Bioavailability Studies and in vitro Profiling of the Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitor UCPH-102

Article abstract of DOI:10.1002/cmdc.201500527

Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH-101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research field, its inability to penetrate the blood-brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg^-1) of the closely related analogue UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH-102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH-102 (10 μm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH-102 (20 mg kg^-1) did not induce acute effects or any visible changes in behavior. EAAT1 inhibition beyond the BBB: In the present study, oral administration (40 mg kg^-1) of the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. In vitro profiling of UCPH-102 (10 μm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is fully selective for EAAT1.

Full text of DOI:10.1002/cmdc.201500527

DOI: 10.1002/cmdc.201500527
Full Papers
Bioavailability Studies and in vitro Profiling of the
Selective Excitatory Amino Acid Transporter Subtype 1
(EAAT1) Inhibitor UCPH-102
Isabell Haym,[a] Tri H. V. Huynh,[a] Stinne W. Hansen,[a] Martin H. F. Pedersen,[b] Josep A. Ruiz,[a]
Mette N. Erichsen,[a] Mikko Gynther,[c] Walden E. Bjørn-Yoshimoto,[a] Bjarke Abrahamsen,[a]
Jesper F. Bastlund,[d] Christoffer Bundgaard,[d] Anette L. Eriksen,[a] Anders A. Jensen,[a] and
Lennart Bunch*[a]
Although the selective excitatory amino acid transporter sub-
type 1 (EAAT1) inhibitor UCPH-101 has become a standard
pharmacological tool compound for in vitro and ex vivo stud-
ies in the EAAT research field, its inability to penetrate the
blood–brain barrier makes it unsuitable for in vivo studies. In
the present study, per os (p.o.) administration (40 mgkgÀ1) of
the closely related analogue UCPH-102 in rats yielded respec-
tive plasma and brain concentrations of 10.5 and 6.67 mm after
1 h. Three analogue series were designed and synthesized to
improve the bioavailability profile of UCPH-102, but none dis-
played substantially improved properties in this respect. In vi-
tro profiling of UCPH-102 (10 mm) at 51 central nervous system
targets in radioligand binding assays strongly suggests that
the compound is completely selective for EAAT1. Finally, in
a rodent locomotor model, p.o. administration of UCPH-102
(20 mgkgÀ1) did not induce acute effects or any visible
changes in behavior.
Introduction
In the mammalian central nervous system (CNS), uptake of glu-
tamate (Glu) is mediated by a family of high-affinity Na+-de-
pendent excitatory amino acid transporters (EAAT) comprising
five subtypes: EAAT1–5.[1–4] Although EAAT2 is the most abun-
dant subtype in the adult brain and is believed to be responsi-
ble for more than 90% of total Glu uptake, the EAAT1 and
EAAT3 subtypes are also expressed throughout the CNS, albeit
at lower densities. Finally, EAAT4 is expressed almost exclusive-
ly on GABAergic Purkinje cells of the cerebellum and is consid-
ered to be a low-capacity Glu transporter, and EAAT5 is only
expressed in the retina. Changes in the expression and/or func-
tions of the EAATs have been linked to several disease states
such as amyotrophic lateral sclerosis, Alzheimer’s disease,
chronic pain, and obsessive compulsive disorder.
In the course of our previous work on the EAATs we discov-
ered the first class of selective EAAT1 inhibitors[5,6] and subse-
quently conducted elaborate structure–activity relationship
(SAR) studies to optimize the inhibitory potencies as well as
the pharmacokinetic properties of this inhibitor class.[7–10] The
analogue UCPH-101 has become a standard tool compound in
the EAAT field (Figure 1); however, as it does not penetrate the
blood–brain barrier (BBB), it is not suitable for in vivo stud-
ies.[11–14] Herein we report that the close analogue UCPH-102, in
contrast to UCPH-101, is able to cross the BBB, and we present
[a] Dr. I. Haym, Dr. T. H. V. Huynh, Dr. S. W. Hansen, Dr. J. A. Ruiz,
Dr. M. N. Erichsen, W. E. Bjørn-Yoshimoto, Dr. B. Abrahamsen, A. L. Eriksen,
Prof. A. A. Jensen, Prof. L. Bunch
Department of Drug Design and Pharmacology
Faculty of Health and Medical Sciences
University of Copenhagen, 2100 Copenhagen Ø (Denmark)
E-mail: lebu@sund.ku.dk
[b] Dr. M. H. F. Pedersen
Technical University of Denmark, Center for Nuclear Technologies
DTU Nutech/Hevesy Laboratory
Frederiksborgvej 399, Building 202, 4000 Roskilde (Denmark)
[c] Dr. M. Gynther
School of Pharmacy, Faculty of Health Sciences
University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio (Finland)
Figure 1. General formula I of the first class of selective EAAT1 inhibitors and
structures of three of the most potent analogues UCPH-101, UCPH-102, and
UCPH-103.
[d] Dr. J. F. Bastlund, Dr. C. Bundgaard
H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark)
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Table 1. Bioavailability in rat (p.o. administration, n=3) of UCPH-101, UCPH-102, and UCPH-103.
Compd
Dose
[mgkgÀ1
Plasma concentration [mm] (ngmLÀ1
0.5 h 1 h
)
Brain concentration [mm] (ngmLÀ1
)
]
2 h
0.5 h
1 h
2 h
UCPH-101[7]
UCPH-103[6]
UCPH-102
10
10
40
1.66
0.67
0.66
–
0.16
1.24
–
0
–
0
0
0.75
–
[a]
[b]
[a]
–
[a]
[a]
9.30 (3070Æ125)
10.5 (3457Æ324)
4.02 (1327Æ48)
6.67 (2203Æ269)
[a] Not determined. [b] Below detection limit of 250 ngmLÀ1
.
SAR studies aimed at improving the potency, bioavailability,
and pharmacokinetic properties of this EAAT1 inhibitor (com-
pound series 1a–k, 2a–e, and 3a–d). Finally, we describe triti-
um labeling of UCPH-102 ([3H]UCPH-102) for potential use in in
vitro studies.
Results and Discussion
We previously published data on the oral bioavailability and
BBB penetration for the two analogues UCPH-101[6] and UCPH-
103[8] in rats (Figure 1, Table 1). No BBB penetration could be
detected for UCPH-101 (2 h, 10 mgkgÀ1), and the brain con-
centration of UCPH-103 after 2 h (10 mgkgÀ1) was determined
to be only 0.75 mm. We speculated that the different abilities
to penetrate the BBB could be due to the size of the R2 sub-
stituent (4-methoxyphenyl versus methyl). We therefore inves-
tigated the oral bioavailability and BBB penetration of UCPH-
102 at a dose of 40 mgkgÀ1. As can be seen from Table 1, this
resulted in a brain concentration of 6.67 mm after 1 h (Table 1).
We next determined brain protein binding (fraction unbound,
FU [%]) of UCPH-102 by in vitro equilibrium dialysis, and found
the unbound fraction of the compound to be 3.8% (Table 2).
Scheme 1. Synthesis of quinoline and isoquinoline analogues 1a–i by a one-
pot three-component reaction. Reagents and conditions: a) sBuLi, DMF, THF,
À788C!RT (58 and 76%); b) diethyl (2-oxopropyl)phosphonate, NaH, THF,
RT (60–83%); c) diethyl malonate, NaOEt/EtOH, then NaOH (reflux), then
H2SO4 (reflux) (18–84%); d) malononitrile, acetaldehyde N-methylmorpho-
line, EtOH, 08C!RT (43–68%).
ride salt.[16] These were then cyclized to their corresponding
benzisoxazoles by treatment with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) and triphenylphosphine in high yields.[17]
The free phenol of series j was then converted into its triflate
form by use of triflic anhydride in good yield.[18] Heck coupling
of analogues 9j[19] and 9k afforded the ketones 5j,k, which
were next converted into the 1,3-diketones 6j,k by reaction
with diethylmalonate followed by aqueous ester hydrolysis
and acidic decarboxylation. Finally, the one-pot, three-compo-
nent reaction using malononitrile and acetaldehyde gave the
final compounds 1j,k (Scheme 2).
The pharmacological properties of the 11 analogues 1a–k
were characterized at stable EAAT1-, EAAT2-, and EAAT3-
HEK293 cells in a conventional [3H]d-Asp uptake assay,[20] and
the results are presented in Table 2. All of the analogues were
inactive at EAAT2 and EAAT3 in the concentration ranges
tested (Table 2). Analogues 1a–g displayed between 5- and
100-fold decreases in inhibitory potency at EAAT1 relative to
UCPH-102, depending on the position of the introduced nitro-
gen atom. Analogously, analogues 1h,i also exhibited ~10-fold
lower inhibitory potencies in comparison with a similar 2-naph-
thyl analogue previously reported.[5,6] Finally, the two benzisox-
azoles 1j,k displayed distinct inhibitory potencies (respective
IC50 values of 14 and 100 mm) depending on the substitution
point. This difference was not unexpected, as we have previ-
ously observed a similar pattern for 1-/2-naphthyl groups as
substituents at the 7-position of this scaffold.[5,6]
Design and synthesis of UCPH-102 analogues
Given the inverse correlation between lipophilicity and %FU,[15]
we next sought to optimize the physicochemical properties of
UCPH-102 by decreasing its lipophilic character, while main-
taining (or improving) its inhibitory potency at EAAT1. This was
attempted by the systematic introduction of a nitrogen atom
into the highly lipophilic naphthyl group of UCPH-102. The
nine quinoline/isoquinoline analogues 1a–i were designed and
their synthesis pursued in accordance with the previously de-
scribed one-pot three-component reaction (Scheme 1). For an-
alogues 1a,f commercially available bromoisoquinolines were
firstly converted into their corresponding carbaldehydes 4a,f.
All nine carbaldehydes 4a–i were condensed with diethyl (2-
oxopropyl)phosphonate to afford enones 5a–i in good yields.
Subsequent reaction with diethyl malonate followed by basic
ester hydrolysis and acidic decarboxylation afforded the corre-
sponding 1,3-diketones 6a–i in moderate to good yields. The
target compounds 1a–i were finally obtained by a one-pot
three-component reaction by addition of malononitrile and
acetaldehyde.
The synthesis of target analogues 1j,k commenced with
conversion of the corresponding acetophenones 7j,k into hy-
droxylamines 8j,k by reaction with hydroxylamine hydrochlo-
The unbound fraction of the most potent analogues 1a,e,g
in the series were determined to be 4.7, 6.9, and 3.8%, respec-
tively. Thus, for 1a and 1e this property was improved relative
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to UCPH-102 (Table 2). However, in light of their ~5-fold lower
inhibitory potencies at EAAT1, the modestly increased %FU
values were not sufficiently prominent for these analogues to
be considered superior to UCPH-102 as in vivo candidates. In
summary, the 1-series did not lead to a better in vivo candi-
date than UCPH-102.
Table 2. Inhibitory potencies of analogues 1a–k at EAAT1, calculated
logP values, and percent fraction unbound (FU) of selected analogues.[a]
Compd
R1
IC50 [mm]
(pIC50 ÆSEM)[b]
cLogP FU [%][c]
Design and synthesis of analogues of 2a
Recently, we have shown that analogue 2a (Table 3) displays
low-micromolar inhibitory potency at EAAT1. However, per-
centage unbound to brain protein (%FU) of this analogue was
determined to be 10-fold lower than UCPH-102 (0.3%, Table 3).
Nevertheless, we decided to follow the same strategy as that
outlined for 1a–i and incorporated a nitrogen atom into the
synthetically feasible positions of the naphthyl group in 2a
(analogues 2b–d). Further to this, we also synthesized two an-
UCPH-102
0.42[7]
2.71
1.44
3.8
4.7
1a
1b
2.5 (5.61Æ0.06)
4.6 (5.34Æ0.08)
1.47
–
Table 3. Inhibitory potencies of analogues 2a–f at EAAT1, calculated
logP values, and percent fraction unbound (FU) of selected analogues.[a]
1c
1d
1e
11 (4.95Æ0.09)
~50 (~4.3)
1.69
1.69
1.48
–
–
Compd
R2
IC50 [mm]
(pIC50 ÆSEM)[b]
cLogP
FU [%][c]
2.2 (5.65Æ0.07)
6.9
2a
1.6[21]
3.89
0.3
1 f
~30 (~4.5)
1.48
1.69
–
2b
2c
2d
35 (4.63Æ0.21)
8.8 (5.09Æ0.09)
15 (4.90Æ0.13)
2.87
2.87
1.40
–
–
–
1g
2.1 (5.67Æ0.06)
3.8
1h
1i
~100 (~4.0)
~100 (~4.0)
1.66
1.73
–
–
1j
14 (4.88Æ0.06)
~100 (~4.0)
1.51
1.55
–
–
2e
2 f
~100 (<4.0)
~100 (<4.0)
1.40
–
–
–
1k
[a] All analogues displayed no inhibitory potency at EAAT2 or EAAT3 at
concentrations of 100 mm (IC50 >100 mm). [b] Values are the mean ÆSEM
of n=3 experiments. [c] Residual brain protein binding.
[a] All analogues displayed no inhibitory potency at EAAT2 or EAAT3 at
concentrations of 100 mm (IC50 >100 mm). [b] Values are the mean ÆSEM
of n=3 experiments. [c] Residual brain protein binding.
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Scheme 2. Synthesis of target analogues 1j,k by a one-pot three-component reaction. Reagents and conditions: a) hydroxylamine hydrochloride, EtOH/H2O,
708C (80 and 99%); b) PPh3, DDQ, CH2Cl2, RT, 10 min, (63 and 84%), for 8j also triflic anhydride, pyridine, CH2Cl2, 08C!RT, 18 h, 87%; c) 5j: PdCl2(PPh3)2, NEt3,
DMF, 1108C, 18 h, quant., 5k: Pd(OAc)2, P(o-tol)3, K2CO3, DMF, MW, 1008C, 1 h, 53%; d) diethyl malonate, NaOEt/EtOH, then NaOH (reflux), then H2SO4 (reflux)
(47 and 49%); e) malononitrile, acetaldehyde, N-methylmorpholine, EtOH, 08C!RT (both 21%).
Scheme 3. Synthesis of 2b–f starting from commercially available 5-isopro-
pylcyclohexa-1,3-dione and the appropriate commercially available aldehyde
R2CHO, by use of a one-pot three-component reaction. Reagents and condi-
tions: a) malononitrile, appropriate commercial aldehyde (R2CHO), amine
base, EtOH, RT (22–62%).
Scheme 4. Synthesis of pyridine analogues 3a–d. Reagents and conditions:
a) NH4OAc, 3 Š MS, EtOH, reflux (84%); b) RCHO, CH2(CN)2, nPrOH, 110 8C.
alogues with a lower relative carbon content (pyridine ana-
logues 2e,f). The five analogues 2b–f were synthesized follow-
ing the standard route (Scheme 3) and subsequently character-
In vitro profiling of UCPH-102
ized as inhibitors at EAAT1, EAAT2, and EAAT3 in the [3H]d-Asp
uptake assay (Table 3).[20] Analogues 2b–d displayed 5- to 20-
fold decreases in inhibitory potencies at EAAT1 relative to 2a,
and a complete loss of inhibitory activity was observed for pyr-
idine analogues 2e,f. Given these results, the %FU values were
not determined for this 2-series.
UCPH-101 and UCPH-102 were shown in previous studies to
be completely selective inhibitors of EAAT1 over the four other
EAAT subtypes.[5,6,21] To elucidate the overall selectivity profile
of UCPH-102, the compound was subjected to an elaborate in
vitro screening on a selection of neurotransmitter receptors
and transporters in a total of 51 radioligand binding assays
(performed by the US National Institute of Mental Health’s Psy-
choactive Drug Screening Program) and at other CNS targets
in in-house functional assays. When tested at an assay concen-
tration of 10 mm in the competition binding assays, UCPH-102
did not display significant binding affinity to the majority of re-
ceptors and transporters for serotonin, dopamine, norepi-
nephrine, histamine, acetylcholine, GABA, glutamate, and
opioids included in the screenings (Table 4). However, UCPH-
102 inhibited radioligand binding to the a2C norepinephrine re-
ceptor, the norepinephrine transporter (NET), and the hista-
mine H2 receptor, as well as to the benzodiazepine binding
sites of native rat GABAA receptors, by approximately 50% (i.e.,
IC50 ~10 mm at these targets). In the functional assays, UCPH-
102 was found to be completely inactive at GABA transporters,
GABAB receptors, and the a4b2 nicotinic acetylcholine receptor
when tested at an assay concentration of 100 mm (Table 4).
A caveat associated with the use of the competition binding
assays in this screening is that ligands acting through allosteric
Design and synthesis of pyridine analogues
As a final attempt to optimize the in vivo properties of UCPH-
102, the 4H-pyran ring was substituted for a pyridine ring. The
four analogues 3a–d were synthesized, varying the 3-substitu-
ent from hydrogen to alkyl groups of increasing length
(Scheme 4). None of the four analogues displayed inhibitory
activity at EAAT1–3 at concentrations up to 300 mm (IC50 >
300 mm) in the [3H]d-Asp uptake assay.[20]
In summary, we pursued the optimization of the brain pro-
tein binding properties of UCPH-102 with the aim of nominat-
ing an in vivo candidate for animal studies. Three compound
series 1–3 were designed and synthesized, but none of the an-
alogues displayed improved properties in terms of potency
versus %FU.
