Establishment of a structure-activity relationship of 1 H-imidazo[4,5-c]quinoline-based kinase inhibitor NVP-BEZ235 as a lead for African sleeping sickness

Article abstract of DOI:10.1021/jm500361r

Compound NVP-BEZ235 (1) is a potent inhibitor of human phospoinositide-3- kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using 1 as a starting point for ant

Full text of DOI:10.1021/jm500361r

Article
pubs.acs.org/jmc
Establishment of a Structure−Activity Relationship of
1H‑Imidazo[4,5‑c]quinoline-Based Kinase Inhibitor NVP-BEZ235 as a
Lead for African Sleeping Sickness
Joao D. Seixas,†,‡ Sandra A. Luengo-Arratta,†,‡ Rosario Diaz,‡ Manuel Saldivia,‡ Domingo I. Rojas-Barros,‡
̃
Pilar Manzano,§ Silvia Gonzalez,§ Manuela Berlanga,§ Terry K. Smith,∥ Miguel Navarro,*,‡
and Michael P. Pollastri*,†
†Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
‡Instituto de Parasitología y Biomedicina “Lop
́
ez-Neyra”, Granada 18100, Spain
§Tres Cantos Medicines Development Campus, DDW and CIB, GlaxoSmithKline, 28760 Tres Cantos, Spain
∥Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, United Kingdom
S
* Supporting Information
ABSTRACT: Compound NVP-BEZ235 (1) is a potent
inhibitor of human phospoinositide-3-kinases and mammalian
target of rapamycin (mTOR) that also showed high inhibitory
potency against Trypanosoma brucei cultures. With an eye
toward using 1 as a starting point for anti-trypanosomal drug
discovery, we report efforts to reduce host cell toxicity, to
improve the physicochemical properties, and to improve the
selectivity profile over human kinases. In this work, we have
developed structure−activity relationships for analogues of 1
and have prepared analogues of 1 with improved solubility
properties and good predicted central nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g as the most
promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert
their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases.
repurposing” approach, existing drugs and drug-like com-
pounds serve as early hits or leads from which to optimize
parasite-specific therapeutics.4
INTRODUCTION
■
Human African trypanosomiasis (HAT), also known as
sleeping sickness, is caused by an infection with a subspecies
of the eukaryotic protozoan parasite, Trypanosoma brucei.
Trypanosoma brucei gambiense occurs in Western and Central
Africa and is responsible for over 90% of all reported cases of
infection, whereas Trypanosoma brucei rhodesiense is found in
Eastern and Southern Africa. Both subspecies are transmitted
by the bite of infected tsetse flies, and it is estimated that about
60 million people are at risk in sub-Saharan Africa, with at least
10 000 cases occurring annually.1 Established therapies were
introduced in the mid-to-late 20th century and have severe
safety and efficacy limitations, and drug resistance is emerging
against some treatments.2
Kinase inhibitors represent one promising class of com-
pounds in both humans and parasites. As a pivotal class of
enzymes central to cellular signaling, kinases have been
identified as key targets for inflammation,5,6 cancer,7 and a
wide range of other therapeutic indications. Indeed, kinases are
estimated to represent 22% of the druggable human genome.8
The genome of T. brucei encodes 176 kinases, and the kinome
of the related parasites Trypanosoma cruzi and Leishmania spp.
consists of highly orthologous enzymes,9,10 some of which are
beginning to emerge as druggable targets of potential
intervention for such parasitic infections.11−15
We recently reported that NVP-BEZ235 (1, Figure 1A),
currently a phase III clinical candidate for cancer, showed a
subnanomolar growth inhibitory phenotype in T. brucei and
good-to-modest activities against cultures of T. cruzi and
Leishmania major.12 Recognizing, of course, that 1 is a potent
human kinase inhibitor, we started to study the structure−
activity relationships (SAR) of this class of compounds in an
Thus, there is an urgent need to develop new safe, effective,
and affordable therapeutics that can be orally administered and
are stable under tropical conditions.3 However, financial
incentives for drug discovery against HAT are quite limited
because of the economically disadvantaged regions where this
disease is endemic. As a strategy to overcome this disincentive
for drug discovery, we have hypothesized that medicinal
chemistry knowledge against classes of human drug targets
could be repurposed to facilitate rapid and cost-effective drug
discovery against parasite drug targets. In this “target
Received: March 7, 2014
Published: May 7, 2014
© 2014 American Chemical Society
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Figure 1. (A) Compound 1 and its proposed interactions with human PI3K-γ.16 (B) General regions of the compound’s core and the structure of 2,
a recently disclosed mTOR/PI3K inhibitor.17
a
Scheme 1
a
Reagents and conditions: (a) ArB(OH)2, Pd(PPh3)4, K2CO3, glyme/EtOH/H2O; (b) NH3/H2O, CuO, N1,N2-diisopropyloxalohydrazide, K3PO4,
TBAB, H2O; (c) AcCl, K2CO3, DCM; (d) morpholine or N-methylpiperazine, butyl di-1-adamantylphosphine, Pd(OAc)2, toluene; (e) 3-
acetyleneylpyridine, Pd(PhCN)2Cl2, t-Bu3P, CuI, dioxane/NMP; (f) MeZnCl, Pd(PPh3)4, THF; (g) Pd(PPh3)4, K2CO3, glyme/EtOH/H2O.
attempt to reduce the inherent host cellular toxicity and to
allow assessment and subsequent improvement of the
selectivity profile over human kinases. Furthermore, the
physicochemical properties of 1 do not lend it to CNS
exposure (a requirement for HAT therapeutics), as suggested
by GSK internal models of CNS penetration and by other
predictive models recently disclosed, such as the central
nervous system multiparameter optimization (CNS MPO)
score.18
potent growth inhibition. We therefore divided the structure of
1 into three regions (R1, R2, and R3), as shown in Figure 1B, for
systematic modulation.
The docking studies mentioned above showed that the
nitrogen atom of the quinoline substituent in the R3 position
could play an important role in binding to Asp933. Very
recently, a new mTOR/PI3K dual inhibitor, PF-04979064 (2,
Figure 1B), was disclosed by Pfizer that contains a 3-pyridine in
the same position as the 3-quinolinyl, reinforcing the
importance of this substituent for activity against the human
targets.17 To evaluate the importance of this region of the
molecule to confer activity against the parasite, we replaced the
quinoline with a variety of aromatic and nonaromatic
substituents.
RESULTS AND DISCUSSION
■
In order to establish the SAR of this chemotype, we looked
toward the docking studies of 1 that were previously reported
using a homology model of the human kinase domain of PI3Kγ,
showing that the binding of the inhibitor to the hinge region of
the kinase is made mainly through three H-bond interactions
(Figure 1A).16 We anticipated that such interactions would also
be important for the parasitic kinase(s) by which 1 effected its
Preparation of compounds 4−10 was accomplished via
various cross-coupling reactions using common intermediate 3
(Scheme 1); Suzuki conditions were employed in the
preparation of compounds 4, direct amination using a
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Table 1. Screening Data for R3 Variants of 1
a
d
b
c
Data not obtained due to low solubility. EC50 values are an average of two replicates, with SD less than 0.1 log unit. SD was less than 0.2 log units.
e
f
SD was over 1 log unit. Selectivity = TC50/EC50. Stock concentration: 2.5 mM.
a b
,
Scheme 2.
a
Reagents and conditions: (a) R1NH2, AcOH; (b) Fe, NH4Cl, EtOH/H2O; (c) Cl3OCOCl, Et3N, DCM; (d) MeI, 0.15 M NaOH(aq), DCM,
b
TBAB; (e) ArB(OH)2, Pd(PPh3)4, K2CO3, 1,2-DME, EtOH, H2O; (f) Pd(PPh3)4, K2CO3, 1,2-DME, EtOH, H2O. See the tables for the R1
substituents.
Buchwald−Hartwig reaction for compounds 5, or copper-
catalyzed conditions in aqueous ammonia for compound 6.
Compound 10 was prepared utilizing Sonogashira coupling
conditions. Palladium-mediated dehalogenation of 3 provided
9. Compound 8 was easily obtained utilizing Negishi coupling
conditions with chloro(methyl)zinc and palladium catalyst.
Biological assessments of these compounds are summarized in
Table 1 and are discussed below.
