Identification of a Potent Phosphoinositide 3-Kinase Pan Inhibitor Displaying a Strategic Carboxylic Acid Group and Development of Its Prodrugs

Article abstract of DOI:10.1002/cmdc.201700340

Activation of the phosphoinositide 3-kinase (PI3K) pathway is a key signaling event in cancer, inflammation, and other proliferative diseases. PI3K inhibitors are already approved for some specific clinical indications, but their systemic on-target toxicity limits their larger use. In particular, whereas toxicity is tolerable in acute treatment of life-threatening diseases, this is less acceptable in chronic conditions. In the past, the strategy to overcome this drawback was to block selected isoforms mainly expressed in leukocytes, but redundancy within the PI3K family members challenges the effectiveness of this approach. On the other hand, decreasing exposure to selected target cells represents a so-far unexplored alternative to circumvent systemic toxicity. In this manuscript, we describe the generation of a library of triazolylquinolones and the development of the first prodrug pan-PI3K inhibitor.

Full text of DOI:10.1002/cmdc.201700340

DOI: 10.1002/cmdc.201700340
Full Papers
Identification of a Potent Phosphoinositide 3-Kinase Pan
Inhibitor Displaying a Strategic Carboxylic Acid Group and
Development of Its Prodrugs
Tracey Pirali+,[a] Elisa Ciraolo+,[b] Silvio Aprile,[a] Alberto Massarotti,[a] Alex Berndt,[c]
Alessia Griglio,[a] Marta Serafini,[a] Valentina Mercalli,[a] Clarissa Landoni,[a]
Carlo Cosimo Campa,[b] Jean Piero Margaria,[b] Rangel L. Silva,[d] Giorgio Grosa,[a]
Giovanni Sorba,[a] Roger Williams,[c] Emilio Hirsch,*[b, e] and Gian Cesare Tron*[a, e]
Activation of the phosphoinositide 3-kinase (PI3K) pathway is a
key signaling event in cancer, inflammation, and other prolifer-
ative diseases. PI3K inhibitors are already approved for some
specific clinical indications, but their systemic on-target toxicity
limits their larger use. In particular, whereas toxicity is tolerable
in acute treatment of life-threatening diseases, this is less ac-
ceptable in chronic conditions. In the past, the strategy to
overcome this drawback was to block selected isoforms mainly
expressed in leukocytes, but redundancy within the PI3K
family members challenges the effectiveness of this approach.
On the other hand, decreasing exposure to selected target
cells represents a so-far unexplored alternative to circumvent
systemic toxicity. In this manuscript, we describe the genera-
tion of a library of triazolylquinolones and the development of
the first prodrug pan-PI3K inhibitor.
Introduction
The discovery of phosphoinositide 3-kinases (PI3Ks) dates back
to 1985 when, with the seminal work of Cantley et al.,[1] the
specific lipid kinases able to phosphorylate the alcoholic group
selectively at the 3-position of phosphatidylinositols were re-
ported. Three subfamilies of PI3Ks have been identified so far,
referred to as classes I, II, and III. Class I is the most studied and
comprises four isoforms, PI3Ka, PI3Kb, PI3Kd, and PI3Kg, that
share some structural features but that are distinct for protein
domains, regulatory subunits, and activation mechanism.[2] All
class I PI3Ks can catalyze the in vivo conversion of phosphati-
dylinositol 4,5-bisphosphate (1, PIP2) into phosphatidylinositol
3,4,5-trisphosphate (2, PIP3) (Figure 1). PIP3 production induces
the recruitment to the plasma membrane of 3-phosphoinosi-
tide-dependent protein kinase-1 (PDK1), which in turn activates
protein kinase B (Akt) through phosphorylation on Thr308.
Thirty years of basic research on PI3Ks have unequivocally
demonstrated their involvement in a plethora of biological
processes such as cell growth, proliferation, differentiation, and
motility.[3] Furthermore, hyperactivation of the PI3K/Akt signal-
ing pathway has been linked to different pathologies, such as
cancer and autoimmune diseases.[4,5] Studies performed with
the use of animal models and PI3K inhibitors have established
PI3Ka and PI3Kb as promising targets for the treatment of
human cancer.[6] Concurrently, the central role of PI3Kg and
PI3Kd in leukocyte biology sustains their inhibition as a promis-
ing therapeutic approach in a variety of inflammatory diseas-
es.[5] These findings have prompted both academia and indus-
try research to develop inhibitors targeting all PI3K isoforms.
Most of these molecules act by antagonizing the binding of
ATP to the PI3K catalytic pocket. To date, hundreds of pan-
and tens of isoform-selective PI3K inhibitors have been discov-
ered, and some of them are in clinical evaluation for tumor
treatment (Figure 2).[6–8] These efforts culminated in 2014 with
the FDA approval of idelalisib (8), a PI3Kd-selective inhibitor for
the treatment of patients with relapsed follicular B-cell non-
Hodgkin lymphoma or relapsed small lymphocytic lymphoma
(SLL).[9]
[a] Prof. T. Pirali,+ Dr. S. Aprile, Dr. A. Massarotti, Dr. A. Griglio, Dr. M. Serafini,
Dr. V. Mercalli, Dr. C. Landoni, Prof. G. Grosa, Prof. G. Sorba, Prof. G. C. Tron
Dipartimento di Scienze del Farmaco, Universitꢀ degli Studi del Piemonte
Orientale “A. Avogadro”, Largo Donegani 2, 28100 Novara (Italy)
E-mail: giancesare.tron@uniupo.it
[b] Dr. E. Ciraolo,+ Dr. C. C. Campa, Dr. J. P. Margaria, Prof. E. Hirsch
Department of Molecular Biotechnology and Health Sciences, University of
Torino, Via Nizza 52, 10126 Torino (Italy)
E-mail: emilio.hirsch@unito.it
[c] Dr. A. Berndt, Prof. R. Williams
MRC Laboratory of Molecular Biology, Medical Research Council, Cambridge
CB2 0QH (UK)
[d] Dr. R. L. Silva
Department of Pharmacology, Ribeir¼o Preto Medical School, University of
S¼o Paulo, Avenida Bandeirantes 3900, 14049-900 Ribeir¼o Preto (Brazil)
[e] Prof. E. Hirsch, Prof. G. C. Tron
Kither Biotech S.r.l., Molecular Biotechnology Center, Via Nizza 52, 10126
Torino (Italy)
[+] These authors contributed equally to this work.
A more accurate analysis of the structure of already-reported
PI3K inhibitors[10] highlighted the lack of molecules displaying
a carboxylic acid group that we here report to be fundamental
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201700340.
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Figure 1. Class I PI3Ks phosphorylate the 3-hydroxy group of phosphatidylinositol 4,5-bisphosphates. PTEN=phosphatase and tensin homologue.
Figure 2. A selection of PI3K inhibitors in clinical trials, and the molecular structure of idelalisib.
for binding to the PI3 kinase structural motif. Furthermore, this
functional group can undergo chemical modifications with the
intent to develop useful prodrugs. Indeed, this approach
might circumvent nonspecific targeting and avoid systemic
side effects associated with the use of a pan-PI3K inhibitor, es-
pecially if proposed for topical use. Therefore, in the present
manuscript we report the discovery of 37, a potent pan-PI3K
inhibitor displaying a strategic and pivotal carboxylic acid
group, which can be esterified to provide prodrugs. The syn-
thesis of 37 and the metabolic stability of its prodrugs, along
with their full biological characterization, are discussed herein.
A hydrogen bond is formed between the morpholino
oxygen atom and the backbone amide group of residue
Val882 of the hinge region, and this bond mimics the interac-
tion that ATP establishes with the enzyme. There is also a puta-
tive hydrogen bond between Lys833 and the carbonyl group.
The phenyl ring is located in a pocket, usually occupied by the
ribose of ATP, formed by Met804 and Trp812 on one side and
Met953 on the other, and it protrudes toward the solvent-ac-
cessible part of the binding pocket. Interestingly, 9 does not
extend into the phosphate-binding region.
We therefore reasoned that the phenyl ring could be re-
placed with a 1,4-disubstituted 1,2,3-triazole by a click-chemis-
try approach.[13] In this case, the heterocycle ring would
behave as a mere linker, delivering an extra site R (Figure 4),
that might establish additional interactions with the close
amino-acid residues, either enhancing the potency of 9 or im-
parting selectivity. Indeed, it is important to highlight that the
rim of the binding site of class I PI3Ks is the region of the
enzyme for which there is major variability in the amino-acid
composition among all the four isoforms,[14] and therefore, it is
more amenable to be targeted in the search for selectivity.
