PdII-catalyzed conjugate addition of boronic acids to ketoglutaconic esters toward the synthesis of functionalized pyridazin-3(2H)-ones with neuroprotective activity

Article abstract of DOI:10.1002/ejoc.201200734

The development of the regioselective conjugate addition of boronic acids to ketoglutaconic esters under transition metal catalysis is reported. Among the different catalysts tested for this transformation, the dicationic Pd ^II catalysts generated with Pd(OCOCF₃)₂, dppben, and HBF₄ performed best in terms of yields, regioselectivities and avoidance of Heck-type by-products. The resulting 4-aryl-2-oxopentadienoates were transformed into pyridazin-3(2H)-ones, potentially useful for the therapy of neurodegenerative diseases. These compounds simultaneously exhibited β-secretase activity, inhibition of β-amyloid (βA) aggregation, and disaggregation of pre-formed βA fibrils, and also had a good scavenging profile for intracellular reactive oxygen species (ROS). Copyright

Full text of DOI:10.1002/ejoc.201200734

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FULL PAPER
DOI: 10.1002/ejoc.201200734
PdII-Catalyzed Conjugate Addition of Boronic Acids to Ketoglutaconic Esters
toward the Synthesis of Functionalized Pyridazin-3(2H)-ones with
Neuroprotective Activity
Silvia Roscales,[a] Andrea Ortega,[b] Sagrario Martín-Aragón,[b] Paloma Bermejo-Bescós,[b]
and Aurelio G. Csákÿ*[a]
Keywords: Boron / Palladium / Regioselectivity / Homogeneous catalysis / Michael addition / Nitrogen heterocycles /
Medicinal chemistry / Neuroprotection
The development of the regioselective conjugate addition of
boronic acids to ketoglutaconic esters under transition metal
catalysis is reported. Among the different catalysts tested for
this transformation, the dicationic PdII catalysts generated
with Pd(OCOCF3)2, dppben, and HBF4 performed best in
terms of yields, regioselectivities and avoidance of Heck-type
by-products. The resulting 4-aryl-2-oxopentadienoates were
transformed into pyridazin-3(2H)-ones, potentially useful for
the therapy of neurodegenerative diseases. These com-
pounds simultaneously exhibited β-secretase activity, inhibi-
tion of β-amyloid (βA) aggregation, and disaggregation of
pre-formed βA fibrils, and also had a good scavenging profile
for intracellular reactive oxygen species (ROS).
Introduction
Among the different types of unsaturated carbonyl com-
The conjugate addition reaction constitutes one of the pounds used as substrates, ene-dicarbonyl compounds have
most powerful methods available for the construction of C– not received much attention. The RhI-catalyzed conjugate
C bonds.[1] Among the different approaches reported for addition of arylboronic acids to maleimides constitutes one
the addition of carbon nucleophiles in this type of reaction, of the more studied examples,[6] but ene-diesters[7] and ene-
the use of boronic acids under transition metal catalysis has diketones[8] have been less considered. On the other hand,
become a general method for the introduction of aryl and the RhI-catalyzed additions to electronically differentiated
alkenyl groups. Boronic acids are readily available chemi- 1,4-unsaturated dicarbonyl compounds, such as 4-oxobut-2-
cals, have low toxicity, and do not require manipulation in enamides[9] and 4-oxobut-2-enoates,[10] have been much less
anhydrous solvents.[2] This gives an advantage over other developed. These types of substrates are challenging, due
more conventional reagents.
to the possibility of two alternative regiochemistries in the
RhI complexes have become the most popular catalysts formation of the new C–C bond. Regarding PdII catalysis,
for this type of reaction since their first introduction in the conjugate addition of arylboronic acids to ene-di-
1997.[3,4] Despite their widespread use, and due to the high carbonyl compounds has been reported only in the case of
price of Rh, alternative transition metal catalysts have been maleimides.[11]
sought. In particular, dicationic PdII complexes and some
In this paper, we have centered our attention (Scheme 1)
palladacycles have proved useful in these reactions, with
minor competition from the Mizoroki–Heck reaction, typi-
cal of other Pd-based systems.[5] However, the number of
examples reported for the Pd-catalyzed conjugate addition
reaction of boronic acids remains scarce in comparison with
those using RhI catalysis.
on the conjugate addition of boronic acids to ketoglut-
aconic esters 1 (R3 = CO2R2) as a simple route for the con-
struction of functionalized pyridazin-3(2H)-ones 5.[12]
There are no previous literature reports for the conjugate
addition of boronic acids to ketoglutaconic esters 1 (R3 =
CO2R2). In addition, the RhI-catalyzed addition of boronic
acids to other α,β-unsaturated α-keto esters is known to
take place in a 1,2-fashion to give alcohols 3,[13] and under
PdII catalysis, boronic acids give 1,2-addition to the keto
group of α-keto esters.[14]
Pyridazin-3-(2H)-ones 5 were interesting to us due to
their potential neuroprotective activities.[15] The inhibition
of β-secretase activity,[16] the prevention of β-amyloid (βA)
aggregation, and the disaggregation of preformed βA fi-
brils[17] constitute three major target processes in the devel-
[a] Laboratorio de Síntesis Orgánica, Unidad de Cartografía
Cerebral, Instituto Pluridisciplinar, Universidad Complutense
de Madrid,
28040 Madrid, Spain
Fax: +34-91 3943225
E-mail: csaky@quim.ucm.es
[b] Departamento de Farmacología, Facultad de Farmacia,
Universidad Complutense de Madrid,
28040 Madrid, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200734.
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A. G. Csákÿ et al.
FULL PAPER
cell-death and neurodegenerative diseases;[21] 6-methyl-2-[4-
(naphthylpiperazin-1-yl)-butyl]-3-(2H)-pyridazinone has
been described as a mixed dopamine D2-antagonist and 5-
HT1A-partial agonist in functional in vitro and in vivo as-
says;[22] and some 3(2H)-pyridazinone derivatives have
shown in vitro inhibition of the activity of acetylcho-
linesterase (AChE), a proposed drug target for the palliative
treatment of AD.[23,24]
Due to their reported pharmacological activities in the
context of neuroprotection, and in the course of our explor-
atory research targeting the aggregation or deposition of
the βA peptide, we have profiled pyridazinones 5 against
the βA aggregation process, β-secretase activity, and the in-
tracellular generation of ROS.
Scheme 1. Conjugate addition of boronic acids to α,β-unsaturated
α-keto esters.
