Structure-activity relationship study of first selective inhibitor of excitatory amino acid transporter subtype 1: 2-Amino-4-(4-methoxyphenyl)-7- (naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4 H -chromene-3-carbonitrile (UCPH-101)

Article abstract of DOI:10.1021/jm1009154

The excitatory amino acid transporters (EAATs) are expressed throughout the central nervous system, where they are responsible for the reuptake of the excitatory neurotransmitter (S)-glutamate (Glu).(1)Recently, we have reported the discovery of the first subtype selective EAAT1 inhibitor 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile (UCPH-101) (1b) and presented an introductory structure-activity relationship (SAR) study.(2)Here, we present a detailed SAR by the design, synthesis, and pharmacological evaluation of analogues 1g-1t. By comparison of potencies of 1b, 1h, and 1i versus 1j, it is evident that potency is largely influenced by the chemical nature of the R¹ substituent. The study also demonstrates that any chemical change of the functional groups or a change to the parental scaffold results in the complete loss of inhibitory activity of the compounds at EAAT1. Finally, a bioavailability study of UCPH-101 determined the half-life to be 30 min in serum (rats) but also that it was not able to penetrate the blood-brain barrier to any significant degree.

Full text of DOI:10.1021/jm1009154

7180 J. Med. Chem. 2010, 53, 7180–7191
DOI: 10.1021/jm1009154
Structure-Activity Relationship Study of First Selective Inhibitor of Excitatory Amino Acid
Transporter Subtype 1: 2-Amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (UCPH-101)
Mette N. Erichsen,† Tri H. V. Huynh,† Bjarke Abrahamsen,† Jesper F. Bastlund,‡ Christoffer Bundgaard,‡ Olja Monrad,†
Anders Bekker-Jensen,† Christina W. Nielsen,† Karla Frydenvang,† Anders A. Jensen,† and Lennart Bunch*,†
†Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2,
DK-2100 Copenhagen, Denmark, and ‡H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
Received July 21, 2010
The excitatory amino acid transporters (EAATs) are expressed throughout the central nervous system,
where they are responsible for the reuptake of the excitatory neurotransmitter (S)-glutamate (Glu).1
Recently, we have reported the discovery of the first subtype selective EAAT1 inhibitor 2-amino-4-(4-
methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101)
(1b) and presented an introductory structure-activity relationship (SAR) study.2 Here, we present a
detailed SAR by the design, synthesis, and pharmacological evaluation of analogues 1g-1t. By
comparison of potencies of 1b, 1h, and 1i versus 1j, it is evident that potency is largely influenced by
the chemical nature of the R1 substituent. The study also demonstrates that any chemical change of
the functional groups or a change to the parental scaffold results in the complete loss of inhibitory
activity of the compounds at EAAT1. Finally, a bioavailability study of UCPH-101 determined the
half-life to be 30 min in serum (rats) but also that it was not able to penetrate the blood-brain barrier
to any significant degree.
Introduction
As a part of our medicinal chemistry research program
within glutamatergic neurotransmission, we are interested in
the development of subtype selective ligands for the EAATs.2
Besides the therapeutic potential of such ligands, they are
also highly interesting as pharmacological tools, enabling
the explorations of the respective roles of the EAAT subtypes
in physiology and in disease. Recently we reported the dis-
covery of the first class of compounds acting as subtype
selective inhibitors of EAAT1.2 By screening of a 3040
compound library at the EAAT1-3, compound 1a was found
to selectively inhibit EAAT1 over EAAT2 and EAAT3,
displaying inhibitory activities in low micromolar range at
this subtype (IC50 = 4.7 μM, Table 1). An initial structure-
activity relationship (SAR) study was subsequently car-
ried out by variation of the R1 and R2 groups, and the
compound 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-
1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile
(UCPH-101, 1b), which is now commercially available,11
In the central nervous system (CNSa), the excitatory amino
acid transporters (EAATs) are believed to be primarily re-
sponsible for the uptake of the excitatory neurotransmitter
(S)-glutamate (Glu) into glial cells and neurons, hereby
maintaining synaptic as well as extra-synaptic Glu concentra-
tions below levels of neurotoxicity.1 To this day, five EAAT
subtypes have been identified and named EAAT1-5 in
humans. For reasons of tradition, the rodent orthologues are
named GLAST, GLT-1, EAAC1, EAAT4, and EAAT5. The
five EAAT subtypes exhibit distinct expression patterns in the
CNS: While EAAT1-3 (GLAST, GLT-1, EAAC1) are ex-
pressed throughout the CNS, the EAAT4,5 subtypes are
expressed exclusively in Purkinje cells of the cerebellum and
in the retina, respectively.3 At the cellular level, EAAT1,2 are
expressed predominantly in glia cells and astrocytes, whereas
EAAT3,4 are expressed almost exclusively on neurons.4 With
respect to function, EAAT1-3 are high-capacity Glu trans-
porters, while EAAT4,5 are considered to be low-capacity
Glu transporters, functioning primarily as Glu-gated chloride
channels.4 Malfunction of the EAATs has been suggested to
be an underlying factor in neurodegenerative diseases such as
Alzheimer’s,5 Huntington’s,6 amyotrophic lateral sclerosis
(ALS),7 cerebral stroke,8 and epilepsy.1,9,10
turned out to be the most potent analogue in the series (IC50
=
0.66 μM) (Figure 1). In the same study, it was also concluded
that an aromatic ring was mandatory in the 7-position (R1) of
the parental skeleton and that an alkyl or aryl substituent in the
4-position (R2) was essential for the inhibitory activity of the
analogues at EAAT1. In this full paper, we present a compre-
hensive SAR study of this compound class as well as pharma-
cokinetic data for UCPH-101.
*To whom correspondence should be addressed. Phone: þ45
35336244. Fax: þ45 35336041. E-mail: lebu@farma.ku.dk.
aAbbreviations: BBB, blood-brain barrier; CNS, central nervous
system; EAAT(s), excitatory amino acid transporter(s); HMBC, hetero-
nuclear multiple bond coherence; HMQC, heteronuclear multiple quan-
tum coherence; SAR, structure-activity relationship.
Results and Discussion
Building on the first reported SAR, a series of new ana-
logues were designed in order to explore the importance of the
r
pubs.acs.org/jmc
Published on Web 09/21/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7181
Table 1. Pharmacological Characterization of 1a-s as Potential Inhibitors at hEAAT1 in a [3H]-D-Aspartate Uptake Assaya

7182 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 1. Continued
Erichsen et al.
a Data are given as IC50 values in μM with pIC50 ( SEM in brackets. All analogues were without inhibitory activity at EAAT2 and EAAT3 when
tested at the highest possible concentration (75 μM for analogue 1l, 100 μM for analogues 1a, 1g, 1i, 1m, 1n, 1p, 1q, 300 μM for analogues 1b-f, 1h, 1k,
syn-1r, anti-1r, syn-1s, anti-1s, and 1000 μM for analogues 1j, 1o).
Scheme 1. Reagents and Conditions Used for the Synthesis
of 1k-m as Analogues of the Cyano Functionalitya
a Reagents and conditions: (a) For 3a: benzaldehyde, piperidine,
Figure 1. Lead structure 1a identified from screening of a small
MeOH, reflux, 70 min, 93%; for 3b: benzaldehyde, piperidine, i-PrOH,
reflux, 70 min, 67%; for 3c, benzaldehyde, piperidine, EtOH, rt., 3 h,
42%. (b) For 1k,l: 5-phenylcyclohexane-1,3-dione, H2O/EtOH, MW,
85 °C, 30 min, 67% and 72%, respectively; for 1m: 5-phenylcyclohexane-
1,3-dione, dioxane, piperidine, rt, overnight, 57%.
compound library and 1b (UCPH-101) as the most potent analogue
from the initial SAR study.2
and the malononitrile components form an intermediate
condensation product, which then reacts with the diketone
to form the desired product. The compounds may also be
synthesized in a two-step process, wherein the intermediate
condensation product is first isolated and then reacted with
the diketone component. The diketone components used in
the synthesis of 1g and 1i were synthesized in accordance
with literature.2,12
Synthesis of Analogues in the 3-Position (3-Cyano Group).
To investigate the significance of the cyano group in the
3-position of parental skeleton and to explore the pharma-
cological effect of extending the ligand in this direction,
esters and amide analogues 1k-m were designed. The strat-
egy for their synthesis seemed straightforward; exchange of
the malononitrile component used in the three-component
reaction with the corresponding 2-cyano analogues was
believed to give access to the desired products. Despite
several attempts, the three component reaction led to a
condensation product between the aldehyde and diketone.
However, performing the reaction in two steps by first
synthesis and isolation of the condensation product (3a,
3b, 3c) of benzaldehyde and 2a, 2b, or 2c followed by reaction
with the diketone gave the amide and ester analogues in good
yields (Scheme 1).
Figure 2. Generally used three component reaction of diketone,
aldehyde, and malononitrile for the preparation of the 2-amino-5-
oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile parental skele-
ton with various substituents R2 and R1 in the 4- and 7-positions,
respectively.
cyano-, amino-, and keto functionalities for the inhibitory
activity at EAAT1. Furthermore, additional variations of the
substituents in the R1 and R2 positions were performed.