Owing to the versatility and functional-group tolerance of the
Fokin–Sharpless reaction,[15] a combinatorial approach was ex-
ploited that would allow exploration of a vast array of different
functional groups in the R portion (Figure 4). It is important to
highlight that modification of the phenyl ring of 9 was already
Results and Discussion
Chemistry
Design, synthesis, and enzyme inhibition
On the basis of the considerations discussed above, we decid-
ed to start our project by modifying compound LY294002 (9,
Figure 3), one of the first pan-PI3K inhibitors discovered.[11] This
quercetin analogue competes with ATP in the ATP-binding site
of PI3Ks, with an IC50 value of ~0.5 mm against all of the iso-
forms. The X-ray structure of 9 bound to PI3Kg is also avail-
able,[12] and it shows that the morpholino ring partially over-
laps the volume occupied by the adenine in the ATP–enzyme
complex (Figure 3).
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Figure 4. LY294002 (9), TGX155 (11), and design of new analogues by a
click-chemistry approach.
to diversify the chemical nature of the R group by exploiting
different azides 13. The azides were preferred as decorating
groups rather than alkynes because their chemical preparation,
starting from commercially available amines, is easier than the
more challenging synthesis of alkynes.
Once the goal was fixed, we proceeded with the synthesis
of the pivotal alkyne. The preparation of 12 started with the
reaction between Meldrum’s acid (14) and CS2 (15). The inter-
mediate was then methylated in situ to give 16.[17] Subse-
quently, this compound underwent a first nucleophilic dis-
placement with 2-iodoaniline (17) at reflux to give 18, which
was then treated with morpholine (19) in THF at reflux to give
the second nucleophilic substitution to lead to compound 20.
Heating compound 20 in diphenyl ether at 1708C triggered in-
tramolecular aromatic nucleophilic substitution followed by a
decarboxylation reaction to afford iodo intermediate 21. Sono-
gashira coupling with trimethylsilyl acetylene gave 22 and sub-
sequent deprotection under basic conditions afforded desired
alkyne 12 (Scheme 1).
Once alkyne 12 was obtained, a combinatorial parallel syn-
thesis with 30 azides was performed. To this point, before
starting the construction of the library, a preliminary model re-
action with phenyl azide (23) was attempted, which revealed
that under the standard conditions of the Fokin–Sharpless click
reaction (CuSO4·5H2O, sodium ascorbate, tBuOH/H2O) unex-
pected compound 24 was obtained instead of desired triazole
25. It was reasonable to suppose that copper triggered intra-
molecular cyclization between the N1 atom and the triple
bond, with the formation of a 6-oxopyrrolquinolinic ring
(Scheme 2).[18] In light of this event, we reasoned that in the
presence of an already-chelated copper ion, this transforma-
tion might be suppressed. The ligand tris[(1-benzyl-1H-1,2,3-tri-
azol-4-yl)methyl]amine (TBTA, also known as the Chan ligand)
was our first choice. It was previously shown that TBTA was
indeed able to stabilize the oxidation state of the copper(I) ion
by encapsulating it in its structure and preventing other inter-
actions.[19] The use of the Chan ligand was successful, and
under optimized reaction conditions (THF/H2O, Cu(OAc)2,
Figure 3. X-ray structures of pig PI3Kg in complex with a) ATP (PDB ID: 1E8X)
and c,d) 9 (PDB ID: 1E7V). b) 2D structure of 9. Protein is shown in pale-
green cartoon; ligands atoms are shown as sticks. Hydrogen-bonding inter-
actions are plotted as yellow dotted lines. The 9–protein complex is shown
from two different points of view: b) front and c) side.
attempted and described. For example, TGX155 (11), in which
the phenyl ring is replaced with a 4-fluoro-2-methylphenoxy
moiety, was shown to be a selective, potent inhibitor of the b
isoform of PI3K and a potential therapeutic agent for cardio-
vascular diseases.[16] This example corroborates our hypothesis
to replace the phenyl ring of 9. In our specific case, we opted
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Scheme 1. Synthesis of alkyne 12. TEA=triethylamine, TMS=trimethylsilyl, DIPEA=N,N-diisopropylethylamine.
Table 1. Inhibitory activity against different PI3K isoforms.[a]
Compd
R
PI3K isoform
a
b
g
d
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
phenyl
3-hydroxyphenyl
+
À
À
À
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
+
À
À
+
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
+
À
À
+
À
À
À
À
À
À
+
À
À
À
À
À
À
À
À
À
+
À
+
À
À
À
À
À
À
À
+
À
À
+
À
À
À
À
À
+
3-hydroxy-4-methoxyphenyl
4-chlorophenyl
4-(trifluoromethoxy)phenyl
4-methoxy-3-nitrophenyl
2-(4-(methoxycarbonyl)benzyl
4-aminophenyl
4-(cyanomethyl)phenyl
naphthalen-2-yl
3-methoxyphenyl
Scheme 2. Optimization of the click reaction.
sodium ascorbate, TBTA), desired 1,2,3-triazole 25 was ob-
tained in excellent yield (Scheme 2).
quinolin-3-yl
3-carboxybenzyl
With this procedure in hand, we proceeded with our combi-
natorial strategy. The azides were selected to introduce both
electron-donating and electron-withdrawing groups, hydro-
gen-bond donors and acceptors, and ionizable functions. They
were already present in our database or prepared according to
standard procedures. The feasibility of this approach was dem-
onstrated by precipitation of the resultant products, which
were purified by filtration and subsequent water/diethyl ether
washes. This procedure resulted in the formation of 30 com-
5-acetamido-2-methoxyphenyl
5-methoxy-2-(trifluoromethyl)phenyl
4-hydroxy-2-methylphenyl
2-(hydroxymethyl)phenyl
4-(methoxycarbonyl)phenyl
3,4-dimethoxyphenyl
4-methoxyphenyl
4-carboxyphenyl
benzo[d][1,3]dioxol-5-yl
3,5-dimethoxyphenyl
benzyl
3-(methoxycarbonyl)phenyl
4-sulfamoylphenyl
2-methoxybenzyl
3-carboxyphenyl
naphthalen-1-yl
2-hydroxyphenyl
1
pounds, which were submitted to MS and H NMR spectrosco-
py analysis to verify their purity (>95%) and structural identity.
Then, the 1,2,3-triazoles were evaluated for their ability to in-
hibit PI3K isoforms of class I at a fixed concentration of 1 mm,
as shown in Table 1. The synthesized 1,2,3-triazoles along with
their ability to inhibit PI3K isoforms at 1 mm concentration are
reported. Possibly, a minute concentration of copper salts
might have precipitated with the products and might have
been retained in the purified compounds, and so, we also eval-
[a] PI3K inhibition determined at 1 mm with at least triplicate determina-
tions in multiple experiments; ’+’ refers to those compounds able to in-
hibit PI3K isoform activity by more than 50%.
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uated the PI3K inhibitory activity of these salts. Nei-
ther copper(II) acetate nor copper(I) iodide displayed
significant inhibition of PI3Ks up to 10 mm.
Only those compounds able to suppress the
kinase activity by 50% of at least one isoform (i.e.,
25, 35, 37, 45, 48, and 54) at 1 mm were resynthe-
sized, purified by column chromatography, and re-
evaluated to obtain a precise IC50 value.
Compound 37 emerged from the screening as the
most-potent inhibitor with higher selectivity against
PI3Ka (IC50 =18 nm) and PI3Kd (IC50 =19 nm) than
against PI3Kb (IC50 =59 nm) and PI3Kg (IC50 =
186 nm). Its inhibitory nature was tested on other
PI3Ks of classes II and III (Table 2 and Figure 5), dem-
onstrating its selectivity over class I isoforms.
Figure 5. Inhibition profile of 37 against a) class I and b) classes II and III PI3Ks. Each PI3K
isoform (30 ng) was incubated with various concentrations of 37 in the presence of ATP
(10 mm for PI3Ka, PI3Kb, PI3Kg, PI3Kd, PI3K-C2a, and PI3K-C3; 100 mm for PI3K-C2g and
PI3K-C2b).[20] ATP loss and consequent ADP production were measured though a lumino-
metric assay. The residual PI3K activity after treatment with different concentrations of
37 was calculated as a percentage of the untreated control (recombinant protein+DM-
SO). The dose–response curve was obtained by plotting the PI3K activity [%] versus the
logarithms of the inhibitor concentrations. Data are the meanÆSD of at least two inde-
pendent experiments each performed in triplicate.
These multiple experiments highlight the pivotal
role played by the carboxylic acid if positioned in the
meta position on the benzyl ring in increasing the
potency of this class of compounds. Therefore, start-
ing from 37, we decided to replace the 3-methylben-
zoic acid group with three linear aliphatic carboxylic
acids, namely, propionic acid, butyric acid, and pentanoic acid
(i.e., to give compounds 55, 56, and 57, respectively), and the
carboxyl moiety with its canonical bioisosteres such as sulfona-
mide (i.e., compound 58); primary (i.e., compound 59), secon-
dary (i.e., compounds 60 and 61), and tertiary (i.e., compound
62) amides; and finally tetrazole (i.e., compound 63). All the at-
tempted substitutions were detrimental to inhibition activity
(Table 3).
