Design, synthesis, and biological evaluation of 1-phenylpyrazolo[3,4- e ]pyrrolo[3,4- G ]indolizine-4,6(1 H,5 h)-diones as new glycogen synthase kinase-3β inhibitors

Article abstract of DOI:10.1021/jm401466v

Compound 5 was selected from our in-house library as a suitable starting point for the rational design of new GSK-3β inhibitors. MC/FEP calculations of 5 led to the identication of a structural class of new GSK-3β inhibitors. Compound 18 inhibited GSK-3β with an IC₅₀ of 0.24 μM and inhibited tau phosphorylation in a cell-based assay. It proved to be a selective inhibitor of GSK-3 against a panel of 17 kinases and showed >10-fold selectivity against CDK2. Calculated physicochemical properties and Volsurf predictions suggested that compound 18 has the potential to diffuse passively across the blood-brain barrier.

Full text of DOI:10.1021/jm401466v

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Article
Design, Synthesis, and Biological Evaluation of 1-
Phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-
diones as New Glycogen Synthase Kinase-3# Inhibitors
Valeria La Pietra, Giuseppe La Regina, Antonio Coluccia, Valeria Famiglini, Sveva
Pelliccia, Batya Plotkin, Hagit Eldar-Finkelman, Andrea Brancale, Carlo Ballatore, Alex
Crowe, Kurt R Brunden, Luciana Marinelli, Ettore Novellino, and Romano Silvestri
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Dec 2013
Downloaded from http://pubs.acs.org on December 10, 2013
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Design, Synthesis, and Biological Evaluation of 1-Phenylpyrazolo[3,4-e]pyrrolo[3,4-
g]indolizine-4,6(1H,5H)-diones as New Glycogen Synthase Kinase-3β Inhibitors
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Valeria La Pietra,fi Giuseppe La Regina,†,* Antonio Coluccia,† Valeria Famiglini,† Sveva Pelliccia,†
Batya Plotkin,§ Hagit Eldar-Finkelman,§ Andrea Brancale,¶ Carlo Ballatore†,‡, Alex Crowe‡, Kurt R.
Brunden‡, Luciana Marinelli,fi,* Ettore Novellino,fi and Romano Silvestri†
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† Istituto Pasteur – Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco,
Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy
fiDipartimento di Farmacia, Università di Napoli Federico II, Via Domenico Montesano 49, I-80131
Napoli, Italy
§Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv
University, IL-69978 Tel Aviv, Israel
¶Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, UK
†Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, 231 South 34th
Street, Philadelphia, Pennsylvania 19104, United States
‡Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine,
Perelman School of Medicine, University of Pennsylvania, 3600 Spruce Street, Philadelphia,
Pennsylvania 19104, United States
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ABSTRACT
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Compound 5 was selected from our in-house library as a suitable starting point for the rational design
of new GSK-3β inhibitors. MC/FEP calculations of 5 led to identify a structural class of new GSK-3β
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inhibitors. Compound 18 inhibited GSK-3β with IC50 of 0.24 µM, and inhibited tau phosphorylation in
a cell-based assay. It proved to be a selective inhibitor of GSK-3 against a panel of 17 kinases, and
showed >10-fold selectivity against CDK2. Calculated physico-chemical properties and Volsurf
predictions suggested that compound 18 has the potential to passively diffuse across the blood-brain
barrier.
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INTRODUCTION
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Alzheimer’s disease (AD) is a leading cause of death worldwide. Currently, more than five
million people are estimated to suffer from AD and this number will grow significantly over the
coming decades. By 2050, there is expected to be nearly a million new cases per year.1 AD is a
devastating dementia condition characterized by a progressive age-related loss of memory and
impairment of cognitive capability. The development of AD is accompanied by neuronal loss, which
appears to correlate with the appearance of intracellular aggregates of the microtubule-associated
protein (MAP) tau (e.g., neurofibrillary tangles, or NFTs).2 In addition, the AD brain is characterized
by the presence of extracellular senile plaques comprised of Aβ peptides.3,4 Current treatments of AD
are not disease-modifying and are merely palliative. There is an unmet need for newer drugs in the
treatment of AD as the current options show significant limitations.5
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Glycogen synthase kinase-3 (GSK-3) is a proline-directed serine/threonine kinase that is
expressed as α and β isoforms and which is responsible for the phosphorylation of a variety of cellular
substrates and is involved in the molecular pathogenesis of severe human diseases.6 In the brain, a
principal consequence of dysregulation of GSK-3 is the hyperphosphorylation of tau proteins.7 GSK-3
plays a key role in glucose metabolism as its inhibitory phosphorylation reduces the rate of conversion
of glucose to glycogen, thus elevating the glucose concentration in the blood.8 It is also recognized that
that GSK-3 is involved in cell-fate control,9 insulin signaling cascade,10 differentiation and
proliferation,11 apoptosis regulation (its inhibition may have a neuroprotective effect),12 and it may
serve as a drug target for amyotrophic lateral sclerosis.13
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Chart 1. Reference Compounds 1-5, and new Derivatives 6-18.a
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a6, R1 = 2-Cl, R2 = R3 = H; 7, R1 = 3-Cl, R2 = R3 = H; 8, R1 = 4-Cl, R2 = R3 = H; 9, R1 = 2-Me,
R2 = R3 = H; 10, R1 = 3-Me, R2 = R3 = H; 11, R1 = 4-Me, R2 = R3 = H; 12, R1 = 2-MeO, R2 =
R3 = H; 13, R1 = H, R2 = Me, R3 = H; 14, R1 = R2 = H, R3 = Me; 15, R1 = 2-Me, R2 = Me, R3 =
H; 16, R1 = 2-Cl, R2 = Me, R3 = H; 17, R1 = 4-Cl,2-Me, R2 = Me, R3 = H; 18, R1 = 2,4-Cl2, R2 =
Me, R3 = H;
GSK-3α,
-3βGSsKhare 84% overall identity and >98% identity within their catalytic
domains.14 Both isoforms are highly expressed in the brain and testicles and share similar functions in
several cellular processes.15 GSK-3β represents the predominant isoform present in most CNS areas,
where it is localized primarily in neurons.
GSK-3β-mediated hyperphosphorylation of tau has been implicated in the pathogenesis of AD,
as hyperphosphorylation promotes the detachment of tau from the microtubules and renders the protein
more prone to misfolding and aggregation, thus leading to the formation of NFTs and, ultimately,
neuronal death.16 There is evidence of a correlation between an increase in GSK-3β activity and NFT
formation, altered integrity of the microtubule network, and neurodegeneration.17 As GSK-3β showed
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connections to mechanisms involved in AD and other CNS disorders, it has become an attractive target
for medicinal chemists both in academia and pharmaceutical companies.18
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Over the past decades, a number of diverse GSK-3β inhibitors have been reported.19 We
observed that compound 5, previously synthesized by us,20 shared some chemical features common
with known GSK-3β inhibitors (1,21 2,22 3,23 4,24 ) (Chart 1). In a preliminary screening for GSK-3β
inhibitory activity, compound 5 showed an IC50 of 1.69 µM. These results prompted us to analyse the
potential for productive modifications of 5 in a quest to discover new potent GSK-3β inhibitors. Thus,
optimization of 5 was initiated using a computationally driven approach, primarily guided by results of
free energy perturbation (FEP) calculations for complexes of analogues of 5 with GSK-3β.25
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CHEMISTRY
The synthesis of 1-(phenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione derivatives 5-
18 is depicted in Scheme 1. Sodium dicyanoalk-1-en-1-olates 19 or 20 were obtained by reaction of
succinonitrile with an ester R2COOEt (R2 = H, Me) in the presence of sodium hydride and iso-amyl
alcohol. Treatment of 19 or 20 with phenylhydrazine or phenylhydrazine hydrochloride in the presence
of triethylamine 21-30 in ethanol at reflux provided 2-[5-amino-1-(phenyl)-1H-pyrazol-4-
yl]acetonitriles 31-43. Compounds 31-43 were converted into the corresponding 5-pyrryl derivatives
44-56 by Klauson-Kaas26 reaction with 2,5-dimethoxytetrahydrofuran in boiling acetic acid.
