Compounds were drawn and minimized under the Tripos force-
field26 in Sybylx1.2. (Tripos Inc., St Louis, MO USA). The docking of
the synthetic analogues into the IRAP model was performed using
Fred V.2.2.5,27 ensuring that the Zn2+ ion was included into the
docking site. Then, 4 Å was added to the docking box, and the top 20
poses were retained for further analysis in Vida (OpenEye). Docking
solutions as described were all present in a large cluster of solutions
and/or were highly ranked using the G-score scoring function. All
EXPERIMENTAL SECTION
Computer Modeling. A molecular model of the catalytic site of
IRAP was generated based on the structure of the homologous enzyme
■
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Ethyl 2-Amino-4-(2-cyanophenyl)-7-hydroxy-4H-chromene-3-
carboxylate (16b). Piperidine (46 μL, 0.46 mmol) was added to a
solution of ethyl cyanoacetate (100 mg, 0.89 mmol) and 2-
cyanobenzaldehyde (119 mg, 0.91 mmol) in EtOH (5 mL), and the
mixture stirred at ambient temperature for 20 h. The resulting
precipitate was collected and washed with cold EtOH to afford ethyl 2-
1
cyano-3-(2-cyanophenyl)acrylate as a yellow solid (70 mg, 35%). H
NMR (300 MHz, CDCl3) δ 13.08 (s, 1H), 8.66 (dd, J = 5.8, 3.1 Hz,
2H), 7.80 (dd, J = 5.9, 3.1 Hz, 2H), 4.45 (q, J = 7.1 Hz, 2H), 1.42 (t, J
= 7.1 Hz, 3H).
Piperidine (60 μL, 0.6 mmol) was added to a solution of ethyl 2-
cyano-3-(2-cyanophenyl)acrylate (70 mg, 0.3 mmol) and resorcinol
(34 mg, 0.3 mmol) in EtOH (5 mL) and the mixture stirred at reflux
for 12 h. Removal of the solvent in vacuo followed by purification on
silica (CH2Cl2/Et2O, 9:1) afforded 16b as a cream solid (30 mg,
30%).1H NMR (300 MHz, CDCl3) δ 9.75 (s, 1H), 8.52 (d, J = 7.8 Hz,
1H), 7.69−7.46 (m, J = 23.2, 7.4 Hz, 4H), 7.26 (s, 1H), 6.12 (d, J =
8.9 Hz, 1H), 5.76 (d, J = 9.1 Hz, 1H), 4.34−4.19 (m, 2H), 1.33 (td, J =
7.0, 1.4 Hz, 3H). MS (ESI+) m/z: 337.5 (M + H)+ (20%); 169.3 (M +
2H)2+ (100%).
Ethyl 2-Amino-7-hydroxy-4-pyridin-2-yl-4H-chromene-3-carbox-
ylate (16c). Piperidine (93 μL, 0.94 mmol) was added to a solution of
ethyl cyanoacetate (0.25 g, 2.21 mmol), resorcinol (0.25 g, 2.25
mmol), and 2-pyridine carboxaldehyde (0.24 g, 2.25 mmol) in EtOH
(10 mL) and stirred at ambient temperature for 20 h. The resulting
precipitate was removed by filtration and the filtrate purified on silica
(EtOAc/CH2Cl2, 1:1). Recrystallization from EtOH gave 16c as an
off-white solid (42 mg, 6%). HRMS (ESI+): found m/z 313.1197 (M +
H)+; C17H17N2O4 requires m/z 313.1188. 1H NMR (300 MHz,
DMSO) δ 9.56 (s, 1H), 8.37 (ddd, J = 4.7, 1.7, 0.8 Hz, 1H), 7.67−7.60
(m, 1H), 7.23 (dd, J = 4.8, 3.9 Hz, 1H), 7.09 (ddd, J = 7.5, 4.8, 1.1 Hz,
1H), 7.02 (d, J = 8.5 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 6.39 (d, J
= 2.4 Hz, 1H), 4.91 (s, 1H), 3.99−3.79 (m, 2H), 0.95 (t, J = 7.1 Hz,
3H). MS (ESI+) m/z: 313.2 (M + H)+ (100%).