Results and Discussion
opment of small-molecule lipophilic drugs for the treatment
of Alzheimer’s disease (AD). In addition, several lines of
We began our work with substrates 1a and 2a (Table 1)
evidence show that mitochondrion-derived reactive oxygen by screening several catalytic systems which have proved to
species (ROS) result in enhanced amyloidogenic processing be of general use in conjugate addition reactions of aryl-
of the amyloid precursor protein (APP), and this process boronic acids. We did not observe any reaction when using
could be partly reduced by antioxidants.[18] Since AD is a either neutral or cationic RhI catalysts (Table 1, entries 1–
complex neurodegenerative disorder resulting from multiple 3), and with the exception of the Pd2(dba)3CHCl3 / Ph3P /
molecular abnormalities, strategies to develop new drugs Cs2CO3 (dba
that simultaneously affect multiple biological targets is (Table 1, entry 4), the addition of 2a in the presence of dif-
highly important.[19] ferent types of PdII catalysts (Table 1, entries 5–11) took
=
dibenzylideneacetone) system[25]
Different pyrazolopyridinepyridazinones have shown to place with low regioselectivity to give variable yields of con-
be good phosphodiesterase (PDE) inhibitors;[20] 5-(benzyl- jugate addition product 4a together with furanone 7.[26] Di-
sulfonyl)-4-bromo-2-methyl-3(2H)-pyridazinones have been rect addition of the nucleophile to the keto group of com-
identified as inhibitors of permeability transition pores pound 1a (1,2-addition, resulting in the formation of
(PTP), mitochondrial megachannels involved in neuronal alcohols 3) was not observed in any case. In particular, di-
Table 1. Conjugate addition of 1a with phenylboronic acid (2a).[a]
Entry
Catalyst[b]
Additives [equiv.][b]
Solvent
4a [%][c]
7 [%][c]
1
2
3
4
5
6
[Rh(cod)Cl]2
[Rh(cod)Cl]2
[Rh(cod)2]BF4
Pd2(dba)3CHCl3
Pd(OAc)2
K3PO4 [1.0]
Et3N [1.0]
Et3N [1.0]
PPh3 [0.05] Cs2CO3 [1.0]
2,2Ј-bpy [0.2]
dioxane/H2O [10:1]
dioxane/H2O [10:1]
dioxane/H2O [10:1]
toluene
AcOH/THF/H2O [1.0:0.5:0.1]
CH3NO2
–
–
–
–
16
5
–
–
–
–
28
8
Pd(OAc)2
2,2Ј-bpy [0.2]
7
8
9
10
11
Pd(OCOCF3)2
Pd(acac)2
Pd(acac)2
Pd(acac)2
Pd(acac)2
dppben [0.055] HBF4 [1.0]
dppben [0.05] Cu(BF4)2 [0.2]
dppben [0.05] Cu(BF4)2 [0.2]
dppben [0.05] Cu(BF4)2 [0.2]
dppethy [0.05] Cu(BF4)2 [0.2]
dioxane/H2O [8:2]
dioxane/H2O [8:2]
Dioxane/H2O [10:1]
THF/H2O [8:2]
18
40
38
16
10
35
60
52
29
26
dioxane/H2O [10:1]
[a] Reactions carried out at room temp. for 18 h. [b] Dppben = 1,2-bis(diphenylphosphanyl)benzene; dppethy = 1,2-bis(diphenylphos-
phanyl)ethylene; cod = 1,5-cyclooctadiene; 2,2Ј-bpy = 2,2Ј-bipyridine. [c] Isolated yields after purification by column chromatography.
2
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Pd-Catalyzed Conjugate Addition of Boronic Acids to Ketoglutaconic Esters
cationic PdII catalysts generated either with Pd(OCOCF3)2, Table 2. [PdII]2+-catalyzed conjugate addition of 1b with boronic
acids 2.[a]
1,2-bis(diphenylphosphanyl)benzene (dppben), and HBF4
(Table 1, entry 7),[27] or Pd(acac)2 (acac = acetylacetone),
Cu(BF4)2, and dppben,[28] resulted in good conversions of
the starting materials (Table 1, entries 8–10).[29] The best re-
sults were obtained for the Pd(acac)2, Cu(BF4)2, dppben
system when working in dioxane/H2O (8:2) as solvent
(Table 1, entry 8). This can be compared with the results
obtained using other solvent mixtures (Table 1, entries 9
and 10) or other bisphosphane ligands (Table 1, entry 11).
The formation of compound 4a (Scheme 2) can be
understood by the addition of the Ph-[PdII]+ species, gener-
ated by transmetallation of 2a, to the β position with re-
spect to the ketone group, followed by protonation of the
corresponding oxa-π-allyl-species I. The formation of fur-
anone 7 can be explained by the addition of the Ph-[PdII]+
species with the alternative regiochemistry, i.e., at the β po-
sition with respect to the 4-ester moiety, in a Heck-type
reaction via intermediate II. Compound 6a (R1 = Ph, R2 =
Me) was not detected, but rather it cyclized in the reaction
medium to give 7 directly.[26]
Entry
2
R1
Conditions[b] 4/8 (ratio,[c] yield [%][d]
)
1
2
3
4
5
6
7
8
9
2a Ph
A
A
A
B
A
A
A
B
4b/8a (90:10, 65)
4c/8b (95:05, 63)
4d/8c (95:05, 53)
4e/8d (65:35, 74[e])
4f/8e (90:10, 64)
4g (100:0, 61)
4h (100:0, 48)
4b/8a (75:25, 86)
4c/8b (75:25, 80)
2b 4-F-C6H4
2c 4-CF3-C6H4
2d 4-MeO-C6H4
2e 3,4-(O-CH2-O-)C6H3
2f 4-Br-C6H4
2g 3,4-di-Cl-C6H3
2a Ph
2b 4-F-C6H4
B
[a] Reactions carried out at room temp. for 18 h. [b] Conditions A:
2 (2.5 equiv.), Pd(OCOCF3)2 (5 mol-%), dppben (5 mol-%), HBF4
(1.0 equiv.), dioxane/H2O (8:2); Conditions B: 2 (1.5 equiv.), Pd-
(acac)2 (5 mol-%), dppben (5 mol-%), CuBF4 (0.2 equiv.), dioxane/
1
H2O (8:2). [c] Determined by integration of the H NMR (CDCl3,
300 MHz) spectra of the crude reaction mixtures. [d] Isolated yields
of 4b–4h after purification by column chromatography. [e] Com-
bined isolated yield of the mixture 4e/8d, not separated.
Table 3. Conjugate addition of 1b with boronic acids 2 catalyzed
by PdII-palladacycle 9.[a]
Scheme 2. Reaction course.
We found that switching to α,ω-diisopropyl ester 1b as
starting material (Table 2) allowed us to improve the re-
gioselectivity of the reaction, which was the major flaw ob-
served in the previous reactions with α,ω-dimethyl ester 1a.
The best results for the conjugate addition of 2a to 1b
were observed (Table 2, entries 1–3) when using the dicat-
ionic PdII complex formed with Pd(OCOCF3)2 (5 mol-%),
dppben (5 mol-%), and HBF4 (1.0 equiv.) in dioxane/H2O
(8:2) (Conditions A). These conditions were used for the
reaction of 1b with other arylboronic acids substituted
either with electron-donating or electron-withdrawing sub-
stituents (Table 2, entries 2–7), and the formation of
alcohols 3 was not observed. The use of the catalytic system
Entry
2
R1
4/8/10 (ratio,[b] yield [%][c])
1
2
3
4
5
6
2a
2b
2c
2e
2f
Ph
4b/8a/10a (75:10:15, 70)
4c/8b/10b (75:10:15, 68)
4d/8c/10c (75:10:15, 60)
4-F-C6H4
4-CF3-C6H4
3,4-(O-CH2-O-)C6H3 4f/8e/10d (70:10:20, 59)
4-Br-C6H4
4g/8f/10e (75:15:10, 68)
4h/8g/10f (70:15:15, 64)
2g
3,4-di-Cl-C6H3
[a] Reaction conditions: 2 (2.0 equiv.), palladacycle 9 (5 mol-%),
K3PO4 (1.0 equiv.), toluene, room temp., 18 h. [b] Determined by
integration of the 1H NMR (CDCl3, 300 MHz) spectra of the crude
reaction mixtures. [c] Isolated yields of 4b–4h after purification by
formed with Pd(acac)2 (5 mol-%), dppben (5 mol-%), and column chromatography.
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Table 4. Activities of pyridazinones in the pharmacological assays.