To investigate the binding of 37 in the enzyme active site,
the crystal structure of 37 bound to the murine d isoform of
PI3K was resolved (see the Supporting Information for techni-
cal details). Compound 37 binds in the ATP binding site in a
canonical mode (Figure 7), as described for other type I kinase
inhibitors. In detail, the morpholine ring establishes a typical
hydrogen bond with Val882 in the hinge region, analogously
to 9; the quinolone ring is in a central pocket with an orienta-
tion very similar to that of 9 (Figure 7a), and the carbonyl
group is involved in a putative hydrogen-bonding interaction
with Asp911. The 1,2,3-triazole appears as fundamental to ori-
entate the carboxylic acid group properly. Indeed, the X-ray
structure of 9 shows a perpendicular orientation of the phenyl
ring to the central core, whereas 37 is characterized by copla-
nar orientation of the 1,2,3-triazole to the quinolinone core.
This different spatial disposition allows for a pivotal ionic salt-
We then proceeded to evaluate the ATP competitive inhibi-
tory nature of 37 through an enzymatic kinetic study by using
an in vitro assay to measure the PI3Kd activity at various con-
centrations of ATP and 37. As shown in Figure 6, Lineweaver–
Burk plot analysis revealed that 37 acts as an ATP-competitive
inhibitor for PI3Kd, as the plots (straight lines) intersect on the
1/v axis (v=rate of ADP production).
Table 2. IC50 values of 25, 35, 37, 45, 48 and 54 on different PI3K isoforms.[a]
IC50 [mm]
TGX155
>10
25
35
37
45
48
54
PI3Ka
PI3Kb
PI3Kg
PI3Kd
PI3Kc2a
PI3Kc2b
PI3Kc2g
PI3Kc3
0.74Æ0.31
5.5Æ2.5
3.0Æ1.0
0.61Æ0.27
n.d.
1.3Æ0.7
9.3Æ5.8
4.2Æ2.1
0.81Æ0.36
n.d.
0.018Æ0.004
0.059Æ0.015
0.186Æ0.043
0.019Æ0.004
3.7Æ1.1
0.32Æ0.2
0.30Æ0.2
0.30Æ0.2
0.17Æ0.1
n.d.
n.d.
n.d.
n.d.
2.65Æ0.8
0.10Æ0.04
1.0Æ0.5
0.47Æ0.26
n.d.
0.26Æ0.12
1.1Æ0.5
1.4Æ0.44
0.35Æ0.17
n.d.
0.096Æ0.007
9.40Æ1.2
0.41Æ0.13
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>20
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2.8Æ0.7
n.d.
0.59Æ0.2
[a] Values are the means of at least three determinations in multiple experiments, with 95% confidence intervals; n.d.: not determined.
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Table 3. IC50 values of analogues of 37 against PI3Ka.[a]
Compd
R
IC50 [mm]
>1
>1
>1
>1
55
56
57
58
59
60
61
62
63
3-carboxypropyl
4-carboxybutyl
5-carboxypentyl
3-[(methylsulfonyl)carbamoyl]benzyl
3-carbamoylbenzyl
3-(benzylcarbamoyl)benzyl
3-(cyclopropylcarbamoyl)benzyl
3-(diethylcarbamoyl)benzyl
3-(1H-tetrazol-5-yl)benzyl
0.800Æ0.74
0.140Æ0.09
0.250Æ0.08
0.290Æ0.08
>1
[a] Values are the means of at least three determinations in multiple ex-
periments, with 95% confidence intervals; n.d.: not determined. See the
Experimental Section for the synthesis of compound 63.
Figure 6. Inhibition mode of 37 against PI3Kd isoform. Lineweaver–Burk
plot, 1/v versus 1/[ATP]. The inhibition assay was performed by using vary-
ing concentrations of ATP (5, 10, 25, 50, and 100 mm) and fixed concentra-
tions of PI3Kd recombinant protein (2.4 mgmLÀ1) and lipids (1 mgmLÀ1 PI/PS
lipid micelles mixture) in the presence or absence of different concentrations
of 37 (5, 10, and 20 nm).
Figure 7. X-ray structures of murine PI3Kd in complex with 37 (PDB ID:
5NGB). Protein is shown in pale-green cartoon; ligand is shown as sticks
with carbon atoms depicted in orange. Hydrogen-bonding and ionic interac-
tions are plotted as yellow and pink dotted lines, respectively. The ligand–
protein complex is shown from different points of view: a) top with the crys-
tal structure of 9 (PDB ID: 1E7V) superposed as pink sticks, b) front, and
c) side.
bridge interaction between the carboxyl moiety and Lys708,
which is consistent with the observed potent inhibitory activi-
ty.
Figure 8, 37 did not affect the PI3K signaling pathway, as it did
not decrease Akt phosphorylation.
Biological assays
We therefore reasoned that the lack of activity of 37 in cell-
based experiments could be ascribed to its inability to cross
cell membranes owing to the increased polarity imparted by
the ionized carboxylic acid. For this reason, we prepared corre-
sponding methyl ester 64. A lipid kinase assay confirmed that
64 was not able to inhibit PI3Ka activity at 100 nm (see the
Supporting Information), which confirmed the crucial role
played by the carboxyl group in the interaction with PI3Ks.
Nevertheless, given its hydrophobic nature, it was conceivable
that the methyl ester could cross the membrane and, after in-
Cellular inhibitory activities
After identifying 37 as a promising candidate, we started to
evaluate it in cell-based assays to define its inhibitory activity
on the PI3K signaling pathway. Therefore, we selected an in vi-
tro insulin model to assess the inhibitory effect of 37 in PI3K
signaling. NIH3T3 cells were treated with different concentra-
tions of 37, stimulated with insulin, and the amount of phos-
phorylated Akt was detected. Nonetheless, as shown in
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Figure 8. Treatment with 64 prodrug inhibited PI3K/Akt signaling. NIH3T3
cells were harvested for 12 h and were treated with the indicated concentra-
tions of 37 and 64. Cells were next stimulated with 1 mm insulin for 5 min,
and total cell lysates were prepared. Immunoblot analysis was conducted
for the expression levels of p-AKT and GAPDH. The immunoblot is represen-
tative of three independent experiments.
tracellular enzymatic hydrolysis, explicate its inhibitory activity.
To demonstrate that 64 could act as a prodrug, we treated
NIH3T3 cells with different concentration of 64, and we then
analyzed Akt phosphorylation after insulin stimulation.
As shown in Figure 8, methyl ester 64 was able to inhibit
the PI3K-signaling pathway in a dose-dependent manner.
To determine whether 64 could inhibit PI3K/Akt in tumor
cells, in which the PI3K signaling is hyperactive, PC3 prostatic
tumor cells were incubated with different concentrations of 64
(Figure 9). Treatment with 64 attenuated, in a dose-dependent
manner, the levels of p-Akt, as well as the levels of some Akt
effectors such us p-PRAS40, p-p70S6K, p-GSK3a/b, and p-
FOXO3A.
Figure 9. Treatment with 64 dose dependently inhibited PI3K/Akt signaling
in PC3 cells. PC3 cells were treated with the indicated concentrations of 64
for 12 h. At the end of treatment, cells were harvested and total cell lysates
were prepared. Immunoblot analysis was performed to analyze the expres-
sion levels of p-Akt (T308 and S473), p-PRAS40 (T246; a target of Akt), as
well as the levels of p-p70S6K (T389), p-GSK3a/b (S9), and p-FOXO3A (T32).
The figure is representative of at least three independent experiments.
We next determined the effect of 64 on cell proliferation,
cell cycle, and apoptosis. As shown in Figure 10a, 72 h of treat-
ment with 64 dose-dependently decreased proliferation of PC3
cells. In addition, after 48 h of treatment, 64 was able to
induce cell-cycle arrest by significantly increasing the percent-
age of cells in the G0–G1 phase with concomitant reduction in
the percentage in the S and G2/M phases (Figure 10b). On the
other hand, 64 did not show any effect in inducing cell apop-
tosis (see the Supporting Information).