Synthesis of Analogues in 4- and 7-Positions. To further
address the influence of the chemical nature of the substit-
uents in the 4-/7-position, the four analogues 1g-j were
designed and prepared following a three-component reac-
tion strategy (Figure 2) as reported for UCPH-101.2 In
general, an approximately 1:1 ratio of diastereomers was
1
observed based on H NMR analyses. By monitoring the
three component reaction, it is observed that the aldehyde
Elimination of the 3-cyano group will induce a significant
change in the electronic distribution of the skeleton; thus

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7183
Scheme 3. Synthesis of Analogues in the 2-Positiona
Scheme 2. Mechanism of Unexpected Decarboxylation of 1l
under Acidic Conditions to Give Analogue 1na
a Reagents and conditions: (a) triethyl orthoformate, acetic anhy-
dride, reflux, 3.5 h, 86%; (b) NaBH4, EtOH, rt, 3 h, 34%; (c) formic acid,
EtOH, rt to 60 °C, 2 days, quant.
a Reagents and conditions: (a) HCl in dioxane, 50 °C, 25 h, 85%.
analogue 1n is of interest to evaluate the impact of such a
change for the inhibitory activity at EAAT1. On its synthesis,
applying the strategy used for the synthesis of 1k-m was not
productive. However, in an attempt to synthesize the 3-car-
boxylic acid analogue (brackets Scheme 2), the treatment of
1l with HCl in dioxane led to the expected cationic elimina-
tion of the tert-butyl group but also the immediate decar-
boxylation to give 1n. (Scheme 2). Even at room temperature
or with the use of different acids (TFA, acetic acid, dichloro-
acetic acid), these two sequential reactions could not be
controlled as to produce the free 3-carboxylic acid analogue.
Synthesis of Analogues in the 2-Position (2-Amino Group).
The 2-amino functionality is intriguing as it is in conjugation
with the electron withdrawing 3-cyano group but also in
competition with the electron donating ring oxygen. Given
this analysis, the pKa value of the 2-amine is comparable to
an amide and thus not protonated under physiological
conditions, making it capable of hydrogen bond donation
and acceptation. Three analogues, 4, 1p, and 1q, were
designed to address this matter. To explore the possibility
to expand the structure in direction of the amino group to
obtain analogues of higher potency, the obvious choice of
design is the N-methylated analogue (4). Different strategies
were tried for synthesis of 4 (Scheme 3): Direct methylation
of the amino group using dimethyl sulfate as the methylating
agent with sodium hydride as base13 did not lead to the
desired product but rather a complex mixture of products
including 5-methylated analogues. As a consequence, alter-
native strategies were explored. The tactic that seemed most
promising was introduction of an ethoxymethylidene group
(dCHOCH2CH3) on the amino functionality by reaction of
1j with triethyl orthoformate to give 1o. The subsiding
reduction14-16 to give 4 failed despite the fact that several
reduction conditions (NaBH4, NaB(OAc)3H, NaBH3CN,
formic acid)16,17 were tried. Either full reduction of the
ethoxymethylidene group was not achieved or the reaction
gave complex reactions mixtures. Interestingly, treating 1o
with formic acid yielded 1j. Reduction of 1o using NaBH4 in
ethanol at room temperature resulted in 5, with the desired
methylated amine, but also reduction of the 5-keto group to
the alcohol as the main product. An LC-MS analysis of
Scheme 4. Synthetic Pathway for the Preparation of 1qa
a Reagents and conditions: (a) benzaldehyde, NaOEt, EtOH, rt, 20 h;
(b) 6N HCl, rt, 1 h, 40% over two steps; (c) 5-phenylcyclohexane-1,3-
dione, triethylamine, CH2Cl2, rt, 2 h, then add DMAP, MsCl, rt., 4 days,
67%.
recrystallized 5 confirmed the structure. Reoxidation of the
alcohol to give 4 was attempted by several methods (TPAP/
NMO, Dess-Martin periodinane, MnO2, DDQ, Swern),18-22
however, without success.
The acetylated analogue also explores the possibility
to extend the structure in the direction of the amino
group. Treating 1j with acetic anhydride gave the acetylated
compound.14
Removal of the amino group will introduce major
changes in the electronic distribution of the parental skele-
ton as well as deplete the possibility of ligand hydrogen
donation and acceptance in this position. To synthesize 1q
by use of the three-component reaction (Scheme 4), the
malononitrile component must be replaced with a compo-
nent that can introduce a leaving group in the 2-position.
On the basis of the observation made with the synthesis of
the analogues in the 3-position, it was decided to synthesize

7184 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Erichsen et al.
Scheme 5. Synthetic Pathway towards β-Ketolactam 13a
a Reagents and conditions: (a) formic acid, methanol/H2O (1:2), rt,
3 days, 78%; (b) Cs2CO3, THF, 60 °C, 30 min 100%; (c) LDA, THF,
-50 °C, then add 11, 3 h, 80%; (d) TFA, dichloromethane, rt, 3 h;
(e) NaHCO3, dichloromethane, rt, 6 h HCl (pH = 3), 89%.
Figure 3. Chemical structure of new heterocyclic analogues of 1j:
lactam 1r and lactone 1s.
Scheme 6. Synthetic Pathway Towards β-Ketolactone 15a
Figure 4. Three point superimposition of low-energy conformation
of 1j with lactam 1r. (A) syn-Diastereomers: 1j (green) and 1r (type
code). (B) anti-Diastereomers: 1j (green) and 1r (type code).
the intermediate product rather than forming it in situ to
better be able to monitor and control the reaction (Scheme 4).
3,3-Diethoxypropionitrile (6) was reacted with benzalde-
hyde to form 7, which then underwent hydrolysis to form
8.23 The initial strategy was that the reaction between 8
and 5-phenylcyclohexane-1,3-dione in the presence of catalytic
amounts of base would produce condensation product 1q
simply due to the driving force for formation of the 2,3-
conjugated system. However, these conditions only led to
formation of the hemiacetal intermediate 9 (Scheme 4). To
overcome this problem, conversion of the alcohol into the
much better leaving group, the mesylate, by addition of
DMAP and mesyl chloride in situ, cleanly gave 1q in 67%
yield.
Synthesis of Analogues in the 5,6-Position (Lactone and
Lactam). To address the influence of the 5-oxo group on the
selective EAAT1 inhibitory activity, we designed the corre-
sponding lactam 1r and lactone 1s analogues by introduction
of a nitrogen or an oxygen in the 6-position of 1j (Figure 3).
An in silico study was conducted to assess the influence of
the planned chemical changes. The study disclosed that 1r
and 1s do not occupy a volume outside what is defined by 1j.
Furthermore, the low-energy conformations of the two new
heterocyclic skeletons did not deviate considerably from the
low energy conformation of 1j (Figure 4, only data for the
study of 1r is shown).
a Reagents and conditions: (a) NaH, n-BuLi, THF, rt -20 °C, 1.5 h,
57%; (b) KOH, rt, 30 min HCl (pH = 3), 90%.
The synthesis of β-ketolactam 13 commenced with the
preparation of sulfone 10 from benzaldehyde, sodium ben-
zenesulfinate, and tert-butyl carbamate (Scheme 5), which
was subsequently converted to imine 11 in quantitative yield
using cesium carbonate in THF.24,25 Because of its suscept-
ibility, the imine 11 was used immediately or stored in the
freezer under a nitrogen atmosphere. The imine was reacted
with 2.5 equiv of 1,3-dicarbonyl dianion to afford the
corresponding δ-amino-β-keto ester 12 in a 80% yield.26
Deprotection of the δ-amino-β-keto ester 12 with 5 equiv of
TFA in dichloromethane removed the Boc-protecting group
and gave the amine (not shown), which was reacted with
saturated NaHCO3 in dichloromethane to afford β-ketolactam
13 in 89% yield.27,28
β-Ketolactone 15 was synthesized by first trapping the
dianion of methyl acetoacetate with benzaldehyde to give
hydroxyketoester 14 (Scheme 6). Subsequent hydrolysis
using potassium hydroxide followed by treatment with
hydrochloric acid afforded the desired β-ketolactone 15 in
90% yield.29,30
With β-ketolactam 13 and β-ketolactone 15 in hand, the
final step toward the synthesis of target compounds 1r and 1s
was to be investigated. To better control this exploratory
step, 2-benzylidenemalononitrile (16) was prepared first31
rather than generating it in situ. Finally, conjugate addition
of β-ketolactam 13 with the 2-benzylidene-malononitrile
1632 in absolute ethanol with a catalytic amount of piperidine
for 30 min at room temperature afforded a mixture of two
The two new diheteroatomic scaffolds found in 1r and 1s
have not been reported earlier. Despite the at-first-glance
chemical complexity, we believed they could be readily pre-
pared following the same strategy used for the synthesis of
1a-j, (Figure 2) only by substitution of the 1,3-cyclohexa-
dione component for β-ketolactam 13 or β-ketolactone 15.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7185
Scheme 7. Synthetic Pathway and Mechanism for the Prepara-
tion of Lactam 1r, Lactone 1s, and Chemical Structures of
8-N/O Regioisomers 17 and 18 (Not Formed)a
Figure 5. X-ray crystal structures of syn/anti-1r and syn/anti-1s;
ellipsoids of the non-hydrogen atoms are represented at 50%
probability. Hydrogen atoms are represented by spheres of arbi-
trary size.
similarity of the chemical shifts and coupling patterns for the
four analogues. Furthermore, it is evident that the NMR
experiments reported here could not be used as to unam-
biguously assign the syn/anti stereochemistry of 1r and 1s
(see Supporting Information).