Ethyl 2-Amino-7-hydroxy-4-quinolin-2-yl-4H-chromene-3-car-
boxylate (16d). Piperidine (46 μL, 0.46 mmol) was added to a
solution of ethyl cyanoacetate (100 mg, 0.88 mmol) and 2-
quinolinecarboxaldehyde (139 mg, 0.88 mmol) in EtOH (5 mL)
and the mixture stirred at ambient temperature for 24 h. The resulting
precipitate was collected and washed with cold EtOH to afford the
intermediate ethyl 2-cyano-3-(quinolin-2-yl)acrylate as a yellow solid
(83 mg, 37%). 1H NMR (300 MHz, CDCl3) δ 8.49 (s, 1H), 8.31 (d, J
= 8.6 Hz, 1H), 8.22 (d, J = 8.6 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.88
(d, J = 8.4 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.70−7.62 (m, 1H), 4.44
(q, J = 7.1 Hz, 1H), 1.43 (t, J = 7.1 Hz, 1H).
Piperidine (16 μL, 0.16 mmol) was added to a solution of ethyl 2-
cyano-3-(quinolin-2-yl)acrylate (80 mg, 0.32 mmol) and resorcinol
(35 mg, 0.32 mmol) in EtOH (5 mL) and the mixture stirred at
ambient temperature for 24 h. Removal of the solvent in vacuo
followed by purification on silica (EtOAc/hexane, 3:2) and trituration
with CH2Cl2 afforded afforded 16d as a white solid (14 mg, 12%).
HRMS (ESI+): found m/z 363.1349 (M + H)+; C21H19N2O4 requires
m/z 363.1345. 1H NMR (300 MHz, DMSO) δ 9.63 (s, 1H), 8.21 (d, J
= 8.5 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H),
7.79−7.64 (m, 3H), 7.50 (t, J = 6.9 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H),
7.07 (d, J = 9.3 Hz, 1H), 6.47−6.39 (m, 2H), 5.12 (s, 1H), 3.84 (q, J =
7.0 Hz, 2H), 0.82 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 363.6 (M + H)+
(100%).
■
□
Figure 6. B:P ratio of 3 ( ) and 1 ( ) following intravenous
administration (2 mg/kg) of 3 to male Sprague−Dawley rats. Data are
presented as the mean SD (n = 3). ND − 3 was not detected in the
plasma or brain homogenate.
figures were constructed using PyMOL (The PyMOL Molecular
Graphics System, version 1.3, Schrodinger, LLC).
̈
Chemical Synthesis. All reagents and solvents were used as
received. Proton nuclear magnetic resonance (1H NMR) spectra were
recorded at 300 MHz with a Bruker Advance DPX-300. The 1H NMR
spectra refer to solutions in deuterated solvents as indicated. The
residual solvent peaks have been used as an internal reference, with
each resonance assigned according to the following convention:
chemical shift (δ) measured in parts per million (ppm) relative to the
residual solvent peak. High resolution mass spectometry (HRMS)
analyses were collected on a Bruker Apex II Fourier transform ion
cyclotron resonance mass spectometer fitted with an electrospray ion
source (ESI). Low resolution mass spectrometry analyses were
performed using a Micromass Platform II single quadropole mass
spectrometer equipped with an atmospheric pressure (ESI/APCI) ion
source. Analytical HPLC was performed on a Waters 2690 HPLC
system incorporating a diode array detector (254 nm), employing a
Phenomenex column (Luna C8(2), 100 × 4.5 mm ID) eluting with a
gradient of 16−80% acetonitrile in 0.1% aqueous trifluoroacetic acid,
over 10 min at a flow rate of 1 mL/min. Analytical thin layer
chromatography (TLC) was performed on Merck aluminum sheets
coated in silica gel 60 F254 and visualization accomplished with a UV
lamp. Column chromatography was carried out using silica gel 60
(Merck). The purity of compounds (≥95%) was established by reverse
phase HPLC. Compounds were also custom synthesized by Epichem
Pty Ltd. as indicated.