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Table 2. Biological Data for R1 and R3 Variations
a
b
c
Data were not obtained due to low solubility. EC50 average was obtained from two replicates, with SD lower than 0.1 log units. SD lower than 0.2
d
e
f
log. TC50 average was obtained from two replicates, with SD lower than 0.5 log units. Selectivity = TC50/EC50. Stock concentration: 2.5 mM.
g
h
i
Stock concentration: 0.35 mM. Stock concentration: 0.75 mM. Stock concentration: 2 mM.
Synthesis of analogues with variations of both R1 and R3 was
but it displays even worse selectivity. In fact, 10 is a clinical
achieved starting from 11, which was prepared via a three-step
sequence starting from 2-amino-5-iodobenzoic acid with an
overall yield of 51% (Scheme 2).19 Preparation of anilines 12
was accomplished via nucleophilic aromatic substitution with
the requisite amine, and nitro group reduction using iron
provided intermediates 13. Cyclization of the imidazolidinone
ring was obtained with diphosgene (compounds 14), and N-
methylation gave intermediates 15. Final compounds (16 and
17) were synthesized from 14 and 15 using Suzuki cross-
couplings in a microwave reactor. We also prepared the ring-
opened analogue 20 from 13 using Suzuki conditions. Structure
and biological activity of the compounds prepared in Scheme 2
are summarized in Table 2.
Biological Assessments. As shown in Table 1, replacing
the quinoline with a 3-pyridine (4b), with the nitrogen atom in
the same spatial region, decreased potency by 8-fold (EC50 = 16
nM) compared to that of 1 and exhibited high HepG2
cytotoxicity (TC50 = 575 nM). The same 3-pyridine with an
ethyne spacer (10) only loses 4-fold activity (EC50 = 8 nM),
backup molecule for 1 from Novartis,20 so this host cell activity
is not unexpected.
However, introducing a trifluoromethyl group in the para
position of the 3-pyridine (4d) eliminated HepG2 cytotoxicity
(TC50 > 25 μM) while maintaining a good activity against the
parasite (EC50 = 53 nM).
Installation of a 4-pyridine (4a) at the R3 position also
rendered a potent molecule (EC50 = 136 nM), with 25-fold
selectivity over host cells. Overall, the pyridine substituents
presented a more favorable profile than an unadorned aromatic
phenyl ring (4c), which has lower potency and reduced
selectivity.
Following the promising profile exhibited by the pyridine-
substituted analogues, other heteroaromatic substituents were
explored. For example, the five-membered ring methyl-
imidazole afforded a highly potent compound, 4e (EC50 = 51
nM), albeit with only a 12-fold selectivity window. However,
more bulky benzothiophene 4f was similarly potent (54 nM),
yet with no HepG2 toxicity observed.
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Table 3
a
b
EC50 average was obtained from two replicates, with SD lower than 0.1 log. TC50 average was obtained from two replicates, with SD lower than 0.5
c
f
g
log. Selectivity = TC50/EC50. Stock concentration: 2.5 mM. Stock concentration: 1.25 mM.
Introducing cyclic or linear amines or amides at the R3
position led to complete loss of activity against T. brucei and
HepG2. Eliminating R3 substituents from 1 altogether (9)
resulted in a ∼300-fold loss of potency, although this
μM) (Supporting Information, Table S2).22 The high number
of aromatic rings surely contributes to the establishment of
favorable π-stacking interactions, resulting in low solubility. Not
unexpectedly, R3 quinolinyl compounds 16a−c also presented
very low solubility. However, with a 4-pyridyl substituent at R3,
a drastic reduction in cLogP was observed (compare 1 vs 4a
and 4c), and one of these compounds (4a) maintained good
antiparasitic activity (EC50 = 136 nM, TC50 = 3.43 μM).
Using 4a as reference, increase of potency was achieved by
removing the two α-methyl groups (16d) or by replacing the
−C(CH3)2CN group by a dimethyl amine (16h). We
hypothesize that the improved positioning of a nitrogen lone
pair for hydrogen bonding lends 16h to having improved
potency. However, introduction of a methyl (16e) slightly
decreases potency relative to that of 4a, and incorporation of a
methoxy group (16g) has little impact on activity. Addition of a
larger, nonaromatic heterocycle or replacement of the R1 group
with methyl reduces or abrogates activity (16i−k).
compound still remained in submicromolar range (EC50
=
624 nM). We observe that an aromatic system (preferably
heteroaromatic) is needed in the R3 region to afford potent
anti-trypanosomal activity (cf. 8). Nevertheless the choice of
this group influences HepG2 cytotoxicity and the overall
physicochemical profile of the molecule.
In the R1 position, we first evaluated the importance of the
substituents in the para position of the aromatic ring in R1 with
16a−c (Table 2). These derivatives exhibited a 10−15-fold
decrease in potency when compared to that of 1. Surprisingly,
the most potent compound was the one without a para
substituent (16c, EC50 = 24 nM), and both the nitrile (16b)
and trifluoromethyl (16a) analogues showed similar anti-
trypanosomal potency (EC50 = 91 and 103 nM, respectively).
In addition, these three compounds exhibited an excellent
selectivity profile against HepG2. This data shows that when R3
= 3-quinolinyl the presence of a substituent in the para position
of the phenyl ring of the R1 position is important, but not
essential, for anti-trypanosomal activity. Interestingly, when the
R3 group is 4-pyridyl, the presence of a nitrile group (16d and
16f) or amine moiety (16h) in the R1 para position seems to
afford improved potency over p-methyl or methoxy (16e and
16g). This is perhaps consistent with the predicted binding of 1
to the human p110α orthologue, where the nitrile established
hydrogen bonds with Ser774, contributing to a strong binding
to the human target.16 We observe a loss in activity when
substituents on both the R1 and R3 substituents are eliminated
simultaneously (23, Table 3).
The R2 substituent on the urea ring adjacent to the quinoline
core was also explored in terms of methylation versus
nonmethylation of the free NH for compounds 16/17b, 16/
17d, 16/17h, and 18/19 (Table 3). However, no clear trends
were evident. Compound 16h is also the only example where
methylation afforded toxicity in HepG2 (TC50 = 2.34 μM).
Opening the imidazolone ring the carbonyl group in the “urea-
imidazo” ring seems to play an important role. Disruption of
the aromatic ring, affording the free amine (20), led to a
significant loss in potency (EC50 = 0.767 μM). From a
physicochemical property perspective, we do not observe a
trend in solubility and permeability when comparing R2 = Me
versus H.
As mentioned above, we note that 1 does not penetrate the
blood−brain barrier (BBB), a crucial requirement for new
drugs in order to be effective in the second stage of HAT. In
evaluating analogue designs, we employed the multiparameter
optimization (MPO) algorithm developed by Pfizer scientists
One of the medicinal chemistry goals of this work was to
improve the physicochemical properties of the new com-
pounds. Compound 1 is very lipophilic (cLogP = 5.81,
chromLogD = 4.75)21 and displays very poor solubility (19
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Table 4. Comparison of Properties of 1 and Prioritized Compounds
1
4e
9
16e
0.200
16g
0.166
Potencies
T. brucei EC50 (μM)
HepG2 TC50 (μM)
0.0025
0.051
0.631
0.30
0.617
>50
b
nd
4.786
0.33
11.220
0.32
c
LE
0.32
0.33
mTOR IC50 (μM)
PI3Kα IC50 (μM)
PI3Kβ IC50 (μM)
PI3Kγ IC50 (μM)
PI3Kδ IC50 (μM)
Properties
0.005
0.020
0.316
0.100
0.013
1.175
0.398
7.943
0.126
0.794
1.622
3.981
>30
0.468
0.631
31.623
0.251
2.512
0.380
0.501
7.943
0.316
2.512
e
1.995
15.849
MPO score
3.22
4.08
422.5
5.3
4.56
342.4
5.62
(4.69)
63.61
0
4.07
366.4
5.86
3.5 (4.95)
52.71
0
4.57
382.4
5.1
MW
469.6
5.81
cLogP
d
21
,
chromLogD
TPSA
4.75 (6.44)
76.52
0
(5.3)
81.43
0
3.03 (5.32)
61.94
0
HBD
solubility (μM)22
a
a
19
nd
nd
17
22
a
a
permeability
nd
nd
570
470
860
a
b
c
d
nd = data not obtained. Not obtained due to low solubility. LE = −Log(pEC50) × 1.37/number of heavy atoms. LogD values in parentheses are
e
calculated values. One replicate experiment showed an IC50 >30 μM.
for CNS-active drugs.18,23 This aforementioned publication
showed a correlation between six fundamental physicochemical
properties (cLogP, cLogD, MW, TPSA, hydrogen-bond
donors, and the pKa of the most basic center) and their
impact on CNS-acting agents as well as other critical ADME,
toxicity, and binding efficiencies. By summing scores for each of
these six properties, one can ascertain the likelihood that a
given compound should penetrate the CNS. Importantly, a
subsequent report successfully applied this approach prospec-
tively for design of CNS-penetrant PI3K-α inhibitors.24
As a point of reference, the CNS MPO score of 1 is 3.22,
which is lower than the desirable range of CNS-active
molecules (≥4). From the new analogues synthesized and
tested here, only four shown in Tables 1−3 scored equal to or
worse than 1, and these were primarily due to unfavorable
LogP, LogD, and molecular weight values. The MPO score of
our most potent analogue (4b) improved to a score of 3.9,
which is a direct effect of MW and cLogP decrease compared to
those of 1. However, although 16d is slightly less potent than
4b, we observe significant improvement in cellular selectivity
and predicted CNS activity (MPO score = 4.8).