Compounds 44-54 afforded 3-cyano-2-hydroxyacrylates 57-67 on treatment with diethyl oxalate in the
presence of sodium ethoxide, that underwent intramolecular cyclization by heating at 90 °C in
polyphosphoric acid to provide the desidered 1-(phenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-
4,6(1H,5H)-diones 5-13, 15 and 16.
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Scheme 1. Synthesis of Compounds 5-18. a
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5-18, see Chart 1; 19, R2 = H; 20, R2 = Me; 21, R1 = H; 22, R1 = 2-Cl; 23, R1 = 3-Cl; 24: R1 = 4-Cl; 25, R1 = 2-Me; 26, R1 =
3-Me; 27, R1 = 4-Me; 28, R1 = 2-MeO; 29, R1 = 2,4-Cl2; 30, R1 = 2-Me,4-Cl; 31, 44, 57, R1 = R2 = H; 32, 45, 58, R1 = 2-Cl,
R2 = H; 33, 46, 59, R1 = 3-Cl, R2 = H; 34, 47, 60, R1 = 4-Cl, R2 = H; 35, 48, 61, R1 = 2-Me, R2 = H; 36, 49, 62, R1 = 3-Me,
R2 = H; 37, 50, 63, R1 = 4-Me, R2 = H; 38, 51, 64, R1 = 2-MeO, R2 = H; 39, 52, 65, R1 = H, R2 = Me; 40, 53, 66, R1 = 2-Me,
R2 = Me; 41, 54, 67, R1 = 2-Cl, R2 = Me; 42, 55, 68, 70, R1 = 2,4-Cl2, R2 = Me; 43, 56, 69, 71, R1 = 4-Cl,2-Me, R2 = Me.
aReagents and reaction conditions: (a) NaH, iso-amyl alcohol, toluene, 0 °C to room temp., 16 h; (b) ethanol, reflux, 4 h; (c)
2,5-dimethoxytetrahydrofuran, acetic acid, reflux, 30 min; (d) diethyl oxalate, sodium ethoxide, absolute ethanol, room
temp., 5 h; (e) 2-chloro-2-oxoacetate, toluene, reflux, 6 h; (f) potassium carbonate, DMF, 100 °C, 4 h; (g) PPA, 90 °C, 1 h;
(h) trifluoroacetic acid, sulfuric acid, room temp., 2 h; (i) potassium carbonate, DMF, room temp., 3 h.
To improve the overall yield, 2-[5-(1H-pyrrol-1-yl)-1-(4-methylphenyl)-1H-pyrazol-4-
yl]acetonitriles 55 and 56 were transformed into the corresponding pyrryl-2-oxoacetates 68 and 69 with
2-chloro-2-oxoacetate in hot toluene. Intramolecular cyclization of 68 or 69 in DMF at 100 °C in the
presence of potassium carbonate gave 1-(phenyl)-4-cyano-3-methyl-1H-pyrazolo[3,4-e]indolizine-5-
carboxylates 70, 71 that on treatment with trifluoroacetic acid and sulfuric acid mixture furnished
indolizines 17 or 18, respectively (Scheme 1).
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Figure 1. Binding pose of 5 in the active site of GSK-3β shown as cornflower stick and tan cartoon,
respectively. Interacting residues are shown as sticks and coloured by atom type, while the H-bonds are
represented by a black dashed line.
RESULTS AND DISCUSSION
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Docking and MC/FEP Studies. Docking calculations of 5 into the GSK-3β active site suggested
the binding pose depicted in Figure 1. Key interactions included: (i) two H-bonds established by the
maleimide moiety with D133 and V135 backbone of the hinge-region; (ii) multiple hydrophobic
contacts between the four planar rings and A83, V70, L132, I62 (N-lobe), L188, V110, C199, V135
(C-lobe); (iii) hydrophobic interactions of the pendant 1-phenyl ring with V70, L188, C199 and Q185
side chains.
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Table 1. Computed Change in Free Energy of Binding for Introducing a
Chlorine Atom or a Methyl Group at the 1-Phenyl Ring of 5.
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H to Cl
H to Me
b
b
Positiona
C2’
ΔΔGb
σc
Positiona
C2’
ΔΔGb
σc
-0.7
-1.1
-1.3
0.84
0.11
0.21
0.18
0.20
0.19
0.19
-0.24
-0.4
0.19
0.22
0.23
0.18
0.18
C3’
C3’
C4’
C4’
-0.48
0.57
C5’
C5’
C6’
C6’
-1.754
aPosition on the 1-phenyl ring of 5. bΔΔGb, computed change in free energy of binding
(kcal/mol). c±σ, computed uncertainty.
Given the aforesaid binding mode, we carried out MC/FEP calculations with a chlorine and a methyl
scan on the pendant 1-phenyl ring of 5 (see Table 1 for numbering). Replacement of hydrogen by
chlorine was predicted to be favourable (more negative free energy of binding, ΔΔGb, Table 1) at C2’,
C3’, and C4’ (average computed uncertainty,σ ± 0.2 kcal/mol). As for the methyl scan, a favourable
replacement was predicted at C2’, C3’, C4’ and C6’ (Table 1).
Some positions were found to be equivalent, for example, both C2’ and C6’ (as it occurred for the
2’-Me analogue). When only one position was favourable (C3’-Cl and C3’-Me analogues), a penalty of
RT ln 2 (0.6 kcal/mol) was due to the breakage of one rotameric state upon binding.. Thus, from the
FEP scan resulted a particularly favourable replacement at C2’ or C6’, followed by C3’and the
symmetrical position C4’.
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Table 2. GSK3β Inhibition of Compounds 5-14 and Reference Compound 1.
5
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GSK-3β
Compd
R1
R2
R3
IC50 (µM)
1.69
0.81
nd
H
2-Cl
3-Cl
4-Cl
2-Me
3-Me
4-Me
2-OMe
H
H
H
H
H
5
6
H
H
7
H
H
nd
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H
H
1.21
LMRa
LMR
1
9
H
H
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14
1
H
H
H
H
Me
H
H
0.69
nd
H
Me
—
—
—
0.174
aLMR: low micromolar range.
Taking into account the synthetic accessibility of each analogue predicted to be more active than 5
by the MC/FEP calculation, a number of compounds were synthesized (Table 2). As regards the
substitutions at the pendant 1-phenyl ring, the 2’-Cl (6) and 2’-Me (9) substitutions were consistent
with the computed results, as compounds 6 and 9 were found potent GSK-3β inhibitors. In contrast, a
discrepancy was observed for the substitutions at position 3’ or 4’ (7, 8, 10, 11), which were predicted
to be more active than 5. Given the result of compound 9, the corresponding 2’-OMe analogue (12)
was synthesized, and it also proved to inhibit the GSK-3β.