5, 11
βA aggregation inhibition[a]
βA fibril disaggregation[b]
β-secretase inhibition[c]
ROS inhibition[d]
5a
8.302Ϯ0.422
6.888Ϯ0.197
7.132Ϯ0.390
6.307Ϯ0.388
6.909Ϯ0.239
6.215Ϯ0.155
8.345Ϯ0.593
8.200Ϯ0.255
7.153Ϯ0.505
7.759Ϯ0.569
7.482Ϯ0.141
7.885Ϯ0.777
38.89Ϯ1.704
27.62Ϯ0.831
22.81Ϯ2.638
20.49Ϯ0.816
–
1.508Ϯ0.326
0.444Ϯ0.135
6.116Ϯ0.161
2.415Ϯ0.391
3.035Ϯ0.190
1.879Ϯ0.508
5b
5c
5e
11a
11c
25.83Ϯ1.952
[a] Inhibition of βA aggregation, IC50 [μm]. [b] Disaggregation of βA fibrils, IC50 [μm]. [c] Inhibition of β-secretase activity at 10 μm
concentration [%]. [d] Inhibition of ROS generation, IC50 [μm]. Treatment of APPswe cells with pyridazinones 5a–11c for 24 h did not
show any significant toxicity up to 20 μm in comparison with untreated cells.
CuBF4 (0.2 equiv.) (Conditions B) gave higher ratios of 10 μm), all the compounds tested were active in the μm
compounds 8 (Table 2, entries 8 and 9).
range. In addition, they also exhibited good inhibition of
In the search for other PdII catalysts active in this type intracellular ROS (IC50 = 3.0–0.4 μm). Compound 5b (IC50
of transformation, we also tested the reaction between 1b = 0.444Ϯ0.135 μm) was particularly good in this respect.
and boronic acids 2 using palladacycle 9 (Table 3).[30] In
this case, we observed good overall conversions and the for-
Conclusions
mation of compounds 4 as major products, but we detected
the formation of minor amounts of Heck-type products 8,
together with conjugate addition products 10.
In summary, we have developed the synthesis of the new
pyridazin-3(2H)-ones 5 using the conjugate addition reac-
tion of arylboronic acids to ketoglutaconic esters as a key
step. Among the different catalysts tested for this transfor-
mation, the dicationic PdII catalysts generated with Pd(OC-
OCF3)2, dppben, and HBF4 performed best in terms of
yields, regioselectivities, and avoidance of Heck-type by-
products. These pyridazin-3(2H)-ones are presented as
promising candidates in the development of small-molecule
lipophilic drugs for the treatment of neurodegenerative dis-
eases simultaneously targeting the βA peptide aggregation
process and β-secretase activity, while also having a good
intracellular ROS scavenging profile.
The conversion of compounds 4 into pyridazin-3(2H)-
ones 5 was carried out by reaction with hydrazines, followed
by aromatization of the corresponding dihydro intermedi-
ates (i.e., 11)[31] (Scheme 3).
Experimental Section
General Methods: All starting materials were commercially avail-
able research-grade chemicals, and were used without further puri-
fication. The catalysts were commercially available. All solvents
were dried by standard methods and distilled under argon. Silica
gel 60 F254 was used for TLC, and the spots were detected with
UV light or vanillin solution. Flash column chromatography was
1
carried out on silica gel 60. H NMR spectra were recorded at 300
MHz and 13C NMR spectra were recorded at 75 MHz, both in
CDCl3 solution, unless otherwise stated. Compound 1a was pre-
pared following a previously reported procedure.[32]
(E)-Diisopropyl 4-Oxopent-2-enedioate (1b): 2-Ketoglutaric acid
(2.1 g, 14.6 mmol) and 2-propanol (3.4 mL, 43.9 mmol) were
Scheme 3. Synthesis of pyridazin-3(2H)-ones 5.
Table 4 summarizes our results on the inhibition of βA heated at reflux in toluene (44 mL) with p-toluenesulfonic acid
(140 mg, 0.7 mmol) as a catalyst in a Dean Stark apparatus for
18 h. After cooling to room temp., H2O (10 mL) was added. The
organic layer was separated, and the aqueous phase was extracted
with Et2O (3ϫ10 mL). The combined organic phases were dried
with MgSO4, filtered, and concentrated in vacuo. The residue was
dissolved in CH2Cl2 (73 mL) and a solution of bromine (1.1 mL,
22.0 mmol) in CH2Cl2 (0.5 mL) was added. The solution was
stirred at reflux for 3 h. After cooling to room temp., the solvent
and residual HBr were evaporated under vacuum to yield di-
isopropyl bromoglutarate as an orange oil. The diisopropyl bromo-
aggregation, the disaggregation ability towards pre-formed
βA fibrils, the inhibition of β-secretase activity, and the in-
hibition of intracellular ROS for pyridazinones 5a–c,e,f and
dihydropiridazinones 11a–c. We observed that the two N-
benzylated derivatives 5f and 11b showed cytotoxicity on
the neuroblastoma APPswe cells up to 20 μm, and so they
were discarded for the in vitro assays undertaken.
With respect to inhibition of βA aggregation (IC50 = 8.3–
6.2 μm), disaggregation activity of preformed βA fibrils
(IC50 = 8.3–7.1 μm), and β-secretase inhibition (39–20% at glutarate was dissolved in diethyl ether (88 mL), and triethylamine
4
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Pd-Catalyzed Conjugate Addition of Boronic Acids to Ketoglutaconic Esters
(2.3 mL, 16.1 mmol) was added. After stirring at room temp. for
30 min, the mixture was filtered twice through a pad of silica gel.
The ether solution was evaporated to give an oil, which was puri-
fied by column chromatography (hexane/EtOAc = 9:1). Yellow oil
(OMe), 100.6 (C-2), 116.6 (C-4), 127.8 (2 C-2Ј), 128.4 (C-1Ј), 129.4
(2 C-3Ј), 132.0 (C-4Ј), 160.8 (C-3), 168.6 (C-1), 169.4 (C-5) ppm.
MS (70 eV, EI): m/z (%) = 235 (10) [M + 1], 234 (48) [M], 202 (72),
176 (84), 175 (100), 148 (73), 147 (99), 105 (80), 102 (99), 90 (41).
C12H10O5 (234.21): calcd. C 61.54, H 4.30; found C 61.62, H 4.25.
1
3
(2.3 g, 70%). H NMR (300 MHz, CDCl3, 25 °C): δ = 1.33 (d, J
3
= 6.3 Hz, 6 H, CH3 iPr), 1.39 (d, J = 6.3 Hz, 6 H, CH3 iPr), 5.14
General Procedure for the Dicationic PdII/dppben-Catalyzed Conju-
gate Addition of Boronic Acids 2 to Ketoglutaconic Ester 1b (Table2)
3
3
(q, J = 6.3 Hz, 1 H, CH iPr), 5.22 (q, J = 6.3 Hz, 1 H, CH iPr),
3
3
6.92 (d, Jtrans = 16.1 Hz, 1 H, CH), 7.57 (d, Jtrans = 16.1 Hz, 1
H, CH) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ = 21.5 (CH3
iPr), 21.6 (CH3 iPr), 69.3 (CH iPr), 71.2 (CH iPr), 133.9 (C-3),
136.1 (C-4), 160.3 (CO), 164.1 (C-5), 183.1 (CO) ppm. C11H16O5
(228.24): calcd. C 57.88, H 7.07; found C 57.99, H 7.14.