Biochemical Kinase Selectivity. On the basis of the
cellular potency and selectivity profiles, we prioritized 14
compounds, plus 1, to be tested against PI3K-α, -β, -γ, and -δ as
well as mTOR, and this data is shown in the Supporting
Information (Table S1). We note that although seven of these
compounds retain nanomolar levels of mTOR activity, in most
cases we do observe decreases in potency against the
mammalian kinases tested.
Next, on the basis of an evaluation of anti-trypanosomal
activity, selectivity, MPO score, and the biochemical selectivity
assays, we selected four compounds (plus compound 1) to
initiate mechanism of action studies. These four prioritized
compounds are shown in Table 4.
potential mechanism of action against T. brucei cells, we
performed a lipidomic analysis to discern if PI kinases are likely
targets of these analogues. Lipid extracts of cells grown in the
presence of the best compounds at sublethal doses (200 nM)
for 12 h were analyzed by ES-MS. Survey scans in positive and
negative ion mode between 600 and 1000 m/z showed a wide
range of expected phospholipid species (Supporting Informa-
tion Figures S1 and S2, respectively). There were no significant
differences in the phosphatidylcholine (PC) and sphingomylein
(SM) species between the control and cells grown in the
presence of the other compounds (Figure S1). Additionally,
only minor differences were observed in the negative ion mode
survey scans between the control and the treated cells (Figure
S2). However, variations were observed in the relative ratios of
the PI species 862 m/z (18:0/18:2), 886 m/z (18:0/20:4), and
912 m/z (18:0/22:5), as well as relative to 826 m/z PG (18:0/
22:4). This could be a reflection in changes in either the
formation of PI (hence, PG increases as PI decreases) because
they have a common lipid donor (cytidine-diphosphate-
diacylglycerol) or variation in the usage of PI (i.e., for PIP
formation). Alternatively, as is often the case, changes in the
lipid profile could be a reflection in the cells succumbing to (or
adapting to) a defect in a major cellular process, such as endo-
or exocytosis or cell cycle arrest.
Because these compounds are thought to target PI3K,
negative ion mode scans between 950 and 1300 m/z were
conducted to focus upon singly and multiply phosphorylated
PIs. As seen in Figure 2A for the control cells, PIP species are
observed at ∼992 m/z and ∼1040 m/z, corresponding to 40:5-
8 and 44:7-9, respectively, as well as PIP3 (44:9-12) species at
∼1215 m/z.
Compounds 1 and 16g (Figure 2B,C) show almost complete
absence of all phosphorylated PIs, clearly indicating that these
compounds are targeting PI kinases (e.g., PI3K). Compounds
4e and 16e (Figure 2C,E) also show significant (albeit
incomplete) reduction in PIP levels, although this reduction
is significant compared to that in the control cells (Figure 2A),
indicating that they are affecting metabolism of PIPs, most
likely phosphorylation of PI.
Phospholipidomics Analysis. We originally selected 1 to
investigate its potential as an trypanocidal agent on the basis of
its activity as a PI3K/mTOR inhibitor in mammalian cells and
the fact that PI3K activity is essential for the bloodstream form
of the T. brucei parasite.25 In the interest of elucidating the
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Val855, and Asp933, were observed. To resolve the possible
target(s) of compound 1, we compared the phenotype of cells
treated with 1 with the previously described phenotypes caused
by RNAi of these various kinases. TbPI3KIII depletion in T.
brucei induced a phenotype characteristic of a defective
endocytosis,25 similar to the phenotype observed in cells
treated with 1. Furthermore, ES-MS analyses show complete
absence of PIPs, suggesting that 1 is indeed targeting one or
more of the PI/PIP kinases, including TbPI3KIII (Figure 2).
Highly efficient and rapid endocytosis is one of the most
important cellular processes for this infective parasite to survive
and multiply within the bloodstream of the host, and the
activity of TbPI3KIII is essential for this process.25 To gain
insight on the possible target(s) of our compounds, we carried
out transferrin uptake experiments to measure receptor-
mediated endocytosis after drug treatment. The analogues of
1 showed varied trypanocidal potency, so we decided to use
concentrations twice that of the EC50 of each of the four
compounds in Table 4 over a short treatment period (18 h).
These transferrin uptake assays showed that 1 inhibited
endocytosis dramatically, as did compounds 16e and 16g
(Figure 3). In addition, the fact that PIP3 levels were
Figure 3. Transferrin uptake of the bloodstream form of T. brucei
treated with BEZ235 derivative compounds. The histogram shows the
percentage of transferrin uptake relative to that in untreated cells
(DMSO) as a control. Mean
ments is shown.
SD of three independent measure-
dramatically reduced after treatment with compounds 1 and
16g further supports the hypothesis they are affecting PIK
activity, including TbPI3KIII (Figure 2). Compound 16e
induced a phenotype defective in endocytosis (Figure 3);
however, the PIP3 molecular species identified (42:7-9) was not
the same PIP3 molecular species identified in WT cells (44:9-
12). These data suggest that 16e reduces PIP3 44:9-12
formation by inhibiting similar target(s) to that of compounds
1 and 16g, but it may have an additional mode of action
because it causes PIP3 42:7-9 to be observed, which may be
present in a different subcellular compartment or organelle.
Conversely, compound 4e showed a different phenotype, as
it did not significantly reduce transferrin endocytosis compared
to that of the control (Figure 3). In addition, compound 4e
affected cell cycle progression by reducing the proportion of
cells in G1 and increasing multinucleated cells, whereas
compounds 1, 16g, and 16e did not significantly alter cell
cycle progression over this short treatment time (18 h)
(Supporting Information Figure S3).
Figure 2. Negative ion mode survey scans from 950 to 1300 m/z: (A)
DMSO (control), (B) 1, (C) 4e, (D) 16g, and (E) 16e.
Cell Phenotype Analysis. BEZ235 is known to inhibit
human PI3K/mTOR; thus, we used the human kinase domain
of PI3K-γ (including Ser774, Val855, and Asp933, mentioned
above) to search in the T. brucei genome geneDB for
homologous enzymes. Two high-homology PI3Ks were
identified: the previously characterized TbPI3KIII
(Tb927.8.6210), which is orthologous to the yeast Vps34,
and an uncharacterized, potentially pseudo TbPIK
(Tb927.11.15330). In addition, three of the four TORs
described in trypanosomes, TbTOR1, TbTOR2, and
TbTOR3, were also identified. In the sequences of these five
proteins, similar positions for the important residues, Ser774,
Collectively, these data suggest that compound 4e has a
different mode of action than 1, 16e, and 16g because
endocytosis was not affected, although the induction of changes
in the PIP3 species (Figure 2) suggests PIP metabolism was
affected.
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this mixture was added iron (10 equiv) followed by a solution of
NH4Cl (1.42 g, 26 mmol) in H2O (11 mL). The resulting suspension
was heated at reflux for 2 h. The hot mixture was then filtered through
a Celite pad, and the filtrate was evaporated under vacuum. The
residue was dissolved in EtOAc (30 mL) and washed with H2O (30
mL), and the aqueous phase was further extracted with ethyl acetate (2
× 20 mL). The organic extracts were combined, dried over MgSO4,
filtered, and evaporated under vacuum.