SAR. The synthesized analogues 1g-q, syn-1r, anti-1r, syn-
1s, and anti-1s were characterized as ligands at EAAT1-3
in a [3H]-D-aspartic acid uptake assay, and the results are
presented in Table 1.33
On the comparison of UCPH-101, which comprises a
1-naphthyl group in the 7-position, a 5-fold decrease in
inhibitory potency is observed for analogue 1h, which holds
a 2-naphthyl group in this position (IC50 values 0.66 μM vs
3.2 μM, respectively). Analogue 1i, a structural hybrid of
UCPH-101 and 1c, is equipotent with UCPH-101 and under-
lines the importance of the R1 group (7-position) being a
1-naphthyl moiety, while the chemical nature of R2 group
(4-position) is less important. However, a substituent in the
R2 position is mandatory for inhibitory activity, as 1d shows
no inhibitory activity in the concentration ranges tested
(>300 μM). The requirement of an aromatic group in R1
was previously established by the observed loss of inhibitory
activity of analogue 1e (R1 = Me, >300 μM). It is interesting
to see that inhibitory activity is reestablished, when a benzyl
group is introduced as the R1 substituent, as 1g displays an
IC50 value of 5.1 μM.
Analogues 1k-1n, which address the importance of the
cyano group in the 3-position, were all inactive at EAAT1,2,3.
Hence, it appears from this SAR that the cyano functionality
is mandatory for the inhibitory activity at EAAT1. The
importance of the amino group is addressed by the two
analogues 1o and 1p. Introduction of substituents on the
2-amino group leads to inactive compounds, and therefore
it is concluded that there is no room for substituents in this
direction. Removal of the amino (analogue 1q) also results in
the loss of activity. Thus, the amino group is clearly of crucial
importance for the inhibitory activity at EAAT1 by its
hydrogen bonding and accepting character.
a Reagents and conditions: Piperidine, abs ethanol, rt, 30 min 90%
(syn/anti-1r, 2:3 ratio) and 91% (syn/anti-1s, 1:1 ratio).
compounds in 90% yield and in a ratio of 3:2. This mixture
was readily separated by column chromatography. The same
outcome was observed for the reaction of β-ketolactone 15
with 16 to give a mixture of two compounds in 91% yield
with a ratio of 1:1 (Scheme 7). A mechanistic analysis of the
formation of 1r (Scheme 7) suggests that the two isolated
compounds could be the respective syn/anti stereoisomers or
the 8-regioisomer 17.
X-ray Crystallography. To resolve the syn/antistereo-
chemical configuration, a structural characterization of the
four compounds by X-ray crystallography was carried out.
To obtain high quality crystals, several solvents were tried
out of which acetonitrile gave the best results. The four X-ray
structures (Figure 5) revealed that the 6-hetero analogues 1r
and 1s were formed and furthermore that the two products
from each reaction were the syn- and anti-stereoisomers.
Because this is the first disclosure on these two heterocyclic
skeletons, a detailed NMR study (heteronuclear multiple
quantum coherence (HMQC) and heteronuclear multiple
bond coherence (HMBC)) was conducted. In summary,
the 2D NMR experiments HMQC and HMBC and their
detailed analyses (see Experimental Section) highlight the
The new heterocyclic analogues syn-1r, anti-1r, syn-1s, and
anti-1s were all found to be without inhibitory activity at the
EAAT1 subtype (IC50 > 300 μM), and no activity was
observed for any of the compounds at neither EAAT2 nor
EAAT3 (IC50 >300 μM). Whereas the latter finding certainly
was expected, the inactivity of the syn-1r, anti-1r, syn-1s, and
anti-1s at EAAT1 is indeed in contrary to our expectations

7186 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Erichsen et al.
Figure 6. (A) Shows superimposition of syn-stereoisomer (4S,7R)-1j
in green with the antistereoisomer (4S,7S)-1j in purple. (B) Shows
superimposition of the syn-stereoisomer (4R,7S)-1j in green with the
antistereoisomer (4R,7R)-1j in purple.
Figure 7. Plasma concentration-time profile of UCPH-101 in
male Sprague-Dawley rats administered 10 mg/kg orally. Data
points represent mean ( SEM of three individual animals. Brain
levels not shown as these were below limit of quantification at all
time points.
Scheme 8. Synthesis of 4,4-Dimethyl Analogue 1ta
one of the two pharmacophores A or B is descriptive for the
ligands inhibitory activity at EAAT1. It remains to be
elucidated which one, an objective that could be met by the
enantioselective synthesis of the four stereoisomers of
UCPH-101.
Bioavailability Study. Exposure studies with UCPH-101 in
rats showed that high plasma concentrations could be ob-
tained following an oral dose of 10 mg/kg (Figure 7). Follow-
ing a rapid absorption, the compound was eliminated in a
first-order fashion with a terminal half-life of approximately
30 min. Despite the fact that UCPH-101 was shown to be
orally absorbed with high systemic exposure, the concentra-
tion in the brain was found to be below the limit of quantifica-
tion (10 ng/g) at all evaluated time-points. This suggests that
UCPH-101 possess limited brain penetration capabilities.
a Inhibitory activity at EAAT1-3: >300 μM.
based on the modeling study. The study concluded that there
were no immediate concerns as to spacial clashes or loss of
hydrogen bonding potential. However, the induced change in
electronic distribution of the parental structure may be the
underlying cause of the inactivity. Yet, further elucidation of
this question is awaiting future studies.
Stereochemistry and 3D Pharmacophore. The parental
skeleton contains the two stereogenic centers of C4 and C7
(R1 and R2 ¼ H), and the compounds in this series are
generally obtained as an approximate 1:1 mixture of the two
diastereomers as determined by 1H NMR. Given the spacial
orientation of the R1 and R2 groups relative to the parental
skeleton, the two diastereomers may be designated the 4,7-
syn enantiomers and 4,7-anti enantiomers. The only exemp-
tion from this is in the case R1 is a 1-naphthyl group 1b
(UCPH-101) and 1i, which gave a 9:1 mixture of the 4,7-syn/
4,7-anti diastereomers. Despite extensive efforts using chiral
HPLC, we have only been able to separate the syn/anti
diastereomers of 1j by flash chromatography. Both diaster-
eomers, syn-1j and anti-1j, were found to be inhibitors of
EAAT1 of comparable IC50 values (3.8 and 7.2 μM, Table 1),
values which were also in concordance with the inhibitory
potency of 1j (3.9 μM, Table 1). To address this observation,
we performed a modeling study commencing with the loca-
lization of the low-energy conformations of the four stereo-
isomers of 1j by a stochastic conformational search (for
details see Experimental Section). A subsequent superimpo-
sition of all four stereoisomers suggests that the stereochem-
istry at C7 is likely not important for inhibitory activity as the
four R1 groups (phenyl) occupies the same area of space (not
shown). On the other hand, superimposition of the two pairs
of diastereomers suggest that the stereochemistry at C4 is
crucial as the R2 group (phenyl) points either up (A, Figure 6)
or down (B, Figure 6). This finding led us to hypothesize that
the EAAT1 protein only recognizes one of the two pharma-
cophore models A and B (Figure 6).
Conclusion
In conclusion, a series of analogues of UCPH-101 has
been designed, synthesized, and characterized pharmacologi-
cally as EAAT inhibitors. On the basis of the functional
properties of these analogues, the following SAR conclusions
may be drawn: the R1-substituent (7-position) must contain
an aromatic group, whereas the R2-substituent (4-position)
must be an alkyl or aryl group. Interestingly, a 1-naphthyl
group as the substituent in the 7-position as in analogue
UCPH-101(1b) appears to be superior to a 2-naphthyl group,
analogue 1h, in terms of EAAT1 activity. Furthermore, the
finding that 1i (R1 = 1-nathpthyl, R2 = Me) is equipotent to
UCPH-101 is evidence that the inhibitory potency of this
compoundclass ismainly dependent onthe chemical natureof
the R1-substituent over the R2-substituent. In this connec-
tion, the fact that two substituents in the 4-position is
clearly not allowed (compound 1t), together with finding
that syn-1j and anti-1j exhibit similar inhibitory activities,
strongly suggest that only one of the two pharmacophore
models described in Figure 6 is valid. Any of the chemical
changes introduced to the scaffold (lactam 1r and lactone
1s) or in the form of changing the 2-amino and the 3-cyano
groups resulted in loss of EAAT1 inhibitory activity. This
SAR information will be valuable for future explorations of
this first class of EAAT1 inhibitors. Finally, UCPH-101
was shown to be bioavailable in rats after oral administra-
tion with a plasma half-life of 30 min and furthermore the
compound was found to be not able to penetrate the BBB.
While this clearly limits the use of UCPH-101 for animal
models using systemic administration, the compound is
nevertheless highly valuable for a wide range of in vitro
and ex vivo experiments.
To challenge this hypothesis, the 4,4-dimethyl analogue 1t
(Scheme 8) was designed and synthesized by the three
component reaction by simple exchange of the aldehyde
for a ketone (acetone). This analogue exhibited no inhibitory
activity at any of the three EAAT subtypes (IC50 >300 μM
for EAAT1-3). This result allowed us to conclude that only

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7187
Experimental Section
for 46.5 h at room temperature, saturated NH4Cl(aq) (20 mL)
was added to the solution whereby all precipitated material
dissolved. The aqueous layer was extracted with diethyl ether
(3 ꢀ 20 mL), dried (MgSO4), filtrated, and evaporated in vacuo to
give a white solid. Purification by dry column vacuum chroma-
tography (10:1 heptanes/EtOAc) afforded the title compound
(2.0 g, 80%) as a white solid. H NMR (300 MHz, CDCl3) δ:
7.94 (s, 1H), 7.87-7.81 (m, 3H), 7.69-7.64 (m, 2H), 7.54-7.48
(m, 2H), 6.82 (d, 1H), 2.43 (s, 3H).