2-Amino-4-[4-(dimethylamino)phenyl]-7-hydroxy-4H-chromene-
3-carbonitrile (15g). Piperidine (2 drops) was added to a solution of
malononitrile (122 mg, 1.84 mmol) and 4-dimethylaminobenzalde-
hyde (250 mg, 1.68 mmol) in EtOH (5 mL) and stirred at ambient
temperature for 4 h. The resulting precipitate was collected and
washed with cold EtOH to afford the intermediate 2-(4-
(dimethylamino)benzylidene)malononitrile as a bright orange solid
1
(274 mg, 83%). H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 9.1 Hz,
2H), 7.47 (s, 1H), 6.69 (d, J = 9.2 Hz, 2H), 3.14 (s, 6H).
Piperidine (3 drops) was added to a solution of 2-(4-
(dimethylamino)benzylidene) malononitrile (50 mg, 0.25 mmol)
and resorcinol (28 mg, 0.25 mmol) in EtOH (3 mL) and the mixture
stirred at reflux for 24 h. The resulting precipitate was collected and
washed with cold EtOH. Purification on silica (CH2Cl2 followed by
EtOAc/hexane, 1:1) and trituration with cold CH2Cl2 gave 15g as a
beige solid (20 mg, 26%). HRMS (ESI+): found m/z 308.1405 (M +
H)+; C18H18N3O2 requires m/z 308.1399. 1H NMR (300 MHz,
DMSO) δ 9.62 (s, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.80−6.69 (m, 3H),
6.64 (d, J = 8.6 Hz, 2H), 6.46 (dd, J = 8.4, 2.2 Hz, 1H), 6.37 (d, J = 2.1
Hz, 1H), 4.46 (s, 1H), 2.84 (s, 6H). MS (ESI+) m/z: 308.4 (M + H)+
(100%).
Ethyl 2-Amino-7-hydroxy-4-(2-nitrophenyl)-4H-chromene-3-car-
boxylate (16e). Piperidine (184 μL, 1.84 mmol) was added to a
solution of ethyl cyanoacetate (100 mg, 0.89 mmol), resorcinol (100
mg, 0.91 mmol), and 2-nitrobenzaldehyde (138 mg, 0.91 mmol) in
EtOH (5 mL) and the mixture stirred at reflux for 18 h. Removal of
the solvent in vacuo followed by purification on silica (CH2Cl2/EtOAc,
9:1) and trituration with CH2Cl2 afforded 16e as a yellow solid (102
mg, 32%). HRMS (ESI+): found m/z 357.1090 (M + H)+;
1
C18H17N2O6 requires m/z 357.1087. H NMR (300 MHz, CDCl3) δ
7.64 (d, J = 7.9 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.29−7.11 (m, 3H),
6.57−6.49 (m, J = 11.1, 2.6 Hz, 2H), 5.57 (s, 1H), 4.01−3.87 (m, 2H),
0.99 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 357.3 (M + H)+ (100%).
G
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Journal of Medicinal Chemistry
Article
Ethyl 2-Amino-4-(2,6-dichloropyridin-3-yl)-7-hydroxy-4H-chro-
mene-3-carboxylate (16h). Piperidine (184 μL, 1.84 mmol) was
added to a solution of ethyl cyanoacetate (100 mg, 0.89 mmol),
resorcinol (100 mg, 0.91 mmol), and 2,6-dichloropyridine-3-
carboxaldehyde (160 mg, 0.91 mmol) in EtOH (5 mL) and the
mixture stirred at reflux for 10 h. Removal of the solvent in vacuo
followed by purification on silica (CH2Cl2/Et2O, 9:1) and trituration
with CH2Cl2 afforded 16h as a yellow solid (61 mg, 18%). HRMS
(ESI+): found m/z 381.0413 (M + H)+; C17H1535Cl2N2O4 requires m/
z 381.0409; found m/z 383.0396 (M + H)+; C17H1535Cl37ClN2O4
tert-Butyl 2-amino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-car-
boxylate (17d). Piperidine (3 drops) was added to a solution of t-
butyl cyanoacetate (128 mg, 0.91 mmol), resorcinol (100 mg, 0.91
mmol), and 3-pyridinecaroxaldehyde (96 mg, 0.90 mmol) in EtOH (5
mL) and the mixture stirred at ambient temperature for 20 h. The
solvent was removed in vacuo and the residue treated with Et2O. The
resulting precipitate was collected and washed with Et2O. Purification
1
on silica (Et2O) afforded 17d as a white solid (114 mg, 37%). H
NMR (300 MHz, CDCl3) δ 9.65 (s, 1H), 8.44 (d, J = 1.7 Hz, 1H),
8.32 (dd, J = 4.7, 1.6 Hz, 1H), 7.59 (s, 2H), 7.47−7.41 (m, 1H), 7.24
(dd, J = 7.8, 4.7 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.46 (dd, J = 8.3,
2.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 4.76 (s, 1H), 1.24 (s, 9H). MS
(ESI+) m/z: 341.5 (M + H)+ (100%).