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Table 4. Pharmacological properties of UCPH-102 at a selection of neuro-
transmitter receptors and transporters.[a]
Table 4. (Continued)
Target
Assay
IC50 [nm] (inhibi-
Target
Assay
IC50 [nm] (inhibi-
tion [%])
tion [%])
k (h)
m (h)
[3H]U69593 binding
[3H]DAMGO binding
>10000 (13)
>10000 (À0.7)
>10000 (15)
>10000 (0.2)
>10000 (À13)
>10000 (À1.7)
>10000 (À9.6)
>10000 (34)
>10000 (23)
>10000 (3.2)
>10000 (3.0)
>10000 (29)
>10000 (À13)
>10000 (À6.6)
Sigma
s1 (rat brain) [3H]pentazocine(+) binding
>10000 (À14)
>10000 (À0.9)
s2 (rat,
PC12)
[3H]DTG binding
[a] The binding properties of UCPH-102 (10 mm) at various receptors and
transporters were determined in competition binding assays (using radio-
ligand concentrations at or near the KD value for the specific target) per-
formed by the NIMH Psychoactive Drug Screening Program (PDSP). Per-
cent inhibition or percent potentiation of control radioligand binding at
10000 nm UCPH-102 is given in parentheses (positive and negative
values represent % inhibition and % potentiation, respectively). Inhibition
of control binding by >50% is considered significant by the PDSP. Data
are based on four independent determinations. [b] The functional proper-
ties of UCPH-102 at selected transporters and receptors were determined
in in-house functional assays (IC50 and EC50 values given in nm); h:
human, gp: guinea pig, m: mouse, r: rat.
Dopamine (DA)
D1 (h)
[3H]SCH23390 binding
>10000 (18)
>10000 (20)
>10000 (22)
>10000 (À0.9)
>10000 (16)
>10000 (À12)
D2 (h)
D3 (h)
D4 (h)
D5 (h)
[3H]N-methylspiperone binding
[3H]N-methylspiperone binding
[3H]N-methylspiperone binding
[3H]SCH23390 binding
DAT (h)
[3H]WIN35428 binding
Norepinephrine (NE)
a1A (h)
a1B (h)
a1D (h)
a2A (h)
a2B (h)
a2C (h)
b1 (h)
b2 (h)
b3 (h)
NET (h)
[3H]prazosin binding
>10000 (8.9)
>10000 (12)
>10000 (À0.7)
>10000 (À3.0)
>10000 (32)
~10000 (46)
>10000 (À0.9)
>10000 (À2.8)
>10000 (9.2)
~10000 (44)
[3H]prazosin binding
sites will not necessarily compete with, or modulate, orthoster-
ic radioligand binding to a target. However, the majority of tar-
gets assayed by radioligand binding in this study are class A 7-
transmembrane receptors, and to our knowledge few (if any)
allosteric modulators of these receptors have been reported to
not affect orthosteric radioligand binding. Hence, although we
cannot exclude the possibility that UCPH-102 could target an
allosteric site in some of the receptors, the inactivity of the
drug in the binding assays is likely to be a true reflection of its
pharmacology at them. In conclusion, considering the consid-
erably higher functional potency displayed by UCPH-102 as an
EAAT1 inhibitor (IC50 =0.42 mm) relative to the targets included
in this screening, it is reasonable to conclude that the com-
pound is quite selective for EAAT1.
[3H]prazosin binding
[3H]rauwolscine binding
[3H]rauwolscine binding
[3H]rauwolscine binding
[
125I]pindolol binding
[3H]CGP12177 binding
[3H]CGP12177 binding
[3H]nisoxetine binding
Acetylcholine
m1 (h)
m2 (h)
m3 (h)
m4 (h)
[3H]QNB binding
>10000 (0.2)
>10000 (À20)
>10000 (À5.7)
>10000 (À7.5)
>10000 (8.7)
>100000
[3H]QNB binding
[3H]QNB binding
[3H]QNB binding
m5 (h)
a4b2[b] (m)
[3H]QNB binding
FLIPR membrane potential
(EC50 >100000)
Following the in vitro profiling, we continued with an in vivo
study of possible acute behavioral effects 30–90 min after p.o.
administration of UCPH-102 at doses of 1, 5, and 20 mgkgÀ1
on mouse locomotor activity (Figure 2). None of the three
drug doses induced significant changes in activity counts in
comparison with vehicle (Figure 2). Furthermore, visual inspec-
tion of the mice did not disclose any changes in basal behav-
ior.
Histamine
H1 (h)
H2 (h)
H3 (gp)
H4 (h)
[3H]pyrilamine binding
>10000 (9.0)
~10000 (54)
>10000 (4.2)
>10000 (12)
[
125I]tiotidine binding
[3H]a-methylhistamine binding
[3H]histamine binding
GABA
Rat forebrain [3H]muscimol binding (GABA-A)
>10000 (22)
Rat brain
[3H]flunitrazepam binding (GABA- ~10000 (52)
Based on these results it seems reasonable to conclude that
UCPH-102 has no high-affinity off-targets that result in acute
visible effects. The free brain concentration of the two active
diastereomers of UCPH-102 after 40 mgkgÀ1 p.o. administra-
tion can be calculated to 0.13 mm (6.67/2”0.038). In view of
this, EAAT1 target occupancy must be expected to be lower
than the in vitro IC50 value determined for UCPH-102 in the
[3H]d-Asp uptake assay. This fact should be considered when
evaluating the effects of EAAT1 inhibition in the aforemen-
tioned in vivo study as well as in the planning of further stud-
ies.[22]
A)
GAT1[b] (h)
BGT1[b] (h)
GAT2[b] (h)
GAT3[b] (h)
[3H]GABA uptake
[3H]GABA uptake
[3H]GABA uptake
[3H]GABA uptake
>100000
>100000
>100000
>100000
>100000
(EC50 >100000)
>100000
GABAB1a,2[b] (r) Ca2+/Fluo4 assay (with Gqi5)
GABAB1b,2[b] (r) Ca2+/Fluo4 assay (with Gqi5)
(EC50 >100000)
Opioid
d (h)
[3H]DADLE binding
>10000 (10)
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azotization by use of sodium nitrite, concentrated sulfuric acid,
and potassium iodide in water gave diiodonaphthalene
11.[23,24] Under optimized conditions, treatment of 11 with two
equivalents of tert-butyllithium in tetrahydrofuran (THF) at
À788C, followed by quenching with one equivalent of N,N-di-
methylformamide, afforded the desired carbaldehyde 12 in
58% yield. This intermediate was then converted into enone
13 by reaction with diethyl (2-oxopropyl)phosphonate and
sodium hydride in dry THF.[6] Subsequent reaction with diethyl
malonate, 21% w/w sodium ethoxide in absolute ethanol fol-
lowed by aqueous ester hydrolysis and acidic decarboxylation
provided 1,3-diketone 14. Finally, a three-component reaction
of diketone 14, malononitrile, and acetaldehyde gave the de-
sired precursor 15 in 72% yield.[6] To explore reaction condi-
tions for the tritiation step, hydrodehalogenation of iodine 15
under palladium on carbon and hydrogen gas afforded UCPH-
102 in quantitative yield. Next, tritium labeling of iodine 15
was performed on a tritium manifold system using carrier-free
tritium gas and Pd/C as the catalyst to afford [3H]UCPH-102.
The radioligand was stable when dissolved and stored in 10%
ethanol in dimethyl sulfoxide.
Figure 2. Locomotor activity 0–30 min and 35–60 min after p.o. dosing of 1,
5, or 20 mg of UCPH-102. Data are the mean ÆSD of n=8 mice. Activity
was assessed using standard assay conditions and custom-made equipment,
as described in the Experimental Section.
Conclusions
Synthesis of [3H]UCPH-102
In the present study we identified the selective EAAT1 inhibitor
UCPH-102 as a better-suited candidate for in vivo studies than
UCPH-101, due to its BBB-penetration ability. We pursued the
structural optimization of UCPH-102 with respect to its lipophi-
licity and brain protein binding properties. Despite the synthe-
sis and evaluation of series 1–3, UCPH-102 remained the best-
suited in vivo candidate in evaluating the parameters of oral
bioavailability, BBB penetration, and brain protein binding
against potency. An elaborate in vitro screening of UCPH-102
(10 mm) at 51 relevant CNS targets advocated that UCPH-102 is
EAAT1-selective. Furthermore, p.o. administration of UCPH-102
at 1, 5, and 20 mgkgÀ1 in a rodent locomotor model produced
no significant effects. Also, visual inspection of the mice dis-
closed no changes in basal behavior.
We were interested in obtaining a tritium-labeled analogue of
UCPH-102 for potential use in numerous in vitro studies. A ret-
rosynthetic analysis suggested the iodinated compound 15 as
a suitable precursor for the synthesis of [3H]UCPH-102 by palla-
dium-catalyzed reduction with tritium gas (Scheme 5). Starting
from commercially available naphthalene-1,5-diamine (10), di-
Experimental Section
Chemistry
All reactions involving dry solvents or sensitive agents were per-
formed under a nitrogen or argon atmosphere, and glassware was
dried prior to use. Commercially available chemicals were used
without further purification. THF, DMF, and CH2Cl2 were dried using
an SG water solvent purification system. Reactions were monitored
by analytical thin-layer chromatography (TLC, Merck silica gel
60 F254 aluminum sheets). Flash chromatography and dry vacuum
chromatography was carried out using Merck silica gel 60A (35–70
and 15–30 mm, respectively). 1H NMR spectra were recorded on
a 300, 400, or 600 MHz Avance Bruker instrument, and 13C NMR
spectra on a 75, 100, or 150 MHz Avance Bruker instrument. HPLC
was performed with a Dionex UltiMate 3000 pump and photo-
diode array detector (l 200 and 210 nm, respectively) installed with
an XTerra MS C18 column (3.5 mm, 4.6 mm”150 mm), using a 5–
95% MeCN gradient in H2O containing 0.1% TFA. LC–MS spectra
were recorded using an Agilent 1200 series solvent delivery system
Scheme 5. Synthesis of iodine precursor 15 and its application to the syn-
thesis of [3H]UCPH-102. Reagents and conditions: a) NaNO2, KI, conc. H2SO4,
H2O, 808C, 21 h, (38%); b) tBuLi, DMF, THF, À788C, 1.5 h, (58%); c) diethyl
(2-oxopropyl)phosphonate, NaH, THF, 20.5 h, (73%); d) diethyl malonate,
21% w/w NaOEt, absolute EtOH, reflux, 15 h; e) 2m NaOH, H2O, 858C, 3 h,
2m H2SO4 (pHꢀ1), 1008C, 1 h, (71%); f) malononitrile, acetaldehyde, N-
methylmorpholine, absolute EtOH, RT, 23 h, (72%); g) 10% Pd/C, Et3N, H2(g)
,
3
~101 kPa, DMSO, RT, 90 min, quant.; h) 10% Pd/C, H2(g), 40 kPa, DMSO,
60 min.
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Full Papers
equipped with an auto-injector coupled to an Agilent 6400 series
triple quadrupole mass spectrometer equipped with an electro-
spray ionization source. Gradients of 5% aqueous MeCN+0.1%
HCO2H (solvent A), and 95% aqueous MeCN+0.05% HCO2H (sol-
vent B) were used. Melting points were measured using an MPA
100 Optimelt automatic melting point system and are uncorrected.
The purity of compounds submitted for pharmacological character-
ization was determined by HPLC to be >95%.
(8 mL) and malononitrile (15 mg, 0.24 mmol) was added at RT. The
mixture was cooled to 08C and acetaldehyde (27 mL, 0.50 mmol)
was added. The mixture was stirred for 10 min and then N-methyl-
morpholine (5 mL, 0.04 mmol) was added. The mixture was
warmed to RT and stirred for 18 h. The solvent was evaporated and
the residue purified by flash column chromatography (CH2Cl2/
EtOAc 9:1 to 5:1) to give the title compound as a mixture of dia-
stereomers as a white solid (45 mg, 0.13 mmol, 81%); Rf =0.24 and
1
0.34 (CH2Cl2/EtOAc, 5:1); H NMR (600 MHz, [D6]DMSO): d=8.46 (d,
Apparatuses and general setup for the tritiation experiment:
The tritiation reaction was performed on a custom-designed triti-
um manifold system manufactured by RC Tritec AG, Switzerland.
Tritium gas stored as uranium tritide on a uranium bed and was re-
leased by heating to ~5008C. HPLC analysis and purification of tri-
tium samples were performed using a Waters 1524 binary pump,
a Waters 2487 dual absorbance detector (set at l 220 and 254 nm),
equipped with Bioscan Hidex liquid scintillation counter. HPLC-
grade water obtained by purification using an Aurium 611 UV
system (Sartorius, Germany) was used. Safety handling with tritium
gas was monitored by a Canberra T100DSi Tritium air monitor.
Liquid scintillation counting was performed by RackBeta 1214 and
Hidex 300SL scintillation counters.
J=5.6 Hz, 1H), 8.45 (d, J=8.5 Hz, 1H), 8.38 (t, J=8.1 Hz, 1H), 8.00
(d, J=8.1 Hz, 1H), 7.81–7.77 (m, 1H), 7.75 (d, J=5.6 Hz, 1H), 7.74–
7.69 (m, 2H), 6.87 (s, 2H), 6.80 (s, 0.8H), 4.64 (d, J=6.3 Hz, 0.4H),
4.60–4.54 (m, 1H), 3.19–3.14 (m, 1H), 3.11 (dd, J=17.5, 10.2 Hz,
1H), 2.94 (qd, J=17.3, 7.0 Hz, 1H), 2.85 (t, J=4.0 Hz, 1H), 2.84–2.79
(m, 1H), 2.76 (dd, J=17.8, 4.5 Hz, 1H), 2.69 (dd, J=16.3, 4.6 Hz,
1H), 2.64 (d, J=3.8 Hz, 2H), 1.18 (d, J=4.5 Hz, 3H), 1.05 ppm (d,
J=7.0 Hz, 1.2H); 13C NMR (150 MHz, CDCl3): d=195.4, 163.0, 160.4,
158.8, 141.4, 135.9, 130.3, 127.7, 127.5, 125.5, 124.7, 119.9, 114.7,
57.8, 56.0, 42.2, 35.1, 31.9, 24.8, 23.0, 22.3, 18.5 ppm; mp: 117.3–
120.88C (decomp.); LC–MS: (m/z) calcd for C20H18N3O2 [M+H]+:
332.1, found: 332.1.
2-Amino-7-(isoquinolin-4-yl)-4-methyl-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (1b). 5-(Isoquinolin-4-yl)cyclohexan-
1,3-dione (49.0 mg, 0.21 mmol) was dissolved in absolute EtOH
(10 mL) and malononitrile (17 mg, 0.26 mmol) was added at RT.
The mixture was cooled to 08C and acetaldehyde (35 mL,
0.63 mmol) was added. The mixture was stirred for 10 min and
then N-methylmorpholine (5 mL, 0.04 mmol) was added. The mix-
ture was warmed to RT and stirred for three days. The formed pre-
cipitate was then filtered off and washed with cold EtOH (3 mL)
and dried to give the title compound as a white solid (30 mg,
2-Amino-7-(5-3H-naphth-1-yl)-4-methyl-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile ([3H]UCPH-102). Iodine 15 (3.4 mg)
was dissolved in DMSO (1.0 mL) then added to a round-bottomed
reaction vessel (1 mL) already loaded with the palladium catalyst
(3.8 mg, 10% Pd/C). The reaction vessel was mounted onto the tri-
tium manifold system and frozen with liquid nitrogen, then evacu-
ated to <5”10À3 mbar. The vessel was then filled with an atmos-
phere of argon before it was allowed to warm to RT. It was stirred
vigorously for a couple of minutes before the freeze; the evacua-
tion and stir procedure was repeated three times to effectively
ensure deoxygenation of the reaction mixture. The flask was yet
again frozen with liquid nitrogen, evacuated to <5”10À3 mbar
before carrier-free tritium gas (8.9 Ci, 3H2, 400 mbar) was added.