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Table 3. Computed Change in Free Energy of Binding for Introducing a
Chlorine Atom or a Methyl Group at the 1-Phenyl Ring of 13.
6
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H to Cl
H to Me
b
b
Positiona
C2’
ΔΔGb
σc
Positiona
C2’
ΔΔGb
σc
-2.765
0.015
-1.523
1.509
-0.089
0.15
0.13
0.17
0.14
0.16
-0.83
-0.731
-1.127
0.66
0.27
0.21
0.22
0.28
0.29
C3’
C3’
C4’
C4’
C5’
C5’
C6’
C6’
-0.059
aPosition on the 1-phenyl ring of 13. ΔΔGb, computed change in free energy of
b
binding (kcal/mol). c± σ, computed uncertainty.
We speculated about the replacement of the hydrogen with a chlorine or a methyl group at the
position 3 of the pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione ring. The resultant ΔΔGb
values were -3.8 and -2.83 kcal/mol, respectively with uncertainty of ± 0.2 kcal/mol. Consistent with
the computational predictions, the introduction of a methyl group at the position 3 led to 13, a more
potent GSK-3β inhibitor than the parent compound 5. Unfortunately, the introduction of the chlorine at
this position was unsuccessful. inaccessible. We calculated the ΔΔGb (6.06 kcal/mol) for the inactive
methyl analogue 14, whose prediction was in line with the experimental data.
Inspection of the complexes of 13 and 14 with the GSK-3β built using BOMB (data not shown)
allows to rationalize the FEP predictions. For example, the methyl group at position 3 of 13 plunges
into the N-lobe of the enzyme enhancing the hydrophobic contacts within the pocket. On the contrary,
the introduction of a methyl group at the maleimide function of 14 disrupts the hydrogen bonds with
the hinge region which are crucial for the binding process.
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Table 4. Computed Change in Free Energy of
Binding for Introducing Two Chlorine Atoms
at the 1-Phenyl Ring of 13.
7
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9
10
11
12
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18
19
20
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H to Cl
b
Positiona
C2’/C3’
C2’/C4’
C2’/C5’
C2’/C6’
C3’/C4’
C3’/C5’
C3’/C6’
C4’/C5’
C4’/C6’
C5’/C6’
ΔΔGb
-2.765
-4.988
-1.402
-0.816
-3.026
-1.354
-1.098
0.731
σc
0.21
0.31
0.27
0.23
0.27
0.25
0.25
0.22
0.24
0.22
-1.426
-2.541
aPosition on the 1-phenyl ring of 13. bΔΔGb,
computed change in free energy of binding
(kcal/mol). c±σ, computed uncertainty.
A second round of MC/FEP-based optimization was attempted on the highly active compound
13, perturbing its structure again with chlorine or methyl groups (Table 3). The replacement of
hydrogen by a chlorine atom or a methyl group was predicted to be particularly favourable at C2’, C4’
and C6’ positions. Since results for double substitutions might not be addictive, MC/FEP calculations
were also accomplished for all 10 dichloro combinations, as summarized in Table 4.
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Table 5. GSK3β Inhibition of Compounds 13, 15-18 and Reference Compound 1.
5
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GSK-3β
IC50 (µM)
Compd
R1
R2
R3
H
2-Me
2-Cl
Me
Me
Me
Me
Me
—
H
H
H
H
H
—
0.69
13
15
16
17
18
1
0.44
0.94
LMRa
4-Cl,2-Me
2,4-Cl
—
0.24
0.174
aLMR: low micromolar range.
The results supported the viability of substitutions at C2’, C3’ and C4’, with the most favourable
substitution being C2’/C4’. Compound 15 inhibited GSK-3β with IC50 of 440 nM (Table 5). In line
with the computational predictions, substitution at both the C2’ and C4’ positions with chlorine atoms
led to compound 18. It was the most potent GSK-3β inhibitor within the series, with an inhibitor
activity of 240 nM, which is almost 10-fold more active than the parent compound 5. Such an
inhibitory activity was not preserved when the 2’-chlorine atom was replaced with a methyl group
(compare 18 with 15 and 17).
A picture of the computed structure for 18 bound to GSK-3β is provided in Figure 2. The
methyl group at the C7 position is inserted in the small N-Lobe pocket where it establishes
hydrophobic interactions with L132, M101, V70, and K85 side chains. As for the 1-phenyl ring, the
presence of the two electron withdrawing chlorine atoms remarkably boosts the hydrophobic contacts
with the aforementioned alkylic side chains of the C-lobe residues.
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Figure 2. Putative binding pose of 18 in the active site of GSK-3β shown as cornflower stick and tan cartoon,
respectively. Interacting residues are shown as sticks and colored by atom type, while the H-bond is
represented by a black dashed line
Kinase Selectivity. The most potent GSK-3β inhibitor 18 was measured for the inhibition of CDK2,
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a proline directed Ser/Thr kinase showing high sequence similarity27 to GSK3β in the ATP binding site.
Compound 18 inhibited CDK2 with IC50 of 2.8 µM, and showed >10-fold selectivity.
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Figure 5. Histogram of % inhibition of 17 human protein kinases by compound 18. Test
concentration was 1 µM. The data represent an average of two independent measurements.
Compound 18 was tested for selectivity at a concentration of 1 μM against a panel of 17 human
protein kinases selected from homologous kinases (AurA/Aur2, Fyn kinase, PKAα, RET kinase,
ROCK1, SGK1), neurodegeneration kinases (CaMK2α, CDK5/p35, ERK1, GSK-3α, LRRK2, P38δ
kinase, PKA), tumor kinases (FLT3 kinase, KDR kinase (VGFR2), PDGFRβ), and RAF-1/MEK1
kinase. Compound 18 displayed strong inhibition of GSK-3α(98.3%); it weakly inhibited FLT3
kinase (30%) and PDGFRβ (13.7%), and 14 kinases of in this panel showed an inhibition lower than
10% (additional results are shown in the Supporting Information). Therefore, in summary, within this
test panel compound 18 proved to be selective inhibitor of GSK-3 (Figure 5). Compound 18 was
evaluated in the ovarian carcinoma cell lines OVCAR-8 and its cognate P-glycoprotein (Pgp)
overexpressing line NCI/ADR-RES, and showed no activity up to 5 µM concentration in both cell lines.
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Figure 3. Compound 18 inhibits tau phosphorylation. HEK293 cells expressing human tau were treated with synthetic tau
fibrils to induce the formation of intracellular tau with increased phosphorylation. This can be seen by an increase in the
slower migrating species of tau using the AT180 antibody that recognizes the p231T and p235S phospho-epitopes of tau.
Treatment with 1 µM 18 led to decrease in the slow mobility AT180 band and an increase in the faster migrating AT180
band, as well as an increase in the higher mobility band recognized by the 17025 antibody to total tau. The combined gel is
a composite of the AT180 and 17025 immunoreactivity, and GAPDH is glyceraldehydes 3-phosphate dehydrogenase,
which is utilized as a housekeeping protein to demonstrate equal protein loading.