Ethyl 2-Amino-4-pyridin-3-yl-4H-benzo[h]chromene-3-carboxy-
late (18g). Acetic acid (100 μL) and piperidine (40 μL) were added
to a stirred solution of ethyl cyanoacetate (2.3 g, 20.3 mmol) and 3-
pyridinecarboxaldehyde (2.2 g, 20.5 mmol) in toluene (20 mL). The
reaction mixture was heated to reflux for 6 h. The resulting precipitate
was collected and washed with toluene to afford the intermediate ethyl
2-cyano-3-(pyridin-3-yl)acrylate as a beige solid (3.69 g, 90%). 1H
NMR (300 MHz, CDCl3) δ 8.92 (d, J = 2.3 Hz, 1H), 8.76 (dd, J = 4.8,
1.6 Hz, 1H), 8.58 (dt, J = 8.1, 1.8 Hz, 1H), 8.26 (s, 1H), 7.48 (dd, J =
8.2, 4.8 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). MS
(ESI+) m/z: 203.3 (M + H)+ (100%).
1
requires m/z 383.0379. H NMR (300 MHz, CDCl3) δ 8.68 (s, 2H),
7.44 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.4 Hz,
1H), 6.54−6.45 (m, 2H), 5.41 (s, 1H), 4.06−3.91 (m, 3H), 1.10 (t, J =
7.1 Hz, 2H). MS (ESI+) m/z: 381.3 (M (35Cl) + H)+ (100%), 382.9
(M (35Cl)(37Cl) + H)+ (40%).
Ethyl 2-Amino-7-hydroxy-4-(4-nitrophenyl)-4H-chromene-3-car-
boxylate (16o). Piperidine (184 μL, 1.84 mmol) was added to a
solution of ethyl cyanoacetate (100 mg, 0.89 mmol), resorcinol (100
mg, 0.91 mmol), and 4-nitrobenzaldehyde (138 mg, 0.91 mmol) in
EtOH (5 mL) and the mixture stirred at reflux for 18 h. Removal of
the solvent in vacuo followed by purification on silica (CH2Cl2/Et2O,
9:1) and trituration with CH2Cl2 afforded 16o as a yellow solid (15
mg, 5%). HRMS (ESI+): found m/z 357.1077 (M + H)+; C18H17N2O6
1
requires m/z 357.1087. H NMR (300 MHz, CDCl3) δ 8.76 (s, 1H),
Piperidine (2 drops) was added to a solution of ethyl 2-cyano-3-
(pyridin-3-yl)acrylate (100 mg, 0.49 mmol) and 1-naphthol (71 mg,
0.49 mmol) in EtOH (3 mL) and the mixture stirred at ambient
temperature for 24 h. The resulting precipitate was collected and
washed with cold EtOH to give 18g as a beige solid (110 mg, 65%).
HRMS (ESI+): found m/z 347.1396 (M + H)+; C21H19N2O3 requires
m/z 347.1396. 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 8.38 (d, J
= 4.5 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H),
7.62−7.44 (m, 4H), 7.17−7.05 (m, J = 9.6 Hz, 2H), 5.10 (s, 1H), 4.10
(q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 374.4
(M + H)+ (100%).
8.03 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 9.0 Hz,
1H), 6.50 (d, J = 6.7 Hz, 1H), 4.93 (s, 1H), 4.00 (q, J = 7.1 Hz, 2H),
1.09 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 357.2 (M + H)+ (100%).