Chemistry. All reactions involving dry solvents or sensitive
agents were performed under a nitrogen atmosphere, and
glassware was dried prior to use. Solvents were dried according
to standard procedures, and reactions were monitored by
analytical thin-layer chromatography (TLC) analysis. TLC
was carried out using Merck silica gel 60 F254 aluminum sheets.
Flash chromatography was carried out using Merck silica gel
60A (35-70 μm). Dry column vacuum chromatography34 was
performed using Merck silica gel 60 (20-45 μm). 1H NMR
spectra were recorded on a 300 MHz Varian Mercury 300BB
and 13C NMR spectra on the 300 MHz Varian Gemini 2000BB.
The 2D NMR spectra were recorded on a 600 MHz Bruker
Avance spectrometer. MS spectra were recorded using LC-MS
performed using an Agilent 1200 series solvent delivery system
equipped with an autoinjector coupled to an Agilent 6400 series
triple quadrupole mass spectrometer equipped with an electro-
spray ionization source. Gradients of 10% aqueous acetonitrile
þ0.05% formic acid (buffer A) and 90% aqueous acetonitrile
þ0.046% formic acid (buffer B) were employed. Melting points
were measured with a MPA 100 Optimelt automatic melting
point system. All purchased chemicals were used without further
purification. Single crystals suitable for X-ray diffraction studies
were grown from a solution in acetonitrile, and the data of syn/
anti-1r and syn/anti-1s isomers were collected using graphite-
1
5-(Naphthalen-2-yl)cyclohexane-1,3-dione. To a 21 wt % solu-
tion of sodium ethoxide in denatured ethanol (8.34 mL, 22.3
mmol) was added diethyl malonate (3.4 mL, 22.3 mmol) fol-
lowed by (E)-4-(naphthalene-2-yl)but-3-en-2-one (4.38 g, 22.3
mmol) under a nitrogen atmosphere. The solution was heated
under reflux for 6 h, allowed to cool to room temperature
overnight, and filtered to provide a crude solid. The filtrate
was concentrated in vacuo to a gum which was suspended in
water (30 mL) and washed with DCM (2 ꢀ 40 mL). The aqueous
layer was concentrated in vacuo to a solid, which was then
combined with the solid obtained above. The combined solid
was treated with NaOH (16 mL, 2 N), and the resulting solution
heated under reflux for 2 h. After cooling to room temperature,
sulphuric acid (16 mL, 5 N) was added, and the suspension was
heated under reflux for 4.5 h. After cooling to room tempera-
ture, filtration provided the crude product, which was triturated
with toluene to give the title compound (4.07 g, 77%) as a white
˚
1
monochromated Mo KR radiation (λ = 0.71073 A) on a κ CCD
powder. H NMR (300 MHz, CD3OD) δ: 7.92-7.76 (m, 4H),
diffractometer with area detector. The purity of all tested com-
pounds was determined by elementary analysis to be >95%.
2-Amino-7-benzyl-4-(4-methoxyphenyl)-5-oxo-5,6,7,8-tetra-
hydro-4H-chromene-3-carbonitrile (1g). Equivalent molar amounts
of 5-benzylcyclohexane-1,3-dione (0.10 g, 0.49 mmol) and 2-(4-
methoxybenzylidene)malononitrile (0.90 g, 0.49 mmol) were dis-
solved in EtOH (2 mL). Piperidine (4 μL, 0.04 mmol) was added,
and the mixture was heated to 50 °C and stirred overnight. The
reaction mixture was evaporated and recrystallized from EtOH to
afford the title compound (0.09 g; 47%) as a yellow crystalline
solid. Rf 0.41(1:1 EtOAc/heptane). 1H NMR(300MHz, CDCl3) δ:
7.38-7.18 (m, 3H), 7.18-7.04 (m, 4H), 6.87-6.75 (m, 2H), 4.48
(br s, 2H), 4.36 (s, 1H), 3.77 (s, 1.5H), 3.76 (s, 1.5H), 2.79-2.58 (m,
2H), 2.58-2.27 (m, 4H), 2.23-2.07 (m, 1H). 13C NMR (75 MHz,
DMSO-d6) δ: 196.2, 164.4, 163.6, 159.2, 158.8, 139.9, 137.6, 129.9,
129.8, 129.3, 129.2, 129.1, 127.0, 120.7, 114.8, 114.7, 114.5, 59.3,
59.2, 55.9, 43.39, 43.32, 35.7, 35.5, 35.2, 35.0, 33.2, 32.6; mp 178.6-
180.1 °C.
2-Amino-4-(4-methoxyphenyl)-7-(naphthalene-2-yl)-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (1h). The product
was prepared according to a procedure for the synthesis of
UCPH-101 described previously by us.2 The crude product was
purified by recrystallization from EtOH (96%, 248 mL) to give
the title compound (1.40 g, 79%) as a white powder. Rf 0.35 and
0.41 (3:1 ratio of diastereomers, 1:1 EtOAc/heptane).1H NMR
(300 MHz, DMSO-d6) δ: 7.90-7.66 (m, 4H), 7.60-7.42 (m, 3H),
7.14 (d, 1.5H, J = 8.5Hz), 6.99 (s, 2H), 6.97 (d, 0.5H, J =8.5 Hz),
6.86 (d, 1.5H, J = 8.5 Hz), 6.70 (d, 0.5H, J = 8.5 Hz), 4.20 (s,
0.75H), 4.18 (s, 0.25H), 3.72 (s, 2.25H), 3.67 (s, 0.75H), 3.60-3.45
(m, 1H), 3.19-2.98 (m, 1H), 2.93-2.70 (m, 2H), 2.67-2.50 (m,
1H). 13C NMR(75MHz, DMSO-d6) δ: 195.7, 164.4, 163.4, 159.3,
159.1, 158.7, 158.6, 141.0, 140.9, 137.7, 137.3, 133.8, 133.7, 132.8,
132.7, 129.1, 129.0, 128.9, 128.5, 128.4, 128.3, 128.2, 127.0, 126.9,
126.7, 126.6, 126.5, 125.9, 125.8, 120.7, 114.7, 114.6, 114.4, 59.4,
59.3, 56.0, 55.9, 44.2, 44.1, 38.43, 38.37, 35.8, 35.6, 34.7, 34.3; mp
216.3-219.2 °C.
7.56-7.43 (m, 3H), 3.65-3.50 (m, 1H), 2.83 (dd, 2H, J = 17.0,
12.0 Hz), 2.68 (dd, 2H, J = 17.0, 5.0 Hz). 13C NMR (75 MHz,
DMSO-d6) δ: 141.0, 133.0, 131.9, 127.9, 127.5, 127.4, 126.0,
125.6, 125.5, 124.9, 103.6, 40.1, 39.8, 39.5, 39.2, 39.0; mp 208.2-
211.3 °C.
2-Amino-4-methyl-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahy-
dro-4H-chromene-3-carbonitrile (1i). 5-(Naphthalen-1-yl)cyclo-
hexane-1,3-dione (50 mg, 0.20 mmol), malononitrile (14 mg,
0.20 mmol), and acetaldehyde (35 μL, 0.60 mmol) was stirred
in absolute ethanol (3 mL) at room temperature for 5 min.
N-Methylmorpholine (3 μL, 27 μmol) was added, and the reac-
tion mixture was stirred for 5 h. The reaction mixture was
concentrated with silica. The crude/silica mixture was purified
by column chromatography on silica gel (eluent: 3:4 EtOAc/
heptane). This afforded the title compound (62 mg, 90%) as
colorless solid. Rf 0.30 (3:4 EtOAc/heptane). 1H NMR (300
MHz, CDCl3) δ: 8.01 (dd, 1H, J = 13.5, 8.1 Hz), 7.91-7.86 (m,
1H), 7.78 (dd, 1H, J = 8.1, 3.0 Hz), 7.59-7.24 (m, 4H), 4.46 (s,
2H), 4.33-4.09 (m, 1H), 3.48-3.39 (m, 1H), 2.96-2.69 (m, 4H),
1.35 (d, 2H, J = 6.6 Hz), 1.26 (d, 1H, J = 6.6 Hz). 13C NMR (75
MHz, CDCl3) δ: 196.0, 162.8, 157.6, 137.4, 134.0, 130.8, 129.2,
127.9, 126.5, 125.9, 125.4, 122.8, 122.3, 118.8, 116.3, 43.6, 34.4,
33.4, 24.9, 22.9, 22.2; mp 204-206 °C.
2-Amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-chromene-
3-carbonitrile (1j). Equimolar amounts of 5-phenylcyclohexane-
1,3-dione (0.94 g, 5.0 mmol), benzaldehyde (0.55 mL, 5.0 mmol),
and malononitrile (0.33 g, 5.0 mmol) were mixed together in
H2O/EtOH (1:1, 20 mL) and were allowed to react under
microwave irradiation (reaction time, 30 min; temp, 85 °C;
prestirring, 3 min; vial size, 10-20 mL; absorption level,: high;
fixed hold time, on). The reaction mixture was allowed to cool to
room temperature. The solvent was filtered off, and the solid
obtained was washed with H2O to give the title compound
(1.28 g, 72%) as a white crystalline powder. Rf 0.16 (2:8
EtOAc/toluene). 1H NMR (300 MHz, DMSO-d6) (∼1:1 ratio of
diastereomers) δ: 7.39-7.12 (m, 10H), 7.11-6.96 (m, 2H), 4.22
(s, 0.5H), 4.21 (s, 0.5H), 3.58-3.46 (0.5H), 3.45-3.34 (m, 0.5H),
(E)-4-(Naphthalene-2-yl)but-3-en-2-one. To a suspension of
sodium hydride 60% dispersion in mineral oil (0.572 g, 14.3
mmol) in dry diethyl ether (44 mL) was added a solution of
diethyl 2-oxopropylphosphonate (2.8 mL, 14.6 mmol) in dry
diethyl ether (10 mL). The solution was stirred for 1 h at room
temperature, after which a solution of 2-naphthaldehyde (2.00,
12.8 mmol) in dry diethyl ether (10 mL) was added. After stirring
3.11-2.90 (m, 1H), 2.80-2.60 (m, 2H), 2.52-2.36 (m, 1H). 13
C
NMR (75 MHz, DMSO-d6) δ: 195.9, 164.8, 164.0, 159.4, 159.2,
145.6, 145.3, 143.54, 143.48, 129.4, 129.34, 129.25, 129.1, 128.1,
128.0, 127.8, 127.7, 127.6, 127.5, 127.4, 120.6, 114.4, 114.3, 59.1,
59.0, 44.24, 44.15, 38.5, 38.2, 36.5, 36.3, 34.6, 34.4; mp 178 °C
(decomposes).