General Procedure C: Formation of the C ring. A solution of
diamine 13 (10.5 mmol) and triethylamine (1.2 equiv) in CH2Cl2
(175 mL) was added dropwise at 0 °C to a solution of trichloromethyl
chloroformate (1.1 equiv) in CH2Cl2 (50 mL), and the resultant
solution was stirred for 2 h while being allowed to warm slowly to rt.
The reaction was quenched with a saturated aqueous solution of
NaHCO3 (150 mL) and stirred for 2 h. Phases were separated, and the
aqueous layer was further extracted with CH2Cl2 (2 × 60 mL).
Organic phases were combined, dried over MgSO4, filtered, and
concentrated
Previously published functional analysis showed that
TbTOR2 depletion affected endocytosis and cytokinesis,26
whereas rapamycin (a specific inhibitor of TbTOR2 in
trypanosomes) treated cells induce different changes in the
lipid profile than those from compound 1 (data not shown).
The RNAi of additional TbTOR1 and TOR3 protein
kinases26,27 does not resemble the defect in endocytosis
observed by the treatment with 1, suggesting that the
TbTOR family of PIKK is not the main target of 1.
These studies cannot determine unequivocally which
member of the trypanosome PI3K family may be the actual
molecular target; however, our data strongly suggests that
TbPI3KIII is the main target of BEZ235 as well as of derivative
compounds 16e and 16g. Regardless, it is possible that
additional molecular target(s) in the parasite may be affected
In summary, we have developed initial SAR for analogues of
1 as anti-trypanosomal agents. We have identified regions of the
molecule that allow improvement of selectivity over selected
kinases and HepG2 cells, and we have measured and computed
physicochemical properties that place analogues in a CNS-
penetrant region of properties space, as predicted by the CNS
MPO scoring regime. With this in mind, we highlight the
compounds in Table 4 as our most promising leads in this
series, as they possess significant overall improvements over 1.
We now have preliminary lipidomics and cell phenotype data to
suggest primary targets of action within the lipid kinase
pathways, and additional target identification work is
continuing using orthogonal methods. Work is also ongoing
to establish whether the predicted favorable properties hold
true in pharmacokinetic and animal efficacy studies, which will
be reported in due course.
General Procedure D: N1 Methylation. An aqueous solution of
NaOH (88 mL, 0.15 M) was added to a mixture of intermediate 14
(8.8 mmol), methyl iodide (1.5 equiv), and tetrabutylammonium
bromide (0.1 equiv) in CH2Cl2 (180 mL). The resultant mixture was
stirred for 24 h at rt in a sealed tube. The reaction was quenched with
H2O (100 mL), phases were separated, and the aqueous layer was
further extracted with CH2Cl2 (3 × 180 mL). Volatiles were combined
and evaporated under reduced pressure.
General Procedure E: Suzuki Coupling. A microwave vessel was
loaded with aryl iodide 3 (0.149 mmol), K2CO3 (1.5 equiv),
Pd(PPh3)4 5 mol %), and boronate (1.1 equiv) in 1,2-dimethoxy-
ethane (2 mL), EtOH (1 mL), and H2O (0.5 mL). The vessel was
evacuated, backfilled with nitrogen, and sealed. The mixture was
heated in a microwave reactor at 175 °C for 15 min (including the
ramp time). After cooling, the solids were removed by filtration, H2O
(10 mL) and CH2Cl2 (10 mL) were added, phases were separated, and
the aqueous layer was further extracted with CH2Cl2 (10 mL).
Volatiles were combined and evaporated under reduced pressure.
Purification was carried out by flash column chromatography over
silica gel.
General Procedure F: Buchwald Coupling. A sealed vial was
charged with intermediate 3 (0.256 mmol), morpholine (1.2 equiv),
butyl(ditricyclo[3.3.1.1∼3,7∼]dec-1-yl)phosphane (0.1 equiv), sodium
tert-butoxide (1.2 equiv), Pd(OAc)2 (5 mol %), and toluene (2.3 mL).
The resultant mixture was purged with argon for 5 min and then
heated at 115 °C for 3 days. The reaction mixture was quenched with
H2O (3 mL) and extracted with CH2Cl2 (2 × 10 mL). The organics
were combined, dried over MgSO4, filtered, and concentrated under
vacuum. Purification was carried out by flash column chromatography
over silica gel.
General Procedure G: Negishi Coupling. A microwave vessel was
loaded with aryl iodide 3 (80 mg, 171 μmol), methylzinc chloride
solution 2 M in THF (4 equiv), and palladium tetrakis(triphenyl-
phosphine) (0.1 equiv) in THF (4 mL). The vessel was evacuated,
backfilled with nitrogen, and sealed. The mixture was heated in a
microwave reactor at 75 °C for 1 h (including the ramp time). After
cooling, the solids were removed by filtration. Saturated solutions of
ammonium chloride (5 mL) and EtOAc (10 mL) were added, phases
were separated, and the aqueous layer was further extracted with
EtOAc (10 mL). Volatiles were combined and evaporated under
reduced pressure. Purification was carried out by washing the resultant
solid with MeOH.
EXPERIMENTAL SECTION
■
Chemistry. Starting materials were obtained from commercial
suppliers and used without further purification unless otherwise stated.
Flash column chromatography was carried out using prepacked Isolute
Flash or Merck Si60 (15−40 μm) silica gel columns as the stationary
phase and analytical grade solvents as the eluent unless otherwise
stated. Proton magnetic resonance (1H NMR) spectra were recorded
at 400 MHz on a Bruker AMX400 spectrometer and are reported as
follows: chemical shifts δ (ppm) (multiplicity, coupling constant in J
(Hz), number of protons). Multiplicities are labeled s, singlet; d,
doublet; t, triplet; m, multiplet; br, broad; or a combination of these.
Total ion current traces were obtained for electrospray positive and
negative ionization (ES+/ES−) on a Waters SQ detector. Analytical
chromatographic conditions used for the LC/MS analysis were as
follows. The column was an Acquity UPLC BEH C18 1.7 μm, 3 × 50
mm. Solvent A was an aqueous solvent consisting of 25 mM
ammonium acetate. Solvent B was 90% acetonitrile + 10% water (pH
6.6). Additional chromatographic parameters were as follows: flow
rate, 0.8 mL/min; injection volume, 2 μL; column temperature, 40 °C;
and UV wavelength range, 200−330 nm. The purity of all tested
compounds was ≥95% using the analytical method described above
unless otherwise stated. Compounds 10 and 1120 were prepared as
previously described, and their characterization is included in the
Supporting Information.
General Procedure A: Aryl Halide Displacement. To a suspension
of 4-chloro-6-iodo-3-nitroquinoline (11) (8 g, 24 mmol) in AcOH
(100 mL) was added a solution of amine (1.1 equiv) in 70 mL of
AcOH. The suspension was stirred for 3 h at rt under N2 and
quenched with H2O (150 mL). The resulting yellow precipitate was
recovered by filtration, dissolved in CH2Cl2, washed with a saturated
aqueous solution of NaHCO3 (100 mL) and then H2O (100 mL),
dried over MgSO4, filtered, and concentrated.
2-(4-(8-Iodo-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-1-yl)phenyl)-2-methylpropanenitrile (3). Prepared
using General Procedure D from 14 to give 3 as a yellow solid
1
(yield: 81%). H NMR (400 MHz, DMSO-d6) δ ppm: 9.01 (s, 1H),
7.85 (d, J = 8.5 Hz, 2H), 7.76 (m, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.14
(s, 1H), 3.58 (s, 3H), 1.81 (s, 6H). LCMS found, 469 [M + H]+.
2-Methyl-2-(4-(3-methyl-2-oxo-8-(pyridin-3-yl)-2,3-dihydro-
1H-imidazo[4,5-c]quinolin-1-yl)phenyl)propanenitrile (4b).
Prepared from 3-pyridinyl boronic acid using General Procedure E.
Purification was carried out by flash column chromatography over
General Procedure B: Reduction of Nitroarenes. A suspension of
nitroarene 12 (2.6 mmol) in ethanol (37 mL) was heated at reflux. To
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silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 4b as an off-
white solid (yield: 40%). 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.01
(s, 1H), 8.54 (m, 2H), 8.15 (d, J = 8.8 Hz, 1H), 7.97 (dd, J = 8.8, 2.0
Hz, 1H), 7.88 (m, 2H), 7.75 (m, 3H), 7.41 (m, 1H), 7.12 (d, J = 1.8
Hz,1H), 3.62 (s, 3H), 1.83 (s, 6H). LCMS found, 420 [M + H]+.