The reaction mixture was stirred at RT for 1 h, then carefully pulled
out of the vessel and diluted with H2O (2.0 mL). The dilution was
filtered through a disposable Whatman glass microfiber filter
(grade GF/C, 1.2 mm pore size, 13 mm Ø) for removal of the solid
catalyst. The filter was flushed with additional H2O (1.0 mL). The
crude mixture was subjected to three consecutive freeze pump
thaw repetitions for the removal of labile tritium. After freeze-
drying of the crude mixture the product was purified by RP-HPLC
using a C18 column and a gradient of MeCN/H2O as the eluent. The
fraction containing the product was lyophilized and then re-solubi-
lized in 25 mL 10% EtOH in DMSO. The radiochemical yield was an-
alyzed to be 75 mCi, with a specific activity of 18.1 CimmolÀ1. The
radioligand was chemically stable in this solvent mixture, while
water should be avoided due to hydrolysis over time. Preparative
HPLC: column: Princeton Spher C30 (250 mm”10 mm, 5 mm); flow
rate: 4 mLminÀ1; eluents: A=99.9% purified H2O, 0.1% TFA; B=
90% MeCN, 9.9% purified H2O, 0.1% TFA; gradient: 0–1 min 30%
B, 1–20 min 30–100% B, 20–21 min 100% B, 21–22 min 100–30%
B, 22–25 min 30% B. The product [3H]UCPH-102 was collected at
8.8 min. Analytical HPLC: column: Phenomenex Luna C18 (2)
(250 mm”4.6 mm); flow rate: 1.5 mLminÀ1; eluents: A=94.9% pu-
rified H2O, 5% MeCN, 0.1% TFA; B=99.9% MeCN, 0.1% TFA; gradi-
ent: 0–0.50 min 10% B, 0.5–18 min 10–75% B, 18–19 min 75–100%
B, 19–20 min 100% B, 20–21 min 100–10% B, 21–23 min 0% B.
The retention time of [3H]UCPH-102 was detected at 11.3 min.
1
0.09 mmol, 43%); Rf =0.45 (EtOAc/MeOH/AcOH 100:10:1); H NMR
(600 MHz, [D6]DMSO): d=9.24 (d, J=4.8 Hz, 1H), 8.52 (d, J=
42.1 Hz, 1H), 8.30 (dd, J=27.4, 8.5 Hz, 1H), 8.16 (d, J=8.2 Hz, 1H),
7.86–7.82 (m, 1H), 7.72 (t, J=7.3 Hz, 1H), 6.91 (d, J=3.4 Hz, 2H),
4.37–4.30 (m, 0.35H), 4.25–4.18 (m, 0.6H), 3.19 (dt, J=13.1, 6.5 Hz,
1H), 3.10–3.02 (m, 1H), 2.94 (ddd, J=24.2, 16.1, 12.3 Hz, 1H), 2.81–
2.73 (m, 1H), 2.71–2.61 (m, 1H), 1.19 (d, J=6.5 Hz, 3H), 1.12 ppm
(d, J=6.5 Hz, 1H); 13C NMR (150 MHz, CDCl3): d=195.5, 163.2,
158.8, 148.4, 142.7, 140.0, 135.8, 130.3, 125.8, 125.7, 124.8, 120.9,
120.0, 114.8, 57.8, 43.3, 33.7, 31.7, 24.8, 22.9 ppm; LC–MS: (m/z)
calcd for C20H18N3O2 [M+H]+: 332.1, found: 332.1.
2-Amino-3-cyano-4-methyl-5-oxo-7-(4-quinolinyl)-5,6,7,8-tetrahy-
dro-4H-chromene (1c). 5-(Quinolin-4-yl)cyclohexane-1,3-dione
(50 mg, 0.21 mmol) was suspended in absolute EtOH (10 mL) and
malononitrile (14 mg, 0.21 mmol) was added. The suspension was
evacuated and backfilled with N2 several times, cooled to 08C and
acetaldehyde (34 mL, 0.63 mmol) was added. The reaction mixture
was stirred at 08C for 20 min before N-methylmorpholine (5 mL,
0.04 mmol) was added. The reaction mixture was left in the ice-
bath to gradually warm up to RT during 16 h during which further
precipitation occurred. The suspension was filtered and the result-
ing white solid was washed with cold EtOH (3 mL) and dried to
give the title compound as a white solid (43 mg, 0.13 mmol, 62%)
containing 6% EtOH w/w. Rf =0.5 (two spots, EtOAc/MeOH/AcOH
100:10:1); 1H NMR (400 MHz, [D6]DMSO): d=8.90–8.86 (m, 1H),
8.36 (d, J=8.3 Hz, 0.3H), 8.31 (d, J=8.3 Hz, 0.7H), 8.06 (d, J=
8.4 Hz, 1H), 7.81–7.75 (m, 1H), 7.68–7.62 (m, 1H), 7.55 (d, J=
4.6 Hz, 0.3H), 7.48 (d, J=4.6 Hz, 0.7H), 6.90 (s, 2H), 4.49–4.39 (m,
1H), 3.24–3.15 (m, 1H), 3.07–2.95 (m, 1H), 2.95–2.52 (m, 3H), 1.19
(d, J=6.5 Hz, 0.9H), 1.12 ppm (d, J=6.5 Hz, 2.1H); 13C NMR
(100 MHz, [D6]DMSO): d=195.4, 162.6, 158.8, 150.3, 148.0, 147.9,
2-Amino-7-(isoquinolin-1-yl)-4-methyl-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (1a). 5-(Isoquinoline-1-yl)cyclohexan-
1,3-dione (40.0 mg, 0.17 mmol) was dissolved in absolute EtOH
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409
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
129.9, 129.3, 126.8, 126.0, 123.4, 119.9, 118.4, 114.9, 57.6, 42.4, 32.6,
32.6, 24.9, 22.3 ppm; mp: 174.6–178.88C (decomp.); LC–MS: (m/z)
calcd for C20H18N3O2 [M+H]+: 332.1, found: 332.1.
J=5.6 Hz, 1H), 7.79–7.75 (m, 1H), 7.70 (d, J=7.2 Hz, 1H), 6.90 (s,
2H), 4.53–4.47 (m, 1H), 3.19 (dd, J=12.4, 5.9 Hz, 1H), 3.01 (dd, J=
17.1, 10.3 Hz, 1H), 2.88 (dd, J=16.2, 13.1 Hz, 1H), 2.75 (dd, J=17.6,
4.2 Hz, 1H), 2.64–2.59 (m, 1H), 1.20 ppm (d, J=6.5 Hz, 3H);
13C NMR (150 MHz, CDCl3): d=195.5, 163.2, 158.8, 148.4, 142.7,
140.0, 135.8, 130.3, 125.8, 125.7, 124.8, 120.9, 120.0, 114.8, 57.8,
43.3, 33.7, 31.7, 24.8, 22.9 ppm; mp: 208.2–220.38C (decomp.); LC–
MS: (m/z) calcd for C20H18N3O2 [M+H]+: 332.1, found: 332.1.
2-Amino-3-cyano-4-methyl-5-oxo-7-(quinolin-5-yl)-5,6,7,8-tetra-
hydro-4H-chromene (1d). 5-(Quinolin-5-yl)cyclohexane-1,3-dione
(50 mg, 0.21 mmol) was suspended in absolute EtOH (20 mL) and
malononitrile (14 mg, 0.21 mmol) was added. The suspension was
evacuated and backfilled with N2 three times, cooled to 08C and
acetaldehyde (34 mL, 0.63 mmol) was added. The reaction mixture
was stirred at 08C for 30 min before N-methylmorpholine (5 mL,
0.04 mmol) was added. The reaction mixture was left in the ice-
bath to gradually warm up to RT during 16 h during which further
precipitation occurred. The volume was decreased in vacuo to
~5 mL and the resulting suspension was filtered and the white
solid was washed with cold EtOH (3 mL) and dried to give the title
compound as a white solid (47 mg, 0.142 mmol, 68%) containing
12% EtOH w/w. Rf =0.5 (EtOAc/MeOH/AcOH 100:10:1); 1H NMR
(400 MHz, [D6]DMSO): d=8.95–8.88 (m, 1H), 8.74 (dd, J=18.5,
8.6 Hz, 1H), 7.94 (dd, J=8.4, 2.7 Hz, 1H), 7.80–7.70 (m, 1H), 7.68–
7.52 (m, 2H), 6.89 (s, 2H), 4.44–4.35 (m, 0.6H), 4.31–4.23 (m, 0.4H),
3.25–3.15 (m, 1H), 3.05–2.79 (m, 2H), 2.78–2.54 (m, 2H), 1.20 (d,
J=6.5 Hz, 1.8H), 1.12 ppm (d, J=6.5 Hz, 1.8H); 13C NMR (100 MHz,
[D6]DMSO): d=195.8, 195.5, 163.3, 162.8, 158.8, 158.8, 150.2, 148.1,
148.1, 139.5, 139.4, 131.8, 131.7, 129.1, 129.0, 128.3, 128.2, 125.8,
125.8, 123.7, 123.7, 121.4, 121.4, 119.9, 114.8, 114.8, 57.8, 57.6, 43.2,
33.6, 33.5, 32.7, 32.2, 24.9, 24.9, 23.0, 22.4 ppm; mp: 201.8–204.68C
(decomp.); LC–MS: (m/z) calcd for C20H18N3O2 [M+H]+: 332.1,
found: 332.1.
2-Amino-4-methyl-5-oxo-7-(quinolin-8-yl)- 5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (1g). 5-(Quinoline-8-yl)cyclohexan-1,3-
dione (50.0 mg, 0.21 mmol) was dissolved in absolute EtOH
(10 mL) and malononitrile (16 mg, 0.24 mmol) was added at RT.
The mixture was cooled to 08C and acetaldehyde (35 mL,
0.63 mmol) was added. The mixture was stirred for 10 min and
then N-methylmorpholine (5 mL, 0.04 mmol) was added. The mix-
ture was warmed to RT and stirred for 18 h. The formed precipitate
was filtered off and washed with cold EtOH (3 mL) and dried to
give the title compound as a mixture of two diastereomers as
a white solid (38 mg, 0.11 mmol, 55%); ratio: 1:1.27; Rf =0.64
(EtOAc/MeOH/AcOH 100:10:1); 1H NMR (600 MHz, [D6]DMSO): d=
8.96 (d, 1H), 8.54 (d, J=6.0 Hz, 1H), 8.11 (d, J=6.1 Hz, 1H), 8.04 (d,
J=8.1 Hz, 1H), 7.75 (d, J=7.1 Hz, 1H), 7.69–7.65 (m, 1H), 6.90 (s,
2H), 4.38–4.30 (m, 1H), 3.20 (dd, J=12.9, 6.5 Hz, 1H), 2.97 (dd, J=
17.1, 4.9 Hz, 1H), 2.90 (dd, J=15.9, 11.7 Hz, 1H), 2.75 (dd, J=17.0,
4.9 Hz, 1H), 2.65 (dd, J=15.7, 3.9 Hz, 1H), 1.12 ppm (d, J=6.5 Hz,
3H); 13C NMR (150 MHz, CDCl3): d=195.8, 162.7, 158.8, 153.2,
143.3, 137.9, 133.2, 128.6, 127.5, 127.1, 126.9, 119.9, 116.1, 114.8,
57.6, 42.9, 33.2, 32.7, 24.9, 22.4 ppm; mp: 208.7–222.88C
(decomp.); LC–MS: (m/z) calcd for C20H18N3O2 [M+H]+: 332.1,
found: 332.1.
2-Amino-7-(isoquinolin-5-yl)-4-methyl-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (1e). 5-(Isoquinoline-5-yl)-cyclohexan-
1,3-dione (52.0 mg, 0.22 mmol) was dissolved in absolute EtOH
(10 mL) and malononitrile (16 mg, 0.24 mmol) was added at RT.
The mixture was cooled to 08C and acetaldehyde (40 mL,
0.64 mmol) was added. The mixture was stirred for 10 min and
then N-methylmorpholine (5 mL, 0.04 mmol) was added. The mix-
ture was warmed to RT and stirred for 18 h. The formed precipitate
was then filtered off and washed with cold EtOH (3 mL) and dried
to give the title compound as one diastereomer as a white solid
(42 mg, 0.13 mmol, 58%); Rf =0.48 (EtOAc/MeOH/AcOH 100:10:1);
1H NMR (600 MHz, [D6]DMSO): d=9.33 (s, 1H), 8.54 (d, J=6.0 Hz,
1H), 8.11 (d, J=6.1 Hz, 1H), 8.04 (d, J=8.1 Hz, 1H), 7.75 (d, J=
7.1 Hz, 1H), 7.69–7.65 (m, 1H), 6.90 (s, 2H), 4.38–4.30 (m, 1H), 3.20
(dd, J=12.9, 6.5 Hz, 1H), 2.97 (dd, J=17.1, 4.9 Hz, 1H), 2.90 (dd,
J=15.9, 11.7 Hz, 1H), 2.75 (dd, J=17.0, 4.9 Hz, 1H), 2.65 (dd, J=
15.7, 3.9 HZ, 1H), 1.12 ppm (d, J=6.5 Hz, 3H); 13C NMR (150 MHz,
CDCl3): d=195.8, 162.7, 158.8, 153.2, 143.3, 137.9, 133.2, 128.6,
127.5, 127.1, 126.9, 119.9, 116.1, 114.8, 57.6, 42.9, 33.2, 32.7, 24.9,
22.4 ppm; mp: 208.6–223.18C (decomp.); LC–MS: (m/z) calcd for
C20H18N3O2 [M+H]+: 332.1, found: 332.1.
2-Amino-4-methyl-5-oxo-7-(quinolone-2-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (1h). 5-(Quinolin-2-yl)cyclohexan-1,3-
dione (36 mg, 0.15 mmol) was dissolved in absolute EtOH (8 mL)
and malononitrile (12 mg, 0.18 mmol) was added at RT. The mix-
ture was cooled to 08C and acetaldehyde (50 mL, 0.90 mmol) was
added. The mixture was stirred for 10 min and then N-methylmor-
pholine (10 mL, 0.08 mmol) was added. The mixture was warmed
to RT and stirred for 20 h. The solvent was evaporated and the resi-
due purified by flash column chromatography (CH2Cl2/EtOAc 5:1)
to give the title compound as a white foam (10.27 mg, 0.03 mmol,
21%); Rf =0.14 and 0.11 (CH2Cl2/EtOAc 5:1); 1H NMR (600 MHz,
[D6]DMSO): d=8.73 (s, 1H), 8.54 (d, J=5.6 Hz, 1H), 7.89 (d, J=
8.1 Hz, 1H), 7.85 (d, J=5.6 Hz, 1H), 7.79–7.75 (m, 1H), 7.70 (d, J=
7.2 Hz, 1H), 6.90 (s, 2H), 4.53–4.47 (m, 1H), 3.19 (dd, J=12.4,
5.9 Hz, 1H), 3.01 (dd, J=17.1, 10.3 Hz, 1H), 2.88 (dd, J=16.2,
13.1 Hz, 1H), 2.75 (dd, J=17.6, 4.2 Hz, 1H), 2.64–2.59 (m, 1H),
1.20 ppm (d, J=6.5 Hz, 3H); 13C NMR (150 MHz, CDCl3): d=195.4,
195.3, 163.2, 161.6, 161.5, 158.8, 146.9, 146.6, 136.8, 129.6, 128.6,
127.8, 126.8, 126.7, 126.2, 120.6, 119.9, 115.2, 115.0, 57.8, 57.6, 41.9,
41.1, 31.7, 31.6, 24.8, 24.7, 23.0, 22.4 ppm; mp: 79.0–98.88C
(decomp.); LC–MS: (m/z) calcd for C20H18N3O2 [M+H]+: 332.1,
found: 332.1.
2-Amino-7-(isoquinolin-8-yl)-4-methyl-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (1 f). 5-(Isoquinoline-8-yl)cyclohexan-
1,3-dione (30.0 mg, 0.12 mmol) was dissolved in absolute EtOH
(8 mL) and malononitrile (9 mg, 0.12 mmol) was added at RT. The
mixture was cooled to 08C and acetaldehyde (25 mL, 0.49 mmol)
was added. The mixture was stirred for 10 min and then N-methyl-
morpholine (5 mL, 0.04 mmol) was added. The mixture was
warmed to RT and stirred for two days. The formed precipitate was
then filtered off and washed with cold EtOH (3 mL) and dried to
give the title compound as a mixture of two diastereomers as
a white solid (20 mg, 0.06 mmol, 51%); Rf =0.72 (EtOAc/MeOH/
AcOH 100:10:1); 1H NMR (600 MHz, [D6]DMSO): d=9.75 (s, 1H),
8.73 (s, 1H), 8.54 (d, J=5.6 Hz, 1H), 7.89 (d, J=8.1 Hz, 1H), 7.85 (d,
2-Amino-4-methyl-5-oxo-7-(quinolin-7-yl)-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (1i). 5-(Quinolin-7-yl)cyclohexan-1,3-dione
(40 mg, 0.15 mmol) was dissolved in absolute EtOH (8 mL) and ma-
lononitrile (15 mg, 0.19 mmol) was added at RT. The mixture was
cooled to 08C and acetaldehyde (50 mL, 0.90 mmol) was added.