7188 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Erichsen et al.
Methyl 2-Amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-
chromene-3-carboxylate (1k). Equimolar amounts of 5-phenyl-
cyclohexane-1,3-dione (0.94 g, 5.0 mmol) and (E)-methyl 2-cy-
ano-3-phenylacrylate (3a) (0.94 g, 5.0 mmol) were mixed
together in H2O/EtOH (1:1, 20 mL) and were allowed to react
under microwave irradiation (reaction time, 30 min; temp,
85 °C; prestirring, 3 min; vial size, 10-20 mL; absorption level,
high; fixed hold time, on). The reaction mixture was allowed to
cool to room temperature. The solvent was filtered off and the
solid obtained was washed with H2O to give a white powder.
Recrystallization from absolute EtOH gave the title compound
(1.25 g, 67%) as white crystals. Rf 0.33 (2:8 EtOAc/toluene). 1H
NMR (300 MHz, DMSO-d6) (∼1:1 ratio of diastereomers) δ: 7.57
(d, 2H, J = 7.6 Hz), 7.38-7.02 (m, 10H), 4.58 (s, 0.5H), 4.55 (s,
0.5H), 3.49 (s, 3H), 3.32-3.20 (m, 1H), 3.06-2.87 (m, 1H), 2.82-
2.52 (m, 2H), 2.47-2.33 (m, 1H). 13C NMR (75 MHz, DMSO-d6)
δ: 196.0, 169.2, 164.4, 163.5, 160.3, 160.2, 147.3, 146.9, 143.6, 129.4,
129.3, 128.8, 128.6, 128.5, 128.4, 127.9, 127.8, 127.6, 127.5, 126.8,
126.7, 117.6, 78.5, 78.3, 51.4, 44.3, 44.1, 38.8, 38.4, 34.5, 34.3, 34.1,
33.9; mp 189.2-189.8 °C (decomposes).
tert-Butyl 2-Amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-
chromene-3-carboxylate (1l). Equimolar amounts of 5-phenyl-
cyclohexane-1,3-dione (0.94 g, 5.0 mmol) and (E)-tert-butyl
2-cyano-3-phenylacrylate (3b)(1.15 g, 5.0 mmol) were mixed
together in H2O/EtOH (1:1, 20 mL) and were allowed to react
under microwave irradiation (reaction time, 30 min; temp, 85 °C;
prestirring, 3 min; vial size, 10-20 mL; absorption level, high;
fixed hold time, on). The reaction mixture was allowed to cool to
room temperature. The mixture was evaporated to give an off-
white solid. Recrystallization from EtOH (absolute) gave the title
compound (1.50 g, 72%) as white crystals. Rf 0.68 (2:8 EtOAc/
toluene). 1H NMR (75 MHz, DMSO-d6) (∼1:1 ratio of dia-
stereomers) δ: 7.53-7.44 (m, 2H), 7.37-7.02 (m, 10H), 4.49 (s,
0.5H), 4.47 (s, 0.5H), 3.56-3.44 (m, 0.5H), 3.29-3.21 (m, 0.5H),
3.04-2.87 (m, 1H), 2.80-2.53 (m, 2H), 2.47-2.33 (m, 1H), 1.27
(s, 9H). 13C NMR (75 MHz, DMSO-d6) δ: 196.1, 168.8, 164.0,
163.2, 159.8, 159.7, 147.6, 147.1, 143.6, 129.4, 129.3, 128.84,
128.79, 128.5, 128.3, 127.8, 127.6, 127.5, 126.63, 126.56, 117.3,
79.9, 79.7, 79.4, 44.3, 44.2, 38.8, 38.3, 34.8, 34.6, 34.5, 34.4, 28.9;
mp 184.1-184.4 °C.
10.20 (s, 0.66H), 7.44-7.00 (m, 10H), 4.18 (br t, 1H), 3.55-3.37
(m, 1H), 3.02-2.36 (m, 6H), 2,29 (from toluene). 13C NMR (75
MHz, DMSO-d6) δ: 195.0, 194.7, 171.2, 171.0, 155.0, 154.7, 144.2,
144.1, 143.5, 143.4, 129.8, 129.42, 129.35, 129.3, 129.1, 127.9, 127.6,
127.5, 127.3, 126.2, 113.9, 113.8, 44.5, 44.0, 39.9, 39.4, 39.3, 35.0,
34.6, 34.5, 34.0, 22.0 (from toluene); mp >180 °C (decomposes).
(E)-Ethyl N-3-Cyano-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-
4H-chromen-2-ylformimidate (1o). Under a nitrogen atmosphere,
2-amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-chromene-3-
carbonitrile (1j) (0.86 g, 2.5 mmol) was suspended in acetic
anhydride (5.0 mL). Triethyl orthoformate was added, and the
mixture was refluxed (150 °C) for 3.5 h. The reaction mixture was
cooled to room temperature and then evaporated to give a light-
brown solid. Recrystallization from ethanol gave the title com-
pound (0.856 g; 86%) as off-white crystals. Rf 0.59 (2:8 EtOAc/
toluene). 1H NMR (300 MHz, CDCl3) (∼1:1 ratio of dia-
stereomers) δ: 8.22 (s, 0.5H), 8.21 (s, 0.5H), 7.40-7.15 (m,
10H), 4.60 (s, 0.5H), 4.58 (s, 0.5H), 4.40 (q, 1H, J = 7.15 Hz),
4.39 (q, 1H, J = 7.15 Hz), 3.58-3.46 (m, 0.5H), 3.45-3.31 (m,
0.5H), 3.02-2.55 (m, 4H), 1.38 (t, 1.5H, J = 7.15 Hz), 1.37 (t,
1.5H, J = 7.15 Hz). 13C NMR (75 MHz, CDCl3) δ: 195.6, 195.4,
163.6, 162.8, 159.6, 156.3, 156.1, 142.4, 142.1, 142.0, 129.4, 129.3,
129.2, 129.1, 128.3, 128.0, 127.9, 127.8, 127.7, 127.1, 127.0, 117.5,
114.5, 114.4, 84.6, 84.4, 64.9, 64.8, 44.2, 44.0, 39.0, 38.6, 38.1, 38.0,
35.2, 35.1, 14.3; mp 143.4-147.6 °C.
N-(3-Cyano-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-chromen-
2-yl)acetamide (1p). Under a nitrogen atmosphere, 2-amino-5-oxo-
4,7-diphenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (1j
(1.00 g, 2.92 mmol) was suspended in acetic anhydride (2.76
mL, 29.2 mmol) and the mixture was refluxed (150 °C) for 2 h.
The reaction mixture was then cooled to room temperature and
evaporated to give a light-brown oil. Purification of the crude
product by dry column vacuum chromatography (eluent: 1:9
EtOAc/toluene) gave the title compound (0.095 g, 8%) as off-
white crystals. Rf 0.24 and 0.31 (two pairs of diastereomers) (1:1
1
EtOAc/heptane). H NMR (300 MHz, CDCl3) (∼1:1 ratio of
diastereomers) δ: 7.49-7.20 (m, 11H), 4.69 (s, 0.5H), 4.68 (s,
0.5H), 3.66-3.54 (m, 0.5H), 3.51-3.37 (m, 0.5H), 3.08-2.90 (m,
2H), 2.84-2.63 (m, 2H), 2.24 (s, 1.5H), 2.23 (s, 1.5H), 1.50 (br s,
1H). 13C NMR(75MHz, DMSO-d6) δ: 195.6, 195.5, 171.0, 165.1,
164.1, 151.0, 150.9, 143.03, 142.96, 141.8, 141.5, 129.4, 129.21,
129.17, 128.88, 128.85, 128.4, 128.3, 127.64, 127.61, 127.5, 115.2,
112.8, 112.7, 97.0, 96.9, 44.2, 38.3, 38.2, 38.0, 34.1, 33.8, 25.8;
mp >146 °C (decomposes).
2-Amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-chromene-
3-carboxamide (1m). Under a nitrogen atmosphere, 5-phenyl-
cyclohexane-1,3-dione (0.57 g, 3.0 mmol) and (E)-2-cyano-3-
phenylacrylamide (3c) (0.52 g, 3.0 mmol) were suspended in dry
dioxane (12 mL). Piperidine (39 μL, 0.39 mmol) was added, and
the mixture was stirred at room temperature overnight. The
reaction mixturewasallowed to stand withoutstirring overnight.