2-Methyl-2-(4-(3-methyl-2-oxo-8-phenyl-2,3-dihydro-1H-
imidazo[4,5-c]quinolin-1-yl)phenyl)propanenitrile (4c). Pre-
pared from phenylboronic acid using General Procedure E.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 4c as an off-
white solid (yield: 51%). 1H NMR (400 MHz, CDCl3) δ ppm: 8.81 (s,
1H), 8.19 (d, J = 9.0 Hz, 1H), 7.86 (dd, J = 9.0, 1.9 Hz, 1H), 7.79 (m,
2H), 7.63 (m, 2H), 7.35 (m, 5H), 7.29 (d, J = 1.9 Hz, 1H), 3.71 (s,
3H), 1.85 (s, 6H). LCMS found, 419 [M + H]+.
2-Methyl-2-(4-(3-methyl-2-oxo-8-(6-(trifluoromethyl)-
pyridin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl)-
phenyl)propanenitrile (4d). Prepared from 2-(trifluorimethyl)-
pyridin-5-ylboronic acid using General Procedure E. Purification was
carried out by flash column chromatography over silica gel (eluent:
CH2Cl2/MeOH 100:0 to 95:5) to give 4d as a yellow-orange solid
(yield: 25%). 1H NMR (400 MHz, CDCl3) δ ppm: 8.87 (s, 1H), 8.65
(s, 1H), 8.25 (d, J = 8.5 Hz, 1H), 7.80 (m, 5H), 7.71 (d, J = 8.1 Hz,
1H), 7.63 (d, J = 8.5 Hz, 2H), 3.73 (s, 3H), 1.86 (s, 6H). LCMS
found, 488 [M + H]+.
CH2Cl2/MeOH 100:0 to 95:5) to give 6 as a white solid (18.4 mg, 51
μmol, 28%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.58 (s, 1H),
7.74 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 9.1 Hz, 1H), 7.54 (m, 2H), 6.94
(dd, J = 9.1, 2.3 Hz, 1H), 5.87 (d, J = 2.5 Hz, 1H), 5.39 (brs, 2H), 3.50
(s, 3H), 1.80 (s, 6H). LCMS found, 358 [M + H]+.
1
N-(1-(4-(2-Cyanopropan-2-yl)phenyl)-3-methyl-2-oxo-2,3-di-
hydro-1H-imidazo[4,5-c]quinolin-8-yl)acetamide (7). Acetyl
chloride (10 μL, 0.14 mmol) was added to a mixture of compound
6 (50 mg, 0.14 mmol) and potassium carbonate (20 mg, 0.14 mmol)
in CH2Cl2 (4 mL), and the resultant mixture was stirred for 24 h at rt.
The mixture was filtered, and volatiles were removed under vacuum.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 93:7) to give 7 as a white
solid (4.7 mg, 12 μmol, 8%). 1H NMR (400 MHz, DMSO-d6) δ ppm:
9.97 (s, 1H), 8.84 (s, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.79 (m, 1H), 7.74
(m, 2H), 7.55 (m, 2H), 7.33 (dd, J = 9.0, 2.6 Hz, 1H), 3.56 (s, 3H),
1.91 (s, 3H), 1.81 (s, 6H). LCMS found, 400 [M + H]+.
2-(4-(3,8-Dimethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-1-yl)phenyl)-2-methylpropanenitrile (8). Prepared from
intermediate 3 using General Procedure G. Compound 8 was obtained
without further purification as an orange solid (yield: 90%, purity:
94%). 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.93 (s, 1H), 7.92 (d, J
= 8.8 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.39
(dd, J = 8.8, 1.7 Hz, 1H), 6.64 (s, 1H), 3.58 (s, 3H), 2.16 (s, 3H), 1.82
(s, 6H). LCMS found, 357 [M + H]+.
2-Methyl-2-(4-(3-methyl-8-(1-methyl-1H-pyrazol-4-yl)-2-
oxo-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl)phenyl)-
propanenitrile (4e). Prepared from 1-methylpyrazole-4-boronic acid
pinacol ester using General Procedure E. Purification was carried out
by flash column chromatography over silica gel (eluent: CH2Cl2/
2-Methyl-2-(4-(3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-
c]quinolin-1-yl)phenyl)propanenitrile (9). Prepared using General
Procedure E, reacting intermediate 3 in the absence of boronic acid.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 9 as a pale
1
MeOH 100:0 to 95:5) to give 4e as a yellow solid (yield: 49%). H
NMR (400 MHz, CDCl3) δ ppm: 8.75 (s, 1H), 8.10 (d, J = 8.8 Hz,
1H), 7.81 (m, 2H), 7.68 (dd, J = 8.8 and 2.0 Hz, 2H), 7.63 (m, 2H),
7.41 (s, 1H), 7.34 (s, 1H), 7.14 (d, J = 1.78 Hz, 1H), 3.91 (s, 3H), 3.70
(s, 3H), 1.90 (s, 6H). LCMS found, 423 [M + H]+.
1
yellow solid (yield: 11%, purity: 93%). H NMR (400 MHz, DMSO-
d6) δ ppm: 9.00 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.8 (d, J = 8.6 Hz,
2H), 7.66 (d, J = 8.6 Hz, 2H), 7.55 (m, 1H), 7.31 (m, 1H), 7.03 (d, J =
7.8 Hz, 1H), 3.58 (s, 3H), 1.80 (s, 6H). LCMS found, 343 [M + H]+.
2-(4-((6-Iodo-3-nitroquinolin-4-yl)amino)phenyl)-2-methyl-
propanenitrile (12). Prepared using General Procedure A, reacting
intermediate 11 with 2-(4-amino-phenyl)-2-methylpropionitrile (Fig-
ure S2, Supporting Information). Compound 12 was isolated as a
2-(4-(8-(Benzo[b]thiophen-2-yl)-3-methyl-2-oxo-2,3-dihy-
dro-1H-imidazo[4,5-c]quinolin-1-yl)phenyl)-2-methylpropane-
nitrile (4f). Prepared using General Procedure E, from 3, providing 4f
1
as as an off-white solid (yield: 25%). H NMR (400 MHz, CDCl3) δ
ppm: 8.79 (s, 1H), 8.14 (d, J = 9.1 Hz, 1H), 7.94 (dd, 9.1, J = 2.0 Hz,
1H), 7.86 (dd, J = 6.6, 2.0 Hz, 2H), 7.75 (m, 2H), 7.64 (dd, J = 6.6, 2.0
Hz, 2H), 7.44 (s, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.33 (m, 2H), 3.71 (s,
3H), 1.93 (s, 6H). LCMS found, 475 [M + H]+.
2-Methyl-2-(4-(3-methyl-8-(4-methylpiperazin-1-yl)-2-oxo-
2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl)phenyl)-
propanenitrile (5a). Prepared from intermediate 3 using General
Procedure F. Purification was carried out by flash column
chromatography over silica gel (eluent: CH21Cl2/MeOH 100:0 to
95:5) to give 5a as a tan solid (yield: 17%). H NMR (400 MHz,
DMSO-d6) δ ppm: 8.72 (s, 1H), 7.81 (m, 3H), 7.66 (d, J = 8.6 Hz,
2H), 7.39 (dd, J = 9.5, 2.5 Hz, 1H), 6.05 (d, J = 2.5 Hz, 1H), 3.54 (s,
3H), 2.82 (m, 4H), 2.31 (m, 4H), 2.16 (s, 3H), 1.78 (s, 6H). LCMS
found, 441 [M + H]+.
1
yellow solid (yield: 73%). H NMR (400 MHz, DMSO-d6) δ ppm:
10.1 (s, 1H), 9.10 (s, 1H), 8.80 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.75
(d, J = 8.6 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H),
1.67 (s, 6H). LCMS found, 459 [M + H]+.
6-Iodo-3-nitro-N-(4-(trifluoromethyl)phenyl)quinolin-4-
amine (12a). Prepared using General Procedure A, reacting
intermediate 11 with 4-(trifluoromethyl) aniline. Compound 12a
was isolated as a yellow solid (yield: 78%). 1H NMR (400 MHz,
DMSO-d6) δ ppm: 10.21 (s, 1H), 9.11 (s, 1H), 8.9 (s, 1H), 8.18 (d, J
= 8.7 Hz 1H), 7.8 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.21
(d, J = 8.3 Hz, 2H). LCMS found, 458 [M − H]+.