The mixture was stirred for 10 min and then N-methylmorpholine
(5 mL, 0.04 mmol) was added. The mixture was warmed to RT and
stirred for 20 h. The solvent was evaporated and the residue puri-
fied by prep. TLC (CH2Cl2/EtOAc 2:1) to give the title compound as
an off-white solid (10.31 mg, 0.03 mmol, 19%); Rf =0.16 (CH2Cl2/
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1
EtOAc 2:1); H NMR (600 MHz, [D6]DMSO): d=8.73 (s, 1H), 8.54 (d,
pound as yellowish crystals (10 mg, 0.03 mmol, 33%); Rf =0.19
(CH2Cl2/EtOAc 10:1); 1H NMR (400 MHz, CDCl3): d=8.45–8.35 (m,
2H), 7.54 (tdd, J=10.3, 2.4, 1.6 Hz, 1H), 7.15 (dddd, J=7.8, 5.0, 4.1,
0.9 Hz, 1H), 4.61 (d, J=3.9 Hz, 2H), 4.37 (d, J=1.6 Hz, 1H), 2.52
(ddd, J=17.8, 4.7, 1.6 Hz, 1H), 2.49–2.24 (m, 3H), 2.08–1.87 (m,
3H), 1.80 (dddd, J=17.8, 8.5, 4.6, 2.5 Hz, 1H), 1.61–1.47 (m, 1H),
1.19 (s, 1H), 0.91–0.74 ppm (m, 7H); 13C NMR (100 MHz, CDCl3): d=
196.1, 163.9, 157.9, 149.2, 149.2, 148.4, 138.7, 135.5, 123.4, 123.4,
114.1, 77.3, 77.2, 77.0, 76.7, 62.3, 40.7, 39.8, 38.7, 33.5, 31.8, 31.7,
30.9, 19.5, 19.4, 19.4 ppm; mp: 184.1–184.48C; LC–MS: (m/z) calcd
for C18H20N3O2 [M+H]+: 310.1, found: 310.1; HPLC: >96% purity.
J=5.6 Hz, 1H), 7.89 (d, J=8.1 Hz, 1H), 7.85 (d, J=5.6 Hz, 1H),
7.79–7.75 (m, 1H), 7.70 (d, J=7.2 Hz, 1H), 6.90 (s, 2H), 4.53–4.47
(m, 1H), 3.19 (dd, J=12.4, 5.9 Hz, 1H), 3.01 (dd, J=17.1, 10.3 Hz,
1H), 2.88 (dd, J=16.2, 13.1 Hz, 1H), 2.75 (dd, J=17.6, 4.2 Hz, 1H),
2.64–2.59 (m, 1H), 1.20 ppm (d, J=6.5 Hz, 3H); 13C NMR (150 MHz,
CDCl3): d=195.4, 195.3, 163.2, 161.6, 161.5, 158.8, 146.9, 146.6,
136.8, 129.6, 128.6, 127.8, 126.8, 126.7, 126.2, 120.6, 119.9, 115.2,
115.0, 57.8, 57.6, 41.9, 41.1, 31.7, 31.6, 24.8, 24.7, 23.0, 22.4 ppm;
mp: 180.1–204.88C (decomp.); LC–MS: (m/z) calcd for C20H18N3O2
[M+H]+: 332.1, found: 332.1.
2-Amino-4-methyl-7-(3-methylbenzo[d]isoxazol-4-yl)-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (1j). 5-(3-Methyl-
benzo[d]isoxazol-4-yl)cyclohexan-1,3-dione (70 mg, 0.29 mmol) was
dissolved in absolute EtOH (15 mL) and malononitrile (26 mg,
0.39 mmol) was added at RT. The mixture was cooled to 08C and
acetaldehyde (50 mL, 0.90 mmol) was added. The mixture was
stirred for 10 min and then N-methylmorpholine (10 mL,
0.08 mmol) was added. The mixture was warmed to RT and stirred
for 20 h. The solvent was evaporated and the residue purified by
flash chromatography (CH2Cl2/EtOAc 2:1) to give the title com-
pound as a white solid (10.3 mg, 0.03 mmol, 21%); Rf =0.14 and
2-Amino-7-isopropyl-5-oxo-4-(pyridin-4-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (2c). Carried out as described for 2b
starting with pyridine-4-aldehyde. Recrystallization from CH2Cl2/Pe-
troleum ether afforded the title compound as white crystals
(10 mg, 0.03 mmol, 33%); Rf =0.29 (CH2Cl2/EtOAc 10:1); 1H NMR
(400 MHz, CDCl3): d=8.45 (t, J=4.8 Hz, 2H), 7.10 (dd, J=14.7,
5.1 Hz, 2H), 4.73 (d, J=4.4 Hz, 2H), 4.34 (s, 1H), 2.66–2.22 (m, 3H),
2.14–1.87 (m, 1H), 1.54 ppm (dq, J=12.8, 6.5 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d=195.1, 195.0, 163.3, 162.6, 157.1, 157.0, 150.7,
150.5, 149.0, 149.0, 121.8, 121.7, 117.2, 112.6, 112.5, 76.3, 76.2, 76.0,
75.7, 60.4, 60.3, 40.1, 39.7, 38.8, 37.7, 34.2, 34.0, 30.8, 30.7, 29.9,
29.7, 18.5, 18.4, 18.4, 18.3 ppm; mp: 177.5–178.38C; LC–MS: (m/z)
calcd for C18H20N3O2 [M+H]+: 310.1, found: 310.1; HPLC: >98%
purity.
1
0.11 (CH2Cl2/EtOAc 5:1); H NMR (600 MHz, [D6]DMSO): d=7.56 (d,
J=8.0 Hz, 1H), 7.32 (t, J=7.8 Hz, 1H), 7.27 (d, J=7.5 Hz, 1H), 6.90
(s, 2H), 3.89–3.81 (m, 1H), 3.59 (d, J=5.7 Hz, 1H), 3.47 (s, 1H), 3.16
(q, J=6.4 Hz, 1H), 3.11 (dd, J=17.2, 11.6 Hz, 1H), 2.88 (dd, J=16.3,
13.4 Hz, 1H), 2.77 (dd, J=17.3, 4.1 Hz, 1H), 2.63 (s, 3H), 1.15 ppm
(d, J=6.5 Hz, 3H); 13C NMR (150 MHz, CDCl3): d=195.4, 195.3,
163.2, 161.6, 161.5, 158.8, 146.9, 146.6, 136.8, 129.6, 128.6, 127.8,
126.8, 126.7, 126.2, 120.6, 119.9, 115.2, 115.0, 57.8, 57.6, 41.9, 41.1,
31.7, 31.6, 24.8, 24.7, 23.0, 22.4 ppm; LC–MS: (m/z) calcd for
C19H18N3O3 [M+H]+: 336.1, found: 336.1.
2-Amino-7-isopropyl-5-oxo-4-(quinolin-7-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (2d). Carried out as described for 2b
starting with quinoline-7-carboxaldehyde. Recrystallization from
CH2Cl2/Petroleum ether afforded the title compound as white crys-
1
tals (8 mg, 0.02 mmol, 22%); Rf =0.07 (CH2Cl2/EtOAc 10:1); H NMR
(400 MHz, CDCl3): d=8.79 (ddd, J=4.6, 2.9, 1.7 Hz, 2H), 8.06 (dq,
J=8.5, 1.5 Hz, 2H), 7.81–7.67 (m, 4H), 7.55 (ddd, J=13.7, 8.3,
1.9 Hz, 2H), 7.28 (ddd, J=8.3, 4.2, 1.4 Hz, 2H), 4.52 (dd, J=19.6,
3.5 Hz, 7H), 2.55–2.25 (m, 6H), 2.09–1.82 (m, 5H), 1.50 (s, 16H),
1.19 (s, 1H), 0.87 ppm (td, J=8.9, 7.3 Hz, 16H); 13C NMR (100 MHz,
CDCl3): d=196.3, 163.9, 157.7, 150.5, 145.4, 136.0, 128.0, 127.6,
127.2, 120.9, 114.6, 77.3, 77.2, 77.0, 76.7, 40.8, 38.6, 35.5, 31.7, 30.9,
19.5, 19.4, 19.4 ppm; mp: 179.1–179.48C; LC–MS: (m/z) calcd for
C22H22N3O2 [M+H]+: 360.2, found: 360.1; HPLC: >99% purity.
2-Amino-4-methyl-7-(3-methylbenzo[d]isoxazol-5-yl)-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (1k). 5-(3-methyl-
benzo[d]isoxazol-5-yl)cyclohexan-1,3-dione
hydrate
(29.8 mg,
0.11 mmol) was dissolved in absolute EtOH (6 mL) and malononi-
trile (8 mg, 0.11 mmol) was added at RT. The mixture was cooled to
08C and acetaldehyde (20 mL, 0.34 mmol) was added. The mixture
was stirred for 10 min and then N-methylmorpholine (3 mL,
0.08 mmol) was added. The mixture was warmed to RT and stirred
for 20 h. The solvent was evaporated and the residue purified by
flash chromatography (CH2Cl2/EtOAc 5:1 to 2:1) to give the title
compound as a yellow solid (8.0 mg, 0.024 mmol, 21%); Rf =0.57
2-Amino-7-isopropyl-5-oxo-4-(quinolin-3-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (2e). Carried out as described for 2b
starting with quinoline-3-carboxaldehyde. Filtration of the reaction
mixture afforded the title compound as white crystals (18 mg,
1
(CH2Cl2/EtOAc 2:1); H NMR (600 MHz, [D6]DMSO): d=9.64 (s, 1H),
1
0.05 mmol, 51%); Rf =0.14 (CH2Cl2/EtOAc 10:1); H NMR (400 MHz,
9.32 (s, 1H), 7.62 (s, 1H), 6.94 (dd, J=8.3, 2.0 Hz, 1H), 6.88 (s, 1H),
6.81 (d, J=8.3 Hz, 2H), 3.44 (dd, J=7.0, 5.1 Hz, 1H), 3.29–3.22 (m,
2H), 3.12 (dt, J=6.6, 5.2 Hz, 1H), 2.73 (dd, J=16.4, 11.0 Hz, 2H),
2.62–2.54 (m, 3H), 2.45 (dd, J=16.3, 3.1 Hz, 1H), 2.08 (s, 3H),
1.11 ppm (d, J=6.5 Hz, 3H); 13C NMR (150 MHz, CDCl3): d=195.7,
169.0, 163.3, 158.8, 146.7, 133.1, 126.2, 122.9, 120.9, 119.9, 115.9,
114.9, 57.7, 43.7, 36.7, 34.0, 24.8, 23.6, 23.0 ppm; mp: 189.1–
221.68C (decomp.); LC–MS: (m/z) calcd for C19H18N3O3 [M+H]+:
336.1, found: 336.1.
CDCl3): d=8.77 (dd, J=14.8, 2.3 Hz, 1H), 8.06 (dd, J=8.8, 2.0 Hz,
2H), 7.82 (dt, J=8.1, 2.0 Hz, 1H), 7.68 (ddd, J=8.4, 6.5, 1.3 Hz, 1H),
7.58–7.35 (m, 1H), 4.72 (d, J=4.2 Hz, 1H), 4.63 (q, J=1.4 Hz, 1H),
2.67–2.25 (m, 3H), 2.16–1.96 (m, 1H), 1.05–0.77 ppm (m, 5H);
13C NMR (150 MHz, CDCl3): d=199.1, 196.2, 196.1, 163.9, 163.2,
157.8, 150.5, 147.6, 135.5, 134.6, 128.6, 128.0, 126.7, 118.3, 113.7,
77.2, 77.0, 76.8, 62.4, 39.8, 38.7, 31.7, 30.7, 19.5 ppm; mp: 210.5–
212.48C; LC–MS: (m/z) calcd for C22H22N3O2 [M+H]+: 360.2, found:
360.1; HPLC: >98% purity.
2-Amino-7-isopropyl-5-oxo-4-(pyridin-3-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (2b). Nicotinaldehyde (10.7 mg,
0.1 mmol) and malononitrile (7.3 mg, 0.11 mmol) were dissolved in
absolute EtOH (2 mL) at RT under N2. 5-Isopropyl-1,3-hexanedione
(16.9 mg, 0.11 mmol) and N-methylmorpholine (1.4 mL, 0.015 mmol)
were added, and the reaction mixture was stirred for 24 h at RT.
The reaction mixture was then evaporated to dryness and purified
by flash column chromatography (CH2Cl2/EtOAc 10:1 to 10:3). Re-
crystallization from CH2Cl2/Petroleum ether afforded the title com-
2-Amino-7-isopropyl-5-oxo-4-(quinolin-6-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (2 f). Carried out as described for 2b
starting with quinoline-6-carboxaldehyde. Filtration of the reaction
mixture afforded the title compound as white crystals (22 mg,
1
0.06 mmol, 62%); Rf =0.10 (CH2Cl2/EtOAc 10:1); H NMR (400 MHz,
CDCl3): d=8.79 (dt, J=4.2, 1.5 Hz, 1H), 8.07 (dddd, J=8.3, 3.5, 1.7,
0.8 Hz, 1H), 7.97 (dd, J=8.8, 5.0 Hz, 1H), 7.67 (dd, J=11.5, 2.0 Hz,
1H), 7.47 (ddd, J=17.1, 8.7, 2.1 Hz, 1H), 7.31 (ddd, J=8.3, 4.2,
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1.1 Hz, 1H), 4.61–4.53 (m, 3H), 2.56 (ddd, J=17.8, 4.7, 1.6 Hz, 1H),
2.52–2.26 (m, 3H), 2.09–1.87 (m, 1H), 1.81 (dddd, J=17.9, 8.4, 6.5,
4.3 Hz, 1H), 1.17 (t, J=7.0 Hz, 1H), 0.91–0.81 ppm (m, 6H);
13C NMR (100 MHz, CDCl3): d=196.2, 163.8, 163.1, 157.8, 150.2,
141.0, 136.3, 130.1, 130.0, 128.9, 128.8, 128.1, 126.7, 126.6, 121.3,
114.5, 77.3, 77.2, 77.0, 76.7, 63.0, 41.3, 40.8, 39.9, 38.7, 35.8, 35.6,
31.8, 31.7, 30.9, 30.8, 19.5, 19.4, 19.4 ppm; mp: 214.0–214.38C; LC–
MS: (m/z) calcd for C22H22N3O2 [M+H]+: 360.2, found: 360.1; HPLC:
>98% purity.
115.4, 90.2, 46.3, 40.9, 32.9, 25.9, 14.1 ppm; mp: 206.3–207.78C;
LC–MS: (m/z) calcd for C22H20N3O [M+H]+: 342.2, found: 342.1.
2-Amino-4-benzyl-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (3d). To a solution of 3-amino-5-(naphtha-
len-1-yl)cyclohex-2-en-1-one (50 mg, 0.21 mmol) and malononitrile
(42 mg, 0.63 mmol) in n-propanol (0.8 mL) was added phenylace-
taldehyde (74 mL, 0.63 mmol). The resulting mixture was then
heated at 110 8C in a sealed flask overnight. The reaction mixture
was cooled to RT and evaporated. The resulting brown oil was pu-
rified by flash column chromatography (0!10% EtOAc in CH2Cl2)
followed by recrystallization in CH2Cl2/MeOH to afford the title
compound as off-white crystals (34 mg, 0.08 mmol, 40%); Rf =0.45
(CH2Cl2/EtOAc 4:1); 1H NMR (400 MHz, [D6]DMSO): d=8.21 (d, J=
8.1 Hz, 1H), 7.98–7.91 (m, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.72 (s, 2H),
7.60–7.50 (m, 2H), 7.50–7.39 (m, 2H), 7.32–7.25 (m, 2H), 7.23–7.12
(m, 3H), 4.71 (d, J=13.9 Hz, 1H), 4.58 (d, J=13.9 Hz, 1H), 4.38–4.27
(m, 1H), 3.39–3.29 (m, 2H), 3.27–3.18 (m, 1H), 3.03 (dd, J=16.3,
11.4 Hz, 1H), 2.87–2.77 ppm (m, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=195.5, 169.3, 160.5, 157.3, 138.9, 138.0, 133.5, 130.6,
128.8, 128.3, 128.3, 127.1, 126.3, 126.1, 125.7, 125.5, 123.1, 122.9,
116.4, 115.7, 91.7, 46.1, 40.9, 36.9, 32.7 ppm; mp: 217.8–219.38C;
LC–MS: (m/z) calcd for C27H22N3O [M+H]+: 404.2, found: 404.1.
2-Amino-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydroquinoline-
3-carbonitrile (3a). To a solution of 3-amino-5-(naphthalen-1-yl)cy-
clohex-2-en-1-one (50 mg, 0.21 mmol) in n-propanol (0.9 mL) was
added malononitrile (620 mg, 9.39 mmol). Additional n-propanol
(0.5 mL) was added to bring all reagents into solution. Formalde-
hyde (37% aqueous solution, 51 mL, 0.63 mmol) was added and
the reaction mixture was heated at 110 8C in a sealed flask. After
6 h, the solvent was evaporated and the resulting residue was puri-
fied by flash column chromatography (0!10% EtOAc in CH2Cl2).