The solvent was filtered off, and the solid obtained was washed
with dioxane, which upon drying gave the title compound (0.62
g, 57%) as white powder. Rf 0.31 (4:6:0.5 EtOAc/heptane/
5-oxo-4,7-Diphenyl-5,6,7,8-tetrahydro-4H-chromene-3-car-
bonitrile (1q). Under a nitrogen atmosphere, (E)-2-formyl-3-
phenylacrylonitrile (8) (0.10 g, 0.64 mmol) and 5-phenylcyclo-
hexane-1,3-dione (0.12 g, 0.64 mmol) were suspended in dry
CH2Cl2 (20 mL). Triethylamine (0.1 mL, 0.7 mmol) was
added, and the mixture became clear. The reaction mixture
was stirred for 2 h before DMAP (0.016 g, 0.127 mmol) and
MsCl (50 μL, 0.64 mmol) were added. The mixture was stirred
for 4 days and then washed with H2O (20 mL ꢀ 2). The organic
phase was dried using MgSO4, filtered and evaporated to give
an orange foamy solid (0.240 g). Purification by dry column
vacuum chromatography (eluent, EtOAc/heptane; gradient,
0-25% EtOAc) gave the title compound (0.14 g, 67%) as
white powder. Rf 0.51 (4:6 EtOAc/heptane). 1H NMR (300
MHz, CDCl3) (∼1:1 ratio of diastereomers) δ: 7.41-7.21 (m,
11H), 4.50 (s, 0.5H), 4.48 (s, 0.5H), 3.56-3.44 (m, 0.5H),
3.43-3.29 (m, 0.5H), 3.00-2.78 (m, 2H), 2.77-2.54 (m, 2H).
13C NMR (75 MHz, CDCl3) δ: 195.8, 195.6, 163.9, 163.0,
149.5, 149.3, 142.1, 142.0, 141.7, 141.4, 129.4, 129.3, 129.2,
128.5, 128.3, 128.2, 127.9, 127.8, 127.1, 127.0, 116.5, 114.0,
98.4, 98.2, 44.2, 44.1, 38.9, 38.5, 36.1, 36.0, 35.0, 34.9; mp
131.9-132.4 °C. LC-MS (m/z) calcd for C22H17NO2 [M þ Hþ],
328.1; found, 328.1.
1
AcOH). H NMR (300 MHz, DMSO-d6) (∼1:1 ratio of dia-
stereomers) δ: 7.63 (br s, 1H), 7.60 (br s, 1H), 7.37-7.03 (m,
10H), 6.49 (br s, 2H), 4.62 (s, 0.5H), 4.60 (s, 0.5H), 3.54-3.44 (m,
0.5H), 3.28-3.15 (m, 0.5H), 2.99-2.86 (m, 1H), 2.78-2.33 (m,
3H). 13C NMR (75 MHz, DMSO-d6) δ: 195.9, 171.4, 164.1,
163.3, 158.4, 147.1, 146.6, 143.7, 129.34, 129.25, 128.8, 128.7,
128.5, 127.85, 127.81, 127.6, 127.5, 126.8, 126.7, 117.5, 80.3, 80.1,
44.4, 44.2, 38.8, 38.4, 34.6, 34.4, 34.2, 34.1; mp 186.3-186.8 °C
(decomposes).
2-Amino-4,7-diphenyl-7,8-dihydro-4H-chromen-5(6H)-one (1n).
Under a nitrogen atmosphere, tert-butyl 2-amino-5-oxo-4,7-di-
phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carboxylate (1l) (0.50 g,
1.2 mmol) was dissolved in a solution of HCl in dioxane (4.0 M,
10 mL). The mixture was heated to 50 °C and then stirred at
that temperature for 25 h. The reaction mixture was cooled to
room temperature and evaporated to give an off-white solid which
was triturated with ether. The crude product (0.32 g, 85%) was
recrystallized from toluene to give the title compound (0.14 g, 37%,
cocrystallized with 0.3 mol equiv toluene). Rf 0.13 (2:8 EtOAc/
2-Amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-pyrano-
[3,2-c]pyridine-3-carbonitrile (1r). 6-Phenylpiperidine-2,4-dione (13)
(115 mg, 600 μmol) and 2-benzylidenemalononitrile (16) (94 mg,
1
toluene). H NMR (300 MHz, DMSO-d6) δ: 10.22 (s, 0.33H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7189
600 μmol) were stirred in absolute ethanol (7 mL) in a sample vial at
room temperature. Piperidine (12 μL, 120 μmol) was added, and the
reaction mixture was stirred for 15 min. The reaction mixture was
concentrated with silica gel in vacuum (using hot ethanol to dissolve)
and purified by column chromatography in silica gel (eluent: 1:5
EtOAc/dichloromethane). This afforded compounds 1r syn:anti
stereoisomers (185 mg in a ratio of 2:3, 89%) as a colorless solid.
Analytical data for anti-1r: Rf 0.53 (1:5 EtOAc/dichloromethane).
1H NMR (300 MHz, DMSO-d6) δ: 7.63 (s, 1H), 7.41-7.16 (m,
10H), 6.92 (s, 2H), 4.72-4.65 (m, 1H), 4.28 (s, 1H), 2.75-2.70 (m,
2H). 13C NMR (75 MHz, DMSO-d6) δ: 35.0, 36.9, 53.7, 58.9, 108.0,
120.8, 127.4, 127.7, 127.8, 128.2, 128.6, 129.1, 129.2, 129.3, 141.9,
124.7, 155.0, 159.6, 166.2; mp 225-227 °C. LC-MS [M þ Hþ] calcd
for C11H11NO2, 344.1321; found, 344.1. Analytical data for syn-1r:
Rf 0.42 (1:5 EtOAc/dichloromethane). 1H NMR (300 MHz,
DMSO-d6) δ: 7.88 (d, 1H, J = 3.9 Hz), 7.25-7.16 (m, 6H), 7.06-
7.03 (m, 2H), 6.98-6.94 (m, 2H), 6.85 (s, 2H), 4.73-4.65 (m, 1H),
the mixture was allowed to stand at room temperature for 2.5 h. The
solvent was filtered off and the solid obtained was washed with ice-
cold MeOH to give the title compound (3.47 g, 93%) as shiny white
crystals. Rf 0.71 (2:8 EtOAc/toluene). 1H NMR (300 MHz,
DMSO-d6) δ: 8.40 (s, 1H), 8.07-8.02 (m, 2H), 7.67-7.54 (m,
3H), 3.86 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 163.2, 156.0,
134.3, 132.2, 131.7, 130.2, 116.4, 103.1, 54.2; mp 89.5-90.0 °C.
(E)-tert-Butyl-2-cyano-3-phenylacrylate (3b). To benzalde-
hyde (2.0 mL, 20 mmol) dissolved in i-PrOH (25 mL) were
added tert-butyl 2-cyanoacetate (2.9 mL, 20 mmol) and piper-
idine (0.26 mL, 2.6 mmol). The reaction mixture was refluxed for
70 min and then allowed to cool to room temperature. The
mixture was evaporated to give an yellow oil that solidifies on
standing. Two successive recrystallizations from EtOH (absolute)
gave the title compound (3.06 g, 67%) as white crystals. Rf 0.77
(2:8 EtOAc/toluene). 1H NMR (300 MHz, DMSO-d6) δ: 8.30 (s,
1H), 8.03-7.98 (m, 2H), 7.64-7.53 (m, 3H), 1.53 (s, 9H). 13C
NMR (75 MHz, DMSO-d6) δ: 161.5, 155.3, 134.0, 132.2, 131.5,
130.1, 116.5, 104.8, 84.2, 28.4; mp 55.7-57.4 °C.
(E)-2-Cyano-3-phenylacrylamide (3c). To a suspension of
2-cyanoacetamide (1.7 g, 20 mmol) in EtOH (25 mL) were
added benzaldehyde (2.0 mL, 20 mmol) and piperidine (0.26
mL, 2.6 mmol). The reaction mixture was allowed to stir at room
temperature for 3 h. During stirring, the reaction mixture
became clear and slightly yellow. The mixture was evaporated
to give a yellow solid. Recrystallization from EtOH (absolute)
gave the title compound (1.4 g, 42%) as slightly yellow crystals.
Rf 0.15 (2:8 EtOAc/toluene). 1H NMR (300 MHz, DMSO-d6) δ:
8.17 (s, 1H), 8.01-7.69 (m, 4H), 7.61-7.50 (m, 3H). 13C NMR
(75 MHz, DMSO-d6) δ: 163.5, 151.5, 133.2, 132.8, 130.9, 130.1,
117.3, 107.5; mp 117.0-117.3 °C.
(E)-2-(Diethoxymethyl)-3-phenylacrylonitrile (7). Under a ni-
trogen atmosphere, benzaldehyde (3.1 mL, 30 mmol) and 3,3-
diethoxypropionitrile (4.5 mL, 30 mmol) were mixed together
and added to a solution of NaOEt in EtOH (11.2 mL, 21 wt %,
30 mmol). The mixture was stirred at room temperature for 1 h,
and then additional dry EtOH (5 mL) was added and the
mixture was stirred for further 20 h. The solvent was removed
by evaporation in vacuo, and the yellow residue was partitioned
between CH2Cl2 (600 mL) and H2O (150 mL). The organic
phase was evaporated to give an yellow oil (8.80 g). The crude
product was used in the next step (8) without further purifica-
tion. Rf 0.63 (2:8 EtOAc/toluene).