4-((6-Iodo-3-nitroquinolin-4-yl)amino)benzonitrile (12b).
Prepared using the General Procedure A, reacting intermediate 11
with 4-aminobenzonitrile. Compound 12b was isolated as a yellow
2-Methyl-2-(4-(3-methyl-8-morpholino-2-oxo-2,3-dihydro-
1H-imidazo[4,5-c]quinolin-1-yl)phenyl)propanenitrile (5b).
Prepared from 3 using General Procedure F, providing 5b as a tan
solid following flash column chromatography over silica gel (eluent:
1
solid (yield: 98%). H NMR (400 MHz, DMSO-d6) δ ppm: 10.36 (s,
1H), 9.11 (s, 1H), 8.85 (s, 1H), 8.16 (dd, J = 8.7, 1.8 Hz, 1H), 7.78 (d,
J = 8.7 Hz, 1H), 7.72 (m, 2H), 7.12 (d, J = 8.6 Hz, 2H). LCMS found,
417 [M + H]+.
1
0−3% MeOH/CH2Cl2) (yield: 12%). H NMR (400 MHz, DMSO-
6-Iodo-3-nitro-N-phenylquinolin-4-amine (12c). Prepared
d6) δ ppm: 8.75 (s, 1H), 7.83 (m, 3H), 7.66 (d, J = 8.6 Hz, 2H), 7.40
(dd, J = 9.5, 2.5 Hz, 1H), 6.06 (d, J = 2.5 Hz, 1H), 3.61 (m, 4H), 3.55
(s, 3H), 2.77 (m, 4H), 1.78 (s, 6H). LCMS found, 428 [M + H]+.
2-(4-(8-Amino-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-
c]quinolin-1-yl)phenyl)-2-methylpropanenitrile (6). A sealed vial
was loaded with CuO (1 mg, 13 μmol), N1,N2-diisopropyloxalohy-
drazide28 (10.2 mg, 51 μmol), intermediate 3 (120 mg, 0.25 mmol),
commercial 25−28% aqueous ammonia solution (0.26 mL), K3PO4
(108 mg, 0.51 mmol), tetra-butyl ammonium bromide (41 mg, 0.13
mmol), and H2O (0.26 mL). The mixture was heated at 110 °C for 3
h. After allowing the mixture to cool to rt, the reaction mixture was
extracted with CH2Cl2 (3 × 10 mL). The combined organic phase was
washed with brine and concentrated under vacuum. Purification was
carried out by flash column chromatography over silica gel (eluent:
using General Procedure A, reacting intermediate 11 with aniline.
1
Compound 12c was isolated as a brown solid (yield: 83%). H NMR
(400 MHz, DMSO-d6) δ ppm: 10.07 (s, 1H), 9.02 (s, 1H), 8.85 (d, J =
1.8 Hz, 1H), 8.11 (dd, J = 8.6, 1.6 Hz, 1H), 7.74 (d, J = 8.6 Hz, 1H),
7.32 (m, 2H), 7.13 (t, J = 7.3 Hz, 1H), 7.08 (d, J = 7.6 Hz, 2H). LCMS
found, 392 [M + H]+.
2-(4-((6-Iodo-3-nitroquinolin-4-yl)amino)phenyl)acetonitrile
(12d). Prepared using General Procedure A, reacting intermediate 11
with 2-(4-aminophenyl)acetonitrile. Compound 12d was isolated as a
1
yellow solid (yield: 93%). H NMR (400 MHz, DMSO-d6) δ ppm:
10.06 (s, 1H), 9.04 (s, 1H), 8.93 (d, J = 1.8 Hz, 1H), 8.15 (dd, J = 8.7,
1.8 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.30 (d, J = 8.3 Hz, 2H), 7.10
(d, J = 8.3 Hz, 2H), 4.02 (s, 2H). LCMS found, 431 [M + H]+.
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6-Iodo-3-nitro-N-(p-tolyl)quinolin-4-amine (12e). Prepared
using General Procedure A, reacting intermediate 11 with p-toluidine.
Compound 12e was isolated as a yellow solid (yield: 95%). H NMR
= 7.3, 1.0 Hz, 2H), 6.69 (t, J = 7.3 Hz, 1H), 6.51 (m, 2H), 1.89 (brs,
2H). LCMS found, 360 [M − H]+.
1
2-(4-((3-Amino-6-iodoquinolin-4-yl)amino)phenyl)-
acetonitrile (13d). Prepared from intermediate using General
Procedure B. Compound 13d was isolated as a tan solid (yield:
(400 MHz, DMSO-d6) δ ppm: 10.03 (s, 1H), 9.01 (s, 1H), 8.87 (d, J =
1.6 Hz, 1H), 8.11 (dd, J = 8.6, 1.6 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H),
7.15 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 2.29 (s, 3H). LCMS
found, 406 [M + H]+.
6-Iodo-N-(4-methoxyphenyl)-3-nitroquinolin-4-amine (12g).
Prepared using General Procedure A, reacting intermediate 11 with p-
anisidine. Compound 12g was isolated as an orange solid (yield: 83%).
1H NMR (400 MHz, DMSO-d6) δ ppm: 10.05 (s, 1H), 8.99 (s, 1H),
1
87%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.60 (s, 1H), 8.05
(s,1H), 7.91 (s, 1H), 7.60 (m, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.52 (d, J
= 8.4 Hz, 2H), 5.40 (s, 2H), 3.85 (s, 2H). LCMS found, 401 [M +
H]+.
6-Iodo-N4-(p-tolyl)quinoline-3,4-diamine (13e). Prepared from
intermediate 12e using General Procedure B. Compound 13e was
1
isolated as a yellow solid (yield: 30%). H NMR (400 MHz, DMSO-
8.77 (s, 1H), 8.08 (dd, J = 8.7, 1.8 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H),
7.06 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.75 (s, 3H). LCMS
found, 422 [M + H]+.
d6) δ ppm: 8.55 (s, 1H), 8.25 (d, J = 1.5 Hz, 1H), 7.83 (dd, J = 8.8, 1.5
Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H), 6.72 (d, J
= 8.2 Hz, 2H), 5.44 (brs, 2H), 2.26 (s, 3H). LCMS found, 376 [M −
H]+.
N1-(6-Iodo-3-nitroquinolin-4-yl)-N4,N4-dimethylbenzene-
1,4-diamine (12h). Prepared using General Procedure A, reacting
intermediate 11 with N,N-dimethyl p-phenylenediamine sulfate.
Compound 12h was isolated as an orange solid (yield: 92%).1H
NMR (400 MHz, DMSO-d6) δ ppm: 10.14 (s, 1H), 9.00 (s, 1H), 8.73
(d, J = 1.5 Hz, 1H), 8.05 (dd, J = 8.7, 1.5 Hz, 1H), 7.68 (d, J = 8.7 Hz,
1H), 6.99 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 2.91 (s, 6H).
LCMS found, 435 [M + H]+.
6-Iodo-N4-(4-methoxyphenyl)quinoline-3,4-diamine (13g).
Prepared from intermediate 12g using General Procedure B.
1
Compound 13g was obtained as a tan solid (yield: 37%). H NMR
(400 MHz, DMSO-d6) δ ppm: 8.55 (s, 1H), 8.09 (m, 1H), 7.56 (m,
3H), 6.75 (d, J = 9.0 Hz, 2H), 6.48 (d, J = 9.0 Hz, 2H), 5.25 (brs, 2H),
3.64 (s, 3H). LCMS found, 392 [M + H]+.
6-Iodo-N-(4-morpholinophenyl)-3-nitroquinolin-4-amine
(12i). Prepared using General Procedure A, reacting intermediate 11
with 4-morpholinoaniline. Compound 12i was isolated as an orange
N4-(4-(Dimethylamino)phenyl)-6-iodoquinoline-3,4-diamine
(13h). Prepared from intermediate 12h using General Procedure B.
Compound 13h was isolated as an orange solid (yield: 84%). 1H NMR
(400 MHz, DMSO-d6) δ ppm: 8.53 (s, 1H), 8.13 (s,1H), 7.58 (d, J =
1.5 Hz, 2H), 7.47 (s, 1H), 6.65 (d, J = 9.0 Hz, 2H), 6.49 (d, J = 9.0 Hz,
2H), 5.18 (s, 2H), 2.76 (s, 6H). LCMS found, 405 [M + H]+.