Further purification by recrystallization from MeOH afforded the
title compound as a beige solid (11 mg, 0.03 mmol, 16%); Rf =0.37
(CH2Cl2/EtOAc 9:1); 1H NMR (400 MHz, CDCl3/[D4]MeOH (1:1)): d=
8.36 (s, 1H), 8.05 (d, J=8.4 Hz, 1H), 7.92–7.85 (m, 1H), 7.77 (d, J=
7.8 Hz, 1H), 7.56–7.38 (m, 4H), 4.37–4.27 (m, 1H), 3.37–3.26 (m,
2H), 3.03–2.95 ppm (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
193.7, 168.1, 160.8, 160.8, 141.6, 139.1, 133.5, 130.6, 128.8, 127.1,
126.3, 125.7, 125.6, 123.0, 117.7, 116.1, 89.0, 44.2, 33.3 ppm; LC–
MS: (m/z) calcd for C20H16N3O [M+H]+: 314.1, found: 314.1.
Isoquinoline-1-carbaldehyde (4a). A solution of 1-bromoisoquino-
line (769 mg, 3.70 mmol) in dry THF (300 mL) was cooled to À788C
under a N2 atmosphere. sBuLi (4.5 mL, 5.80 mmol, 1.4m in cyclo-
hexane) was added dropwise followed by addition of dry DMF
(6.0 mL, 75.9 mmol). The reaction mixture was allowed to warm to
RT and stirred for 30 min. Saturated NH4Cl (300 mL) was added and
the phases were separated. The aqueous layer was extracted with
CH2Cl2 (2”300 mL), dried over MgSO4 and concentrated in vacuo.
Purification by flash column chromatography (heptane/EtOAc 5:1)
afforded the title compound as an orange solid (326 mg,
2-Amino-4-methyl-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (3b). To a solution of 3-amino-5-(naphtha-
len-1-yl)cyclohex-2-en-1-one (42 mg, 0.18 mmol) and malononitrile
(39 mg, 0.59 mmol) in n-propanol (0.8 mL) was added acetaldehyde
(33 mL, 0.60 mmol). The reaction mixture was then heated at 110 8C
in a sealed flask for 6 h. Precipitation occurred upon cooling the re-
action to RT. The solid was filtered off and washed with cold EtOH.
Further purification by trituration in MeOH afforded the title com-
pound as an off-white solid (29 mg, 0.09 mmol, 49%); Rf =0.19
(CH2Cl2/EtOAc 8:2); 1H NMR (400 MHz, [D6]DMSO): d=8.19 (d, J=
7.8 Hz, 1H), 7.99–7.90 (m, 1H), 7.83 (dd, J=2.1, 7.0 Hz, 1H), 7.64
(brs, 2H), 7.60–7.43 (m, 4H), 4.35–4.22 (m, 1H), 3.31 (dd, J=16.6,
11.3 Hz, 1H), 3.19–3.08 (m, 1H), 3.00 (dd, J=16.3, 12.0 Hz, 1H),
2.86–2.76 (m, 1H), 2.74 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO):
d=195.8, 168.6, 160.2, 156.2, 139.0, 133.5, 130.6, 128.8, 127.1,
126.3, 125.7, 125.6, 123.0, 122.8, 116.7, 115.7, 91.1, 46.2, 40.7, 32.9,
20.4 ppm; mp: 257.8–259.38C; LC–MS: (m/z) calcd for C21H18N3O
[M+H]+: 328.1, found: 328.1.
1
2.08 mmol, 58%); Rf =0.20 (heptane/EtOAc 5:1); H NMR (400 MHz,
CDCl3): d=10.40 (s, 1H), 9.34–9.32 (m, 1H), 8.76 (d, J=5.5 Hz, 1H),
1
7.90–7.89 (m, 2H), 7.78–7.76 ppm (m, 2H). The H NMR data are in
agreement with published values.[18]
Isoquinoline-8-carbaldehyde (4 f). A solution of 8-bromoisoquino-
line (1.50 g, 7.21 mmol) in dry THF (400 mL) was cooled to À788C
under a N2 atmosphere. sBuLi (9.5 mL, 6.79 mmol, 1.4m in cyclo-
hexane) was added dropwise followed by addition of dry DMF
(10 mL, 129.98 mmol). The reaction mixture was allowed to warm
to RT and stirred for 30 min. Saturated NH4Cl (400 mL) was added
and the phases were separated. The aqueous layer was extracted
with CH2Cl2 (3”400 mL), dried over MgSO4 and concentrated in va-
cuo. Purification by flash column chromatography (heptane/EtOAc
2-Amino-4-ethyl-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydroqui-
noline-3-carbonitrile (3c). To a solution of 3-amino-5-(naphthalen-
1-yl)cyclohex-2-en-1-one (50 mg, 0.21 mmol, 1 equiv) and malono-
nitrile (42 mg, 0.63 mmol) in n-propanol (0.8 mL) was added pro-
pionaldehyde (55 mL, 0.63 mmol). The resulting clear solution was
then heated at 110 8C in a sealed flask overnight. The reaction mix-
ture was cooled to RT and evaporated. The resulting residue was
suspended in n-propanol (0.3 mL) and cooled in the freezer for fur-
ther precipitation. The solid was filtered off and triturated in MeOH
(1 mL) to afford the title compound as an off-white solid (36 mg,
0.10 mmol, 50%); Rf =0.47 (CH2Cl2/EtOAc 4:1); 1H NMR (400 MHz,
[D6]DMSO): d=8.21 (d, J=7.9 Hz, 1H), 8.02–7.91 (m, 1H), 7.83 (dd,
J=7.1, 1.9 Hz, 1H), 7.65 (brs, 2H), 7.59–7.42 (m, 4H), 4.30 (tt, J=
11.4, 3.6 Hz, 1H), 3.39–3.26 (m, 1H), 3.26–3.09 (m, 3H), 3.02 (dd, J=
16.3, 12.0 Hz, 1H), 2.90–2.74 (m, 1H), 1.20 ppm (t, J=7.4 Hz, 3H);
13C NMR (100 MHz, [D6]DMSO): d=195.4, 169.1, 161.9, 160.3, 139.1,
133.5, 130.7, 128.8, 127.1, 126.3, 125.7, 125.6, 123.1, 122.8, 115.9,
5:1) afforded the title compound as
a yellow solid (0.86 g,
5.48 mmol, 76% yield); Rf =0.21 (heptane/EtOAc 5:1); 1H NMR
(400 MHz, CDCl3): d=10.63 (s, 1H), 10.43 (s, 1H), 8.72 (d, J=5.8 Hz,
1H), 8.19 (dd, J=7.0, 1.2 Hz, 1H), 8.16 (d, J=8.3 Hz, 1H), 7.98 (dd,
J=8.3, 7.0 Hz, 1H), 7.89 ppm (d, J=5.5 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d=192.2, 149.2, 143.0, 137.2, 136.5, 133.5, 132.2, 130.1,
125.2, 121.2 ppm; mp: 81.7–84.38C; LC–MS: (m/z) calcd for
C10H8NO [M+H]+; 158.1, found; 158.1.
4-(Isoquinolin-1-yl)but-3-en-2-one (5a).
A suspension of NaH
(60% w/w in mineral oil, 59 mg, 2.27 mmol) in dry THF (3 mL) was
stirred under a N2 atmosphere for 15 min. A solution of diethyl 2-
oxopropylphosphonate (0.45 mL, 2.27 mmol) in dry THF (1 mL) was
slowly added at 08C over 15 min. The clear yellow solution was
warmed to RT and stirred for 2 h after which 1-isoquinoline-carbal-
dehyde (325 mg, 6.58 mmol) in dry THF (2 mL) was added drop-
wise. The reaction was left to stir for 19 h and then quenched with
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sat. NH4Cl (10 mL). The phases were separated and the aqueous
layer was extracted with Et2O (3”10 mL). The combined organic
phases were washed with H2O (20 mL), dried over MgSO4 and con-
centrated in vacuo. Purification by flash column chromatography
(heptane/EtOAc 3:1) afforded the title compound as green solid
flash column chromatography (CH2Cl2/EtOAc 4:1) afforded the title
compound as yellow solid (338 mg, 1.71 mmol, 81%); Rf =0.15
1
(CH2Cl2/EtOAc 4:1); H NMR (400 MHz, CDCl3): d=9.67 (s, 1H), 8.77
(s, 1H), 8.62 (d, J=5.8 Hz, 1H), 8.36 (d, J=16.0 Hz, 1H), 7.93 (d, J=
8.2 Hz, 1H), 7.89 (d, J=7.2 Hz, 1H), 7.79 (dd, J=10.2, 5.2 Hz, 1H),
6.86 (d, J=16.0 Hz, 1H), 2.50 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=197.6, 147.4, 141.6, 137.5, 136.7, 133.4, 131.8, 131.0,
128.9, 126.8, 126.1, 121.7, 28.0 ppm; mp: 80.8–83.58C; LC–MS: (m/
z) calcd for C13H12NO [M+H]+: 198.1, found: 198.1.
1
(250 mg, 1.27 mmol, 61%); Rf =0.15 (heptane/EtOAc 3:1); H NMR
(400 MHz, CDCl3): d=9.26 (s, 1H), 8.77 (s, 1H), 8.21 (d, J=16.1 Hz,
1H), 8.14 (d, J=8.5 Hz, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.81 (ddd, J=
8.4, 7.0, 1.3 Hz, 1H), 7.71–7.65 (m, 1H), 7.49 (dd, J=8.3, 4.2 Hz, 1H),
6.89 (d, J=16.1 Hz, 1H), 2.47 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=197.5, 154.1, 141.6, 136.9, 133.8, 131.3, 130.4, 128.4,
128.1, 127.7, 125.7, 122.4, 28.4 ppm; LC–MS: (m/z) calcd for
C13H12NO [M+H]+: 198.1, found: 198.1.
4-(Quinolin-8-yl)but-3-en-2-one (5g). Carried out as described for
5a starting with quinoline-8-carbaldehyde. Purification by flash
column chromatography (heptane/EtOAc 3:1) afforded the title
compound as green solid (0.96 g, 4.86 mmol, 76%); Rf =0.15 (hep-
tane/EtOAc 3:1); 1H NMR (400 MHz, CDCl3): d=9.00 (dd, J=4.2,
1.8 Hz, 1H), 8.91 (d, J=16.7 Hz, 1H), 8.20 (dd, J=8.3, 1.6 Hz, 1H),
8.06 (dd, J=7.3, 0.8 Hz, 1H), 7.90 (dd, J=8.2, 1.2 Hz, 1H), 7.60 (t,
J=7.7 Hz, 1H), 7.49 (dd, J=8.3, 4.2 Hz, 1H), 6.97 (d, J=16.7 Hz,
1H), 2.55 ppm (s, 3H). The 1H NMR data are in agreement with
published values.[19]
4-(Isoquinolin-4-yl)but-3-en-2-one (5b). Carried out as described
for 5a starting with isoquinoline-4-carbaldehyde. Purification by
flash column chromatography (CH2Cl2/EtOAc 4:1) afforded the title
compound as yellow–orange solid (1.06 g, 5.35 mmol, 81%); Rf =
1
0.15 (CH2Cl2/EtOAc 4:1); H NMR (400 MHz, CDCl3): d=9.26 (s, 1H),
8.77 (s, 1H), 8.21 (d, J=16.1 Hz, 1H), 8.14 (d, J=8.5 Hz, 1H), 8.03
(d, J=8.2 Hz, 1H), 7.81 (ddd, J=8.4, 7.0, 1.3 Hz, 1H), 7.71–7.65 (m,
1H), 7.49 (dd, J=8.3, 4.2 Hz, 1H), 6.89 (d, J=16.1 Hz, 1H),
2.47 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=197.5, 154.1, 141.6,
136.9, 133.8, 131.3, 130.4, 128.4, 128.1, 127.7, 125.7, 122.4,
28.4 ppm; mp: 107.9–113.78C; LC–MS: (m/z) calcd for C13H12NO
[M+H]+: 198.1, found: 198.1.
4-(Quinolin-2-yl)but-3-en-2-one (5h). Carried out as described for
5a starting with quinoline-2-carbaldehyde. Purification by flash
column chromatography (heptane/EtOAc 3:1) afforded the title
compound as light-brown solid (601 mg, 3.05 mmol, 48%); Rf =
1
0.28 (heptane/EtOAc 3:1); H NMR (400 MHz, CDCl3): d=8.20 (d, J=
8.5 Hz, 1H), 8.11 (d, J=8.5 Hz, 1H), 7.83 (d, J=8.2 Hz, 1H), 7.78–
7.70 (m, 2H), 7.67 (dd, J=8.5, 2.7 Hz, 1H), 7.61–7.55 (m, 1H), 7.16
(dd, J=16.4, 1.6 Hz, 1H), 2.47 ppm (d, J=1.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d=198.7, 153.5, 143.0, 136.9, 131.9, 130.2, 129.7,
128.1, 127.6, 127.5, 120.0, 27.5 ppm; mp: 66.3–71.08C; LC–MS: (m/
z) calcd for C13H12NO [M+H]+: 198.1, found: 198.0.
4-(Quinolin-4-yl)but-3-en-2-one (5c). Carried out as described for
5a starting with quinoline-4-carbaldehyde. Purification by flash
column chromatography (heptane/EtOAc 4:1) afforded the title
compound as beige powder (2.45 g, 12.4 mmol, 86%); Rf =0.45
(heptane/EtOAc 4:1); 1H NMR (400 MHz, CDCl3): d=8.95 (d, J=
7.0 Hz, 1H), 8.22 (d, J=21.1 Hz, 1H), 8.16 (t, J=12.7 Hz, 2H), 7.68
(t, J=11.3 Hz, 1H), 7.61 (t, J=11.3 Hz, 1H), 7.51 (d, J=7.0 Hz, 1H),
6.83 (d, J=22.1 Hz, 1H), 2.49 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=198.1, 151.0, 149.0, 140.5, 137.9, 133.1, 131.0, 130.8,
127.9, 126.2, 123.8, 118.5, 29.1 ppm; mp: 91.7–92.58C; LC–MS: (m/
z) calcd for C13H12NO [M+H]+: 198.1, found: 198.1.
4-(Quinolin-7-yl)but-3-en-2-one (5h). Carried out as described for
5a starting with quinoline-7-carbaldehyde. Purification by flash
column chromatography (CH2Cl2/EtOAc 4:1) afforded the title com-
pound as light-brown solid (442 mg, 2.24 mmol, 47%); Rf =0.17
1
(CH2Cl2/EtOAc 4:1); H NMR (400 MHz, CDCl3): d=8.97 (dd, J=4.2,
4-(Quinolin-5-yl)but-3-en-2-one (5d). Carried out as described for
5a starting with quinoline-5-carbaldehyde. Purification by flash
column chromatography (heptane/EtOAc 1:1) afforded the title
compound as slightly yellow oil (0.36 g, 1.83 mmol, 60%); Rf =0.17
(heptane/EtOAc 1:1); 1H NMR (400 MHz, CDCl3): d=8.99 (dd, J=
4.3, 1.8 Hz, 1H), 8.54 (ddd, J=8.6, 1.5, 0.9 Hz, 1H), 8.29 (d, J=
16.1 Hz, 1H), 8.19 (d, J=8.3 Hz, 1H), 7.86 (d, J=7.0 Hz, 1H), 7.75
(dd, J=8.2, 7.5 Hz, 1H), 7.52 (dd, J=8.8, 4.3 Hz, 1H), 6.87 (d, J=
16.1 Hz, 1H), 2.48 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
197.7, 150.8, 148.5, 138.2, 132.1, 132.0, 131.6, 130.1, 129.1, 126.8,
125.4, 121.7, 28.4 ppm; LC–MS: (m/z) calcd for C13H12NO [M+H]+:
198.1, found: 198.1.
1.5 Hz, 1H), 8.52 (d, J=8.6 Hz, 1H), 8.27 (d, J=16.0 Hz, 1H), 8.17
(d, J=8.5 Hz, 1H), 7.84 (d, J=7.3 Hz, 1H), 7.73 (dd, J=8.4, 7.3 Hz,
1H), 7.49 (dd, J=8.6, 4.2 Hz, 1H), 6.84 (d, J=16.0 Hz, 1H),
2.45 ppm (d, J=0.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=198.1,
150.9, 142.4, 136.2, 130.5, 129.3, 128.8, 128.6, 124.7, 122.0,
27.7 ppm; mp: 61.2–64.58C; LC–MS: (m/z) calcd for C13H12NO [M+
H]+: 198.1, found: 198.0.
4-(3-Methylbenzo[d]isoxazol-4-yl)but-3-en-2-one (5j). 3-Methyl-
benzo[d]isoxazol-4-yl
trifluoromethanesulfonate
(0.38 mg,
1.35 mmol) was dissolved in degassed DMF (15 mL) and
PdCl2(PPh3)2 (0.10 g, 0.13 mmol, 10 mol%), and NEt3 (0.27 mL,
2.03 mmol) were added followed by methyl vinyl ketone (0.55 mL,
6.75 mmol). The reaction was stirred at 110 8C under N2 atm. for
18 h, then cooled to RT and diluted with H2O (30 mL). The mixture
was extracted with EtOAc (3”30 mL) and the combined organic
layers were washed with brine (20 mL), dried over MgSO4, filtered
and concentrated in vacuo. Flash column chromatography (hep-
tane/EtOAc 2:1) afforded the title compound as an orange solid
(271 mg, 1.35 mmol, quant.); Rf =0.18 (heptane/EtOAc 3:1);
1H NMR (600 MHz, CDCl3): d=7.85 (d, J=16.3 Hz, 1H), 7.49 (d, J=
8.1 Hz, 1H), 7.46 (d, J=7.7 Hz, 1H), 7.39 (d, J=16.3 Hz, 1H), 7.31 (t,
J=7.9 Hz, 1H), 2.69 (s, 3H), 2.45 ppm (s, 3H); 13C NMR (150 MHz,
CDCl3): d=198.9, 164.6, 151.3, 140.6, 138.6, 130.4, 126.3, 124.6,
124.5, 111.7, 27.7, 14.6 ppm; mp: 165.5–169.98C; LC–MS: (m/z)
calcd for C12H12NO2 [M+H]+: 202.2, found: 202.1.