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Inhibition of Protein Tau Phosphorylation. To investigate the ability of this class of
compounds to modulate the phosphorylation state of a known GSK3β substrate in a cell-based assay,
we evaluated a representative example, 18, in a HEK-293 cell model in which the full-length four-
repeat tau isoform (T40), bearing the P301L mutation (T40PL) found in a form of inherited
frontotemporal dementia28,29 is expressed under the control of a doxycycline-regulated promoter.
Such cells can be induced to form intracellular inclusions of misfolded tau that bear resemblance
to the hyperphosphorylated NFTs observed in Alzheimer’s disease and related tauopathies, if the cells
are “seeded” with small amounts of synthetic tau fibrils that are created from recombinant tau
protein.30 Here, the HEK-293 cells were treated with doxycycline to induce tau expression, and 48 h
later were treated with synthetic tau fibrils, followed by treatment with 18 or vehicle for an additional
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48 h. Non-fibril treated tau-expressing cells were included as controls. GSK-3β, as well as other
kinases, can phosphorylate tau at residues T231 and S235,31 the extent of tau phosphorylation at these
sites was examined by immunoblot analysis of the cell lysates, employing the AT180 antibody which
recognizes the tau pT231 and pS235 epitopes. In addition, total tau was examined by immunoblotting
with the polyclonal 17025 antibody to full-length tau.
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The total tau immunoblot reveals multiple tau species with slightly differing electrophoretic
mobilities (Figure 3; 17025). As a single isoform of tau is expressed in these cells, the multiple bands
presumably represent tau with different charges that result from post-translational modifications, such
as phosphorylation. In fact, all of the AT180-positive species (Figure 3; AT180) co-migrate with tau
(Figure 3; combined).
Relative to cells that were not seeded with fibrils, the AT180 signal is increased in the slowest
migrating tau species and faster migrating bands are decreased after tau fibril treatment (Figure 3 and
Figure 4-A), with the total AT180 signal remaining unchanged in the fibril-treated cells compared to
non-fibril cells (Figure 4-B). This would suggest that the fibril treatment resulted in increased
phosphorylation at tau residues other than T231 or S235. Interestingly, addition of 18 to the fibril-
treated cells resulted in a diminution of the slowest migrating and an increase in the faster migrating
AT180 species (Figure 3 and Figure 4-A). A somewhat similar shift in distribution can be seen in the
17025 immunoblot of total tau after treatment of the fibril-seeded cells with 18, with an increase in the
percentage of tau with faster electrophoretic mobility relative to the vehicle-treated cells (Figure 3 and
Figure 4-C). As expected, there was no significant difference in the amount of total tau in the different
treatment conditions (Figure 4-D). Taken in totality, these data suggest that 18 inhibited tau
phosphorylation, but at a site(s) other than the pT231 and pS235 sites that are recognized by the AT180
antibody.
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Figure 4. Quantification of the high mobility AT180- and 17025-positive tau species in Figure 3. The fastest migrating
AT180 and 17025 immunoreactive species shown in Figure 3 were quantified as described in the Methods. In addition, the
combined AT180 and 17025 immunoreactive species were quantified and normalized to the housekeeping protein, GAPDH.
GSK-3β is known to phosphorylate tau at sites other than T231/S235, including S202 and
S396,31 and thus the altered electrophoretic profile of tau after 18 treatment may have resulted from
inhibition of phosphorylation at one or both of these residues. It is somewhat surprising that 18 did not
cause a reduction in total AT180 immunostaining, since this antibody recognizes known GSK-3β
phosphorylation sites. However, other kinases can also phosphorylate T231 and S235 of tau,31 and thus
it is possible that one or more of these kinases acts on these residues in the HEK-293 cells, rather than
GSK-3β.
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Evaluation of predicted physico-chemical properties and blood-brain barrier permeability. To
achieve optimum therapeutic efficacy, a GSK-3β inhibitor should display suitable physico-chemical
properties for CNS penetration. Most of the early predictive models for BBB penetration are based on a
multiple linear regression approach and combination of three or more descriptors, i.e. the calculated
LogP, the number of H-bond acceptors in an aqueous medium and the polar surface area.32.33 In our
design strategy, a qualitative prediction of the physico-chemical properties of all the analogues
generated for the MC/FEP calculations has been automatically perfomed by the BOMB program using
the Qikprop software (Schrödinger, LLC New York). These preliminary data suggested that our ligands
have the potential to passively diffuse across the BBB. We herein report the computed physico-
chemical properties of 5 and 18.
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These measurements suggested that compound 18, the most potent GSK-3β inhibitor within the
series, exhibited properties that should favour CNS penetration,32.33 including: (i) a molecular weight
<400 Da; (ii) a log P value comprised between 0 and 5; (iii) less than 3 H-bond donors; (iv) less than 7
H-bond acceptor, (v) the number of rotatable bonds comprised between 0 and 5; (vi) log BB value
comprised between -3 and 1; and (vii) the PSA <90 Å2 (Table 6).
Table 6. Calculated physico-chemical properties of 5 and 18 performed by the Qikprop software
Compd
MW
log P (o/W)
HB donor
HB acceptor
# rotor
logBB
PSA
302
371
2.5
3.4
1
1
4
4
0
0
-0.612
-0.283
94
5
87.47
18
The BBB permeation profiles of our synthesized compounds were also predicted a posteriori using
the BBB VolSurf model, whereby a variety of 3D molecular field descriptors were transformed into a
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new set of descriptors to produce a model by using a discriminant partial least squares (PLS)
procedure.34,35 All our compounds were built in 3D coordinates and then projected in the VolSurf
Crivori model.34 This model was able to discriminate between penetrating (BBB+) and non-penetrating
(BBB–) compounds. Figure 1S of Supporting Infomation shows the BBB profile as predicted by
VolSurf. All of our molecules were located in the BBB+ region of the plot with the exception of 5
which is in the borderline region.
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Aqueous Solubility. The solubility in aqueous pH 7.4 buffer of compound 18 was measured by
Cerep. Compound 18 showed to be moderately soluble in water, with mean solubility of 3.1 µM
(additional results are shown in the Supporting Information).
CONCLUSION
In search for agents to treat neurodegenerative diseases, GSK-3β inhibitors have become an attractive
target as growing evidence correlates an increase in GSK-3β activity with neurodegeneration.
Compound 5 from our in-house library was selected as a suitable starting point for the rational design
of new GSK-3β inhibitors. MC/FEP calculations of 5 led to the identification of a structural class of
new GSK-3β inhibitors. Compound 18 inhibited GSK-3β with IC50 of 0.24 µM, and it was found to be
a selective inhibitor of GSK-3 against a panel of 17 kinases, with >10-fold selectivity against CDK2.
Furthermore, 18 inhibited tau phosphorylation in a cell-based assay. Finally, calculation of physico-
chemical properties suggested that compound 18 exhibits properties that favour passive diffusion
across the BBB In summary, we have developed a new class of GSK-3β inhibitors. Compound 18
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represents a robust lead compound to develop GSK-3β inhibitors with improved activity and selectivity
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that have potential as novel therapeutic agents to treat neurodegenerative disorders.