Figure 10. Compound 64 decreased proliferation and induced cell-cycle
arrest. a) Proliferation curve of PC3 cells. Cell proliferation was analyzed by
using MTT cell-proliferation assay kits. The growth was calculated as the per-
centage of the untreated control and a dose–response curve was generated
by plotting the percentage of growing cells versus the logarithm of inhibitor
concentration. The curve represents the average of three independent ex-
periments. b) PC3 were treated with 10 mm 64 for 48 h. At the end of treat-
ment, cells were washed with 1ꢁ phosphate-buffered saline and were
stained with propidium iodide. The percentage of cells in the G1/G0, S, and
G2/M phases was determined by flow cytometry. Columns represent the
mean of three independent experiments; bars represent the SEM; **p<0.01,
***p<0.001.
Prodrug selection and metabolic stability
The opportunity to generate different prodrugs by acting on
the carboxylic acid group stimulated the design and synthesis
of a panel of different esterified products. It is important to
highlight that by browsing the literature data on PI3K inhibi-
tors it emerged that only one other reported PI3Ka inhibitor
displayed a carboxylic acid, but in this case, the corresponding
methyl ester was still able to inhibit the PI3 kinases and was
only four times less active than the corresponding carboxylic
acid analogue (IC50 on PI3Ka: 46.0 versus 10 nm).[21]
Twelve esters were prepared (see the Experimental Section
for their synthesis and the Supporting Information for chroma-
tographic methods) and evaluated in terms of metabolic stabil-
ity. For this task, a combined approach based on microsomal
and plasmatic stability assays was exploited considering the
relevance of the plasmatic and hepatic tissues in esterase activ-
ity. Microsomal stability of the selected esters was evaluated
by monitoring the disappearance of the substrate incubated in
rat liver microsomes (RLM) at 50 mm concentration, in the pres-
ence of NADPH. In Table 4, the percentage of the remaining
substrate, after incubation (t=60 min), with respect to the ini-
tial amount is reported. Overall, the compounds underwent a
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branched ester 69 and long, linear ester 76, which were inert
to the mouse plasma hydrolases, the other compounds under-
went relevant hydrolytic cleavage with t1/2 values ranging from
<1 to 15 min. In detail, short-linear esters 67 and 73 in addi-
tion to benzyl derivative 65, CH2-pyridin-4-yl derivative 70, and
piperonyl derivative 72 were rapidly hydrolyzed, and their t1/2
values were less than 1 min (see the Supporting Information).
The biological activity of each prodrug was tested by analyz-
ing their ability to decrease PC3 proliferation as a rapid test to
evaluate the PI3Ks inhibitory activity after hydrolysis in a cell-
based assay. PC3 cells were incubated with 10 mm of each pro-
drug: compounds able to decrease proliferation to an extent
more than 50% were defined as active, whereas those reduc-
ing to an extent less than 50% were considered inactive. The
choice of using such a cut-off was due to the intent to identify
rapidly which prodrug could be hydrolyzed in the assay to re-
lease the active compound. As expected, a good correlation
between rate of hydrolysis and cellular activity was observed,
and esters 64–68 and 70–73 were determined to be good can-
didates for topical use. We decided to focus our attention on
ester 64, as its half-life was a good compromise for a prodrug.
The EC50 value of this ester was then evaluated on PC3 cells,
and a value of (2.3Æ0.8) mm was obtained.
Table 4. Synthesized prodrugs, metabolic stability (based on LC–UV anal-
ysis), and effect on PC3 proliferation.[a]
Compd
R
Residual
microsomal
substrate [%]
(t=60 min)
Mouse
plasma
t1/2 [min]
Cell
proliferation
64
65
66
67
68
69
70
71
72
73
74
75
76
Me
Bn
iPr
Et
Bu
tBu
20
53
16
24
53
79
0
18
83
39
60
80
100
13.3
<1
15.1
4.5
<1
N.C.
<1
2.5
<1
<1
4.2
4.4
N.C.
active
active
active
active
active
inactive
active
active
active
active
CH2-pyridin-4-yl
(CH2)2-morpholinyl
piperonyl
isopentyl
1-methylbutanyl
(CH2)10Me
inactive
inactive
inactive
(CH2)17Me
Conclusions
[a] N.C.: not calculable as hydrolytically stable. Active: able to reduce pro-
liferation to an extent greater than 50%; inactive: able to reduce prolifer-
ation to an extent less than 50%.
In conclusion, we report herein the discovery of 3-{[4-(2-mor-
pholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-triazol-1-yl]me-
thyl}benzoic acid (37), a novel, potent pan-phosphoinositide 3-
kinase (PI3K) inhibitor that bears a strategic carboxylic acid,
and the design of its ester prodrugs. Esterification of 37 gener-
ated molecules that were inactive in the enzymatic assay but
that could cross cell membranes, and after ester hydrolysis,
they inhibited the PI3Ks. To date, few prodrug PI3K inhibitors
have been reported. One is a prostate-cancer-specific PI3K in-
hibitor generated by coupling a chemically modified form of
the quercetin analogue LY294002 with the peptide Mu-
LEHSSKLQL, in which the internal sequence HSSKLQ is a sub-
strate for the prostate-specific antigen protease.[23] The second
prodrug was derived by conjugation of LY294002 to the amino
terminus of the Arg-Gly-Asp-Ser integrin binding tetrapeptide
through a morpholinium methylsuccinate.[24] However, in both
prodrugs the biologically active portion was able to cross cell
membranes, which raises questions on side effects mediated
by uncontrolled systemic inhibition of PI3Ks. Recently, Abbott
et al. reported the generation of tetrazole analogues of
LY294002 active on the a isoform of PI3K only if delivered as a
prodrug.[25] Although different cancer cell lines were used, an
EC50 value comparable with the one displayed by our com-
pound, methyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-
8-yl)-1H-1,2,3-triazol-1-yl]methyl}benzoate (64), was reported
[EC50 =(2.1Æ1.1) mm on MCF-7 versus (2.3Æ0.8) mm on PC3].
Although pan-PI3K inhibitors are emerging as a new oppor-
tunity in the treatment of non-cancer-related diseases, data de-
rived from clinical trials has demonstrated that treatment with
pan-PI3K inhibitors causes several side effects, including
nausea, diarrhea, rash, and increased released of insulin in
relevant metabolic transformation, and the substrate depletion
ranged from 0 (for 76) to 100% (for 70). This is mainly due to
hydrolysis of the ester function, whereas the metabolites aris-
ing from the oxidation catalyzed by the NADPH-dependent
monooxygenase system were negligible from a quantitative
point of view. Indeed, the main metabolite formed during the
incubation of the esters was corresponding carboxyl derivative
37. Furthermore, the addition of NADPH did not significantly
increase the oxidative metabolic transformation (see Figure S3
in Supporting Information). As expected, the aliphatic esters
were found to be hydrolytically labile, and ethyl ester 67 was
less stable than methyl ester 64,[22] whereas long, linear alka-
noates 75 and 76 and branched esters 69 and 74 showed
greater stability. Moreover, whereas CH2-pyridin-4-yl ester 70
was completely metabolized, corresponding benzyl derivative
65 and piperonyl derivative 72 were more hydrolytically stable,
and their residual substrates were 53 and 83%, respectively.
The hydrolytic stability of the selected esters was also evaluat-
ed by monitoring the depletion of the substrate incubated at
a 50 mm concentration in mouse plasma over a period of
30 min. As expected, the plasmatic metabolic fate of the esters
was characterized by the formation of 37 as the only metabo-
lite (see Figure S4). The rate constant k [minÀ1], derived for the
substrate peak area versus time curve (single-exponential
decay equation—GraphPad Prism software, Inc., San Diego, CA
(USA); see Figure S5) was used to calculate the in vitro half-life
t
1/2 [min], and the values are reported in Table 4. Except for
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2-iodoaniline (400 mg, 1.83 mmol) in ethanol (4 mL) was heated at
808C for 4 h. During the reaction, the product precipitated. Filtra-
tion yielded 18 as a yellow solid (522 mg, 68%).
plasma. Therefore, the unique properties of the prodrugs de-
scribed in this manuscript might be considered of great rele-
vance in the context of systemic toxicity management. Indeed,
these derivatives might explicate their activity exclusively
within the cell, after conversion into 37, which might in turn
decrease systemic toxicity. Their short half-lives, both in micro-
somes and plasma, highlight the therapeutic potential in topi-
cal administration (e.g., aerosol therapy or epicutaneous treat-
ment), which might overcome toxicity and avoid systemic ex-
posure. Indeed, several pieces of evidence suggest the use of
pan-PI3K inhibitors in topical administration.[26,27] Further stud-
ies are underway and will be performed to demonstrate the ef-
ficacy of compound 64 in different mouse models of chronic
inflammation, for which topical administration would be bene-
ficial, such as asthma and psoriasis, and to evaluate its system-
ic toxicity.