4-(Isoquinolin-5-yl)but-3-en-2-one (5e). Carried out as described
for 5a starting with isoquinoline-5-carbaldehyde. Purification by
flash column chromatography (CH2Cl2/EtOAc 4:1) afforded the title
compound as pale-yellow solid (1.05 g, 5.33 mmol, 83%); Rf =0.11
(CH2Cl2/EtOAc 4:1); 1H NMR (400 MHz, CDCl3): d=9.30 (d, J=
0.7 Hz, 1H), 8.63 (d, J=6.0 Hz, 1H), 8.25 (d, J=16.0 Hz, 1H), 8.05
(d, J=8.2 Hz, 1H), 8.00 (d, J=7.3 Hz, 1H), 7.96 (d, J=6.1 Hz, 1H),
7.65 (t, J=7.8 Hz, 1H), 6.86 (d, J=16.0 Hz, 1H), 2.47 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=197.7, 153.3, 143.9, 137.8, 133.2,
130.9, 130.2, 128.9, 128.8, 127.0, 116.1, 28.2 ppm; mp: 56.1–65.28C;
LC–MS: (m/z) calcd for C13H12NO [M+H]+: 198.1, found: 198.1.
4-(Isoquinolin-8-yl)but-3-en-2-one (5 f). Carried out as described
for 5a starting with isoquinoline-8-carbaldehyde. Purification by
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4-(3-Methylbenzo[d]isoxazol-5-yl)but-3-en-2-one (5k). 5-Bromo-
3-methylbenzo[d]isoxazole (0.98 g, 4.62 mmol) was dissolved in de-
gassed DMF (18 mL) and Pd(OAc)2 (0.11 g, 0.48 mmol, 10 mol%),
tri-o-toluoylphosphine (0.28 g, 0.94 mmol, 20 mol%) and K2CO3
(0.97 g, 6.99 mmol) were added followed by methyl vinyl ketone
(0.45 mL, 5.55 mmol). The microwave vial was sealed and purged
with N2. The reaction was stirred in the microwave at 1008C for
1 h, then cooled to RT and diluted with EtOAc (40 mL) and H2O
(40 mL). The organic phase was washed with H2O (2”20 mL), dried
over MgSO4, filtered and evaporated. Flash column chromatogra-
phy (heptane to heptane/EtOAc 3:1) afforded the title compound
as a yellow solid (522 mg, 2.59 mmol, 56%); Rf =0.17 (heptane/
EtOAc 3:1); 1H NMR (600 MHz, CDCl3): d=7.83 (d, J=1.6 Hz, 1H),
7.61 (d, J=16.2 Hz, 1H), 7.53–7.47 (m, 2H), 6.73 (d, J=16.2 Hz, 1H),
2.66 (s, 3H), 2.40 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=198.2,
165.1, 152.4, 143.3, 142.3, 131.0, 126.8, 125.0, 119.3, 110.7, 27.6,
14.6 ppm; mp: 116.3–118.88C; LC–MS: (m/z) calcd for C12H12NO2
[M+H]+: 202.2, found: 202.1.
then filtered. The mixture was acidified with 2m H2SO4 to pH 5–6
upon which a precipitate formed. Precipitation was allowed to
occur in the fridge overnight. The precipitate was filtered off and
dried to afford the title compound as an off-white solid (360 mg,
1
1.50 mmol, 85%); Rf =0.15 (EtOAc/MeOH/AcOH 100:10:1); H NMR
(400 MHz, [D6]DMSO): d=11.22 (brs, 1H), 9.24 (s, 1H), 8.52 (s, 1H),
8.22 (d, J=8.6 Hz, 1H), 8.16 (d, J=8.1 Hz, 1H), 7.88–7.81 (m, 1H),
7.74–7.68 (m, 1H), 5.36 (s, 1H), 3.09–2.69 ppm (m, 4H); 13C NMR
(150 MHz, CDCl3): d=151.5, 140.3, 133.2, 132.1, 130.9, 128.5, 127.9,
127.2, 122.3, 103.5, 40.1, 32.2 ppm; mp: 127.8–138.28C (decomp.);
LC–MS: (m/z) calcd for C15H14NO2 [M+H]+: 240.1, found: 240.1.
5-(Quinolin-4-yl)cyclohexan-1,3-dione (6c). A solution of NaOEt in
EtOH (2.68m, 2.1 mL, 5.6 mmol) was added dropwise over 40 min
at RT to diethylmalonate (0.85 mL, 5.6 mmol) in absolute EtOH
(50 mL). The solution was stirred at RT for 30 min, after which 4-
(quinolin-4-yl)but-3-en-2-one (1.01 g, 5.0 mmol) in absolute EtOH
(50 mL) was added dropwise over 70 min at RT. The solution was
stirred at reflux for 2 h and then cooled to RT. The solvent was
evaporated and the residue was dried in vacuo. The residue was
dissolved in 2m NaOH (2.8 mL, 5.6 mmol) and the mixture was
heated at 908C and stirred at this temperature for 2.5 h. The mix-
ture was cooled to RT and acidified with 2m H2SO4 to pH 5–6. After
addition of toluene (1.5 mL), the reaction mixture was stirred at
708C for 30 min and then stirred at reflux for 1 h. The brown solu-
tion with the white precipitate was put on the ice-bath to induce
further precipitation. The mixture was then filtered and washed
thoroughly with ice-cold H2O. The mother liquor was evaporated
to dryness and the residue was re-dissolved in EtOH. The brown
mother liquor was evaporated to dryness and the crude residue
was purified by dry column vacuum chromatography (EtOAc/
MeOH/AcOH 100:10:1). The beige precipitate filtered off and yel-
lowish-white powder obtained from dry column chromatography
were both identified as the title compound (865 mg, 3.61 mmol,
61%); Rf =0.18 (EtOAc/MeOH/AcOH 100:10:1); 1H NMR (400 MHz,
[D6]DMSO): d=8.85 (d, J=4.6 Hz, 1H), 8.25 (d, J=8.0 Hz, 1H), 8.03
(d, J=8.3 Hz, 1H), 7.76 (t, J=7.0 Hz, 1H), 7.63 (t, J=7.0 Hz, 1H),
7.50 (d, J=4.5 Hz, 1H), 5.34 (s, 1H), 4.31–4.19 (m, 1H), 2.80–2.66
(m, 3H), 2.58 ppm (s, 2H); 13C NMR (100 MHz, [D6]DMSO): d=150.3,
148.8, 147.9, 129.9, 129.2, 126.7, 126.1, 123.3, 118.3, 103.5,
33.2 ppm; mp: 219.0–222.78C (decomp.); LC–MS: (m/z) calcd for
C15H14NO2 [M+H]+: 240.1, found: 240.1.
5-(Isoquinolin-1-yl)cyclohexan-1,3-dione (6a).
A
solution of
NaOEt in EtOH (2.68m, 0.5 mL) was added dropwise to a solution
of diethylmalonate (0.2 mL, 1.32 mmol) in absolute EtOH (13 mL)
over 10 min at RT and stirred for 30 min. 4-(Isoquinolin-1-yl)but-3-
en-2-one (236 mg, 1.20 mmol) in absolute EtOH (13 mL) was next
added dropwise over 10 min and the mixture was now heated at
reflux and stirred for 4 h. The mixture was cooled to RT and the sol-
vent evaporated in vacuo. The residue was dissolved in H2O
(13 mL) and evaporated. This was repeated three times and then
the residue was dried under vacuum overnight. The solid was sus-
pended in 2m NaOH (3.4 mL), heated at 908C and stirred at that
temperature for 3 h. The mixture was cooled to RT and acidified
with 2m H2SO4 to pH 5–6. The mixture was heated at reflux and
stirred for 2 h before cooling to RT. The reaction was made alkaline
(pH>10) using 5m NaOH. The aqueous phase was washed with
EtOAc (13 mL) and CH2Cl2 (13 mL) and then filtered. The mixture
was acidified with 2m H2SO4 to pH 5–6 upon which a precipitate
formed. Precipitation was allowed to occur in the fridge overnight.
The precipitate was filtered off and dried to afford the title com-
pound as a beige solid (152 mg, 0.63 mmol, 53%); Rf =0.69
(EtOAc/MeOH/AcOH 100:10:1); 1H NMR (400 MHz, [D6]DMSO): d=
11.15 (brs, 1H), 8.46 (d, J=5.6 Hz, 1H), 8.37 (d, J=8.5 Hz, 1H), 7.99
(d, J=8.1 Hz, 1H), 7.81–7.75 (m, 1H), 7.73 (d, J=5.6 Hz, 1H), 7.70
(dd, J=11.3, 4.1 Hz, 1H), 5.32 (brs, 1H), 4.56–4.39 (m, 1H), 2.94–
2.68 (m, 2H), 2.58–2.49 ppm (m, 2H); 13C NMR (150 MHz,
[D6]DMSO): d=161.1, 141.4, 135.9, 130.2, 127.7, 127.5, 125.2, 124.5,
119.7, 103.4, 40.1, 36.2 ppm; mp: 115.2–129.18C (decomp.); LC–MS:
(m/z) calcd for C15H14NO2 [M+H]+: 240.1, found: 240.1.
5-(Quinolin-5-yl)cyclohexan-1,3-dione (6d). Under a nitrogen at-
mosphere diethyl malonate (0.30 mL, 1.97 mmol) was dissolved in
absolute EtOH (20 mL). NaOEt in EtOH (0.75 mL, 1.97 mmol) was
added dropwise and the mixture was stirred at RT for 30 min, after
which 4-(quinolin-5-yl)but-3-en-2-one (354 mg, 1.80 mmol) in abso-
lute EtOH (20 mL) was added slowly over 10 min. The mixture was
heated at reflux and stirred for 3 h after which it was cooled to RT.
The mixture was evaporated to dryness and then suspended in 2m
NaOH (4 mL). The suspension was heated at 908C and stirred at
that temperature for 3 h. The mixture was allowed to cool to RT,
after which it was acidified by addition of 2m H2SO4 to pH 5–6.
The mixture was heated at reflux and stirred for 105 min. The mix-
ture was cooled to RT and then made alkaline (pH>14) by addi-
tion of 5m NaOH. The mixture was washed with EtOAc (20 mL)
and CH2Cl2 (20 mL). The aqueous phase was filtered and the pH
was adjusted to pH 5–6 by addition of 2m H2SO4. Precipitation was
allowed to occur at 58C, and was then filtered off and dried to
give the title compound as beige powder (263 mg, 1.10 mmol,
61%); Rf =0.38 (EtOAc/MeOH/AcOH 100:10:1); 1H NMR (400 MHz,
[D6]DMSO): d=11.22 (brs, 1H), 8.91 (dd, J=4.1, 1.5 Hz, 1H), 8.66
(d, J=8.8 Hz, 1H), 7.93 (d, J=8.5 Hz, 1H), 7.74 (dd, J=8.0, 7.3 Hz,
5-(Isoquinolin-4-yl)cyclohexan-1,3-dione (6b).
A
solution of
NaOEt in EtOH (2.68m, 0.8 mL) was added dropwise to a solution
of diethylmalonate (0.35 mL, 2.15 mmol) in absolute EtOH (20 mL)
over 10 min at RT and stirred for 30 min. 4-(Isoquinolin-4-yl)but-3-
en-2-one (355 mg, 1.77 mmol) in absolute EtOH (20 mL) was next
added dropwise over 10 min and the mixture was now heated at
reflux and stirred for 2.5 h. The mixture was cooled to RT and the
solvent evaporated in vacuo. The residue was dissolved in H2O
(13 mL) and evaporated. This was repeated three times and then
the residue was dried under vacuum overnight. The solid was sus-
pended in 2m NaOH (4 mL), heated at 908C and stirred at that
temperature for 3 h. The mixture was cooled to RT and acidified
with 2m H2SO4 to pH 5–6. The mixture was heated at reflux and
stirred at that temperature for 2 h before cooling to RT. The reac-
tion was made alkaline (pH>10) using 5m NaOH. The aqueous
phase was washed with EtOAc (20 mL) and CH2Cl2 (20 mL) and
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1H), 7.62 (d, J=7.0 Hz, 1H), 7.56 (dd, J=8.8, 4.0 Hz, 1H), 5.35 (s,
1H), 4.27–4.17 (m, 1H), 2.80–2.65 (m, 2H), 2.59–2.45 ppm (m, 2H);
13C NMR (100 MHz, [D6]DMSO): d=150.1, 148.2, 140.2, 131.7, 129.1,
128.0, 125.8, 123.6, 121.4, 103.5, 33.2 ppm; mp: 196.5–209.18C
(decomp.); LC–MS: (m/z) calcd for C15H14NO2 [M+H]+: 240.1,
found: 240.1.
216.18C (decomp.); LC–MS: (m/z) calcd for C15H14NO2 [M+H]+:
240.1, found: 240.1.
5-(Quinolin-8-yl)cyclohexan-1,3-dione (6g). A solution of NaOEt
in EtOH (2.68m, 2.0 mL) was added dropwise over 10 min to a solu-
tion of diethyl malonate (0.8 mL, 5.26 mmol) in absolute EtOH
(54 mL) at RT under N2. The solution was stirred for 30 min before
4-(quinolin-8-yl)but-3-en-2-one (0.94 g, 4.79 mmol) in absolute
EtOH (36 mL) was added dropwise over 10 min. The mixture was
heated at reflux and stirred at that temperature for 2.5 h. The reac-
tion was cooled to RT and the solvent was removed in vacuo. The
residue was dissolved in H2O (30 mL) and concentrated. This was
repeated three times and the residue was dried under vacuum
overnight. The residue was then dissolved in 2m NaOH (15 mL)
and stirred at 908C for 3 h. After cooling to RT the mixture was
acidified with 2m H2SO4 until pH 5–6 and stirred at reflux for 2 h.
The mixture was cooled to RT and treated with aqueous 5m NaOH
until pH>10. The aqueous phase was next washed with EtOAc
(13 mL) and CH2Cl2 (13 mL). The pH of the mixture was adjusted to
pH 5–6 upon which a fine precipitate formed. The suspension was
cooled in an ice bath to induce further precipitation. The precipi-
tate was filtered off and dried. The mother liquor was concentrated
in vacuo, dissolved in 2m NaOH (15 mL) and the pH was adjusted
to pH 5–6. The precipitate formed was filtered and dried. This was
repeated one more time to afford the title compound as a beige
solid (404 mg, 1.69 mmol, 35%); Rf =0.43 (EtOAc/MeOH/AcOH
100:10:1); 1H NMR (600 MHz, [D6]DMSO): d=11.16 (brs, 1H), 8.97
(dd, J=4.1, 1.8 Hz, 1H), 8.40 (dd, J=8.3, 1.7 Hz, 1H), 7.90 (dd, J=
8.2, 1.0 Hz, 1H), 7.75 (d, J=6.7 Hz, 1H), 7.61 (t, J=6.1 Hz, 1H), 7.59
(dd, J=8.2, 4.1 Hz, 1H), 5.34 (s, 1H), 4.60 (m, 1H), 2.80 (dd, J=16.5,
11.8 Hz, 2H), 2.60–2.53 ppm (m, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=149.6, 145.4, 141.1, 136.7, 128.1, 126.8, 126.5, 126.4,
121.4, 103.3, 40.1, 33.2 ppm; mp: 163.9–172.88C (decomp.); LC–
MS: (m/z) calcd for C15H14NO2 [M+H]+: 240.1, found: 240.1.
5-(Isoquinolin-5-yl)cyclohexan-1,3-dione (6e).