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EXPERIMENTAL SECTION
Chemistry. All reagents and solvents are commercially available and were used as purchased,
without further purification. Melting points (mp) were determined on a Stuart Scientific SMP1
apparatus and are uncorrected. Infrared spectra (IR) were run on a Perkin-Elmer SpectrumOne FT-ATR
spectrophotometer. Band position and absorption ranges are given in cm−1. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded on a Bruker 400 MHz FT spectrometer in the indicated
solvent and corresponding fid files processed by MestreLab Research S.L. MestreReNova 6.2.1-769
software. Chemical shifts are expressed in δ units (ppm) from tetramethylsilane. Column
chromatography was performed on columns packed with alumina from Merck (70−230 mesh) or silica
gel from Macherey-Nagel (70-230 mesh). Aluminum oxide thin layer chromatography (TLC) cards
from Fluka (aluminum oxide precoated aluminum cards with fluorescent indicator detectable at 254
nm) and silica gel TLC cards from Macherey-Nagel (silica gel precoated aluminum cards with
fluorescent indicator detectable at 254 nm) were used for TLC. Developed plates were visualized by a
Spectroline ENF 260C/FE UV apparatus. Organic solutions were dried over anhydrous sodium sulfate.
Evaporation of the solvents was carried out on a Büchi rotavapor R-210 equipped with a Büchi V-855
vacuum controller and a Büchi V-700 or V-710 vacuum pump. Elemental analyses of the compounds
were found within ±0.4% of the theoretical values. The purity of tested compounds was >95%.
Phenylhydrazines purchased from commercial sources (Scheme 1): 21, R1 = H; 22, R1 = 2-Cl; 23, R1 =
3-Cl; 24, R1 = 4-Cl; 25, R1 = 2-Me; 26, R1 = 3-Me; 27, R1 = 4-Me; 28, R1 = 2-OMe; 29, R1 = 2,4-Cl2;
30, R1 = 2-Me,4-Cl.
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General procedure for the synthesis of derivatives 5-13, 15 and 16. Example. 1-(4’-
Methylphenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (11). Compound 63 (2.18
g, 0.006 mol) was added in small portions to polyphosphoric acid (PPA) (22 g) pre-heated at 90 °C.
The reaction mixture was stirred at 90 °C for 1 h and then allowed to cool at room temperature. Water
was carefully added (exotermal reaction!) and the mixture was extracted with ethyl acetate. The
combined organic layers were washed with brine and dried, and the solvent was evaporated. The
residue was purified by silica gel column chromatography using ethyl acetate:n-hexane (1:1) as eluent
to give 11 (0.70 g, yield 37%), mp 234-237 °C (from aqueous ethanol). 1H NMR (DMSO-d6)δ 11.09
(s, 1H), 8.36 (d, J = 3.0 Hz, 1H), 7.60 (m, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.09 (dd, J = 1.1 and 4.0 Hz,
1H), 6.93 (dd, J = 2.9 and 4.0 Hz, 1H), 6.85 (dd, J = 1.1 and 2.9 Hz, 1H), 2.48 ppm (s, 3H). IR
ν 1711, 1759, 3199 cm-1. Anal. Calcd. for C18H12N4O2 (316.31), C, H, N
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1-Phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (5). Was prepared as 11 from
57.20
1-(2’-Chlorophenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (6). Was prepared
1
as 11 from 58. Yield 46%, mp 230-232 °C (from aqueous ethanol). H NMR (DMSO-d6)δ 8.48 (s,
1H), 8.32 (s, 1H), 7.95 (dd, J = 7.8, 1.5 Hz, 1H), 7.89 (dd, J = 8.1 and 1.3 Hz, 1H), 7.82 (d, J = 1.6 Hz,
1H), 7.73 (dd, J = 7.6 and 1.4 Hz, 1H), 7.14-7.10 (m, 1H), 7.00-6.92 (m, 1H), 6.72 – 6.67 ppm (m, 1H).
IRν 1707, 1758, 2726, 3034, 3149 cm-1. Anal. Calcd. for C17H9ClN4O2 (336.73), C, H, N, Cl.
1-(3’-Chlorophenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (7). Was prepared
as 11 from 59. Yield 49%, mp 288-290 °C (from aqueous ethanol). 1H NMR (DMSO-d6)δ 11.14 (s,
1H), 8.41 (s, 1H), 7.92 (s, 1H), 7.78 (m, 3H), 7.12 (m, 1H), 7.98 (m, 1H), 6.94 ppm (m, 1H). IRν
1727, 17645, 3235 cm-1. Anal. Calcd. for C17H9ClN4O2 (336.73), C, H, N, Cl.
1-(4’-Chlorophenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (8). Was prepared
1
as 11 from 60. Yield 61%, mp 279-281 °C (from aqueous ethanol). H NMR (DMSO-d6): δ 11.10 (s,
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1H), 8.40 (s, 1H), 7.77 (s, 4H), 7.12 (m, 1H), 6.96 ppm (m, 2H). IRν 1721, 1759, 3240 cm-1. Anal.
Calcd. for C17H9ClN4O2 (336.73), C, H, N, Cl.
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1-(2’-Methylphenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (9). Was prepared
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as 11 from 61. Yield 40%, mp 227-231 °C (from aqueous ethanol). H NMR (DMSO-d6): δ 11.11 (s,
1H), 8.44 (s, 1H), 7.63 (m, 3H), 7.53 (t, J = 7.4 Hz, 1H), 7.10 (m, 1H), 6.90 (m, 1H), 6.60 (m, 1H), 1.95
ppm (s, 3H). IR: ν 1713 cm-1. Anal. Calcd. for C18H12N4O2 (316.31), C, H, N.
1-(3’-Methylphenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione
(10).
Was
1
prepared as 16 from 62. Yield 57%, mp 232-234 °C (from ethyl acetate-n-hexane). H NMR (DMSO-
d6): δ 11.09 (s, 1H), 8.39 (s, 1H), 7.55 (m, 4H), 7.12 (m, 1H), 6.94 (m, 1H), 6.88 (m, 1H), 2.45 ppm (s,
3H). IR: ν 1720, 1758, 1832, 3061, 3171, 3227 cm-1. Anal. Calcd. for C18H12N4O2 (316.31), C, H, N.
1-(2’-Methoxyphenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione
(12).
Was
1
prepared as 11 from 64. Yield 27%, mp 282-284 °C (from aqueous ethanol). H NMR (DMSO-d6): δ
11.04 (s, 1H), 8.37 (s, 1H), 7.70 (m, 2H), 7.38 (d, J = 8.2 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.08 (m,
1H), 6.92 (m, 1H), 6.81 (m, 1H), 3.65 ppm (s, 3H). IR: ν 1720, 1756, 3154 cm-1. Anal. Calcd. for
C18H12N4O3 (332.31), C, H, N.
3-Methyl-1-phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (13). Was prepared
as 11 from 65. Yield 52%, mp >270 °C (from ethanol). 1H NMR (DMSO-d6): δ 11.06 (s, 1H), 7.69 (m,
5H), 7.07 (dd, J = 1.0 and 4.0 Hz, 1H), 6.91 (dd, J = 3.0 and 4.0 Hz, 1H), 6.77 (dd, J = 1.0 and 2.9 Hz,
1H), 2.72 ppm (s, 3H). IR: ν 1724, 1754, 2735, 3062, 3265, 3482 cm-1. Anal. Calcd. for C18H12N4O2
(316.31), C, H, N.
3-Methyl-1-(2’-methylphenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (15).