6-Iodo-N4-(4-morpholinophenyl)quinoline-3,4-diamine
(13i). Prepared from intermediate 12i using General Procedure B.
Purification of the crude was carried out by recrystallization from
EtOH/Et2O to give 13i as a yellow solid (yield: 45%). 1H NMR (400
MHz, DMSO-d6) δ ppm: 8.56 (s, 1H), 8.11 (s,1H), 7.57 (m, 3H),
6.80 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 8.8 Hz, 2H), 6.24 (brs, 2H), 3.70
(t, J = 4.7 Hz, 4H), 2.93 (t, J = 4.7 Hz, 4H). LCMS found, 447 [M +
H]+.
1
solid (yield: 88%). H NMR (400 MHz, DMSO-d6) δ ppm: 10.09 (s,
1H), 9.01 (s, 1H), 8.72 (d, J = 1.8 Hz, 1H), 8.07 (dd, J = 8.8, 1.8 Hz,
1H), 7.70 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 9.0
Hz, 2H), 3.74 (t, J = 4.8 Hz, 4H), 3.11 (t, J = 4.8 Hz, 4H). LCMS
found, 477 [M + H]+.
6-Iodo-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-ni-
troquinolin-4-amine (12j). Prepared using General Procedure A,
reacting intermediate 11 with 4-[(4-methyl-1-piperazinyl)methyl]-
aniline. Compound 12j was isolated as a yellow solid (yield: 78%). 1H
NMR (400 MHz, CDCl3) δ ppm: 10.69 (s, 1H), 9.45 (s, 1H), 7.89
(m, 2H), 7.70 (d, J = 9.3 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.14 (d, J
= 8.3 Hz, 2H), 3.57 (s, 2H), 2.52 (m, 8H), 2.32 (s, 3H). LCMS found,
504 [M + H]+.
6-Iodo-N4-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-
quinoline-3,4-diamine (13j). Prepared from intermediate 12j using
General Procedure B. Compound 13j was isolated as an off-white solid
6-Iodo-N-methyl-3-nitroquinolin-4-amine (12k). To a solution
of intermediate 3 (1 g, 2.9 mmol) in MeOH (250 mL) was bubbled
methylamine, and a precipitate was formed. The reaction mixture was
stirred for another hour, after which the precipitate was recovered by
filtration, washed with MeOH, and dried under vacuum to afford a
yellow solid (0.7 g, 2.1 mmol, 74%). 1H NMR (400 MHz, DMSO-d6)
δ ppm: 8.93 (s, 1H), 8.85 (m, 2H), 8.06 (dd, J = 8.8, 1.9 Hz, 1H), 7.64
(d, J = 8.8 Hz, 1H), 2.98 (s, 3H). LCMS found, 330 [M + H]+.
2-(4-((3-Amino-6-iodoquinolin-4-yl)amino)phenyl)-2-meth-
ylpropanenitrile (13). Prepared from intermediate 12 using General
Procedure B. Purification by flash column chromatography on silica gel
(eluent: CH2Cl2/MeOH 100:0 to 95:5) gave 13 as a yellow solid
(yield: 66%). 1H NMR (400 MHz, CD2Cl2) δ ppm: 8.55 (s, 1H), 8.11
(s, 1H), 7.65 (m, 2H), 7.27 (d, J = 8.7 Hz, 2H), 6.60 (d, J = 8.7 Hz,
2H), 5.60 (brs, 1H), 4.04 (brs, 2H), 1.63 (s, 6H). LCMS found, 429
[M + H]+.
1
(yield: 66%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.59 (s, 1H),
8.06 (s, 1H), 7.78 (s, 1H), 7.59 (m, 2H), 7.03 (d, J = 8.5 Hz, 2H), 6.48
(d, J = 8.5 Hz, 2H), 5.34 (s, 2H), 3.29 (s, 2H), 2.30 (m, 8H), 2.12 (s,
3H). LCMS found, 472 [M − H]+.
6-Iodo-N4-methylquinoline-3,4-diamine (13k). Prepared from
intermediate 12k using General Procedure B. Purification of the crude
was carried out by recrystallization from Et2O to give 13k as a yellow
gummy solid (yield: 49%). 1H NMR (400 MHz, CDCl3) δ ppm: 8.46
(s, 1H), 8.20 (s, 1H), 7.68 (m, 2H), 4.30 (s, 1H), 3.82 (brs, 2H), 2.99
(s, 3H). LCMS found, 300 [M + H]+.
2-(4-(8-Iodo-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]quinolin-
1-yl)phenyl)-2-methylpropanenitrile (14). Prepared from 13
using General Procedure C to give 14 as a brown solid (yield:
91%). 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.77 (s, 1H), 7.83 (d, J
= 8.5 Hz, 2H), 7.74 (m, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.14 (s, 1H),
5.74 (s, 1H), 1.79 (s, 6H). LCMS found, 455 [M + H]+.
6-Iodo-N4-(4-(trifluoromethyl)phenyl)quinoline-3,4-diamine
(13a). Prepared from intermediate 12a using General Procedure B.
8-Iodo-1-(4-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]-
quinolin-2(3H)-one (14a). Prepared from intermediate 13a, using
General Procedure C. Compound 14a was isolated as a white solid
1
Compound 13a was isolated as a tan solid (yield: 94%). H NMR
(400 MHz, DMSO-d6) δ ppm: 8.62 (s, 1H), 8.35 (s, 1H), 8.01 (s,
1H), 7.61 (s, 2H), 7.44 (d, J = 8.2 Hz, 2H), 6.59 (d, J = 8.2 Hz, 2H),
5.55 (brs, 2H). LCMS found 43,0 [M + H]+.
1
(yield: 91%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.73 (s, 1H),
8.03 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 7.9 Hz, 2H), 7.71 (m, 2H), 7.29
(s, 1H), 3.35 (brs, 1H). LCMS found, 454 [M − H]+.
4-((3-Amino-6-iodoquinolin-4-yl)amino)benzonitrile (13b).
Prepared from intermediate 12b using the General Procedure B.
4-(8-Iodo-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-
yl)benzonitrile (14b). Prepared from intermediate 13b using the
General Procedure C. Compound 14b was isolated as a yellow solid
1
Compound 13b was isolated as a tan solid (yield: 84%). H NMR
(400 MHz, DMSO-d6) δ ppm: 8.61 (s, 1H), 8.56 (s, 1H), 7.96 (s,
1H), 7.61 (m, 2H), 7.52 (d, J = 8.8 Hz, 2H), 6.56 (m, 2H), 5.62 (s,
2H). LCMS found, 387 [M + H]+.
1
(yield: 99%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.68 (s, 1H),
8.1 (d, J = 8.6 Hz, 2H), 7.76 (m, 2H), 7.68 (s, 1H), 7.36 (d, J = 1.8 Hz,
1H), 6.58 (d, J = 8.6 Hz, 1H), 6.12 (brs, 1H). LCMS found, 413 [M +
H]+.
6-Iodo-N4-phenylquinoline-3,4-diamine (13c). Prepared from
12c using General Procedure B. Compound 13c was isolated as a tan
1
solid (yield: 98%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.58 (s,
8-Iodo-1-phenyl-1H-imidazo[4,5-c]quinolin-2(3H)-one (14c).
1H), 8.05 (s, 1H), 7.81 (s, 1H), 7.59 (d, J = 1.01 Hz, 2H), 7.12 (dd, J
Prepared from intermediate 13c using General Procedure C.
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1
Compound 14c was isolated as a white solid (yield: 94%). H NMR
(400 MHz, DMSO-d6) δ ppm: 8.76 (s, 1H), 7.75 (m, 2H), 7. 67 (m,
3H), 7.60 (m, 2H), 7.27 (m, 2H). LCMS found, 386 [M − H]+.
2-(4-(8-Iodo-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]quinolin-
1-yl)phenyl)acetonitrile. (14d). Prepared from intermediate 13d
using General Procedure C. Compound 14d was isolated as a yellow
washed with H2O and dried under vacuum to afford 15d (0.37 g, 0.84
mmol, 90%) as a purple solid. H NMR (400 MHz, DMSO-d6) δ
1
ppm: 9.03 (s, 1H), 7.80 (m, 2H), 7.66 (m, 4H), 7.36 (s, 1H), 4.27 (s,
2H), 3.59 (s, 3H). LCMS found, 441 [M + H]+.