5-[(2-Iodophenylamino)(morpholino)methylene]-2,2-dimethyl-
1,3-dioxane-4,6-dione (20): A mixture of 5-[(2-iodophenylamino)-
(methylthio)
methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione
(500 mg, 1.23 mmol) and morpholine (214 mL, 2.46 mmol) in THF
(5 mL) was heated at reflux overnight. The volatiles were evaporat-
ed under reduced pressure. Diethyl ether (10 mL) was added, and
the solid was filtered and washed with EtOAc (10 mL) to give 20 as
a white powder (429 mg, 76%).
8-Iodo-2-morpholinoquinolin-4(1H)-one (21): 5-[(2-Iodophenyla-
mino)(morpholino)methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione
(1 g, 2.18 mmol) was heated in diphenyl ether (10 mL) at reflux for
30 min. The crude brown oil, after purification by column chroma-
tography (petroleum ether/EtOAc 5:5 to 0:1), yielded 21 as a white
powder (519 mg, 67%).
2-Morpholino-8-[(trimethylsilyl)ethynyl] quinolin-4(1H)-one (22):
Under a nitrogen atmosphere, PdCl2(PPh3)2 (6 mg, 0.00843 mmol),
CuI (5 mg, 0.0252 mmol), DIPEA (48 mL, 0.280 mmol), and trimethyl-
silylacetylene (39 mL, 0.280 mmol) were added to a solution of 8-
iodo-2-morpholinoquinolin-4(1H)-one (100 mg, 0.280 mmol) in tol-
uene (2 mL) and DMF (two drops). The mixture was stirred at room
temperature for 2 h. The mixture was diluted with water (10 mL)
and extracted with EtOAc (5ꢁ10 mL). The combined organic layer
was dried with sodium sulfate. The crude material was purified by
column chromatography (EtOAc/MeOH 1:0 to 95:5) to yield 22 as a
yellow solid (66 mg, 72%).
Experimental Section
General procedures
Commercially available reagents and solvents were purchased
from Aldrich or Alfa Aesar and were used without further purifica-
tion. Tetrahydrofuran (THF) and toluene were distilled immediately
before use from Na/benzophenone under a slight positive atmos-
phere of dry nitrogen. Ethylenediamine was distilled immediately
before use from CaH2 under a slight positive atmosphere of dry ni-
trogen. N,N’-Dimethylformamide (DMF) was distilled under vacuum
from KOH and was stored on activated molecular sieves (4 ꢂ). Di-
chloromethane was dried by distillation from P2O5 and was stored
on activated molecular sieves (4 ꢂ). If needed, the reactions were
performed in flame- or oven-dried glassware under a positive pres-
sure of dry nitrogen. Melting points were determined in open
glass capillaries with a Stuart scientific SMP3 apparatus. Com-
pounds were checked by IR (FTIR THERMO-NICOLET AVATAR),
1H NMR and 13C NMR APT (JEOL ECP 300 MHz spectrometer), and
mass spectrometry (Thermo Finnigan LCQ-deca XP-plus) equipped
with an ESI source and an ion-trap detector. Chemical shifts are re-
ported in parts per million (ppm). Flash column chromatography
was performed on silica gel (Kieselgel 60, 230–400 mesh ASTM).
Thin-layer chromatography (TLC) was performed on 5ꢁ20 cm
plates with a layer thickness of 0.25 mm (Merck Silica gel 60 F254). If
necessary, they were developed with KMnO4 reagent. The purity of
the tested compounds was established by elemental analyses. Ele-
mental analyses (C, H, N) of the compounds that underwent bio-
logical evaluation were within Æ0.4% of the calculated values,
confirming ꢀ95% purity.
8-Ethynyl-2-morpholinoquinolin-4(1H)-one (12): Potassium car-
bonate (949 mg, 6.88 mmol) was added to a solution of 2-morpho-
lino-8-[(trimethylsilyl)ethynyl]quinolin-4(1H)-one (1.87 g, 5.74 mmol)
in methanol (20 mL). The mixture was stirred at room temperature
for 3 h. The volatiles were removed in vacuo. The mixture was di-
luted with brine (20 mL) and extracted with CH2Cl2 (4ꢁ10 mL). The
combined organic layer was dried with sodium sulfate. The crude
material was purified by column chromatography (EtOAc/MeOH
1:0 to 9:1) to yield 12 as a yellow solid (1.23 g, 84%).
4-Morpholino-4H-pyrrolo[3,2,1-ij]quinolin-6(5H)-one (24): Alkyne
12 (78 mg, 0.31 mmol) was added to a suspension of phenyl azide
(23; 100 mg, 0.31 mmol) in water (570 mL) and tBuOH (570 mL).
Then, 1m aqueous sodium ascorbate (29 mL) and copper sulfate
pentahydrate (0.72 mg, 0.29 mmol) were added under vigorous
stirring. The product precipitated, and it was filtered and washed
with water (2ꢁ5 mL) and diethyl ether (2ꢁ5 mL) to give 24 as a
yellow solid (49 mg, 62%).
General procedure for the synthesis of compounds 25–54: TBTA
(0.0040 mmol, 0.05 equiv) was added to a solution of Cu(OAc)2
(0.0040 mmol, 0.05 equiv) in THF (0.3 mL), and the resulting mix-
ture was stirred at room temperature for 30 min. A solution of
azide 13 (0.079 mmol, 1 equiv) in THF (0.3 mL), a solution of alkyne
12 (0.079 mmol, 1 equiv) in THF (0.3 mL), and a solution of sodium
ascorbate (0.0079 mmol, 0.1 equiv) in a minimum amount of water
were added. The mixture was stirred at room temperature over-
night. The product precipitated, and it was filtered and washed
with water (2ꢁ5 mL) and diethyl ether (2ꢁ5 mL) to give a solid.
The crude product was then screened, and compounds 25, 35, 37,
45, 48, and 54 were resynthesized and subjected to column chro-
matography (EtOAc and EtOAc/MeOH 9:1).
Synthesis
5-[Bis(Methylthio)methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione
(16): TEA (48.0 mL, 0.346 mmol) and CS2 (10.2 mL, 0.17 mmol) were
added to a solution of Meldrum’s acid (25.0 g, 0.17 mmol) in DMSO
(300 mL). The mixture was stirred for 3 h at room temperature, and
then iodomethane (2 equiv) was slowly added at 08C. The mixture
was stirred overnight at room temperature. Ice was added to the
flask to promote precipitation of the product, which was then fil-
tered to yield compound 16 as a yellow solid (34.1 g, 81%).
5-[(2-Iodophenylamino)(methylthio)methylene]-2,2-dimethyl-
1,3-dioxane-4,6-dione (18): A mixture of 5-[bis(methylthio)methy-
lene]-2,2-dimethyl-1,3-dioxane-4,6-dione (453 mg, 1.83 mmol) and
2-Morpholino-8-(1-phenyl-1H-1,2,3-triazol-4-yl)quinolin-4(1H)-
one (25): Yellow solid (90%): mp: 244–2458C (dec.); 1H NMR
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(300 MHz, [D6]DMSO): d=9.30 (s, 1H), 8.39 (brs, 1H), 7.96–7.91 (m,
4H), 7.63 (t, J=7.7 Hz, 2H), 7.49 (t, J=7.7 Hz, 1H), 7.30 (t, J=
7.7 Hz, 1H), 6.51 (s, 1H), 3.79–3.73 (m, 4H), 3.59–3.53 ppm (m, 4H);
13C NMR (75 MHz, [D6]DMSO): d=160.7, 152.8, 137.3, 136.4, 131.1,
130.6 (2C), 129.2, 128.7, 126.3, 124.3, 121.9, 121.7, 120.6, 91.8, 66.5,
46.4 ppm; IR (KBr): n˜ =n˜3437, 3122, 1636, 1584, 1500, 1237, 1124,
795, 761, 688 cmÀ1; MS (ESI): m/z=374 [M+H]+; elemental analysis
calcd (%) for C21H19N5O2 (373.41): C 67.55, H 5.13, N 18.76; found: C
67.71, H 5.25, N 18.60.
8-{1-[3-Methoxy-5-(trifluoromethoxy)phenyl]-1H-1,2,3-triazol-4-
yl}-2-morpholinoquinolin-4(1H)-one (39): Yellow solid (90%).
8-[1-(4-Hydroxy-2-methylphenyl)-1H-1,2,3-triazol-4-yl]-2-mor-
pholinoquinolin-4(1H)-one (40): Brown solid (31%).
8-{1-[2-(Hydroxymethyl)phenyl]-1H-1,2,3-triazol-4-yl}-2-morpholi-
noquinolin-4(1H)-one (41): Yellow solid (40%).
Methyl 4-[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]benzoate (42): Yellow solid (90%).
8-[1-(3-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoqui-
nolin-4(1H)-one (26): Yellow solid (90%).
8-[1-(3,4-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholino-
quinolin-4(1H)-one (43): Gray amorphous solid (76%).