A
solution of
NaOEt in EtOH (2.68m, 2.0 mL) was added dropwise to a solution
of diethylmalonate (0.8 mL, 5.30 mmol) in absolute EtOH (55 mL)
over 10 min at RT and stirred for 30 min. 5-(Isoquinolin-5-yl)but-3-
en-2-one (950 mg, 4.82 mmol) in absolute EtOH (36 mL) was next
added dropwise over 10 min and the mixture was then heated at
reflux and stirred for 2 h. The mixture was cooled to RT and the sol-
vent evaporated in vacuo. The residue was dissolved in H2O
(30 mL) and evaporated. This was repeated three times and then
the residue was dried under vacuum overnight. The solid was sus-
pended in 2m NaOH (15 mL), heated at 908C and stirred at that
temperature for 3 h. The mixture was cooled to RT and acidified
with 2m H2SO4 to pH 5–6. The mixture was heated at reflux and
stirred at that temperature for 2 h before cooling to RT. The reac-
tion was made alkaline (pH>10) using 5m NaOH. The aqueous
phase was washed with EtOAc (15 mL) and CH2Cl2 (15 mL) and
then filtered. The mixture was acidified with 2m H2SO4 to pH 5–6
upon which a precipitate formed. Precipitation was allowed to
occur in the fridge overnight. The precipitate was filtered off, tritu-
rated with EtOH and dried to afford the title compound as an off-
white solid (511 mg, 2.13 mmol, 44%); Rf =0.17 (EtOAc/MeOH/
AcOH 100:10:1); 1H NMR (400 MHz, [D6]DMSO): d=9.31 (s, 1H),
8.52 (d, J=6.1 Hz, 1H), 8.02 (d, J=2.5 Hz, 1H), 8.01 (d, J=4.8 Hz,
1H), 7.77 (d, J=7.1 Hz, 1H), 7.66 (t, J=7.7 Hz, 1H), 5.18 (brs, 1H),
4.16–4.05 (m, 1H), 2.63 (dd, J=16.5, 11.1 Hz, 2H), 2.55–2.49 ppm
(m, 2H); 13C NMR (150 MHz, [D6]DMSO): d=172.0, 153.2, 143.2,
139.3, 133.3, 128.6, 127.4, 127.1, 126.4, 116.1, 102.6, 33.5, 21.1 ppm;
mp: 203.4–206.58C (decomp.); LC–MS: (m/z) calcd for C15H14NO2
[M+H]+: 240.1, found: 240.1.
5-(Quinolin-2-yl)cyclohexan-1,3-dione (6h). A solution of NaOEt
in EtOH (2.68m, 0.3 mL) was added dropwise to a solution of dieth-
ylmalonate (0.15 mL, 0.80 mmol) in absolute EtOH (8 mL) at RT
under N2. The solution was stirred for 30 min before 4-(quinolin-2-
yl)but-3-en-2-one (140 mg, 0.71 mmol) in absolute EtOH (8 mL)
was added dropwise over 10 min. The mixture was heated at reflux
and stirred at that temperature for 16 h. The reaction was cooled
to RT and the solvent was removed in vacuo and dried under
vacuum. The residue was then dissolved in 2m NaOH (1.6 mL) and
stirred at 908C for 3 h. After cooling to RT the mixture was acidi-
fied with 2m H2SO4 until pH 5–6 and stirred at reflux for 4 h. The
mixture was cooled to RT and treated with aqueous 5m NaOH
until pH>10. The aqueous phase was next washed with EtOAc
(5 mL) and CH2Cl2 (5 mL). The pH of the mixture was adjusted to
pH 5–6 with 2m H2SO4 upon which a fine precipitate formed. The
suspension was cooled in an ice bath to induce further precipita-
tion. The precipitate was filtered off and dried. The mother liquor
was concentrated in vacuo, dissolved in 2m NaOH (15 mL) and the
pH was adjusted to pH 5–6. The precipitate formed was filtered
and dried. This was repeated one more time to afford the title
compound as an off-white solid (58 mg, 0.24 mmol, 34%); Rf =0.67
(EtOAc/MeOH/AcOH 100:10:1); 1H NMR (600 MHz, [D6]DMSO): d=
11.18 (s, 1H), 8.33 (d, J=8.4 Hz, 1H), 7.99–7.92 (m, 2H), 7.74 (ddd,
J=8.3, 6.8, 1.6 Hz, 1H), 7.58 (ddd, J=10.8, 7.1, 4.5 Hz, 2H), 5.30 (s,
1H), 3.69 (ddd, J=11.2, 6.6, 4.6 Hz, 1H), 2.97 (ddd, J=25.6, 12.1,
5.4 Hz, 1H), 2.83 (ddd, J=17.7, 16.0, 9.3 Hz, 1H), 2.73–2.58 ppm (m,
2H); 13C NMR (150 MHz, [D6]DMSO): d=162.3, 147.0, 136.7, 129.5,
128.6, 127.7, 126.8, 126.1, 120.5, 103.3, 41.0, 40.1 ppm; mp: 135.5–
5-(Isoquinolin-8-yl)cyclohexan-1,3-dione (6 f). A solution of NaOEt
in EtOH (2.68m, 0.70 mL) was added dropwise to a solution of di-
ethylmalonate (0.3 mL, 1.88 mmol) in absolute EtOH (19 mL) over
10 min at RT and stirred for 30 min. 5-(isoquinolin-8-yl)but-3-en-2-
one (336 mg, 1.70 mmol) in absolute EtOH (36 mL) was next added
dropwise over 10 min and the mixture was heated at reflux and
stirred for 4 h. The mixture was cooled to RT and the solvent
evaporated in vacuo. The residue was dissolved in H2O (19 mL) and
evaporated. This was repeated three times and then the residue
was dried under vacuum overnight. The solid was suspended in
2m NaOH (3.8 mL), heated at 908C and stirred at that temperature
for 3 h. The mixture was cooled to RT and acidified with 2m H2SO4
to pH 5–6. The mixture was heated at reflux and stirred at that
temperature for 2 h before cooling to RT. The reaction was made
alkaline (pH>10) using 5m NaOH. The aqueous phase was washed
with EtOAc (15 mL) and CH2Cl2 (15 mL) and then filtered. The mix-
ture was acidified with 2m H2SO4 to pH 5–6 upon which a precipi-
tate formed. Precipitation was allowed to occur in the fridge over-
night. The precipitate was filtered off and dried to afford the title
compound as an off-white solid (189 mg, 0.78 mmol, 46%); Rf =
1
0.63 (EtOAc/MeOH/AcOH 100:10:1); H NMR (400 MHz, [D6]DMSO):
d=11.21 (brs, 1H), 9.62 (s, 1H), 8.53 (d, J=5.6 Hz, 1H), 7.89–7.83
(m, 2H), 7.77–7.72 (m, 1H), 7.67 (d, J=7.0 Hz, 1H), 5.36 (s, 1H),
4.47–4.38 (m, 1H), 2.87–2.73 (m, 2H), 2.52–2.49 ppm (m, 2H);
13C NMR (150 MHz, [D6]DMSO): d=148.2, 142.6, 140.6, 135.9, 130.3,
125.7, 125.6, 124.7, 121.0, 103.6, 40.1, 32.8 ppm; mp: 198.5–
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184.58C (decomp.); LC–MS: (m/z) calcd for C15H14NO2 [M+H]+:
240.1, found: 240.1.
and concentrated. This was repeated three times and the final resi-
due was dried under vacuum overnight. The residue was then dis-
solved in 2m NaOH (12 mL) and stirred at RT for 17 h. The mixture
was then acidified with 2m H2SO4 until pH 1–2 and continued to
stir at RT for 5 h. The formed precipitate was filtered off and dried
to afford the title compound as a brown solid (149 mg, 0.61 mmol,
49%); Rf =0.23 (EtOAc/MeOH/AcOH 100:10:1); 1H NMR (600 MHz,
[D6]DMSO): d=11.11 (brs, 1H), 7.57 (s, 1H), 6.91 (d, J=9.9 Hz, 1H),
6.80 (d, J=8.2 Hz, 1H), 5.26 (s, 1H), 3.22–3.15 (m, 1H), 2.67–2.63
(m, 2H), 2.36–2.25 (m, 2H), 2.08 ppm (s, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=169.5, 150.8, 147.1, 134.5, 126.6, 125.8, 123.4, 121.5,
116.4, 104.0, 38.5, 24.0 ppm; mp: 195.4–197.08C (decomp.); LC–
MS: (m/z) calcd for C14H16NO4 [M+H+ +H2O]: 262.1, found: 262.1.
5-(Quinolin-7-yl)cyclohexan-1,3-dione (6i). A solution of NaOEt in
EtOH (2.68m, 0.75 mL) was added dropwise to a solution of dieth-
ylmalonate (0.30 mL, 1.97 mmol) in absolute EtOH (20 mL) at RT
under N2. The solution was stirred for 30 min before 4-(quinolin-7-
yl)but-3-en-2-one (354 mg, 1.79 mmol) in absolute EtOH (20 mL)
was added dropwise over 5 min. The mixture was heated at reflux
and stirred at that temperature for 3 h. The reaction was cooled to
RT and the solvent was removed in vacuo and dried under
vacuum. The residue was then dissolved in 2m NaOH (1.6 mL) and
stirred at 908C for 3 h. After cooling to RT the mixture was acidi-
fied with 2m H2SO4 until pH 5–6 and stirred at reflux for 4 h. The
mixture was cooled to RT and treated with 5m NaOH until pH>10.
The aqueous phase was next washed with EtOAc (5 mL) and
CH2Cl2 (5 mL). The pH of the mixture was adjusted to pH 5–6 with
2m H2SO4 upon which a fine precipitate formed. The suspension
was cooled in an ice bath to induce further precipitation. The pre-
cipitate was filtered off and dried. The mother liquor was concen-
trated in vacuo, dissolved in 2m NaOH (15 mL) and the pH was ad-
justed to pH 5–6 with 2m H2SO4. The precipitate formed was fil-
tered and dried. This was repeated one more time to afford the
title compound as an off-white solid (74 mg, 0.31 mmol, 18%); Rf =
1-(2,6-Dihydroxyphenyl)ethan-1-one oxime (8j). A solution of
2,6-dihydroxyacetophenone (2.01 g, 13.15 mmol) in absolute EtOH
(100 mL) was warmed to 708C. A solution of hydroxylamine hydro-
chloride (25.53 g, 0.37 mol) in H2O (65 mL) was added and the re-
action mixture was stirred at that temperature for 20 h. The solu-
tion was cooled to RT and diluted with H2O (200 mL). The mixture
was extracted with EtOAc (3”100 mL), the combined organic
phases were dried over MgSO4 and concentrated in vacuo. Flash
column chromatography (heptane/EtOAc 3:1) afforded the title
compound as a yellow solid (1.77 g, 10.61 mmol, 81%); Rf =0.33
(heptane/EtOAc 3:1); 1H NMR (600 MHz, [D6]DMSO): d=10.94 (s,
1H), 9.72 (s, 2H), 6.93 (t, J=8.1 Hz, 1H), 6.32 (d, J=8.1 Hz, 2H),
2.10 ppm (s, 3H); 13C NMR (150 MHz, [D6]DMSO): d=156.6, 153.8,
129.3, 111.3, 106.7, 15.4 ppm; mp: 134.8–138.58C; LC–MS: (m/z)
calcd for C8H10NO3 [M+H]+: 168.1, found: 168.1.
1
0.37 (EtOAc/MeOH/AcOH 100:10:1); H NMR (600 MHz, [D6]DMSO):
d=11.28 (brs, 1H), 8.88 (d, J=4.1 Hz, 1H), 8.33 (d, J=8.3 Hz, 1H),
7.98–7.83 (m, 2H), 7.66 (d, J=8.5 Hz, 1H), 7.52–7.46 (m, 1H), 5.34
(s, 1H), 3.62–3.52 (m, 1H), 2.83–2.58 (m, 2H), 2.59 ppm (m, 2H);
13C NMR (150 MHz, [D6]DMSO): d=172.7, 150.6, 147.8, 145.0, 135.6,
128.2, 126.7, 126.4, 126.1, 121.1, 103.6, 48.5, 30.1 ppm; mp: 197.2–
216.98C (decomp.); LC–MS: (m/z) calcd for C15H14NO2 [M+H]+:
240.1, found: 240.1.
1-(5-Bromo-2-hydroxyphenyl)ethan-1-one oxime (8k). A solution
of 5-bromo-2-hydroxyacetophenone (2.02 g, 9.32 mmol) in abso-
lute EtOH (49 mL) was warmed to 708C. A solution of hydroxyla-
mine hydrochloride (19.21 g, 0.27 mol) in H2O (73 mL) was added
and the reaction mixture was stirred at that temperature for 2 h.
The solution was cooled to RT and diluted with H2O (240 mL). The
mixture was extracted with EtOAc (3”150 mL), the combined or-
ganic phases were dried over MgSO4 and concentrated in vacuo to
afford the title compound as an off-white solid (2.14 g, 9.30 mmol,
quant.); Rf =0.18 (heptane/EtOAc 3:1); 1H NMR (400 MHz, CDCl3):
d=11.15 (s, 1H), 7.52 (d, J=2.4 Hz, 1H), 7.34 (dd, J=8.7, 2.4 Hz,
1H), 7.30 (s, 1H), 6.86 (d, J=8.7 Hz, 1H), 2.34 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=158.8, 156.8, 133.5, 130.1, 120.1, 119.2, 110.9,
10.8 ppm; mp: 155.6–158.58C; LC–MS: (m/z) calcd for C8H9BrNO2
[M+H]+: 230.0, 232.0, found: 230.0, 232.0.
4-(3-Methylbenzo[d]isoxazol-4-yl)cyclohexane-1,3-dione (6j).
A
solution of NaOEt in EtOH (2.68m, 0.41 mL) was added dropwise
over 10 min to a solution of diethylmalonate (0.16 mL, 1.09 mmol)
in absolute EtOH (11 mL) at RT under N2. The solution was stirred
for 40 min before 4-(3-methylbenzo[d]isoxazol-4-yl)but-3-en-2-one
(199 mg, 0.99 mmol) in absolute EtOH (11 mL) was added dropwise
over 7 min. The mixture was heated at reflux and stirred at that
temperature for 5 h. The reaction was cooled to RT and the solvent
was removed in vacuo. The residue was dried under vacuum over-
night. The residue was then dissolved in 2m NaOH (2.2 mL) and
stirred at 908C for 4 h. The mixture was cooled to RT and acidified
with 2m H2SO4 until pH 1–2 and continued to stir at RT for 4 h.
The formed precipitate was filtered off, washed with H2O and dried
to afford the title compound as a brown foam (110 mg, 0.45 mmol,
46%); Rf =0.54 (EtOAc/MeOH/AcOH 100:10:1); 1H NMR (400 MHz,
[D6]DMSO): d=11.11 (brs, 1H), 8.98 (s, 1H), 7.11–7.04 (m, 1H), 6.86
(d, J=8.2, 7.7 Hz, 1H), 6.75 (d, J=7.8 Hz, 1H), 3.80–3.73 (m, 1H),
3.38–3.31 (m, 1H), 2.92–2.81 (m, 2H), 2.33–2.24 (m, 2H), 1.99 ppm
(s, 3H); 13C NMR (150 MHz, [D6]DMSO): d=169.4, 168.6, 156.2,
153.3, 142.1, 124.5, 122.3, 121.7, 108.8, 103.5, 100.4, 41.6, 35.2,
14.1 ppm; mp: >2508C; LC–MS: (m/z) calcd for C14H16NO4 [M+H+
+H2O]: 262.1, found: 262.1.
3-Methylbenzo[d]isoxazol-4-ol. PPh3 (2.84 g, 10.83 mmol) was dis-
solved in dry CH2Cl2 (36 mL) and DDQ (2.47 g, 10.88 mmol) was
added at RT. The brown reaction mixture was stirred at that tem-
perature for 3 min before 1-(2,6-dihydroxyphenyl)ethan-1-one
oxime (8j) (1.20 g, 7.18 mmol) was added. The suspension was
stirred for 16 h and the solvent was evaporated in vacuo. Purifica-
tion by flash column chromatography (heptane/EtOAc 3:1) afford-
ed the title compound as a white solid (900 mg, 6.03 mmol, 84%);
1
Rf =0.23 (heptane/EtOAc 3:1); H NMR (600 MHz, CDCl3): d=9.91 (s,
1H), 7.21 (t, J=8.1 Hz, 1H), 7.03 (dd, J=8.1, 0.9 Hz, 1H), 6.88 (dd,
J=8.1, 0.8 Hz, 1H), 2.70 ppm (s, 3H); 13C NMR (150 MHz, CDCl3):
d=163.4, 152.1, 148.1, 129.0, 125.8, 111.1, 101.8, 14.1 ppm; mp:
129.8–131.58C; LC–MS: (m/z) calcd for C8H8NO2 [M+H]+: 150.0,
found: 150.1.
5-(3-Methylbenzo[d]isoxazol-5-yl)cyclohexane-1,3-dione (6k). A
solution of NaOEt in EtOH (2.68m, 0.51 mL) was added dropwise
over 10 min to a solution of diethylmalonate (0.21 mL, 1.37 mmol)
in absolute EtOH (14 mL) at RT under N2. The solution was stirred
for 30 min before 4-(3-methylbenzo[d]isoxazol-5-yl)but-3-en-2-one
(0.25 g, 1.24 mmol) in absolute EtOH (14 mL) was added dropwise
over 10 min. The mixture was heated at reflux and stirred at that
temperature for 4 h. The reaction was cooled to RT and the solvent
was removed in vacuo. The residue was dissolved in H2O (30 mL)
3-Methylbenzo[d]isoxazol-4-yl trifluoromethanesulfonate (9j). 3-
Methylbenzo[d]isoxazol-4-ol (0.80 g, 5.36 mmol) which was pre-
pared as described above was dissolved in dry CH2Cl2 (5 mL) and
pyridine (0.86 mL, 10.72 mmol) was added at RT. The reaction was
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Full Papers
cooled to 08C and Tf2O (1.1 mL, 6.44 mmol) in CH2Cl2 (2.5 mL) was
added dropwise. The reaction was warmed to RT and stirred for
18 h. The reaction was diluted with Et2O (8 mL) and 2m HCl (3 mL).