1
Was prepared as 11 from 66. Yield 19%, mp 227-231 °C (from aqueous ethanol). H NMR (DMSO-
d6): δ 11.04 (s, 1H), 7.50 (m, 4H), 7.07 (m, 1H), 6.89 (m, 1H), 6.55 (m, 1H), 2.75 (s, 3H), 1.96 ppm (s,
3H). IR: ν 1718, 1761, 2722, 3069, 3182 cm-1. Anal. Calcd. for C19H14N4O2 (330.34), C, H, N.
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3-Methyl-1-(2’-chlorophenyl)pyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione
(16).
1
Was prepared as 11 from 67. Yield 67%, mp 200-204 °C (from aqueous ethanol). H NMR (DMSO-
d6): δ 11.11 (s, 1H), 7.91 (d, J = 6.3 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 7.0 Hz, 1H), 7.69 (t,
J = 7.6 Hz, 1H), 7.10 (m, 1H), 6.94 (m, 1H), 6.65 (m, 1H), 2.75 ppm (s, 3H). IR: ν 1707, 1761, 3169
cm-1. Anal. Calcd. for C18H11ClN4O2 (350.76), C, H, N, Cl.
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General procedure for the synthesis of derivatives 17 and 18. 1-(4’-Chloro-2’-methylphenyl)-3-
methylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (17). A solution of 71 (0.0105 g,
0.00026 mol), trifluoroacetic acid (0.24 mL) and sulfuric acid (0.12 mL) was stirred at room
temperature for 2 h. Water was carefully added dropwise (exotermic reaction!) and the mixture was
extracted with ethyl acetate, washed with brine and dried. After evaporation of the solvent, the residue
was purified by silica gel column chromatography using ethyl acetate:n-hexane (1:2) as eluent to obtain
18 (0.03 g, yield 31%), mp 230-233 °C (from aqueous ethanol). 1H NMR (DMSO-d6): δ 11.07 (s, 1H),
7.69 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 10.4 Hz, 1H), 7.08 (d, J = 3.9 Hz, 1H), 6.93 – 6.90
(m, 1H), 6.67 (d, J = 2.5 Hz, 1H), 2.73 (s, 3H), 1.96 ppm (s, 3H). IR: ν 1712, 1765, 3122, 3293 cm-1.
Anal. Calcd. for C19H13ClN4O2 (364.79), C, H, N, Cl.
1-(2’,4’-Dichlorophenyl)-3-methylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione
(18). Was prepared as 18 from 70. Yield 29%, mp 236-240 °C (from ethanol). 1H NMR (DMSO-d6): δ
11.08 (s, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.77 (dd, J = 2.3 and 8.5 Hz, 1H), 7.09
(m, 1H), 6.94 (m, 1H), 6.80 (m, 1H), 2.73 ppm (s, 3H). IR: ν 1708, 1765, 3094 cm-1. Anal. Calcd. for
C18H10Cl2N4O2 (385.20), C, H, N, Cl.
5-Methy-1-Phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-dione (14). A mixture of
5 (0.3 g, 0.001 mol), methyl iodide (0.17 g, 0.0012 mol) and potassium carbonate (0.21 g, 0.0015 mol)
in DMF (5 mL) was stirred at room temperature for 3 h. After dilution with water, the mixture was
extracted with ethyl acetate, washed with brine and dried. Evaporation of the solvent gave a residue
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that was purified by silica gel column chromatography using ethyl acetate:n-hexane (1:2) as eluent to
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obtain 14 (0.26 g, yield 85%). mp 217-221 °C (from ethanol). H NMR (DMSO-d6): δ 3.05 (s, 3H),
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6.85 (d, J = 2.0 Hz, 1H), 6.95 (t, J = 3.0 Hz, 1H), 7.11 (d, J = 3.1 Hz, 1H), 7.72-74 (m, 5H), 8.41 ppm (s,
1H). Anal. Calcd. for C18H12N4O2 (316.31), C, H, N.
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Sodium 2,3-dicyanoprop-1-en-1-olate (19). A suspension of NaH (60 % dispersion in mineral oil,
0.48 g, 0.012 mol) and iso-amyl alcohol (0.2 mL) in anhydrous toluene (10 mL) was heated at reflux
for 30 min. After cooling at room temperature, a solution of succinonitrile (0.80 g, 0.01 mol) and ethyl
formiate (7.79 g, 8.5 mL, 0.105 mol) in anhydrous toluene (5 mL) was added dropwise. The reaction
mixture was stirred at room temperature overnight. The solid product was collected to provide the
desired compound 19 as a solid (1.2 g, yield 77%), mp 180 °C (dec.). 1H NMR (DMSO-d6): δ δ 8.5 (s,
1H), 2.91ppm (s, 2H) ppm. IR: ν 2181, 2246, 3376 cm-1.
Sodium 3,4-dicyanobut-2-en-2-olate (20). Was prepared as 44 using ethyl acetate. Yield 95%, mp
217-221 °C. 1H NMR (DMSO-d6): δ 3.68 (s, 2H), 2.04 ppm (s, 3H). IR: ν 2161, 2255, 3349 cm-1.
General procedure for the synthesis of derivatives 31-43. Example. 2-[5-Amino-1-(4’-
methylphenyl)-1H-pyrazol-4-yl]acetonitrile (37). A mixture of 19 (3.00 g, 0.023 mol), 4-
methylphenylhydrazine hydrochloride (27) (4.38 g, 0.028 mol) in ethanol (23 mL) was heated at reflux
for 2 h. After cooling, the solvent was evaporated and water was added. The mixture was extracted
with ethyl acetate. The combined organic layers were washed with brine and dried. The crude residue
was purified by silica gel column chromatography using dichloromethane as eluent to give 37 (2.7 g,
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yield 56%), mp 115-119 °C (from from aqueous ethanol). H NMR (CDCl3): δ 7.40 – 7.32 (m, 3H),
7.28 (d, J = 8.1 Hz, 2H), 3.96 (s, 2H), 3.47 (s, 2H), 2.40 ppm (s, 3H). IR: ν 2252, 3152, 3376 cm-1.
2-[5-Amino-1-(phenyl)-1H-pyrazol-4-yl]acetonitrile (31). Was prepared as 37 from 19 and
phenylhydrazine.20
2-[5-Amino-1-(2’-chlorophenyl)-1H-pyrazol-4-yl]acetonitrile (32). Was prepared as 37 from 19,
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2-chlorophenylhydrazine hydrochloride and triethylamine. Yield 54%, mp 98-100 °C (from aqueous
ethanol). 1H NMR (DMSO-d6): δ 7.66 (dd, J = 7.8, 1.5 Hz, 1H), 7.52 (dd, J = 9.5 and 1.8 Hz, 2H), 7.48
– 7.42 (m, 1H), 7.34 (s, 1H), 5.28 (s, 2H), 3.67 ppm (s, 2H). IR: ν 2247, 3199, 3315, 3382 cm-1.
2-[5-Amino-1-(3’-chlorophenyl)-1H-pyrazol-4-yl]acetonitrile (33). Was prepared as 37 from 19
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and 3-chlorophenylhydrazine hydrochloride. Yield 28%, mp 144-146 °C (from toluene). H NMR
(CDCl3): δ 7.70 (t, J = 1.9 Hz, 1H), 7.60 – 7.53 (m, 2H), 7.46 (t, J = 8.0 Hz, 1H), 7.40 – 7.34 (m, 1H),
3.99 (s, 2H), 1.58 ppm (s, 2H). IR: ν 3091, 3315, 3400 cm-1.