Conditions A: 1,2-diphenylphosphanyl benzene (dppben, 21 mg,
0.04 mmol) and Pd(OCOCF3)2 (14 mg, 0.04 mmol) were dissolved
in THF (5 mL). The resulting solution was stirred at room temp.
for 20 min. After this time, 1b (190 mg, 0.83 mmol), ArB(OH)2
2
(2.5 mmol), HBF4 (50% aqueous solution, 0.06 mL, 0.83 mmol),
and H2O (0.5 mL) were added. After stirring at room temp. for
18 h, a saturated solution of NaHCO3 was added. The organic
products were extracted with Et2O (3ϫ10 mL). The organic phase
was dried with MgSO4, filtered, and concentrated in vacuo. The
crude products were purified by chromatography on silica gel (hex-
ane/EtOAc = 8:2).
General Reaction Conditions for the Conjugate Addition of 1a with
Phenylboronic Acid 2a (Table 1)
Entries 1, 2: The base (K3PO4 or Et3N, 0.175 mmol) was added
to a mixture of [RhCl(cod)]2 (7 mg, 15ϫ10–3 mmol), 2a (42 mg,
0.35 mmol), and 1a (50 mg, 0.29 mmol) in dioxane/H2O 10:1
(1 mL). The mixture was stirred at room temp. for 40 h and then
filtered through a silica gel pad covered with MgSO4. Filtration
and evaporation under vacuum gave the crude reaction products.
Conditions B: A solution of 1b (230 mg, 1.00 mmol) in dioxane/
H2O (8:2, 3 mL) was added to a mixture of Pd(acac)2 (15 mg,
0.05 mmol), 1,2-diphenylphosphanyl benzene (dppben, 22 mg,
Entry 3: Et3N (25 μL, 0.175 mmol) was added to a mixture of
Rh(cod)2BF4·H2O (4 mg, 8.7ϫ10–3 mmol), 2a (42 mg, 0.35 mmol),
and 1a (31 mg, 0.175 mmol) in dioxane/H2O 10:1 (1 mL). The mix-
ture was stirred at room temp. for 30 h and then filtered through a
silica gel pad covered with MgSO4. Filtration and evaporation un-
der vacuum gave the crude reaction products.
0.05 mmol), Cu(BF4)2 (48 mg, 0.202 mmol), and ArB(OH)2
2
(1.51 mmol). The mixture was stirred at room temp. for 18 h, and
then filtered through a silica gel pad covered with MgSO4. Fil-
tration and evaporation under vacuum gave the crude reaction
products, which were purified by column chromatography (hexane/
EtOAc = 8:2).
Entry 4: A mixture of Pd(dba)3CHCl3 (5 mg, 4.3ϫ10–3 mmol),
PPh3 (2 mg, 0.009 mmol), Cs2CO3 (57 mg, 0.174 mmol), 2a
(42.5 mg, 0.40 mmol), and 1a (31 mg, 0.175 mmol) in toluene
(0.5 mL) was stirred at room temp. for 18 h. Evaporation under
vacuum gave the crude reaction products.
Conjugate Addition Boronic Acids 2 to 1b Catalyzed by PdII-Pallad-
acycle 9 (Table 3): Compound 1b (40 mg, 0.175 mmol), K3PO4
(37 mg, 0.175 mmol), and PdII-palladacycle
9
(14 mg,
9ϫ10–3 mmol) were added to a stirred solution of ArB(OH)2
2
(0.35 mmol) in toluene (0.7 mL). The resulting mixture was stirred
at room temp. for 18 h, then quenched with water, extracted with
CH2Cl2 (3ϫ5 mL), and dried with MgSO4. The filtrate was con-
centrated under reduced pressure. The crude products were purified
by chromatography on silica gel (hexane/EtOAc = 8:2).
Entry 5: A solution of 1a (30 mg, 0.175 mmol) in AcOH/THF/H2O
(1:0.5:0.1, 1.5 mL) was added to a mixture of Pd(OAc)2 (2 mg,
8.7ϫ10–3 mmol), 2,2Ј-bipyridine (5 mg, 0.035 mmol), and 2a
(64 mg, 0.52 mmol). After stirring for 3 d, a saturated solution of
NaHCO3 was added. The organic products were extracted with
Et2O (3ϫ5 mL). The combined organic extracts were washed with
brine, dried with MgSO4, filtrered, and concentrated in vacuo. The
crude product was purified by chromatography on silica gel (hex-
ane/EtOAc = 8:2).
Diisopropyl 2-Oxo-4-phenylpentanedioate (4b): 165 mg, 65% yield.
1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.08 (d, 3J = 6.3 Hz, 3 H,
3
3
CH3iPr), 1.24 (d, J = 6.3 Hz, 3 H, CH3iPr), 1.33 (d, J = 6.3 Hz,
6 H, CH3iPr), 3.11 (dd, 2J = 19.1, 3J = 4.2 Hz, 1 H, 3-H), 3.75 (dd,
2J = 19.1, 3J = 10.5 Hz, 1 H, 3-H), 4.06 (dd, 3J = 10.5, 3J = 4.2 Hz,
Entry 6: A solution of 1a (30 mg, 0.175 mmol) in CH3NO2 (1 mL)
was added to a mixture of Pd(OAc)2 (2 mg, 8.7ϫ10–3 mmol), 2,2Ј-
bipyridine (5 mg, 0.035 mmol), and 2a (42 mg, 0.35 mmol). The re-
sulting mixture was stirred at room temp. for 18 h. Evaporation
under vacuum gave the crude reaction products, which were puri-
fied by column chromatography (hexane/EtOAc = 8:2).
3
3
1 H, 4-H), 4.98 (q, J = 6.3 Hz, 1 H, CHiPr), 5.13 (q, J = 6.3 Hz,
1 H, CHiPr), 7.23–7.37 (m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 21.5 (CH3iPr), 21.7 (2 CH3iPr), 21.8
(CH3iPr), 43.3 (C-3), 46.4 (C-4), 68.9 (CHiPr), 71.0 (CHiPr), 127.7
(C-4Ј), 127.9 (2 C-2Ј), 129.0 (2 C-3Ј), 137.9 (C-1Ј), 160.2 (C-1),
172.4 (C-5), 192.6 (C-2) ppm. C17H22O5 (306.36): calcd. C 66.65,
H 7.24; found C 66.78, H 7.20.
1
Dimethyl 2-Oxo-4-phenylpentanedioate (4a): 37 mg, 18% yield. H
NMR (300 MHz, CDCl3, 25 °C): δ = 3.14 (dd, 2J = 19.2, 3J =
4.3 Hz, 1 H, 3-H), 3.67 (s, 3 H, OMe), 3.79 (dd, 2J = 19.2, 3J =
Diisopropyl 2-(4-Fluorophenyl)-4-oxopentanedioate (4c): 175 mg,
1
3
65% yield. H NMR (300 MHz, CDCl3, 25 °C): δ = 1.13 (d, J =
3
3
10.2 Hz, 1 H, 3-H), 3.88 (s, 3 H, OMe), 4.15 (dd, J = 10.2, J =
4.3 Hz, 1 H, 4-H), 7.19–7.38 (m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): δ = 43.1 (C-3), 46.0 (C-4), 52.6
(OMe), 53.2 (OMe), 127.8 (2 C-2Ј), 127.9 (C-4Ј), 129.1 (2 C-3Ј),
137.5 (C-1Ј), 160.9 (C-1), 173.3 (C-5), 191.8 (C-2) ppm. C13H14O5
(250.25): calcd. C 62.39, H 5.64; found C 62.28, H 5.71.