Ethyl 2-Amino-7-hydroxy-4-(4-pyridin-2-ylphenyl)-4H-chromene-
3-carboxylate (16p). Piperidine (5 drops) was added to a solution of
ethyl cyanoacetate (154 mg, 1.36 mmol) and 4-(2-pyridyl)-
benzaldehyde (250 mg, 1.36 mmol) in EtOH (5 mL) and stirred at
ambient temperature for 20 h. The resulting precipitate was collected
and washed with cold EtOH to afford the intermediate ethyl 2-cyano-
3-(4-(pyridin-2-yl)phenyl)acrylate as a pale yellow solid (278 mg,
1
74%). H NMR (300 MHz, CDCl3) δ 8.74 (d, J = 4.7 Hz, 1H), 8.29
(s, 1H), 8.19−8.07 (m, 4H), 7.84−7.77 (m, 2H), 7.31 (dd, J = 8.8, 4.5
Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H).
Ethyl 3-Amino-1-pyridin-3-yl-1H-benzo[f ]chromene-2-carboxy-
late (18h). Piperidine (2 drops) was added to a solution of ethyl 2-
cyano-3-(pyridin-3-yl)acrylate (100 mg, 0.49 mmol) and 2-naphthol
(71 mg, 0.49 mmol) in EtOH (3 mL) and the mixture stirred at
ambient temperature for 24 h. The resulting precipitate was collected
and washed with cold EtOH to give 18h as a white solid (126 mg,
74%). HRMS (ESI+): found m/z 347.1398 (M + H)+; C21H19N2O3
Piperidine (3 drops) was added to a solution of ethyl 2-cyano-3-(4-
(pyridin-2-yl)phenyl)acrylate (50 mg, 0.18 mmol) and resorcinol (20
mg, 0.18 mmol) in EtOH (3 mL) and the mixture stirred at reflux for
24 h. Removal of the solvent in vacuo followed by purification on silica
(CH2Cl2/EtOAc, 3:1) and recrystallization from EtOH gave 16p as a
cream solid (30 mg, 43%). HRMS (ESI+): found m/z 389.1512 (M +
H)+; C23H21N2O4 requires m/z 3389.1501. 1H NMR (300 MHz,
DMSO) δ 9.64 (s, 1H), 8.65−8.57 (m, 1H), 7.92 (d, J = 8.3 Hz, 2H),
7.89−7.79 (m, 2H), 7.63 (s, 2H), 7.34−7.20 (m, 3H), 7.01 (d, J = 8.3
Hz, 1H), 6.52−6.41 (m, 2H), 4.85 (s, 1H), 3.95 (q, J = 7.1 Hz, 2H),
1.06 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 389.3 (M + H)+ (100%).
Ethyl 2-Amino-4-[4-(dimethylamino)phenyl]-7-hydroxy-4H-chro-
mene-3-carboxylate (16q). Piperidine (5 drops) was added to a
solution of ethyl cyanoacetate (190 mg, 1.68 mmol) and 4-
dimethylaminobenzaldehyde (250 mg, 1.68 mmol) in EtOH (5 mL)
and stirred at ambient temperature for 6 h. The resulting precipitate
was collected and washed with cold EtOH to afford the intermediate
ethyl 2-cyano-3-(4-(dimethylamino)phenyl)acrylate as a bright yellow
1
requires m/z 347.1396. H NMR (300 MHz, CDCl3) δ 8.70 (s, 1H),
8.31 (d, J = 4.7 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.78 (t, J = 7.5 Hz,
2H), 7.54−7.33 (m, 3H), 7.27 (d, J = 8.8 Hz, 1H), 7.13−7.03 (m,
1H), 6.37 (s, 2H), 5.60 (s, 1H), 4.22 (q, J = 7.0 Hz, 2H), 1.36 (t, J =
7.0 Hz, 3H). MS (ESI+) m/z: 374.4 (M + H)+ (100%).
Enzyme Assay. Solubilized, crude membranes isolated from
HEKT cells, transfected with pCI-IRAP, were used as a source of
the enzyme to analyze the efficacy of the candidate compounds to
inhibit its enzymatic activity. As a negative control, membranes from
vector-only transfected cells were used. The enzymatic activity of IRAP
was determined by the hydrolysis of a synthetic aminopeptidase
substrate L-leucine-4-methyl-7-coumarinylamide (Leu-MCA) (Sigma
Aldrich, St Louis, MO, USA) monitored by the release of a fluorogenic
product at excitation and emission wavelengths of 380 and 430 nm,
respectively. Assays were performed in 96-well plates; each well
containing 1 μg of solubilized membrane protein and 25 μM substrate
in a final volume of 100 μL 50 mM Tris-HCl buffer (pH 7.4).