(E)-2-Formyl-3-phenylacrylonitrile (8). (E)-2-(Diethoxymethyl)-
3-phenylacrylonitrile (7) (8.80 g, 30.0 mmol) was dissolved in 6N
HCl (90 mL), and the mixture was stirred at room temperature for
1 h. The yellow precipitate was collected by filtration, washed with
water, and then recrystallized from diethyl ether to give the title
compound (1.90 g, 40% over two steps) as a yellow crystalline
solid. Rf 0.52 (1:1 EtOAc/heptane). 1H NMR (300 MHz, CDCl3)
δ: 9.59 (s, 1H), 8.04 (d, 2H, J=7.7Hz), 7.91(s, 1H), 7.67-7.51 (m,
3H). 13C NMR (75 MHz, CDCl3) δ: 187.4, 159.6, 134.8, 131.8,
131.7, 130.0, 114.5, 112.8; mp 93.6-96.0 °C
4.26 (s, 1H), 3.31-3.22 (m, 1H), 2.55 (dd, 1H, J = 17.1, 2.1). 13
C
NMR (75 MHz, DMSO-d6) δ: 33.7, 36.9, 51.1, 58.7, 108.4, 120.7,
126.9, 127.0, 127.4, 128.0, 128.3, 128.4, 128.9, 129.0, 142.9, 145.0,
153.2, 159.4, 165.6; mp 226-228 °C. LC-MS [M þ Hþ] calcd for
C11H11NO2, 344.1321; found, 344.1.
2-Amino-5-oxo-4,7-diphenyl-4,5,7,8-tetrahydropyrano[4,3-b]-
pyran-3-carbonitrile (1s). 6-Phenyldihydro-2H-pyran-2,4(3H)-
dione (15) (57 mg, 0.3 mmol) and 2-benzylidenemalononitrile
(16) (46 mg, 0.3 mmol) was stirred in absolute ethanol (2 mL) at
room temperature for 10 min. Piperidine (6 μL, 60 μmol) was
added, and the reaction mixture was stirred for 30 min. The
precipitated solid was dissolved in hot ethanol and concentrated
with silica. The crude/silica mixture was purified by column
chromatography on silica gel (eluent: 1:10 EtOAc/dichloro-
methane). This afforded the title compound 1s syn:anti stereo-
isomers (94 mg in a ratio of 1:1, 91%) as a colorless solid.
Analytical data for anti-1s: Rf 0.38 (1:10 EtOAc/dichloro-
1
methane). H NMR (300 MHz, DMSO-d6) δ: 7.55-7.27 (m,
10H), 7.19 (s, 2H), 5.62 (dd, 1H, J = 12.6, 3.6 Hz), 4.38 (d, 1H,
J = 1.5 Hz), 3.32-3.21 (m, 1H), 2.88 (dd, 1H, J = 17.1, 3.9 Hz).
13C NMR (75 MHz, DMSO-d6) δ: 33.0, 37.3, 58.8, 77.2, 105.2,
120.3, 127.5, 127.8, 128.4, 129.3, 129.5, 138.6, 144.6, 158.9,
159.8, 164.9, 175.0; mp 225-227 °C. LC-MS [M þ Hþ] calcd for
C11H10O3, 345.1161; found, 345.2. Analytical data for syn-1s:
1
Rf 0.24 (1:10 EtOAc/dichloromethane). H NMR (300 MHz,
DMSO-d6) δ: 7.34 (s, 5H), 7.24-7.16 (m, 4H), 7.08 (s, 2H), 6.85
(d, 2H, J = 7.8 Hz), 5.73 (dd, 1H, J = 8.1, 5.1 Hz), 4.15 (s, 1H),
3.22 (dd, 1H J = 17.4, 8.5 Hz), 3.02 (dd, 1H, J = 17.4, 5.1 Hz).
13C NMR (75 MHz, DMSO-d6) δ: 31.9, 37.6, 59.3, 76.6, 105.4,
120.2, 126.9, 127.5, 127.9, 129.1, 129.2, 129.3, 139.2, 144.8,
158.8, 159.5, 164.4; mp 222-224 °C. LC-MS [M þ Hþ] calcd for
C11H10O3, 345.1161; found, 345.1.
2-Amino-4,4-dimethyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (1t). Acetone (0.22 mL, 3.0 mmol) was
added to a mixture of 5-phenylcyclohexane-1,3-dione (0.58, 3.1
mmol) and malononitrile (0.20 g, 3.1 mmol) in 99.9% ethanol
(6.0 mL). 4-Methylmorpholine (0.34 mL, 3.1 mmol) was added,
and the mixture was stirred for 30 min, after which it was left to
stand at room temperature for 28 h. The precipitate was filtered
off and washed with ice-cold ethanol and ice-cold heptanes.
Recrystallization from propan-2-ol gave the title compound
tert-Butyl Phenyl(phenylsulfonyl)methylcarbamate (10). tert-
Butyl carbamate (2 g, 17 mmol) and sodium benzenesulfinate
(5.6 g, 34 mmol) were stirred in a mixture of methanol (20 mL)
and H2O (40 mL) at room temperature under an nitrogen
atmosphere. Benzaldehyde (3.4 mL, 34 mmol) and formic acid
(1.2 mL, 34 mmol) were added to the mixture and stirred at room
temperature for 3 days. The white solid material was filtered and
washed several times with water and diethyl ether. This afforded
the title compound (4.6 g, 78% yield) as colorless solid. 1H
NMR (300 MHz, CDCl3) δ: 7.92 (d, 2H, J = 7.2 Hz), 7.67-7.61
(m, 1H), 7.56-7.50 (m, 2H), 7.46-7.36 (m, 5H), 5.94 (d, 1H, J =
10.8 Hz), 5.84 (d, 1H, J = 10.8 Hz), 1.25 (s, 9 H). 13C NMR (75
MHz, CDCl3) δ: 28.4, 74.3, 81.6, 129.1, 129.3, 129.4, 129.9,
130.2, 134.3, 137.3, 153.9; mp 177-178 °C. LC-MS [M þ Hþ]
calcd for C18H21NO4S, 348.1191; found, 348.1.
1
(0.37 g, 42%) as white crystals. H NMR (300 MHz, CDCl3)
δ: 7.38-7.21 (m, 5H), 4.32 (br s, 2H), 3.36 (septet, 1H), 2.58-
2.80 (m, 4H), 1.55 (s, 3H), 1.51 (s, 3H). 13C NMR (75.5 MHz,
CDCl3) δ: 196.5, 161.5, 156.1, 142.0, 129.2, 127.6, 126.8, 118.7,
118.6, 70.3, 45.9, 38.5, 35.5, 32.2, 29.6, 28.7; mp 195.5-196.5 °C.
(E)-Methyl-2-cyano-3-phenylacrylate (3a). To benzaldehyde
(2.1 mL, 20 mmol) dissolved in MeOH (25 mL) were added
methyl 2-cyanoacetate (1.8 mL, 20 mmol) and piperidine
(0.26 mL, 2.6 mmol). Upon addition of piperidine, the reaction
mixture changed color from colorless to yellow. The reaction
mixture was refluxed for 70 min and then allowed to cool to
room temperature. Upon cooling, the product crystallized and
(E)-tert-Butyl Benzylidenecarbamate (11). tert-Butyl phenyl-
(phenylsulfonyl)-methylcarbamate (10) (1 g, 2.9 mmol) and

7190 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Erichsen et al.
Cs2CO3 (2.36 g, 7.25 mmol) were stirred in dry THF (30 mL) at
60 °C under an nitrogen atmosphere for 30 min. The mixture
was cooled to room temperature and filtered through Celite. The
filtrate was concentrated to give the title compound (590 mg,
solution of 0.2 M KOH (8 mL) for 30 min at room temperature. HCl
(4 M) was slowly added (pH ≈ 2), and the precipitated solid was
filtered and dried to afford the title compound (730 mg, 90%) as a
white solid. 1H NMR (300 MHz, CDCl3) δ:7.46-7.35(m,5H),5.69
(dd, 1H, J = 9.3, 3.6 Hz), 3.67 (d, 1H, J = 19 Hz), 3.49 (d, 1H, J =
19 Hz), 2.99-2.83 (m, 2H). 13C NMR (75 MHz, CDCl3) δ: 45.5,
47.4, 76.9, 126.3, 129.5, 129.7, 136.8, 167.2, 199.7; mp 125-127 °C.
LC-MS [M þ Hþ] calcd for C11H10O3, 191.0630; found, 191.1.
2-Benzylidenemalononitrile (16). Benzaldehyde (1 mL, 9.85
mmol), malononitrile (651 mg, 9.85 mmol), and anhydrous zinc
chloride (134 mg, 0.98 mmol) were stirred at 100 °C under
nitrogen atmosphere for 15 min. The reaction mixture was
cooled to room temperature and treated with a solution of
absolute alcohol to obtain a white solid, which was filtered
and dried to afford the title compound (1.42 g, 93%) as a white
solid. 1H NMR (300 MHz, CDCl3) δ: 7.91-7.87 (m, 1H), 7.76
(s, 1H), 7.65-7.59 (m, 1H), 7.55-7.49 (m, 2H). 13C NMR (75
MHz, CDCl3) δ: 82.8, 112.5, 113.7, 129.6, 130.7, 130.9, 134.6,
159.9; mp 85-86 °C. LC-MS [M þ Hþ] calcd for C10H6N2,
155.0531; found, 155.1.
1
100%) as colorless oil. H NMR (300 MHz, CDCl3) δ: 8.88
(s, 1H), 7.93-7.90 (m, 2H), 7.57-7.54 (m, 1H), 7.49-7.45 (m,
2H), 1.59 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ: 28.0, 82.2,
128.7, 130.0, 133.4, 129.8, 162.6, 169.8.