3
6.3 Hz, 3 H, CH3iPr), 1.30 (d, J = 6.3 Hz, 3 H, CH3iPr), 1.38 (d,
3J = 6.3 Hz, 6 H, CH3iPr), 3.16 (dd, J = 19.1, J = 4.6 Hz, 1 H,
2
3
2
3
3
3-H), 3.76 (dd, J = 19.1, J = 10.2 Hz, 1 H, 3-H), 4.10 (dd, J =
10.2, 3J = 4.6 Hz, 1 H, 4-H), 5.03 (q, 3J = 6.3 Hz, 1 H, CHiPr),
3
5.18 (q, J = 6.3 Hz, 1 H, CHiPr), 7.10–7.10 (m, 2 H, 3Ј-H), 7.26–
7.34 (m, 2 H, 2Ј-H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ =
Methyl 2-Hydroxy-5-oxo-3-phenyl-2,5-dihydrofuran-2-carboxylate 21.4 (CH3iPr), 21.6 (2 CH3iPr), 21.7 (CH3iPr), 43.1 (C-3), 45.6 (C-
(7): 68 mg, 35% yield. IR (CH Cl ): ν = 1756, 3317 cm–1. 1H NMR 4), 68.9 (CHiPr), 71.1 (CHiPr), 115.6 (C-3Ј), 115.9 (C-3Ј), 129.4
˜
2
2
(300 MHz CDCl3, 25 °C): δ = 3.82 (s, 3 H, OMe), 5.35 (br. s, 1 H,
(C-2Ј), 129.5 (C-2Ј), 133.5 (C-1Ј), 133.6 (C-1Ј), 160.0 (C-1), 163.9
OH), 6.53 (s, 1 H, 4-H), 7.53–7.39 (m, 3 H, 2Ј-H, 4Ј-H), 7.63–7.55 (C-4Ј), 165.9 (C-4Ј), 172.1 (C-5), 192.3 (C-2) ppm. C17H21FO5
(m, 2 H, 3Ј-H) ppm. 13C NMR (75 MHz CDCl3, 25 °C): δ = 54.8 (324.35): calcd. C 62.95, H 6.53; found C 62.79, H 6.61.
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5

Job/Unit: O20734
/KAP1
Date: 06-08-12 11:56:44
Pages: 9
A. G. Csákÿ et al.
FULL PAPER
3
2
3
Diisopropyl 2-Oxo-4-[4-(trifluoromethyl)phenyl]pentanedioate (4d):
(d, J = 6.2 Hz, 6 H, CH3iPr), 3.12 (dd, J = 18.8, J = 4.8 Hz, 1
1
2
3
3
165 mg, 53% yield. H NMR (300 MHz, CDCl3, 25 °C): δ = 1.10 H, 3-H), 3.69 (dd, J = 18.8, J = 10.0 Hz, 1 H, 3-H), 4.02 (dd, J
3
3
3
3
(d, J = 6.3 Hz, 3 H, CH3iPr), 1.25 (d, J = 6.3 Hz, 3 H, CH3iPr),
= 10.0, J = 4.8 Hz, 1 H, 4-H), 4.99 (q, J = 6.2 Hz, 1 H, CHiPr),
3
3
4
1.34 (d, 3J = 6.3 Hz, 6 H, CH3iPr), 3.14 (dd, 2J = 19.1, 3J = 4.6 Hz, 5.14 (q, J = 6.2 Hz, 1 H, CHiPr), 7.14 (dd, J = 8.3, J = 2.1 Hz,
2
3
4
3
1 H, 3-H), 3.75 (dd, J = 19.1, J = 9.9 Hz, 1 H, 3-H), 4.14 (dd, 3J
1 H, 6Ј-H), 7.39 (d, J = 2.1 Hz, 1 H, 1Ј-H), 7.40 (d, J = 8.3 Hz,
= 9.9, J = 4.6 Hz, 1 H, 4-H), 5.00 (q, J = 6.3 Hz, 1 H, CHiPr), 1 H, 5Ј-H) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ = 21.6
3
3
5.14 (q, 3J = 6.3 Hz, 1 H, CHiPr), 7.41 (d, 3J = 8.1 Hz, 2 H, 3Ј- (CH3iPr), 21.7 (2 CH3iPr), 21.8 (CH3iPr), 42.8 (C-3), 45.5 (C-4),
H), 7.59 (d, 3J = 8.1 Hz, 2 H, 2Ј-H) ppm. 13C NMR (75 MHz,
69.4 (CHiPr), 71.3 (CHiPr), 127.3 (C-6Ј), 130.0 (C-1Ј), 130.9 (C-
CDCl3, 25 °C): δ = 21.5 (CH3iPr), 21.7 (2 CH3iPr), 21.8 (CH3iPr), 5Ј), 132.0 (C-4Ј), 133.1 (C-3Ј), 138.0 (C-1Ј), 160.0 (C-1), 171.3 (C-
42.8 (C-3), 46.2 (C-4), 69.4 (CHiPr), 71.3 (CHiPr), 125.9 (C-3Ј), 5), 192.0 (C-2) ppm. C17H20Cl2O5 (375.25): calcd. C 54.41, H 5.37;
126.0 (C-3Ј), 127.2 (C-2Ј), 127.3 (C-2Ј), 141.8 (C-1Ј), 160.0 (C-1), found C 54.62, H 5.42.
171.6 (C-5), 192.2 (C-2) ppm. C18H21F3O5 (374.35): calcd. C 57.75,
H 5.65; found C 57.59, H 5.58.
General Procedure for the Synthesis of 4,5-Dihydropyridazin-3-(2H)-
ones 11: The corresponding hydrazine (2.0 mmol) and acetic acid
(0.07 mL, 1.2 mmol) were added to a solution of 4 (0.41 mmol) in
CH2Cl2 (5 mL). The solution was heated at refulx for 18 h. After
cooling to room temp., the volatiles were removed in vacuo, and the
resultant residue was purified by column chromatography (hexane/
EtOAc = 1:1).
Diisopropyl 2-(4-Methoxyphenyl)-4-oxopentanedioate (4e) and (E)-
Diisopropyl 2-(4-methoxyphenyl)-4-oxopent-2-enedioate (8d): Mix-
ture 4e:8d = 65:35. 249 mg, 74% combined yield. 1H NMR
3
(300 MHz, CDCl3, 25 °C): δ = 1.08 (d, J = 6.2 Hz, 3 H, CH3iPr),
1.23 (d, 3J = 6.2 Hz, 3 H, CH3iPr), 1.25 (d, 3J = 6.4 Hz, 6 H,
3
2
CH3iPr), 1.32 (d, J = 6.4 Hz, 12 H, CH3iPr), 3.09 (dd, J = 19.1,
Isopropyl 6-Oxo-5-phenyl-1,4,5,6-tetrahydropyridazine-3-carboxyl-
3J = 4.4 Hz, 1 H, 3-H 4e), 3.70 (dd, J = 19.1, J = 10.3 Hz, 1 H,
3-H 4e), 3.78 (s, 3 H, OMe 4e), 3.81 (s, 3 H, OMe 8d), 4.00 (dd, J
= 10.3, J = 4.4 Hz, 1 H, 4-H 4e), 4.90–5.19 (m, 4 H, CHiPr), 6.31
2
3
1
ate (11a): 94 mg, 88% yield. H NMR (300 MHz, CDCl3, 25 °C):
3
3
2
3
δ = 1.36 (d, J = 6.3 Hz, 6 H, CH3iPr), 3.11 (dd, J = 18.0, J =
3
10.5 Hz, 1 H, 3-H), 3.28 (dd, 2J = 18.0, 3J = 7.3 Hz, 1 H, 3-H),
3
3
2
2
3
(s, 1 H, 3-H 8d), 6.84 (d, J = 8.9 Hz, 2 H, 3Ј-H 4e), 6.88 (d, J =
8.9 Hz, 2 H, 3Ј-H 8d), 7.19 (d, 3J = 8.9 Hz, 2 H, 2Ј-H 4e), 7.36 (d, 3J
= 8.9 Hz, 2 H, 3Ј-H 8d) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ
= 21.5 (CH3iPr), 21.6 (CH3iPr), 21.7 (2 CH3iPr), 21.8 (CH3iPr),
21.9 (CH3iPr), 43.3 (C-3, 4e), 45.5 (C-4, 4e), 55.3 (OMe, 4e), 55.5
(OMe, 8d), 68.7 (CHiPr), 69.3 (CHiPr), 70.9 (CHiPr), 71.0
(CHiPr), 114.3 (2 C-3Ј, 4e), 114.6 (2 C-3Ј, 8d), 117.8 (C-3, 8d),
125.5 (C-4, 8d), 128.7 (2 C-2Ј, 8d), 128.9 (2 C-2Ј, 4e), 129.9 (C-4Ј,
4e), 154.3 (C-1Ј, 8d), 159.0 (C-1Ј, 4e), 159.1 (C-4Ј, 4e), 160.1 (C-1,
4e), 161.7 (C-1, 8d), 166.3 (C-5, 8d), 172.5 (C-5, 4e), 189.2 (C-2,
8d), 192.7 (C-2, 4e) ppm.