The reaction mixture was washed with sat. NaHCO3 (5 mL) and
brine (5 mL). The organic phase was dried over MgSO4, filtered and
concentrated in vacuo. Purification by flash column chromatogra-
phy (heptane/EtOAc 6:1) afforded the title compound as a yellow
solid (1.32 g, 4.69 mmol, 87%); Rf =0.33 (heptane/EtOAc 6:1);
1H NMR (400 MHz, CDCl3): d=7.51 (dd, J=8.2, 0.9 Hz, 1H), 7.34 (t,
J=8.2 Hz, 1H), 7.24 (dd, J=8.3, 0.8 Hz, 1H), 2.69 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=165.6, 152.7, 139.0, 134.8, 124.8,
116.9, 110.6, 14.6 ppm; 19F NMR (400 MHz, CDCl3): d=73.0 ppm;
mp: 51.4–55.58C; LC–MS: (m/z) calcd for C9H7F3NO4S [M+H]+:
282.0, found: 282.1.
7.24 ppm (t, J=8.4 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=138.4,
134.5, 133.5, 128.3, 99.7 ppm; mp: 146–1488C.
5-Iodonaphthalene-1-carbaldehyde (12). tBuLi (1.49m, 3.5 mL,
5.26 mmol) was added dropwise to a solution of 1,5-diiodonaph-
thalene (11) (1.00 g, 2.6 mmol) in dry THF (50 mL) at À788C under
a N2 atmosphere. DMF (0.20 mL, 2.6 mmol) was added slowly and
the reaction was stirred at that temperature for 1.5 h. The reaction
mixture was warmed to RT, diluted with H2O (50 mL) and extracted
with EtOAc (3”50 mL). The combined organic phases were
washed with brine (30 mL), dried over MgSO4, filtered and evapo-
rated. Purification by flash column chromatography afforded the
title compound as a yellow solid (371 mg, 1.51 mmol, 58%); Rf =
0.42 (heptane/EtOAc 5:1); 1H NMR (300 MHz, CDCl3): d=10.39 (s,
1H), 9.30 (d, J=8.4 Hz, 1H), 8.43 (d, J=8.4 Hz, 1H), 8.20 (d, J=
7.2 Hz, 1H), 8.02 (d, J=7.2 Hz, 1H), 7.70 (t, J=8.4 Hz, 1H),
7.36 ppm (t, J=8.4 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=192.9,
139.5, 138.9, 137.4, 135.5, 134.6, 131.8, 130.0, 126.7, 125.8,
100.2 ppm; mp: 114–1168C.
5-Bromo-3-methylbenzo[d]isoxazole
(9k).
PPh3
(2.08 g,
7.96 mmol) was dissolved in dry CH2Cl2 (26 mL) and DDQ (1.79 g,
7.88 mmol) was added at RT. The brown reaction mixture was
stirred at that temperature for 5 min before 1-(5-bromo-2-hydroxy-
phenyl)ethan-1-one oxime (1.21 g, 5.23 mmol) was added. The sus-
pension was stirred at RT for 10 min and the solvent was evaporat-
ed in vacuo. Purification by flash column chromatography (hep-
tane/EtOAc 3:1) afforded the title compound as an off-white solid
(E)-4-(1-Iodonaphthalen-5-yl)but-3-en-2-one (13). A solution of di-
ethyl-2-oxopropylphosphonate (0.24 mL, 1.23 mmol) in dry THF
(5 mL) was added dropwise over 20 min to a solution of NaH (60%
dispersion in mineral oil, 49 mg, 1.23 mmol) in dry THF (5 mL) at
RT under a N2 atmosphere. The lightly yellow solution was stirred
for an additional 1.5 h at RT after which a solution of carbaldehyde
12 (330 mg, 1.17 mmol) in dry THF (5 mL) was added at 08C. The
reaction mixture was stirred at RT for 21 h. The reaction mixture
was quenched with H2O (10 mL) and extracted with EtOAc (3”
30 mL). The combined organic phases were washed with brine
(30 mL), dried over Na2SO4 and concentrated in vacuo. Purification
by flash column chromatography afforded the title compound as
a yellow solid (276 mg, 0.85 mmol, 73%); Rf =0.20 (heptane/EtOAc
1
(991 mg, 4.67 mmol, 89%); Rf =0.32 (heptane/EtOAc 3:1); H NMR
(400 MHz, CDCl3): d=7.78 (d, J=1.9 Hz, 1H), 7.41 (dd, J=8.6,
1.9 Hz, 1H), 7.34 (d, J=8.5 Hz, 1H), 2.64 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=165.1, 150.0, 143.2, 127.5, 122.5, 116.8, 111.4,
14.5 ppm; LC–MS: (m/z) calcd for C8H7BrNO [M+H]+: 212.0, 214.0,
found: 212.1, 214.1.
3-Amino-5-(naphthalen-1-yl)cyclohex-2-enone (10). To a solution
of 5-(naphthalen-1-yl)-cyclohexan-1,3-dione (600 mg, 2.52 mmol) in
absolute EtOH (11.5 mL) containing some 3 Š molecular sieves was
added NH4OAc (584 mg, 7.55 mmol) and the reaction mixture was
stirred at reflux overnight. The resulting brown cloudy suspension
was evaporated to dryness. Saturated NaHCO3 (50 mL) was added
and the resulting mixture was extracted with EtOAc (3”75 mL).
The combined organic layers were washed with brine, dried over
MgSO4, filtered and evaporated to afford the title compound as
a yellow solid (505 mg, 84%). The product was used in the follow-
ing steps without any further purification. Rf =0.16 (CH2Cl2/MeOH
95:5); 1H NMR (400 MHz, CDCl3/[D4]MeOH (1:1)): d=8.13 (d, J=
8.3 Hz, 1H), 7.89 (dd, J=8.1, 1.4 Hz, 1H), 7.78 (d, J=7.7 Hz, 1H),
7.60–7.42 (m, 4H), 5.37 (s, 1H), 4.21 (tt, J=10.4, 5.1 Hz, 1H), 2.91–
2.75 (m, 2H), 2.74–2.58 ppm (m, 2H); 13C NMR (100 MHz, CDCl3/
[D4]MeOH (1:1)): d=199.0, 172.5, 140.2, 135.6, 132.5, 130.1, 128.5,
127.3, 126.7, 126.5, 124.0, 123.7, 98.0, 43.3, 36.4, 36.1 ppm; mp:
203.8–205.48C; LC–MS: (m/z) calcd for C16H16NO [M+H]+: 238.1,
found: 238.1.
1
5:1); H NMR (300 MHz, CDCl3): d=8.27 (d, J=15.6 Hz, 1H), 8.17–
8.09 (m, 3H), 7.74 (d, J=6.9 Hz, 1H), 7.52 (t, J=7.8 Hz, 1H), 7.21 (t,
J=7.8 Hz, 1H), 6.76 (d, J=15.6 Hz, 1H), 2.46 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=197.8, 139.4, 138.0, 134.8, 134.3, 132.4, 132.0,
130.2, 127.6, 127.1, 125.9, 124.1, 100.5, 28.1 ppm; mp: 118–1208C;
LC–MS: (m/z) calcd for C14H11IO [M+H]+: 323.1, found: 323.1;
HPLC: purity (l 254 nm) >98%.
5-(1-Iodonaphthalen-5-yl)cyclohexane-1,3-dione (14). A solution
of NaOEt (0.52 mL, 1.39 mmol) in absolute EtOH (5 mL) was added
dropwise over 10 min to a stirred solution of diethylmalonate
(0.22 mL, 1.39 mmol) in absolute EtOH (15 mL) at RT under a N2 at-
mosphere. This yellow solution was stirred at RT for an additional
40 min after which a solution of enone 13 (300 mg, 0.93 mmol) in
a mixture of abs EtOH/THF (12 mL, 3:1) was added dropwise over
10 min. The reaction mixture was stirred at RT for 30 min and then
at reflux for 15 h. The reaction mixture was concentrated and the
brown sticky residue was dried under vacuum. To this residue H2O
(10 mL) and 2m NaOH (2.3 mL, 4.6 mmol) were added and the re-
action mixture was stirred at 858C for 3 h and then cooled to RT.
The solution was adjusted to pH 2 by adding 2m H2SO4 and stirred
at 1008C for 1 h. After concentration in vacuo, the crude product
was dissolved in EtOAc (30 mL) and extracted with 1m NaOH (3”
20 mL). The combined aqueous phases were acidified with conc.
H2SO4 (pHꢀ1) and extracted with EtOAc (3”20 mL). The combined
organic phases were washed with H2O (20 mL) and brine (20 mL).
The organic phase was dried over Na2SO4, filtered and concentrat-
ed in vacuo to afford the title compound as a yellow solid
1,5-Diiodonaphthalene (11). NaNO2 (5.23 g, 75.9 mmol) in H2O
(20 mL) was added dropwise to a solution of naphthalen-1,5-dia-
mine (4.00 g, 25.3 mmol), conc. H2SO4 (25 mL) and H2O (20 mL) at
À158C over 40 min. The reaction mixture was stirred for an addi-
tional 10 min, then KI (12.6 g, 75.9 mmol) in H2O (20 mL) was
added dropwise over 30 min. The reaction mixture was stirred at
808C for 21 h and quenched with KOH (pHꢀ10) at 08C. The black
solid residue was filtered off and the aqueous phase was extracted
with diethyl ether (3”50 mL). The combined organic phases were
washed with 1m HCl (30 mL), sat. Na2S2O3 (30 mL), 1m NaOH
(30 mL) and brine (30 mL). The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. Purification by flash
column chromatography afforded the title compound as a brown
solid (3.68 g, 9.69 mmol, 38%); Rf =0.75 (heptane/CH2Cl2 5:1);
1H NMR (300 MHz, CDCl3): d=8.12 (d, J=2.1 Hz, 2H), 8.09 (s, 1H),
1
(241 mg, 0.66 mmol, 71%); H NMR (300 MHz, [D6]DMSO): d=11.23
(s, 0.5H), 8.27–8.12 (m, 2H), 7.97–7.88 (m, 1H), 7.64–7.55 (m, 2H),
7.35–7.27 (m, 1H), 5.33 (s, 0.5H), 4.24–4.17 (m, 1H), 2.77–2.40 ppm
(m, 4H); 13C NMR (75 MHz, [D6]DMSO): d=197.3, 140.8, 138.0,
ChemMedChem 2016, 11, 403 – 419
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
134.5, 132.2, 131.4, 128.3, 124.8, 104.1, 101.6, 61.2, 34.3 ppm; mp:
162–1648C; LC–MS: (m/z) calcd for C16H13IO2 [M+H]+: 365.2,
found: 365.2; HPLC: purity (l 254 nm) >95%.
Bioavailability studies
Bioavailability studies were performed in rats as described earlier.[6]
All animal experiments were carried out in accordance with Danish
legislation according to the European Union regulation (directive
2010/63 of September 22, 2010), granted by the animal welfare
committee, appointed by the Danish Ministry of Food, Agriculture
and Fisheries—Danish Veterinary and Food Administration.
2-Amino-5,6,7,8-tetrahydro-7-(1-iodonaphthalen-5-yl)-4-methyl-
5-oxo-4H-chromene-3-carbonitrile
(15).
N-Methylmorpholine
(27 mL, 0.24 mmol) was added to a solution of 1,3-dione 14
(180 mg, 0.49 mmol), acetaldehyde (56 mL, 0.98 mmol) and malono-
nitrile (65 mg, 0.98 mmol) in abs EtOH (20 mL) at 08C and the mix-
ture was stirred at RT for 17 h. After concentration in vacuo, the
crude product was purified by column chromatography to afford
the title compound as a yellow solid (161 mg, 0.35 mmol, 72%);
Rf =0.37 (heptane/EtOAc 1:1); 1H NMR (400 MHz, [D6]DMSO): d=
8.38 (d, J=8.0 Hz, 1H), 8.20 (d, J=8.0 Hz, 1H), 8.00 (t, J=4.0 Hz,
1H), 7.65 (d, J=4.0 Hz, 2H), 7.34 (t, J=8.0 Hz, 1H), 6.90 (brs, 2H),
4.45–4.26 (m, 1H), 3.60–3.48 (m, 1H), 2.97–2.59 (m, 4H), 1.20 (d,
J=8.0 Hz, 2H), 1.12 ppm (d, J=8.0 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=195.6, 163.3, 158.8, 139.5, 137.5, 133.9, 131.5, 130.9,
127.6, 124.3, 119.9, 114.8, 100.8, 57.8, 43.7, 32.6, 24.8, 23.0,
17.4 ppm; mp: 202–2048C; LC–MS: (m/z) calcd for C21H17IN2O2 [M+
H]+: 457.3, found: 457.3; HPLC: purity (l 254 nm) >95%.
Locomotor activity studies
Locomotor activity was assessed using standard assay conditions
and custom-made equipment. Briefly, 30 min after dosing with
UCPH-102, mice were placed in a novel test cage, situated in a U-
frame equipped with 2”8 infrared light sources and photocells.
The light beams crossed the cage 1.8 cm above the bottom sur-
face, which was covered by a thin layer of standard bedding mate-
rial. Recording of a motility count required the interruption of adja-
cent light beams, thus avoiding counts induced by stationary
movements of the mouse. All animal experiments were carried out
in accordance with Danish legislation according to the European
Union regulation (directive 2010/63 of September 22, 2010), grant-
ed by the animal welfare committee, appointed by the Danish Min-
istry of Food, Agriculture and Fisheries—Danish Veterinary and
Food Administration.
Nonspecific brain protein binding
Nonspecific brain protein binding of UCPH-102 and its analogues
was determined in rat brain homogenate using in vitro equilibrium
dialysis. Rat brains for nonspecific protein binding studies were
harvested from animals in house according to the European Com-
munity Guidelines and Guide for the Care and Use of Laboratory
Animals (National Institutes of Health publication no. 85-23, revised
in 1985). The procedures were reviewed and approved by the Fin-
nish National Animal Experiment Board (license ESAVI/6317/
04.10.03/2011). Brain tissues were homogenized in three volumes
of phosphate-buffered saline at pH 7.4 on ice by using an ultrason-
ic homogenizer after which the investigated compounds were
added into the homogenate. Equilibrium dialysis was performed
for 400 mL of tissue homogenate and 600 mL of buffer for 4 h at
378C in a single-use RED Plate with an 8 kDa cutoff dialysis mem-
brane (Thermo Scientific, Rockford, IL, USA). After the dialysis con-
centrations were determined by HPLC, the buffer-to-homogenate
concentration ratio of the investigated compounds was calculated.
The concentration ratio was used to calculate the unbound frac-
tion in brain (%FU). A previously described approach to account
for the effect of tissue dilution in the homogenate was used to cal-
culate the %FU in brain.[25]
In silico studies
All in silico studies were performed using the MOE 2013.08 soft-
ware package (Molecular Operating Environment, Chemical Com-
puting Group) using the built-in mmff94x force field and the GB/
SA continuum solvation model. Calculation of the octanol/water
partition coefficient (cLogP) was performed using the built-in func-
tion by the same name.
Acknowledgements
The in vitro binding profiling of UCPH-102 was generously provid-
ed by the US National Institute of Mental Health Psychoactive
Drug Screening Program (NIMH PDSP), Contract #HHSN-271-
2008-025C. The NIMH PDSP is directed by Bryan L. Roth at the
University of North Carolina at Chapel Hill and project officer
Jamie Driscol at NIMH, Bethesda, MD (USA). Kitti Jensen at DTU
Nutech/Hevesy Laboratory is thanked for technical assistance. Fi-
nally, we thank the Novo Nordisk Foundation and the Lundbeck
Foundation for financial support.
Keywords: blood–brain barrier · EAAT1 · inhibitors · in vitro
HPLC analyses
profiling
·
in vivo administration
·
structure–activity
relationships
The purity of the investigated compounds in equilibrium dialysis
samples were determined by an HPLC system, which consisted of
an Agilent 1100 binary pump (Agilent Technologies Inc., Wilming-
ton, DE, USA), a 1100 micro vacuum degasser, an HP 1050 Auto-
sampler, an HP 1050 variable-wavelength detector (operated at
l 254 nm). Chromatographic separations were carried out on an
Agilent Zorbax SB-C18 analytical column (4.6 mm”250 mm, 5 mm;
Agilent Technologies Inc.) by isocratic elution of MeCN and 0.1%
HCO2H in H2O at a ratio of 50:50–70:30 (v/v), depending on the
compound, at a flow rate of 0.8 mLminÀ1 at room temperature. Re-
tention times for the compounds were between 3.0 and 6.0 min.
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Published online on January 21, 2016
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