Methyl5-(tert-Butoxycarbonylamino)-3-oxo-5-phenylpentanoate
(12). Lithium diisopropylamide (14.4 mL, 28.8 mmol) was stirred
in dry THF (50 mL) at -78 °C under an nitrogen atmosphere. A
solution of methyl acetoacetate (1.55 mL, 14.4 mmol) in dry THF
(10 mL) was added with a syringes pump to the reaction mixture
over a period of 3 h. (E)-tert-butyl benzylidenecarbamate (11) (993
mg, 4.84 mmol) in dry THF (10 mL) was added to the reaction
mixture at -50 °C and stirred for 30 min. NH4Cl (10 mL) was
added at -50 °C, and the crude reaction was extracted with EtOAc
(3 ꢀ 30 mL) and the combined organic phases were washed with
H2O (15 mL) and brine (15 mL). The organic phase was dried over
MgSO4. After concentration in vacuo, the crude product was
purified by column chromatography on silica gel (eluent: 1:10
EtOAc/dichloromethane). This afforded the title compound (1.23 g,
80%) as colorless solid. Rf 0.33 (1:10 EtOAc/dichloromethane).
1H NMR (300 MHz, CDCl3) δ: 7.36-7.24 (m, 5H), 5.32 (br s, 1H),
5.10 (d, 1H, J = 6 Hz,), 3.68 (s, 3H), 3.40 (s, 2H), 3.00-3.21 (m,
2H), 1.41 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ: 28.8, 49.8, 51.6,
52.9, 80.2, 126.6, 126.7, 127.9, 128.0, 129.1, 155.5, 167.7, 201.2; mp
95-97 °C. LC-MS [M þ Hþ] calcd for C17H23NO5, 322.1567;
found, 322.1.
Crystallographic Data. For collection and analysis, please see
Supporting Information. CCDC 775129, 775130, 775131, and
775132 contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via http://www.
ccdc.cam.ac.uk.
In Silico Study. The modeling study was performed using the
software package MOE (Molecular Operating Environment,
Chemical Computing Group, 2010) using the built-in mmff94x
forcefield and the GB/SA continuum solvent model. General
procedure: The compound of interest was submitted to a
stochastic conformational search (standard setup) to determine
its low-energy conformation. Superimposition of selected low-
energy conformations was done using the built-in function by
6-Phenylpiperidine-2,4-dione (13). Methyl-5-(tert-butoxycar-
bonylamino)-3-oxo-5-phenylpentanoate (12) (250 mg, 0.78
mmol) was stirred in dichloromethane (10 mL) at 0 °C under a
nitrogen atmosphere. Trifluoroacetic acid (0.3 mL, 3.89 mmol)
was added and stirred at room temperature for 3 h. The reaction
mixture was concentrated and redissolved in dichloromethane
(6 mL) and NaHCO3 (4 mL) and stirred at room temperature for
6 h. The crude reaction was quenched with concentrated HCl
(pH ≈ 3) and extracted with EtOAc (3 ꢀ 20 mL), and the
combined organic phases were washed with H2O (15 mL) and
brine (15 mL). The organic phase was dried over MgSO4. After
concentration in vacuo, the crude product was crystallized with
EtOAc to afford the title compound (131 mg, 89%) as colorless
solid. 1H NMR (300 MHz, CDCl3) δ: 7.27-7.44 (m, 5H), 6.37
(br s, 1H), 4.80 (octet, 1H, J = 9.0, 4.5, 1.5 Hz), 3.37 (s, 2H), 2.89
1
0
fitting the three atoms amino groups and the C -carbons of the
phenyl rings.
Pharmacology. Cell culture of the EAAT-HEK293 cell lines
and the [3H]-D-Asp uptake assay were performed essentially as
previously described.33 The experimental procedures are de-
scribed in detail in Supporting Information.
Pharmacokinetics. Animals. All experimental animals were
allowed to acclimatize at the animal facilities for a minimum of
5 days before experimentation. The animals were kept under a
12 h day/night cycle (lights on at 6:00 a.m.) in a temperature
(21 ( 2 °C) and humidity (60 ( 10%) controlled environment.
Rats were kept in groups of two rats per cage with free access to
rat chow and tap water, ad libitum. All experiments were carried
out in accordance with the ethical rules of the Danish Commit-
tee and Use of Laboratory Animals.
(dd, 1H, J = 17.1, 4.5 Hz), 2.75 (dd, 1H, J = 16.2, 9.0 Hz). 13
C
NMR (75 MHz, CDCl3) δ: 38.8, 47.4, 53.2, 126.1, 129.1, 129.6,
139.2, 168.7, 202.5; mp 167-169 °C. LC-MS [M þ Hþ] calcd for
C11H11NO2, 190.0790; found, 190.1.
Methyl 5-Hydroxy-3-oxo-5-phenylpentanoate (14). Methyl
acetoacetate (1.2 mL, 15 mmol) was added to a suspension of
NaH (720 mg of a 60% dispersion in mineral oil, 18 mmol) in
THF (8 mL) at room temperature and stirred for 30 min under a
nitrogen atmosphere. The reaction mixture was cooled down to
-20 °C, and n-BuLi (11.3 mL of a 1.6 M solution in THF, 18
mmol) was added and stirred for 30 min. Benzaldehyde (1.82 mL,
18 mmol) was added and stirred for an additional 30 min. The
reaction mixture was quenched with saturated NH4Cl (30 mL)
and extractedwithEtOAc(3 ꢀ 30mL), andthe combined organic
phases were washed with H2O (20 mL) and brine (20 mL). The
organic phase was dried over MgSO4. After concentration in
vacuo, the crude product was purified by column chromatogra-
phy on silica gel (eluent: 1:10 EtOAc/dichloromethane). This
afforded the title compound (1.8 g, 57%) as yellow oil. Rf 0.23
(1:10 EtOAc/dichloromethane). 1H NMR (300 MHz, CDCl3) δ:
7.36-7.24 (m, 5H), 5.2-5.14 (m, 1H), 3.72 (s, 3H), 3.49 (s, 2H),
3.04-2.86 (m, 3H). 13C NMR (75 MHz, CDCl3) δ: 49.6, 51.6,
52.5, 69.8, 125.6, 127.8, 128.6, 142.4, 167.4, 202.6; mp 95-97 °C.
LC-MS [M þ Hþ] calcd for C12H14O4, 223.0892; found, 223.1.
6-Phenyldihydro-2H-pyran-2,4(3H)-dione (15). Methyl 5-hydro-
xy-3-oxo-5-phenylpentanoate (950 mg, 4.3 mmol) was stirred in a
Exposure Studies and Bioanalysis. Exposure of UCPH-101 in
plasma and brain was evaluated in male Sprague-Dawley rats
(225 g at arrival). The test compound was formulated in 100%
PEG400 and dosed orally (5 mL/kg). Three groups of rats
(n = 3/group) received a dose of 10 mg/kg, and plasma and
brain were taken at 0.5, 1, and 2 h, respectively, following drug
administration. Under isoflurane anesthesia, cardiac blood was
obtained in EDTA-coated tubes and centrifuged for 10 min at
4 °C, after which plasma was drawn off. Following decapitation,
the brain was removed and brain homogenate was prepared by
homogenizing the whole brain with 70% acetonitrile (1:4 v/v)
followed by centrifugation and collection of the supernatant.
Plasma and brain supernatant samples were frozen at -80 °C
until analysis. Concentrations of UCPH-101 were determined in
plasma and brain homogenate using UltraPerformance LC
chromatography (UPLC) followed by MS/MS detection. Then
150 μL of internal standard (5 ng/mL in acetonitrile containing
0.1% ammonium hydroxide) was added to 25 μL of calibration
standards, QC samples, and test samples. After centrifugation
(6200g, 4 °C, 20 min), 100 μL of supernatant from each sample
was transferred to a new plate and mixed with 100 μL of water

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7191
with 0.1% ammonium hydroxide, and the samples were placed
in the autosampler. Ten μL was injected into the UPLC. The
mobile phase consisted of water/acetonitrile with 0.1% ammo-
nium hydroxide pumped as a gradient through an analytical
column (Acquity UPLC BEH Phenyl 1.7 μM, 2.1 mm ꢀ 30 mm,
Waters, MA). Detection was performed using a Sciex-API 4000
MS (Applied Biosystems, The Netherlands) using electro spray
with positive ionization mode with a parent > daughter molec-
ular mass of 423.1 > 357.2 AMU. Retention time was 0.72 min.
The lower limit of quantification was 5.0 ng/mL in plasma and
10 ng/g in brain (peak S/N>6).
(13) Fuchter, M. J.; Hoffman, B. M.; Barrett, A. G. ROM polymeriza-
tion-capture-release: application to the synthesis of unsymmetrical
porphyrazinedithiols and peripherally metalated derivatives.
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Acknowledgment. We thank Nils Torsson Nyberg and
Jerzy W. Jaroszewski for technical assistance and access to
the Bruker Avance 600 MHz NMR apparatus. The technical
assistance of Niels Vissing Holst, Department of Chemistry,
University of Copenhagen, with collecting X-ray data is
gratefully acknowledged. Shahrokh Padrah and Anette L.
Eriksen are thanked for the technical assistance regarding
parts of the synthetic work. Dr. Susan G. Amara is thanked
for her generous gift of the EAAT cDNAs used for the stable
cell lines. We express our gratitude to the Lundbeck Founda-
tion, the Novo Nordisk Foundation, the Carlsberg Founda-
tion, Drug Research Academy;University of Copenhagen,
and the Danish Medical Research Council for their financial
support.
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Supporting Information Available: X-ray data and 2D NMR
data (HMQC and HMBC) of 1r and 1s. This material is
available free of charge via the Internet at http://pubs.acs.org.
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