3.77 (dd, J = 10.5, J = 7.3 Hz, 1 H, 4-H), 5.21 (q, J = 6.3 Hz, 1
H, CHiPr), 7.19–7.27 (m, 2 H, 3Ј-H), 7.31–7.41 (m, 3 H, 2Ј-H, 4Ј-
H), 8.79 (s, 1 H, NH) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ
= 21.9 (2 CH3iPr), 29.0 (C-3), 42.2 (C-4), 70.4 (CH iPr), 127.9 (2
C-2Ј), 128.1 (C-4Ј), 129.2 (2 C-3Ј), 136.3 (C-1Ј), 143.9 (C-2), 162.6
(CO), 168.2 (CO) ppm. C14H16N2O3 (260.29): calcd. C 64.60, H
6.20; found C 64.73, H 6.29.
Isopropyl 1-Benzyl-6-oxo-5-phenyl-1,4,5,6-tetrahydropyridazine-3-
carboxylate (11b): 144 mg, 83% yield. 1H NMR (300 MHz, CDCl3,
3
2
25 °C): δ = 1.33 (d, J = 6.2 Hz, 6 H, CH3iPr), 3.15 (dd, J = 7.7,
3J = 1.8 Hz, 2 H, 3-H), 3.76 (t, J = 8.4 Hz, 1 H, 4-H), 5.06 (s, 2
3
3
Diisopropyl 2-(Benzo[d][1,3]dioxol-5-yl)-4-oxopentanedioate (4f):
H, CH2Ph), 5.15 (q, J = 6.2 Hz, 1 H, CHiPr), 7.13–7.19 (m, 2 H,
1
186 mg, 64% yield. H NMR (300 MHz, CDCl3, 25 °C): δ = 1.11 3Ј-H), 7.24–7.41 (m, 10 H, Ar-H) ppm. 13C NMR (75 MHz,
3
3
(d, J = 6.2 Hz, 3 H, CH3iPr), 1.25 (d, J = 6.2 Hz, 3 H, CH3iPr),
CDCl3, 25 °C): δ = 21.9 (2 CH3iPr), 29.2 (C-3), 42.5 (C-4), 53.8
1.34 (d, 3J = 6.3 Hz, 6 H, CH3iPr), 3.09 (dd, 2J = 19.2, 3J = 4.4 Hz, (CH2Ph), 70.1 (CHiPr), 127.7 (2 C-2ЈЈ), 127.8 (C-4Ј), 127.9 (C-4ЈЈ),
1 H, 3-H), 3.68 (dd, J = 19.2, J = 10.0 Hz, 1 H, 3-H), 3.97 (dd,
3J = 10.0, 3J = 4.4 Hz, 1 H, 4-H), 4.98 (q, 3J = 6.2 Hz, 1 H, CHiPr),
2
3
128.6 (2 C-3ЈЈ), 128.7 (2 CЈ-2Ј), 129.0 (2 C-3Ј), 136.7 (C-1ЈЈ), 137.1
(C-1Ј), 143.1 (C-2), 162.6 (CO), 166.8 (CO) ppm. C21H22N2O3
(350.42): calcd. C 71.98, H 6.33; found C 72.14, H 6.24.
3
5.14 (q, J = 6.2 Hz, 1 H, CHiPr), 5.95 (s, 2 H, O-CH2-O), 5.75–
6.80 (m, 3 H, 2Ј-H, 5-H, 6Ј-H) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ = 21.6 (CH3iPr), 21.7 (2 CH3iPr), 21.8 (CH3iPr), 43.5 (C-
3), 46.0 (C-4), 68.9 (CHiPr), 71.0 (CHiPr), 101.3 (O-CH2-O), 108.2
(C-2Ј), 108.7 (C-5Ј), 121.2 (C-6Ј), 131.6 (C-1Ј), 147.2 (C-4Ј), 148.2
(C-3Ј), 160.1 (C-1), 172.2 (C-5), 192.4 (C-2) ppm. C18H22O7
(350.37): calcd. C 61.71, H 6.33; found C 61.92, H 6.41.
Isopropyl 1-Methyl-6-oxo-5-phenyl-1,4,5,6-tetrahydropyridazine-3-
1
carboxylate (11c): 84 mg, 75% yield. H NMR (300 MHz, CDCl3,
2
2
25 °C): δ = 1.34 (d, J = 6.3 Hz, 6 H, CH3iPr), 3.16 (dd, J = 7.7,
3J = 3.4 Hz, 2 H, 3-H), 3.53 (s, 3 H, NMe), 3.76 (t, J = 8.6 Hz, 1
H, 4-H), 5.18 (q, J = 6.3 Hz, 1 H, CHiPr), 7.17–7.23 (m, 2 H, 3Ј-
3
3
H), 7.28–7.38 (m, 3 H, 2Ј-H, 4Ј-H) ppm. 13C NMR (75 MHz,
CDCl3, 25 °C): δ = 21.9 (2 CH3iPr), 29.2 (C-3), 38.0 (NMe), 42.4
(C-4), 70.2 (CHiPr), 127.8 (2 C-2Ј), 128.0 (C-4Ј), 129.1 (2 C-3Ј),
136.8 (C-1Ј), 143.0 (C-2), 162.6 (CO), 167.2 (CO) ppm.
C15H18N2O3 (274.32): calcd. C 65.68, H 6.61; found C 65.91, H
6.70.
Diisopropyl 2-(4-Bromophenyl)-4-oxopentanedioate (4g): 195 mg,
1
3
61% yield. H NMR (300 MHz, CDCl3, 25 °C): δ = 1.09 (d, J =
3
6.3 Hz, 3 H, CH3iPr), 1.25 (d, J = 6.3 Hz, 3 H, CH3iPr), 1.33 (d,
3J = 6.3 Hz, 6 H, CH3iPr), 3.10 (dd, J = 19.1, J = 4.6 Hz, 1 H,
3-H), 3.70 (dd, 2J = 19.1, 3J = 9.9 Hz, 1 H, 3-H), 4.03 (dd, 3J =
9.9, 3J = 4.6 Hz, 1 H, 4-H), 4.98 (q, 3J = 6.3 Hz, 1 H, CH3iPr),
2
3
General Procedure for the Transformation of Compounds 11 into
Pyridazin-3-(2H)-ones 5: Br2 (10 μL, 0.21 mmol) was added to 4,5-
dihydropyridazin-3-(2H)-ones 11 (0.14 mmol) dissolved in AcOH
(0.8 mL). After stirring at room temp. for 18 h, a saturated solution
of NaHCO3 was added. The organic products were extracted with
Et2O (3ϫ5 mL). The combined organic layers were dried with
MgSO4, filtered, and concentrated in vacuo. The crude products
were purified by chromatography on silica gel (hexane/EtOAc =
6:4).