2-[5-Amino-1-(4’-chlorophenyl)-1H-pyrazol-4-yl]acetonitrile (34). Was prepared as 37 from 19
and 4-chlorophenylhydrazine hydrochloride. Yield 42% as an oil. 1H NMR (DMSO-d6): δ 7.61 (d, J =
8.8 Hz, 2H), 7.55 (d, J = 8.9 Hz, 2H), 7.39 (s, 1H), 5.51 (s, 2H), 3.69 ppm (s, 2H). IR: ν 2255, 3154,
3369 cm-1.
2-[5-Amino-1-(2’-methylphenyl)-1H-pyrazol-4-yl]acetonitrile (35). Was prepared as 37 from 19
and 2-methylphenylhydrazine hydrochloride. Yield 87% as an oil. 1H NMR (DMSO-d6): δ 7.44 – 7.37
(m, 2H), 7.35 – 7.30 (m, 2H), 7.24 (d, J = 7.5 Hz, 1H), 5.16 (s, 2H), 3.68 (s, 2H), 2.04 ppm (s, 3H). IR:
ν 2248, 3196, 3324 cm-1.
2-[5-Amino-1-(3’-methylphenyl)-1H-pyrazol-4-yl]acetonitrile (36). Was prepared as 37 from 19
and 3-methylphenylhydrazine hydrochloride. Yield 37% as an oil. 1H NMR (DMSO-d6): δ 7.46 – 7.26
(m, 4H), 7.25 – 7.09 (m, 1H), 5.44 (s, 2H), 3.69 (s, 2H), 2.37 ppm (s, 3H). IR: ν 2248, 3197, 3330 cm-
1
.
2-[5-Amino-1-(2’-methoxyphenyl)-1H-pyrazol-4-yl]acetonitrile (38). Was prepared as 37 from
19 and 2-methoxyphenylhydrazine hydrochloride. Yield 10% an an oil. 1H NMR (CDCl3): δ 7.45 (d, J
= 3.2 Hz, 1H), 7.45 – 7.40 (m, 2H), 7.13 – 7.06 (m, 2H), 3.88 (s, 2H), 3.80 (s, 1H), 3.53 ppm (s, 2H).
IR: ν 2247, 2938, 3331 cm-1.
2-[5-Amino-3-methyl-1-(phenyl)-1H-pyrazol-4-yl]acetonitrile (39). Was prepared as 37 from 20
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and phenylhydrazine hydrochloride. Yield 71%, mp 137-140 °C (from aqueous ethanol). H NMR
(DMSO-d6): δ 7.55 (d, J = 7.5 Hz, 2H), 7.47 (t, J = 7.9 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 5.42 (s, 2H),
3.68 (s, 2H), 2.11 ppm (s, 3H). IR: ν 2245, 3175, 3281, 3425 cm-1.
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2-[5-Amino-3-methyl-1-(2’-methylphenyl)-1H-pyrazol-4-yl]acetonitrile (40). Was prepared as
37 from 20 and 2-methylphenylhydrazine hydrochloride. It was used as a crude compound.
2-[5-Amino-1-(2’-chlorophenyl)-3-methyl-1H-pyrazol-4-yl])acetonitrile (41). Was prepared as
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37 from 20 and 2-chlorophenylhydrazine. Yield 51%, mp 140-142 °C (from aqueous ethanol). H
NMR (DMSO-d6) 1H NMR (DMSO-d6): δ 7.64 – 7.60 (m, 1H), 7.52 – 7.39 (m, 3H), 5.22 (s, 2H), 3.64
(s, 2H), 2.08 ppm (s, 3H). IR: ν 3155 cm-1.
2-(5-Amino-1-(2’,4’-dichlorophenyl)-3-methyl-1H-pyrazol-4-yl)acetonitrile (42). Was prepared
as 37 from 20 and 2,4-dichlorophenylhydrazine hydrochloride. Yield 73%, mp 96-98 °C (from aqueous
ethanol). 1H NMR (DMSO-d6): δ 7.80 (d, J = 2.3 Hz, 1H), 7.53 (dd, J = 8.5, 2.3 Hz, 1H), 7.44 (d, J =
8.5 Hz, 1H), 5.34 (s, 2H), 3.63 (s, 2H), 2.07 ppm (s, 3H). IR: ν 2248, 3158, 3282, 3398 cm-1.
2-[5-Amino-1-(4’-chloro-2’-methylphenyl)-3-methyl-1H-pyrazol-4-yl]acetonitrile (43). Was
prepared as 37 from 20 and 4-chloro-2-methylphenylhydrazine hydrochloride. Yield 28% as an oil. 1H
NMR (DMSO-d6): δ 7.45 (s, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 5.20 (s, 2H), 3.63
(s, 2H), 2.07 (s, 3H), 2.04 ppm (s, 3H). IR: ν 2240, 3127, 3392 cm-1.
General procedure for the synthesis of derivatives 44-56. Example. 2-(1-(4’-Methylphenyl)-5-
(1H-pyrrol-1-yl)-1H-pyrazol-4-yl)acetonitrile (50). A mixture of 37 (2.50 g, 0.0118 mol) and 2,5-
dimethoxytetrahydrofuran (1.62 g, 1.59 mL, 0.0123 mol) in acetic acid (12 mL) was heated at reflux
for 30 min. After cooling, the solvent was removed, and the residue was dissolved in water and
extracted with ethyl acetate. The combined organic layers were washed with a solution of sodium
hydrogen carbonate, brine and dried. Removal of the solvent gave a residue that was purified by silica
gel column chromatography using ethyl acetate:n-hexane 1:3 as eluent to obtain 50 (2.0 g, yield 64%),
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mp 74-79 °C (from n-hexane). 1H NMR (DMSO-d6): δ 7.85 (s, 1H), 7.17 (d, J = 8.7 Hz, 2H), 6.99 (d,
J = 8.4 Hz, 2H), 6.90 – 6.80 (m, 2H), 6.33 – 6.23 (m, 2H), 3.82 – 3.72 (m, 2H), 2.30 ppm (s, 3H). IR: ν
2252, 3121 cm-1.
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2-[1-Phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (44). Was prepared as 50 from 31.20
2-(1-(2’-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl)acetonitrile (45). Was prepared as
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50 from 32. Yield 86% as an oil. H (DMSO-d6): δ 7.89 (s, 1H), 7.6 (m, 2H), 7.51 (m, 1H), 7.44 (m,
1H), 6.79 (m, 2H), 6.16 (m, 2H), 3.83 ppm (s, 2H). IR: ν 2251, 3105 cm -1.
2-(1-(3’-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl)acetonitrile (46). Was prepared as
50 from 33. Yield 58% as an oil. 1H NMR (DMSO-d6): δ (CDCl3): δ 7.82 (s, 1H), 7.24 (m, 2H), 7.17 (t,
J = 1.9 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.66(m, 2H), 6.41 (m, 2H), 3.52 ppm (s, 2H). IR: ν 2252,
3105, 3313, 3399 cm-1.
2-[1-(4’-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (47). Was prepared as
50 from 34. Yield 87% as an oil. 1H NMR (DMSO-d6): δ 7.91 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H), 7.09 (d,
J = 8.8 Hz, 2H), 6.91 (t, J = 2.0 Hz, 2H), 6.31 (t, J = 2.0 Hz, 2H), 3.79 ppm. (s, 2H). IR: ν 2252, 3106
cm-1.