8-[1-(3-Hydroxy-4-methoxyphenyl)-1H-1,2,3-triazol-4-yl]-2-mor-
pholinoquinolin-4(1H)-one (27): Yellow solid (40%).
8-[1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoqui-
nolin-4(1H)-one (44): Light-yellow solid (55%).
8-[1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoquino-
lin-4(1H)-one (28): Gray solid (50%).
4-[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tria-
zol-1-yl]benzoic acid (45): Yellow solid (66%): mp: 269–2708C
(dec.); 1H NMR (300 MHz, [D6]DMSO): d=9.39 (s, 1H), 8.42 (d, J=
6.9 Hz, 2H), 8.11–8.09 (m, 2H), 7.97 (d, J=6.9 Hz, 2H), 7.33 (t, J=
8.2 Hz, 1H), 6.54 (s, 1H), 3.79–3.77 (m, 4H), 3.60–3.58 ppm (m, 4H);
13C NMR (75 MHz, [D6]DMSO): d=161.3, 159.8, 138.2, 136.2, 133.3,
132.6, 131.7, 131.4, 128.7, 126.4, 124.1, 123.8, 121.2, 120.6, 111.8,
92.0, 66.3, 46.3 ppm; IR (KBr): n˜ =3336, 3128, 1697, 1620, 1585,
1414, 1238, 811, 768, 750 cmÀ1; MS (ESI): m/z=418 [M+H]+; ele-
mental analysis calcd (%) for C22H19N5O4 (417.42): C 63.30, H 4.59, N
16.78; found: C 63.62, H 4.95, N 16.48.
2-Morpholino-8-{1-[4-(trifluoromethoxy)phenyl]-1H-1,2,3-triazol-
4-yl}quinolin-4(1H)-one (29): Yellow solid (69%).
8-[1-(4-Methoxy-3-nitrophenyl)-1H-1,2,3-triazol-4-yl]-2-morpholi-
noquinolin-4(1H)-one (30): Yellow solid (50%).
Methyl-4-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]methyl}benzoate (31): Gray solid (90%).
8-[1-(4-Aminophenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoquino-
lin-4(1H)-one (32): Brown solid (65%).
8-[1-(Benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-triazol-4-yl]-2-morpholi-
noquinolin-4(1H)-one (46): Yellow solid (91%).
2-{4-[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-
triazol-1-yl]phenyl} acetonitrile (33): Yellow solid (74%).
8-[1-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholino-
quinolin-4(1H)-one (47): Yellow solid (84%).
2-Morpholino-8-[1-(naphthalen-2-yl)-1H-1,2,3-triazol-4-yl]quino-
lin-4(1H)-one (34): Yellow solid (68%).
8-(1-Benzyl-1H-1,2,3-triazol-4-yl)-2-morpholinoquinolin-4(1H)-
one (48): Light-yellow solid (84%): mp: 90–918C; 1H NMR
(300 MHz, CD3OD): d=8.57 (s, 1H), 8.03 (d, J=7.7 Hz, 1H), 7.83 (d,
J=7.7 Hz, 1H), 7.42–7.38 (m, 5H), 7.24 (t, J=7.7 Hz, 1H), 5.81 (s,
1H), 5.68 (s, 2H), 3.88–3.85 (m, 4H), 3.53–3.50 ppm (m, 4H);
13C NMR (75 MHz, [D6]DMSO): d=160.2, 145.2, 136.5, 131.8, 130.4,
129.4, 129.0, 128.2, 127.4, 127.3, 125.1, 122.3, 121.6, 118.6, 91.9,
66.5, 53.6, 45.8 ppm; IR (KBr): n˜ =3264, 3142, 1628, 1586, 1501,
1445, 1255, 1117, 785, 728 cmÀ1; MS (ESI): m/z=388 [M+H]+; ele-
mental analysis calcd (%) for C22H21N5O2 (387.43): C 68.20, H 5.46, N
18.08; found: C 68.18, H 5.72, N 17.83.
8-[1-(3-Methoxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoqui-
nolin-4(1H)-one (35): Yellow solid (57%): mp: 141–1428C (dec.);
1H NMR (300 MHz, CD3OD): d=9.03 (s, 1H), 8.08 (d, J=7.7 Hz, 1H),
7.92 (d, J=7.7 Hz, 1H), 7.47–7.45 (m, 3H), 7.26 (t, J=7.7 Hz, 1H),
7.08–7.04 (m, 1H), 5.79 (s, 1H), 3.89 (s, 3H), 3.84 (t, J=4.9 Hz, 4H),
3.49 ppm (t, J=4.9 Hz, 4H); 13C NMR (75 MHz, [D6]DMSO): d=
160.8, 138.4, 134.5, 133.8, 132.6 (2C), 131.5, 128.7, 123.1, 121.6,
121.2, 115.2, 113.6, 112.5, 110.9, 106.0, 92.1, 66.5, 56.1, 45.7 ppm; IR
(KBr): n˜ =3263, 2965, 1622, 1583, 1495, 1235, 1123, 1003, 871,
791 cmÀ1; MS (ESI): m/z=404 [M+H]+; elemental analysis calcd
(%) for C22H21N5O3 (403.43): C 65.50, H 5.25, N 17.36; found: C
65.22, H 5.27, N 17.33.
Methyl 3-[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]benzoate (49): Yellow solid (62%).
2-Morpholino-8-[1-(quinolin-3-yl)-1H-1,2,3-triazol-4-yl]quinolin-
4(1H)-one (36): Yellow solid (93%).
4-[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tria-
zol-1-yl]benzenesulfonamide (50): Yellow solid (95%).
3-{[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tri-
azol-1-yl]methyl}benzoic acid (37): Yellow solid (70%): mp: 290–
2918C (dec.); 1H NMR (300 MHz, [D6]DMSO): d=8.08 (s, 1H), 8.00
(d, J=7.7 Hz, 1H), 7.93 (d, J=3.3 Hz, 1H), 7.88 (d, J=7.7 Hz, 1H),
7.51 (t, J=7.7 Hz, 1H), 7.34–7.29 (m, 2H), 7.02 (d, J=3.3 Hz, 1H),
6.50 (s, 1H), 5.75 (s, 2H), 3.85–3.81 (m, 4H), 3.26–3.24 ppm (m, 4H);
13C NMR (75 MHz, [D6]DMSO): d=162.4, 159.6, 145.1, 136.2, 134.5,
133.6, 133.1, 130.2, 130.0, 128.2, 126.0, 125.6, 125.2, 122.3, 121.5,
121.2, 118.3, 91.9, 66.5, 53.2, 45.8 ppm; IR (KBr): n˜ =2970, 2866,
1707, 1636, 1582, 1444, 1242, 1117, 789, 739 cmÀ1; MS (ESI): m/z=
432 [M+H]+; elemental analysis calcd (%) for C23H21N5O4 (431.44):
C 64.03, H 4.91, N 16.23; found: C 64.00, H 4.98, N 16.20.
8-[1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoqui-
nolin-4(1H)-one (51): Yellow amorphous solid (90%).
3-[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tria-
zol-1-yl]benzoic acid (52): Yellow solid (89%).
2-Morpholino-8-[1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl]quino-
lin-4(1H)-one (53): Yellow solid (90%).
8-[1-(2-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl]-2-morpholinoqui-
nolin-4(1H)-one (54): Brown solid (77%): mp: 253–2548C (dec.);
1H NMR (300 MHz, [D6]DMSO): d=9.38 (s, 1H), 8.43 (brs, 1H), 7.94
(d, J=7.7 Hz, 1H), 7.78 (d, J=7.1 Hz, 1H), 7.31–7.26 (m, 3H), 7.15
(d, J=7.7 Hz, 1H), 7.00 (t, J=7.7 Hz, 1H), 6.55 (s, 1H), 3.76–3.74 (m,
4H), 3.56–3.54 ppm (m, 4H); 13C NMR (75 MHz, [D6]DMSO): d=
159.5, 154.8, 149.4, 145.2, 141.3, 130.1, 128.3, 127.0, 126.0, 125.2,
124.7, 121.6, 120.3, 117.9, 109.9, 101.3, 92.0, 66.6, 51.9 ppm; IR
N-{4-Methoxy-3-[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-
yl)-1H-1,2,3-triazol-1-yl]phenyl}acetamide (38): Yellow solid
(78%).
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(KBr): n˜ =3230, 2949, 1632, 1586, 1236, 1123, 800, 750 cmÀ1; MS
(ESI): m/z=390 [M+H]+; elemental analysis calcd (%) for
C21H19N5O3 (389.41): C 64.77, H 4.92, N 17.98; found: C 64.88, H
5.17, N 18.21.