3
3
5.00 (q, J = 6.3 Hz, 1 H, CH3iPr), 7.16 (d, J = 8.4 Hz, 2 H, 2Ј-
H), 7.46 (d, 3J = 8.4 Hz, 2 H, 3Ј-H) ppm. 13C NMR (75 MHz,
CDCl3, 25 °C): δ = 21.5 (CH3iPr), 21.7 (2 CH3iPr), 21.8 (CH3iPr),
42.9 (C-3), 45.8 (C-4), 69.2 (CHiPr), 71.2 (CHiPr), 121.8 (C-4Ј),
129.7 (2 C-2Ј), 132.2 (2 C-3Ј), 136.8 (C-1Ј), 160.1 (C-1), 171.9 (C-
5), 192.3 (C-2) ppm. C17H21BrO5 (385.25): calcd. C 53.00, H 5.49;
found C 53.17, H 5.39.
Diisopropyl
2-(3,4-Dichlorophenyl)-4-oxopentanedioate
(4h):
1
3
150 mg, 48% yield. H NMR (300 MHz, CDCl3): δ = 1.12 (d, J
= 6.2 Hz, 3 H, CH3iPr), 1.25 (d, J = 6.2 Hz, 3 H, CH3iPr), 1.34 ones 5 from 4b: The corresponding hydrazine (2.0 mmol) and acetic
General Procedure for the One-Pot Synthesis of Pyridazin-3-(2H)-
3
6
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20734
/KAP1
Date: 06-08-12 11:56:44
Pages: 9
Pd-Catalyzed Conjugate Addition of Boronic Acids to Ketoglutaconic Esters
acid (0.07 mL, 1.2 mmol) were added to solution of
a
4
1 H, CH iPr), 5.49 (s, 2 H, CH2Ph), 7.28–7.29 (m, 3 H, Ar-H),
7.40–7.48 (m, 3 H, Ar-H), 7.50–7.58 (m, 2 H, Ar-H), 7.75–7.86 (m,
2 H, Ar-H) 7.93 (s, 1 H, 3-H) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ = 22.0 (2 CH3 iPr), 57.1 (O-CH2-O), 70.3 (CH iPr), 127.7
(0.41 mmol) in CH2Cl2 (5 mL). The solution was heated at reflux
for 18 h. After cooling to room temp., the volatiles were removed
in vacuo. The residue was dissolved in AcOH (2 mL), and Br2
(0.03 mL, 0.60 mmol) was added. After stirring for 18 h at room (C-3), 128.2 (C-4ЈЈ), 128.6 (2 C-3ЈЈ), 128.7 (2 C-2Ј), 128.8 (2 C-3Ј),
temp., a saturated solution of NaHCO3 was added. The organic
products were extracted with Et2O (3ϫ5 mL). The combined or-
ganic extracts were dried with MgSO4, filtered, and concentrated
in vacuo. The crude product was purified by chromatography on
silica gel (hexane/EtOAc = 6:4).
129.2 (2 C-2ЈЈ), 130.0 (C-4Ј), 133.6 (C-1Ј), 135.7 (C-4), 136.9 (C-
1ЈЈ), 139.2 (C-2), 159.7 (CO), 162.1 (CO) ppm. C21H20N2O3
(348.40): calcd. C 72.40, H 5.79; found C 72.58, H 5.71.
Pharmacological Assays: An overexpressing mutated amyloid pre-
cursor protein (APPswe) cell-line (APP695-transfected neuroblas-
toma SH-SY5Y) was used to select a non-toxic concentration
range, and to examine the potential cytoprotective effect of the
studied compounds by exposing cells to the hydrogen peroxide in-
sult.
Isopropyl 5-Oxo-4-phenyl-5,6-dihydropyridine-2-carboxylate (5a):
32 mg, 89% yield. 1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.42
3
3
(d, J = 6.2 Hz, 6 H, CH3iPr), 5.33 (q, J = 6.2 Hz, 1 H, CHiPr),
7.44–7.54 (m, 3 H), 7.85–7.93 (m, 2 H, 3Ј-H), 8.06 (s, 1 H, 3-H, 2Ј-
H, 4Ј-H), 12.2 (br. s, 1 H, NH) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ = 21.9 (2 CH3iPr), 70.6 (CHiPr), 128.4 (C-3), 128.7 (2 C-
2Ј), 128.8 (2 C-3Ј), 130.4 (C-4Ј), 132.8 (C-1Ј), 138.6 (C-4), 140.0
(C-2), 161.3 (CO), 162.2 (CO) ppm. C14H14N2O3 (258.28): calcd.
C 65.11, H 5.46; found C 65.29, H 5.56.
The mitochondrial-dependent reduction of MTT to formazan was
used to exclude a cytotoxic effect of the tested compound in
APPswe cells. These compounds must be tested at concentrations
non-cytotoxic for brain cells, since they would be eventually admin-
istered to organisms if active. For that purpose, the compounds
were previously tested for cell viability in the APPswe cell-line,
which is widely used for studies of neuroprotection and neurotoxic-
ity.
Isopropyl 4-(4-Fluorophenyl)-5-oxo-5,6-dihydropyridine-2-carboxyl-
1
ate (5b): 61 mg, 54% yield. H NMR (300 MHz, CDCl3, 25 °C): δ
= 1.43 (d, 3J = 6.3 Hz, 6 H, CH3iPr), 5.34 (q, 3J = 6.3 Hz, 1 H,
CHiPr), 7.18 (t, J = 8.7 Hz, 2 H, 3Ј-H), 7.88–7.97 (m, 2 H, 2Ј-H),
8.04 (s, 1 H, 3-H), 12.1 (br. s, 1 H, NH) ppm. 13C NMR (CDCl3,
75 MHz): δ = 21.9 (2 CH3 iPr), 70.6 (CH iPr), 115.7 (C-3Ј), 116.0
(C-3Ј), 128.1 (C-3), 128.8 (C-1Ј), 128.9 (C-1Ј), 130.9 (C-2Ј), 131.0
(C-2Ј), 138.6 (C-4), 138.7 (C-2), 162.4 (CO), 162.1 (CO), 162.3 (C-
4Ј), 165.7 (C-4Ј) ppm. C14H13FN2O3 (276.27): calcd. C 60.87, H
4.74; found C 60.99, H 4.83.
For aggregation inhibition experiments, βA (25–35) peptide was
dissolved at 1 mm in PBS, and 10 μL of this solution was mixed
with the tested compound and incubated at 37 °C for 4 d. For dis-
aggregating experiments, 10 μm βA (25–35) peptide was incubated
at 37 °C for 4 d to generate fibrils. Pre-formed fibrils were mixed
with the tested compound for an additional 4 dat 37 °C. The degree
of βA aggregation and disaggregation was determined using thi-
oflavin-T (Thio-T) fluorescence analyses. Excitation and emission
wavelengths were 448 and 483 nm, respectively. Sample fluores-
cence was determined by subtracting the fluorescence of a Thio-T
blank. Data are shown in Table 4, and are expressed as the 50%
inhibitory concentration (IC50).