2-(1-(2’-Methylphenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl)acetonitrile (48). Was prepared as
50 from 35. Yield 35%, mp 58-63 °C (from toluene). 1H (DMSO-d6): δ 7.86 (s, 1H), 7.32 (m, 4H), 6.77
(m, 2H), 6.15 (m, 2H), 3.82 (s, 2H), 1.95 ppm (s, 3H). IR: ν 2249, 3010, 3117 cm-1.
2-(1-(3’-Methylphenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl)acetonitrile (49). Was prepared as
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50 from 36. Yield 85%, mp 73-77 °C (from aqueous ethanol). H NMR (DMSO-d6 ): δ 7.87 (s, 1H),
7.24 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 6.94 (s, 1H), 6.88 (m, 2H), 6.83 (d, J = 7.8 Hz, 1H),
6.30 (m, 2H), 3.78 (s, 2H), 2.25 ppm (s, 3H). IR: ν 2245 cm-1.
2-[1-(2’-Methoxyphenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (51). Was prepared as
50 from 38. Yield 81% as an oil. 1H NMR (CDCl3): δ 7.81 (s, 1H), 7.37 (m, 1H), 7.31 (dd, J = 1.6 and
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7.8 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.59 (m, 2H), 6.20 (m, 2H), 3.63 (s, 3H),
3.54 ppm (s, 2H). IR: ν 1601, 2250, 2929, 3105 cm-1
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2-[3-Methyl-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (52). Was prepared as 50
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from 39. Yield 58%, mp 100-103 °C (from aqueous ethanol). H NMR (DMSO-d6): δ 7.40-7.25 (m,
3H), 7.05 (m, 2H), 6.87 (dd, J = 3.9 and 1.8 Hz, 2H), 6.30 (dd, J = 3.9 and 1.8 Hz, 2H), 3.70 (s, 2H),
2.34 ppm (s, 3H). IR: ν 2248 cm-1.
2-[3-Methyl-1-(2’-methylphenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (53). Was
prepared as 50 from 40. It was used as a crude compound.
2-[1-(2’-Chlorophenyl)-3-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (54). Was
prepared as 50 from 41. Yield 89% as an oil. 1H NMR (DMSO-d6): δ 7.56 (m, 2H), 7.47 (m, 1H), 7.41
(m, 1H), 6.75 (m, 2H), 6.14 (m, 2H), 3.73 (s, 2H), 2.31 ppm (s, 3H). IR: ν 2252, 3104 cm-1.
2-[1-(2’,4’-Dichlorophenyl)-3-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile (55). Was
prepared as 50 from 42. Yield 93%, mp 105-106 °C (from toluene). 1H NMR (DMSO-d6): δ 7.77 (d, J
= 2.2 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.51 (dd, J = 2.3 and 8.5 Hz, 1H), 6.77 (m, 2H), 6.17 (m, 2H),
3.74 (s, 2H), 2.31ppm (s, 3H). IR: ν 2247, 3092 cm-1.
2-[1-(4’-Chloro-2’-methylphenyl)-3-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]acetonitrile
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(56). Was prepared as 50 from 43. Yield 68% as an oil. H NMR (DMSO-d6): δ 7.40 (s, 1H), 7.26 (s,
2H), 6.78 (m, 2H), 6.16 (m, 2H), 3.72 (s, 2H), 2.31 (s, 3H), 1.95 ppm (s, 3H). IR: ν 2250 cm-1.
General procedure for the synthesis of derivatives 57-67. Example. Ethyl 3-cyano-2-hydroxy-
3-[5-(1H-pyrrol-1-yl)-1-(4’-methylphenyl)-1H-pyrazol-4-yl]acrylate (63). Sodium ethoxide was
freshly prepared from sodium metal (0.13 g, 0.0056 ga) in absolute ethanol (3.74 mL). Diethyl oxalate
(1.07 mL, 0.99 mL, 0.0078 mol) and 50 (1.47 g, 0.0056 mol) were added in sequence. The reaction was
stirred at room temperature for 5 h. The mixture was quenched with water and brought to pH 2 with 1
N HCl. After extraction with ethyl acetate, the combined organic solutions were washed with brine and
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dried. Removal of the solvent afforded 63 (1.6 g, yield 81%), mp 157-162 °C (fom aqueous ethanol).
1H NMR (DMSO-d6): δ 8.08 (s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.79 (m, 2H),
6.20 (m, 2H), 4.28 (q, J = 7.1 Hz, 2H), 2.29 (s, 3H), 1.28 ppm (t, J = 7.1 Hz, 3H). IR: ν 2222, 3236,
3450 cm-1.
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Ethyl 3-cyano-2-hydroxy-3-[5-(1H-pyrrol-1-yl)-1-(phenyl)-1H-pyrazol-4-yl]acrylate (57). Was
prepared as 63 from 44.20
Ethyl 3-[1-(2’-chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]-3-cyano-2-hydroxyacrylate
(58). Was prepared as 63 from 45. Yield 78% as an oil. 1H NMR (DMSO-d6): δ 8.11 (s, 1H), 7.60 (m,
2H), 7.50 (m, 1H), 7.42 (m, 1H), 6.72 (m, 2H), 6.07 (m, 2H), 4.31 (m, 2H), 1.29 ppm (t, J = 7.1 Hz,
3H). IR: ν 1720, 2222 cm-1.
Ethyl 3-[1-(3’-chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]-3-cyano-2-hydroxyacrylate
(59). Was prepared as 63 from 46. Yield 79% as an oil. 1H NMR (DMSO-d6): δ 8.19 (s, 1H), 7.23 (t, J
= 8.0 Hz, 2H), 7.17 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.70 (m, 2H), 6.40 (m, 2H), 4.49 (q, J = 7.1 Hz,
2H), 1.44 ppm (t, J = 7.1 Hz, 3H). IR: ν 1718, 2224 cm-1.
Ethyl 3-[1-(4’-chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazol-4-yl]-3-cyano-2-hydroxyacrylate
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(60). Was prepared as 63 from 47. Yield 62%, mp 59-62 °C (from thyl acetate/n-hexane). H NMR
(DMSO-d6): δ 8.27 (s, 1H), 7.37 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.77 (s, 2H), 6.19 (s, 2H),
4.15 (dd, J = 7.0 and 14.0 Hz, 2H), 1.22 ppm (t, J = 7.2 Hz, 3H). IR: ν 2261, 3361 cm-1.
Ethyl 3-cyano-2-hydroxy-3-[5-(1H-pyrrol-1-yl)-1-(2’-methylphenyl)-1H-pyrazol-4-yl]acrylate
(61). Was prepared as 63 from 48. Yield 76%, mp 137-142 °C (from toluene). 1H NMR (DMSO-d6): δ
8.07 (s, 1H), 7.27 (m, 4H), 6.72 (s, 2H), 6.06 (s, 2H), 4.31 (q, J = 7.0 Hz, 2H), 1.99 (s, 3H), 1.29 ppm (t,
J = 7.1 Hz, 3H). IR: ν 1713, 2221, 3107 cm-1.
Ethyl 3-cyano-2-hydroxy-3-[5-(1H-pyrrol-1-yl)-1-(3’-methylphenyl)-1H-pyrazol-4-yl]acrylate
(62). Was prepared as 63 from 49. Yield 70% as an oil. 1H NMR (DMSO-d6): δ 8.09 (s, 1H), 7.23 (t, J
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