Reactions proceeded at 37 °C for 30 min in a Wallac Victor3 V
Multilabel counter (Perkin-Elmer, Waltham, MA, USA). Inhibitory
constants (Ki) for each of the inhibitors were determined over a range
of concentrations (at 0.01 to 100 μM) and were calculated from the
relationship IC50 = Ki (1 + [S]/KM) with GraphPad Prism 3 software
(GraphPad Software Inc. San Diego, CA, USA).
1
solid (367 mg, 89%). H NMR (300 MHz, CDCl3) δ 8.07 (s, 1H),
7.94 (d, J = 9.1 Hz, 2H), 6.69 (d, J = 9.1 Hz, 2H), 4.33 (q, J = 7.1 Hz,
2H), 3.11 (s, 6H), 1.37 (t, J = 7.1 Hz, 3H).
Piperidine (3 drops) was added to a solution of ethyl 2-cyano-3-(4-
(dimethylamino)phenyl)acrylate (100 mg, 0.41 mmol) and resorcinol
(68 mg, 0.61 mmol) in EtOH (5 mL) and the mixture stirred at reflux
for 24 h. The precipitate obtained on cooling was removed by filtration
and the filtrate reduced in vacuo. Purification on silica (Et2O/hexane,
1:1) gave 16q as a beige solid (10 mg, 7%). HRMS (ESI+): found m/z
1
355.1666 (M + H)+; C20H23N2O4 requires m/z 355.1658. H NMR
(300 MHz, DMSO) δ 9.54 (s, 1H), 7.50 (s, 2H), 6.97−6.87 (m, J =
8.6 Hz, 3H), 6.56 (d, J = 8.7 Hz, 2H), 6.45 (dd, J = 8.3, 2.4 Hz, 1H),
6.40 (d, J = 2.3 Hz, 1H), 4.65 (s, 1H), 4.01−3.88 (m, 2H), 2.79 (s,
6H), 1.09 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 355.6 (M + H)+
(100%).
Physicochemical Characterization. Octanol−water partition
coefficients or effective LogD (ElogD) values for 1 and 2 were
measured using a chromatographic method employing a SUPELCO-
SIL LC-ABZ column with an octanol saturated mobile phase at pH
7.4.28 The values obtained for 1 and 2 were used to provide trained
H
dx.doi.org/10.1021/jm401540f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
partition coefficient values for the corresponding acetylated derivatives,
3 and 4, using the ACD LogD (version 9.0) of software programs.
Pharmacokinetics and Brain Uptake of 3. All animal studies
were performed in accordance with the Australian National Health and
Medical Research Council code of practice for the care and use of
animals for scientific purposes and were approved by the Monash
University (Victorian College of Pharmacy) Animal Ethics committee.
Male Sprague−Dawley rats (7−9 weeks) were intravenously
administered (via an indwelling jugular vein cannula) a 1.0 mL
solution of 3 (2 mg/kg in an aqueous solution containing 30% w/v
hydroxypropyl-β-cyclodextrin and 1% v/v DMSO) over a 5 min
period. In additional studies, rats were intraperitoneally administered a
0.5 mL solution of 3 (10 mg/kg in in 10% DMSO (v/v)) and 30%
hydroxypropyl-β-cyclodextrin (w/v). Samples of arterial blood were
collected (from an indwelling carotid artery cannula) over 24 h using a
Culex automated blood sampler (BASi Instruments, West LaFayette,
IN, USA). Blood samples were collected directly into heparinized
borosilicate vials (at 4 °C) containing Complete protease inhibitor
cocktail (80 mg/mL, Roche, Mannheim, Germany), EDTA (0.1 M),
and potassium fluoride (4 mg/mL) (stabilization mixture). Blood
samples were immediately centrifuged and the plasma supernatant
separated and immediately stored at −20 °C to minimize potential ex
vivo conversion of 3 to 1. All plasma samples were subsequently
assayed for 3 and 1 concentration using a validated LCMS method,
with a lower limit of quantification of 1 ng/mL for 3 and 0.5 ng/mL
for 1. Noncompartmental pharmacokinetic analysis was performed
using WinNonLin software (version 4.0, Pharsight Corporation,
Mountain View, CA, USA) to estimate the terminal elimination half-
life, total plasma clearance, and volume of distribution.