Isopropyl 5-Oxo-4-[4-(trifluoromethyl)phenyl]-5,6-dihydropyridine-2-
carboxylate (5c): 74 mg, 55% yield. 1H NMR (300 MHz, CDCl3,
25 °C): δ = 1.43 (d, 3J = 6.3 Hz, 6 H, CH3iPr), 5.34 (q, 3J = 6.3 Hz,
1 H, CH3iPr), 7.75 (d, J = 8.2 Hz, 2 H, 3Ј-H), 7.99 (d, J = 8.2 Hz,
2 H, 2Ј-H), 8.09 (s, 1 H, 3-H), 11.3 (br. s, 1 H, NH) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 22.0 (2 CH3 iPr), 70.9 (CH iPr), 125.7
(C-3Ј), 128.8 (C-3), 129.1 (C-2Ј), 129.2 (C-2Ј), 138.5 (C-4), 138.8
(C-2), 160.2 (CO), 161.9 (CO) ppm. C15H13F3N2O3 (326.27): calcd.
C 55.22, H 4.02; found C 55.44, H 4.13.
The assay for β-secretase activity was based on the secretase-de-
pendent cleavage of
a specific fluorogenic substrate [H-RE-
(EDANS)EVNLDAEFK(DABCYL)R-OH], which results in the
release of a fluorescent signal. The level of secretase enzymatic ac-
tivity is proportional to the fluorimetric reaction. β-secretase assay
was carried out at 37 °C using 0.24 U of a human recombinant β-
secretase enzyme and 10 μm substrate in 20 mm sodium acetate
buffer (pH 4.5) in a final volume of 100 μL. Wavelengths of exci-
tation and emission were 360 and 528 nm, respectively. The enzyme
activity assay was performed in the absence (control reaction), and
in the presence of compounds 5 or 11. Before the addition of the
substrate, the human recombinant β-secretase enzyme and the
tested compound were pre-incubated at 37 °C for 1 h. The inhibi-
tion ratio of β-secretase activity exerted by 5 or 11 was calculated
as the percentage of the control value after 1 h of incubation, once
the substrate was added. Data are shown in Table 4, and are ex-
pressed as the percentage inhibition at 10 μm.
Isopropyl 4-(4-Methoxyphenyl)-5-oxo-5,6-dihydropyridine-2-carbox-
1
ylate (5d): 61 mg, 52% yield. H NMR (300 MHz, CDCl3, 25 °C):
3
δ = 1.43 (d, J = 6.3 Hz, 6 H, CH3iPr), 3.96 (s, 3 H, OMe), 5.33
(q, 3J = 6.3 Hz, 1 H, CH iPr), 6.99 (d, 3J = 8.9 Hz, 2 H, 3Ј-H),
3
7.98 (d, J = 8.9 Hz, 2 H, 4Ј-H), 8.07 (s, 1 H, 3-H), 11.6 (br. s, 1
H, NH) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ = 22.0 (2 CH3
iPr), 56.6 (OMe), 70.9 (CH iPr), 111.7 (2 C-3Ј), 118.6 (C-1Ј), 126.3
(C-4), 129.6 (2 C-2Ј), 133.4 (C-3), 138.7 (C-2), 157.7 (CO), 162.2
(CO) ppm. C15H16N2O4 (288.30): calcd. C 62.49, H 5.59; found C
62.58, H 5.66.
Isopropyl 4-(Benzo[d][1,3]dioxol-5-yl)-5-oxo-5,6-dihydropyridine-2-
1
carboxylate (5e): 87 mg, 71% yield. H NMR (300 MHz, CDCl3,
25 °C): δ = 1.40 (d, 3J = 6.2 Hz, 6 H, CH3iPr), 5.32 (q, 3J = 6.2 Hz,
1 H, CH iPr), 6.06 (s, 2 H, O-CH2-O), 6.85 (s, 1 H, 5Ј-H), 6.15 (s,
1 H, 6Ј-H), 7.92 (s, 1 H, 3-H), 11.1 (br. s, 1 H, NH) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 22.1 (2 CH3 iPr), 71.2 (CH iPr), 102.3
(O-CH2-O), 110.7 (C-2Ј), 113.8 (C-5Ј), 120.4 (C-3), 124.0 (C-6),
126.5 (C-1Ј), 138.7 (C-4), 143.9 (C-2), 147.9 (C-4Ј), 149.3 (C-3Ј),
160.1 (CO), 162.6 (CO) ppm. C15H14N2O5 (302.29): calcd. C 59.60,
H 4.67; found C 59.88, H 4.75.
The molecular probe dichlorofluorescein diacetate (DCFA-DA)
was used to measure intracellular ROS generation in APPswe cells.
For this assay, cells were subcultured, and 24 h later they were
loaded with 10 μm DCFA-DA, which diffuses through the cell
membrane and is hydrolyzed by intracellular esterases to the
dichlorofluorescein (DCFH). DCFH reacts with intracellular free
radicals to form dichlorofluorescin (DCF), a green fluorescent dye.
Compounds 5 or 11 were added to the cells 30 min prior to the
treatment with 100 μm H2O2, a ROS generator. The fluorescence
caused after a 60 min exposure of the cells to H2O2 was measured,
the wavelengths of excitation and emission being 485 and 520 nm,
Isopropyl
1-Benzyl-6-oxo-5-phenyl-1,6-dihydropyridazine-3-carb-
oxylate (5f): 33 mg, 68% yield. 1H NMR (300 MHz, CDCl3,
25 °C): δ = 1.41 (d, 3J = 6.3 Hz, 6 H, CH3 iPr), 5.30 (q, 3J = 6.2 Hz,
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Pages: 9
A. G. Csákÿ et al.
FULL PAPER
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Acknowledgments
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S. R. is thankful to the Government of Spain for a Formación de
Profesorado Universitario (FPU) grant (AP20090051), and A. O.
to the Government of Chile for an FPU grant (MECESUP,
UCN0604). The Spanish Ministerio de Ciencia e Innovación
(MICINN) is thanked for financial support (project CTQ-2010-
16170). Prof. J. Plumet (Departamento de Química Orgánica, Fac-
ultad de Ciencias Químicas, Universidad Complutense de Madrid)
is thanked for useful comments and suggestions. The APP695-
transfected neuroblastoma SH-SY5Y cell line was kindly donated
by Dr. Weihong Song from the Department of Psychiatry, Brain
Research Center, at the University of British Columbia, 2255 Wes-
brook Mall, Vancouver, BC, V6T 1Z3, Canada.
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Received: May 31, 2012
Published Online: ᭿
8
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20734
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Date: 06-08-12 11:56:44
Pages: 9
Pd-Catalyzed Conjugate Addition of Boronic Acids to Ketoglutaconic Esters
Palladium Catalysis
S. Roscales, A. Ortega,
S. Martín-Aragón, P. Bermejo-Bescós,
A. G. Csákÿ* .................................... 1–9
Dicationic PdII catalysts were found to be
efficient for the regioselective conjugate ad-
dition of boronic acids to ketoglutaconic
esters. The resulting 4-aryl-2-oxopentadi-
enoates were transformed into pyridazin-
3(2H)-ones, which simultaneously exhib-
PdII-Catalyzed Conjugate Addition of
Boronic Acids to Ketoglutaconic Esters
toward the Synthesis of Functionalized
Pyridazin-3(2H)-ones with Neuroprotec-
tive Activity
ited β-secretase activity, inhibition of βA
aggregation, and disaggregation of pre-
formed βA fibrils, and also had an ROS-
scavenging profile.
Keywords: Boron / Palladium / Regioselec-
tivity / Homogeneous catalysis / Michael
addition / Nitrogen heterocycles / Med-
icinal chemistry / Neuroprotection
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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9