130.4, 129.8, 129.7, 129.5, 128.7, 126.4, 123.3, 122.4, 120.8, 118.9,
91.9, 66.3, 54.4, 52.5, 46.3 ppm; IR (KBr): n˜ =3422, 1720, 1582, 1287,
1130, 732 cmÀ1; MS (ESI): m/z=446 [M+H]+; elemental analysis
calcd (%) for C24H23N5O4 (445.47): C 64.71, H 5.20, N 15.72; found: C
64.31, H 5.47, N 15.70.
4-[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tria-
zol-1-yl]butanoic acid (55): Yellow solid (47%).
Benzyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]methyl}benzoate (65): White solid (99%).
5-[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tria-
zol-1-yl]pentanoic acid (56): Yellow solid (40%).
Isopropyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
1H-1,2,3-triazol-1-yl]methyl}benzoate (66): Yellow solid (52%).
6-84-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tri-
azol-1-yl]hexanoic acid (57): Yellow solid (48%).
Ethyl
3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]methyl}benzoate (67): White solid (64%).
N-(Methylsulfonyl)-3-{[4-(2-morpholino-4-oxo-1,4-dihydroquino-
lin-8-yl)-1H-1,2,3-triazol-1-yl]methyl}benzamide (58): Yellow solid
(69%).
Butyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]methyl}benzoate (68): Yellow solid (66%).
tert-Butyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
1H-1,2,3-triazol-1-yl]methyl}benzoate (69): Yellow solid (28%).
3-{[4-(2-Morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-tri-
azol-1-yl]methyl}benzamide (59): Yellow solid (95%).
Pyridin-4-ylmethyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquino-
lin-8-yl)-1H-1,2,3-triazol-1-yl]methyl}benzoate (70): Yellow solid
(79%).
N-Benzyl-3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
1H-1,2,3-triazol-1-yl]methyl}benzamide (60): Yellow solid (24%).
N-Cyclopropyl-3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-
yl)-1H-1,2,3-triazol-1-yl]methyl}benzamide (61): Yellow solid
(31%).
2-Morpholinoethyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquino-
lin-8-yl)-1H-1,2,3-triazol-1-yl]methyl}benzoate (71): Yellow solid
(45%).
N,N-Diethyl-3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
1H-1,2,3-triazol-1-yl]methyl}benzamide (62): Yellow solid (40%).
Benzo[d][1,3]dioxol-5-yl methyl 3-{[4-(2-morpholino-4-oxo-1,4-
dihydroquinolin-8-yl)-1H-1,2,3-triazol-1-yl]methyl}benzoate (72):
Yellow solid (72%).
8-{1-[3-(1H-Tetrazol-5-yl)benzyl]-1H-1,2,3-triazol-4-yl}-2-morpho-
linoquinolin-4(1H)-one (63): 3-{[4-(2-Morpholino-4-oxo-1,4-dihy-
droquinolin-8-yl)-1H-1,2,3-triazol-1-yl]methyl}benzonitrile was pre-
pared following the general procedure for the click reaction.
Isopentyl
3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
1H-1,2,3-triazol-1-yl]methyl}benzoate (73): Yellow solid (78%).
1
Yellow solid (95%). H NMR (300 MHz, [D6]DMSO): d=8.75 (s, 1H),
Pentan-2-yl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
8.36 (d, J=7.1 Hz, 1H), 7.92 (s, 1H), 7.86 (m, 2H), 7.74 (d, J=7.1 Hz,
1H), 7.63 (t, J=7.1 Hz, 1H), 7.28 (t, J=7.3 Hz, 1H), 6.49 (s, 1H), 5.80
(s, 2H), 3.74–3.70 (m, 4H), 3.47–3.41 ppm (m, 4H); MS (ESI): m/z=
413 [M+H]+.
1H-1,2,3-triazol-1-yl]methyl}benzoate (74): Yellow solid (63%).
Undecyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]methyl}benzoate (75): White solid (58%).
Octadecyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-
1H-1,2,3-triazol-1-yl]methyl}benzoate (76): Yellow solid (84%).
Sodium azide (46.8 mg, 0.72 mmol) was added to a solution of 3-
{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-1,2,3-triazol-1-
yl]methyl}benzonitrile (200.0 mg, 0.48 mmol) in DMF (1.2 mL) and
methanol (1.2 mL). The mixture was stirred at 1108C overnight. Di-
ethyl ether (3 mL) was added to the mixture, and the product pre-
cipitated. It was filtered to yield 63 as a brown solid (214 mg,
98%).
Biological methods
In vitro selection, lipid kinase assay: To determine the inhibitory ac-
tivity of the new molecular entities on PI3K enzyme, a lipid kinase
assay was performed: recombinant proteins (30 ng), human PI3Ka
(PIK3CA/PIK3R1, #1161-1165-1 ProQinase–Germany), hPI3Kb
(PIK3CB wt/PIK3R1, #1168-1165-1 ProQinase, Germany), hPI3Kg
(PIK3CG, #1163-0000-1 ProQinase, Germany), hPI3Kd (PIK3CD/
PIK3R1, #1162–1165-1 ProQinase, Germany), hPI3K-C2a (PIK3C2A,
#14-906 Merck Millipore_Merck KGaA, Germany), hPI3K-C2b
(PIK3C2B, #14-907 Merck Millipore_Merck KGaA, Germany), hPI3K-
c2g (PIK3C2G, #1207-0000-1 ProQinase, Germany), and hVps34
(PIK3C3, #1160-0000-1 ProQinase, Germany), were incubated in
kinase buffer {12.5 mL; 20 mm Tris-HCl pH 7.5, 10 mm MgCl2, 10 mm
ATP, 0.05 mgmLÀ1 BSA, 0.5 mm EDTA, 5 mm HEPES pH 7.5, for PI3-
Ka,PI3Kb, PI3Kg, PI3Kd, PI3KC-2a, and PI3KC-2g; 15 mm MnCl2,
5 mm EGTA, 100 mm ATP, 50 mm HEPES pH 7.5, 100 mm NaCl, and
0.03% CHAPS for PI3K-C2b and VPS34 {BSA=bovine serum albu-
min; EDTA=ethylenediaminetetraacetic acid; HEPES=4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid; EGTA=ethylene glycol-
bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid; CHAPS=3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate}. In the
reaction, the ATP concentration used for different PI3Ks was de-
fined on the basis of the Michaelis–Menten kinetics of each
General procedure for the synthesis of ester prodrugs 64–76:
TBTA (0.0040 mmol, 0.05 equiv) was added to
a solution of
Cu(OAc)2 (0.0040 mmol, 0.05 equiv) in THF (0.3 mL), and the result-
ing mixture was stirred at room temperature for 30 min. A solution
of the azide (0.079 mmol, 1 equiv) in THF (0.3 mL), a solution of
alkyne 12 (0.079 mmol, 1 equiv) in THF (0.3 mL), and a solution of
sodium ascorbate (0.0079 mmol, 0.1 equiv) in a minimum amount
of water were added. The mixture was stirred at room temperature
overnight. The product precipitated, and it was filtered and
washed with water (2ꢁ5 mL) and diethyl ether (2ꢁ5 mL) to give a
solid. The crude product was subjected to column chromatography
(EtOAc and EtOAc/MeOH 9:1).
Methyl 3-{[4-(2-morpholino-4-oxo-1,4-dihydroquinolin-8-yl)-1H-
1,2,3-triazol-1-yl]methyl}benzoate (64): White solid (95%): mp:
1
185.5–186.58C; H NMR (300 MHz, [D6]DMSO): d=8.72 (s, 1H), 8.37
(d, J=7.7 Hz, 1H), 8.02 (s, 1H), 7.97–7.90 (m, 2H), 7.71 (d, J=
7.7 Hz, 1H), 7.59 (t, J=7.7 Hz, 1H), 7.28 (t, J=7.9 Hz, 1H), 6.47 (s,
1H), 5.82 (s, 2H), 3.85 (s, 3H), 3.74–3.63 (m, 4H), 3.58–3.24 ppm (m,
4H); 13C NMR (75 MHz, CDCl3): d=166.3, 147.7, 134.4, 132.7, 131.5,
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enzyme, provided by the supplier. Reaction was performed in pres-
ence of 0.5 mgmLÀ1 lipid micelles (1:1 phosphatidylinositol/phos-
phatidylserine) and in the absence (DMSO alone) or presence of in-
creasing concentrations of each compound (from 0.1 nm to
30 mm). The mixture was kept at room temperature (RT) for 30 min
for PI3Ka, PI3Kb, PI3Kg, PI3Kd, PI3KC-2a, and PI3KC-2g and for 1 h
for PI3K-C2b and PI3K-C3. The amount of ADP produced in the re-
action was measured by using the ADP-Glo Kinase Assay kit from
Promega (#V9102) and a luminescence reader (Glomax multi detec-
tion system, Promega, model number 9301-010).