For the assessment of 3 and 1 brain uptake, a 200 μL solution of 3
(in the same formulation used for pharmacokinetic studies) was
injected as a bolus into the jugular vein (via an indwelling cannula) of
rats. At 5, 30, and 240 min postdose, animals were anaesthetized using
inhaled isofluorane (3%) and 500 μL of blood was removed by cardiac
puncture (n = 3 rats per time point), followed by brain removal. Brain
samples were homogenized in 3 parts of water containing stabilization
mixture, and both the brain homogenate and plasma supernatant were
immediately stored at −20 °C until analysis by LCMS (lower limits of
quantitation of 5.0 ng/g brain homogenate for 3 and 0.5 ng/g for 1).
All brain homogenate concentrations were corrected for vascular
contamination of compound, using a vascular volume of 0.026 mL/g
(determined by intravenous administration of the BBB-impenetrable
marker 14C-inulin), and brain-to-plasma (B:P) ratios determined at
each postdose time point.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Health
and Medical Research Council of Australia (NHMRC),
Neurosciences Victoria, and Alzheimer’s Drug Discovery
Foundation/Institute for the Study of Aging, USA. Funding
was also obtained from the Victorian Government Operational
Infrastructure Support Scheme to St. Vincent’s Institute.
M.W.P. is an NHMRC Senior Principal Research Fellow,
S.Y.C. is an NHMRC Senior Research Fellow, and J.K.H. is a
joint Cure Cancer/Leukemia Foundation Post-Doctoral
Fellow. We acknowledge the contribution of Dr. Keith Watson
in the design some of the analogues of 1.
ABBREVIATIONS USED
■
APN, aminopeptidase N; Ang IV, angiotensin IV; AVP,
arginine8 vasopressin; APCI, atmospheric pressure chemical
ionization; BBB, blood−brain barrier; DABCO, 1,4-diazabicy-
clo-octane; DMSO, dimethyl sulfoxide; ESI, electrospray ion;
EtOH, ethanol; EtOAc, ethyl acetate; HRMS, high resolution
mass spectrometry; IRAP, insulin-regulated aminopeptidase;
Leu-MCA, L-leucine-4-methyl-7-coumarinylamide; Leu-Enk,
leu-enkephalin; LTA4H, leukotriene A4 hydrolase; LCMS,
liquid chromatography−mass spectrometry; LVV-H7, LVV-
hemorphin 7; NADPH, reduced form of nicotinamide adenine
dinucleotide phosphate; NMR, nuclear magnetic resonance;
ppm, parts per million; TBAB, tetra-n-butylammonium bro-
mide; TLC, thin layer chromatography; UDPGA, uridine
diphosphate glucuronic acid
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In Vitro Metabolism in Rat and Human Liver Microsomes.
Compound 3 or 1 was incubated at 37 °C with rat or human liver
microsomes at an initial concentration of 1 μM (in 0.1 M phosphate
buffer, pH 7.4). Microsomal reactions were initiated by the addition of
an NADPH-regenerating system (1 mg/mL), and at various time
points, the reaction was terminated by the addition of acetonitrile and
parent compound concentration determined by LCMS. In order to
assess the potential of glucuronidation, additional microsomal samples
were incubated with NADPH and UDPGA (1 mg/mL), the cofactor
required for conjugative phase II metabolism by UGT enzymes. The
concentration of parent compound (3 or 1) versus time was
subsequently modeled to an exponential decay function to determine
the first order rate constant for parent compound depletion (k), which
was ultimately used in the estimation of a hepatic extraction ratio, with
the appropriate assumptions.29 LCMS conditions were also optimized
to detect the +16 (oxygenated) and +176 (glucuronidated)
metabolites of 3 and 1.
AUTHOR INFORMATION
Corresponding Authors
*(P.E.T.) Tel: 613-9903-9672. E-mail: philip.thompson@
monash.edu.
■
*(S.Y.C.) Tel: 613-9905-2515. E-mail: siew.chai@monash.edu.
Notes
The authors declare no competing financial interest.
I
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