Cell proliferation: PC3 cell proliferation was measured with a 3-(4,5-
dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based
colorimetric assay (#11 465 007 001 Roche Diagnostic GmbH, Ger-
many). PC3 cells were seeded in a 96-well plate at a concentration
of 104 cellsmLÀ1. PC3 cells were treated for 72 h with increasing
concentrations of 64, and MTT was added. After ON solubilization
of the MTT metabolite, formazan, the absorbance (l=550–600 nm
absorbance, with a reference wavelength of 650 nm) of each well
was read in a multi-well reader (Glomax multi detection system,
Promega, model number 9301-010). The percentage of proliferat-
ing cells was calculated by relating each absolute value to the con-
trol intended as PC3 cells treated with DMSO alone. An IC50 value
was then derived by plotting all data on a dose–response curve
(Graph Pad software) by using nonlinear regression fit (equation
[log agonist] versus response).
The residual PI3K lipid kinase activity in the presence of each mole-
cule at different concentrations was related to the control, intend-
ed as the recombinant protein in the presence of DMSO alone and
was expressed as a percentage. To derive the IC50 value, all data (%
of lipid kinase activity) were plotted on a dose–response curve
(Graph Pad software), and the IC50 was calculated by using nonlin-
ear regression fit (equation [log agonist] versus response).
Cell-cycle analysis: For DNA content determination, PC3 cells were
seeded at 4ꢁ105 cells per 6 cm dish in triplicate in the presence of
DMEM high glucose GlutaMAXTM (Gibco), 10% fetal bovine serum
(Invitrogen) supplemented with 5000 UmLÀ1 penicillin–streptomy-
cin (Gibco). The day after seeding, cells were treated for 48 h with
10 mm 64 or DMSO. Subsequently, PC3 cells were detached with
0.1% trypsin–EDTA (Gibco), fixed in 70% ethanol overnight at
À208C, and stained for 40 min at 578C with PI solution, containing
0.05% Triton X-100, 0.1 mgmLÀ1 RNase, and 25 mgmLÀ1 propidium
iodide. Sample were analyzed for the DNA content by using a
FACS-Calibur flow cytometer (Becton Dickinson Immunocytometry
Systems, San Jose, USA). The percentages of cells in the G0/G1, S,
and G2 phases were calculated.
Lineweaver–Burk plot: The linear phase of the kinetic reaction was
defined with 2.4 mgmLÀ1 of PI3Kd in a 20 min reaction time. In the
presence of increasing concentrations of 37 (5, 10 and 20 nm), the
PI3K lipid kinase activity (for the assay method see “In vitro selec-
tion, lipid kinase assay” in the Experimental Section) was assayed
at various concentrations of ATP (5, 10, 25, 50, and 100 mm) and in
the presence of 0.5 mgmLÀ1 lipid micelles (1:1 phosphatidylinosi-
tol/phosphatidylserine). A Lineweaver–Burk plot was obtained by
plotting 1/v, for which v represents pmolminÀ1 of ADP produced in
the reaction measured with the ADP-Glo Kinase Assay kit from
Promega (#V9102) versus 1/[ATP] (the inverse of the ATP concen-
tration). The minus-enzyme control was obtained by incubating
phosphatidylinositol in the absence of PI3Kd.
Analysis of apoptosis: For Annexin V staining of apoptotic cells, PC3
cells were seeded at 4ꢁ105 cells per 6 cm dish in triplicate. Cells
were incubated 48 h in the presence or absence of 10 mm 64 and
were then detached with 0.1% trypsin–EDTA, resuspended in An-
nexin buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2),
and stained for Annexin V (FITC Annexin V #640906, BioLegend,
USA). Samples were analyzed by using a FACS-Calibur flow cytome-
ter (Becton Dickinson Immunocytometry Systems, San Jose, USA).
Western blot analysis: PC3 cells were seeded in 96-well plates in
the presence of free Dulbecco’s modified Eagle’s medium (DMEM)
high-glucose GlutaMAXTM (Gibco), 10% fetal bovine serum (Invitro-
gen) supplemented with 5000 UmLÀ1 penicillin–streptomycin
(Gibco). Subsequently, PC3 cells were incubated with increasing
concentrations of 68 for 12 h. Cells were then lysed with a lysis
buffer containing 20 mm Tris-HCl pH 8, 138 mm NaCl, 2.7 mm KCl,
1 mm CaCl2, 5 mm EDTA pH 8, 1 mm MgCl2, 5% glycerol, 1% Triton
X-100, and protein extracts were analyzed by Western blot analysis.
To assess the phosphorylation level of proteins involved in the
PI3K/Akt signaling pathway, the following antibodies were used:
phospho-Akt (Ser473) (#4060 Cell Signaling Technology, USA),
phospho-Akt (Ser308) (#13038 Cell Signaling Technology, USA),
Akt1 (#2967 Cell Signaling Technology, USA), phospho-PRAS40
(Thr246) (#2640 Cell Signaling Technology, USA), PRAS40 (#2610
Cell Signaling Technology, USA), Phospho-p70 S6 Kinase (Thr389)
(Cell Signaling Technology #9205), Phospho-GSK-3a/b (Ser21/9)
(Cell Signaling Technology #8566), GSK-3a/b Cell Signaling Technol-
ogy #5676), phospho-FoxO1 (Thr24)/FoxO3a (Thr32) (Cell Signaling
Technology #9464), FoxO3a (Cell Signaling Technology #2497),
GAPDH (8C2) (Santa Cruz sc-81545), and p70 S6 kinase a (H-160)
(Santa Cruz sc-9027).
Additional biological experiments, crystallographic details, the pdb
file for the X-ray crystal structure of 5NGB, and metabolism stability
studies are available in the Supporting Information.
Acknowledgements
X-ray data were collected on beamline ID23-1 at the European
Synchrotron Radiation Facility (ESRF), Grenoble, France. We are
grateful to Dr. Christoph Mueller-Dieckmann at the ESRF for pro-
viding assistance. Further, we express our thanks to Dr. Fabrice
Gorrec for his help in screen development and maintenance of
the crystallographic robotics and Dr. Minmin Yun for her help
during data collection. Financial support from Universitꢀ del Pie-
monte Orientale and Regione Piemonte (DRUIDI) is gratefully ac-
knowledged. This work was supported by FIRB “Futuro e Ricerca
2010” (RBFR10HP97_004) and by Regione Piemonte “Bando re-
gionale a sostegno di progetti di ricerca industriale e/o sviluppo
sperimentale sulle malattie autoimmuni e allergiche” (PAR FSC
2007/2013; TIPSO project).
NIH3T3 cells were seeded in a 96-well plate and starved overnight
(ON) with serum-free DMEM high glucose GlutaMAXTM (Gibco)
supplemented with 5000 UmLÀ1 penicillin–streptomycin (Gibco).
Cells were then incubated for 1 h with increasing concentrations of
64, 37, or DMSO. Subsequently, cells were stimulated for 5 min
with 1 mm of insulin (#91077C, Sigma–Aldrich, USA). Cells were
lysed and protein extracts were analyzed by western blot to assess
the phosphorylation level of Akt on Ser473.
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Conflict of interest
[14] M. J. Zvelebil, M. D. Waterfield, S. J. Shuttleworth, Arch. Biochem. Bio-
phys. 2008, 477, 404–410.
E.H. and G.C.T. are co-founders of Kither Biotech S.r.l. The other
authors declare no competing interests.
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Keywords: click chemistry
·
inflammation
·
nitrogen
heterocycles · phosphoinositide 3-kinases · prodrugs
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Manuscript received: June 8, 2017
Revised manuscript received: July 25, 2017
Accepted manuscript online: July 25, 2017
Version of record online: && &&, 0000
ChemMedChem 2017, 12, 1 – 14
www.chemmedchem.org
13
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
T. Pirali, E. Ciraolo, S. Aprile,
Pro drugs: Starting from LY294002, a
potent pan-phosphoinositide 3-kinase
(PI3K) inhibitor was identified through a
straightforward click-chemistry ap-
proach. The pivotal carboxylic acid was
esterified to give prodrugs useful as
topical treatments for chronic inflamma-
tion conditions such as psoriasis and
asthma.
A. Massarotti, A. Berndt, A. Griglio,
M. Serafini, V. Mercalli, C. Landoni,
C. C. Campa, J. P. Margaria, R. L. Silva,
G. Grosa, G. Sorba, R. Williams, E. Hirsch,*
G. C. Tron*
&& – &&
Identification of a Potent
Phosphoinositide 3-Kinase Pan
Inhibitor Displaying a Strategic
Carboxylic Acid Group and
Development of Its Prodrugs
&
ChemMedChem 2017, 12, 1 – 14
www.chemmedchem.org
14
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!