8-Iodo-3-methyl-1-(p-tolyl)-1H-imidazo[4,5-c]quinolin-
2(3H)-one (15e). Prepared from intermediate 14e using General
Procedure D. Compound 15e was isolated as a brown solid (yield:
93%). 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.01 (s, 1H), 7.78 (d, J
= 1.5 Hz, 2H), 7.48 (m, 4H), 7.36 (m, 1H), 3.58 (s, 3H), 3.31 (s, 3H).
LCMS found, 416 [M + H]+.
1
brown solid (yield: 62%). H NMR (400 MHz, DMSO-d6) δ ppm:
8.68 (s, 1H), 7.76 (s, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.62 (m, 3H), 7.54
(d, J = 8.6 Hz, 2H), 7.40 (d, J = 1.5 Hz, 1H), 4.23 (s, 2H). LCMS
found, 427 [M + H]+
8-Iodo-1-(4-methoxyphenyl)-3-methyl-1H-imidazo[4,5-c]-
quinolin-2(3H)-one (15g). Prepared from intermediate 14g using
General Procedure D. Compound 15g was obtained as a tan solid
8-Iodo-1-(p-tolyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one
(14e). Prepared from intermediate 13e using General Procedure C.
1
Compound 14e was isolated as a tan solid (yield: 82%). H NMR
1
(yield: 73%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.09 (s, 1H),
(400 MHz, DMSO-d6) δ ppm: 8.70 (s, 1H), 7.69 (m, 2H), 7.44 (m,
6H), 2.47 (s, 3H). LCMS found, 402 [M + H]+.
7.77 (m, 2H), 7.50 (d, J = 9.0 Hz, 2H), 7.35 (m, 1H), 7.21 (d, J = 9.0
Hz, 2H), 3.88 (s, 3H), 3.57 (s, 3H). LCMS found, 432 [M + H]+.
1-(4-(Dimethylamino)phenyl)-8-iodo-3-methyl-1H-imidazo-
[4,5-c]quinolin-2(3H)-one (15h). Prepared from intermediate 14h
using General Procedure D. Compound 15h was isolated as a brown
8-Iodo-1-(4-methoxyphenyl)-1H-imidazo[4,5-c]quinolin-
2(3H)-one (14g). Prepared from intermediate 13g using General
Procedure C. Compound 14g was obtained as a tan solid (yield: 79%).
1H NMR (400 MHz, DMSO-d6) δ ppm: 8.71 (s, 1H), 7.70 (m, 2H),
1
solid (yield: 70%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.98 (s,
7.46 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 1.0 Hz, 1H), 7.19 (d, J = 8.8 Hz,
2H), 3.87 (s, 3H), 3.55 (brs, 1H). LCMS found, 418 [M + H]+.
1-(4-(Dimethylamino)phenyl)-8-iodo-1H-imidazo[4,5-c]-
quinolin-2(3H)-one (14h). Prepared from intermediate 13h using
General Procedure C. Compound 14h was isolated as a brown solid
1H), 7.77 (m, 2H), 7.45 (m, 1H), 7.33 (d, J = 9.0 Hz, 2H), 6.93 (d, J =
9.0 Hz, 2H), 3.57 (s, 3H), 3.03 (s, 6H). LCMS found, 445 [M + H]+.
8-Iodo-3-methyl-1-(4-morpholinophenyl)-1H-imidazo[4,5-
c]quinolin-2(3H)-one (15i). Prepared from intermediate 14i using
General Procedure D. Compound 15i was isolated as a brown solid
1
(yield: 57%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.73 (s, 1H),
1
(yield: 76%). H NMR (400 MHz, DMSO-d6) δ ppm: 8.99 (s, 1H),
7.74 (m, 2H), 7.44 (m, 1H), 7.32 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.9
Hz, 2H), 3.33 (brs, 1H), 3.02 (s, 6H). LCMS found, 431 [M + H]+.
8-Iodo-1-(4-morpholinophenyl)-1H-imidazo[4,5-c]quinolin-
2(3H)-one (14i). Prepared from intermediate 13i using General
Procedure C. Compound 14i was isolated as a brown solid (yield:
39%). 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.64 (s, 1H), 7.66 (d, J
= 8.8 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 1.8 Hz, 1H), 7.32
(d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 3.80 (t, J = 4.7 Hz, 4H),
3.37 (brs, 1H), 3.23 (t, J = 4.7 Hz, 4H). LCMS found, 473 [M + H]+.
8-Iodo-1-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-
imidazo[4,5-c]quinolin-2(3H)-one (14j). Prepared from intermedi-
ate 13j using General Procedure C. Compound 14j was isolated as a
7.78 (m, 2H), 7.41 (m, 3H), 7.20 (d, J = 9.1 Hz, 2H), 3.81 (t, J = 4.8
Hz, 4H), 3.57 (s, 3H), 3.27 (t, J = 4.8 Hz, 4H). LCMS found, 487 [M
+ H]+.
8-Iodo-1,3-dimethyl-1H-imidazo[4,5-c]quinolin-2(3H)-one
(15k). A mixture of intermediate 14k (53 mg, 0.16 mmol), K2CO3 (34
mg, 0.24 mmol), and methyl iodide (15 μL, 0.24 mmol) in acetone
(15 mL) was stirred for 17 h at rt. To complete the reaction another
equiv of K2CO3 (24 mg, 0.16 mmol) and methyl iodide (10 μL, 0.16
mmol) were added, and the resultant solution was left under stirring
for 48 h. The mixture was then taken to dryness, and the solids were
washed with H2O to give 15k as a yellow solid (23 mg, 68 μmol, 42%).
1H NMR (400 MHz, DMSO-d6) δ ppm: 8.89 (s, 1H), 8.60 (d, J = 1.8
1
yellow solid (yield: 87%). H NMR (400 MHz, DMSO-d6) δ ppm:
8.76 (s, 1H), 7.75 (m, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.1
Hz, 2H), 7.20 (s, 1H), 3.61 (s, 2H), 2.36 (m, 9H), 2.16 (s, 3H).
LCMS found, 500 [M + H]+.
8-Iodo-1-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one (14k).
Prepared from intermediate 13k using General Procedure C.
Compound 14k was isolated as a yellow solid (yield: 98%). 1H
NMR (400 MHz, DMSO-d6) δ ppm: 8.54 (s, 1H), 8.51 (d, 1.0 Hz,
1H), 7.64 (m, 2H), 3.71 (s, 3H), 3.18 (brs, 1H). LCMS found, 326 [M
+ H]+.
Hz, 1H), 7.87 (dd, J = 8.8, 1.8 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 3.80
(s, 3H), 3.52 (s, 3H). LCMS found, 340 [M + H]+.
3-Methyl-8-(quinolin-3-yl)-1-(4-(trifluoromethyl)phenyl)-1H-
imidazo[4,5-c]quinolin-2(3H)-one (16a). Prepared using General
Procedure E, reacting intermediate 15a with 3-quinoline boronic acid.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 16a as a
1
yellow solid (yield: 26%). H NMR (400 MHz, DMSO-d6) δ ppm:
9.08 (s, 1H), 8.92 (d, J = 2.3 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.21
(d, J = 8.8 Hz, 1H), 8.14 (m, 3H), 8.03 (d, J = 8.6 Hz, 1H), 7.99 (d, J
= 8.1 Hz, 2H), 7.92 (d, J = 7.3 Hz, 1H), 7.77 (m, 1H), 7.66 (m, 1H),
7.33 (d, J = 1.8 Hz, 1H), 3.63 (s, 3H). LCMS found, 471 [M + H]+.
4-(3-Methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-
imidazo[4,5-c]quinolin-1-yl)benzonitrile (16b). Prepared using
General Procedure E, reacting intermediate 15b with 3-quinoline
boronic acid. Purification was carried out by flash column
chromatography over silica gel (eluent: CH2Cl2/MeOH 100:0 to
8-Iodo-3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-imidazo-
[4,5-c]quinolin-2(3H)-one (15a). Prepared from intermediate 14a
using General Procedure D. Compound 15a was isolated as a pale
1
yellow solid (yield: 75%). H NMR (400 MHz, DMSO-d6) δ ppm:
9.01 (s, 1H), 7.77 (m, 1H), 7.69 (m, 3H), 7.60 (m, 2H), 7.27 (m, 1H),
3.57 (s, 3H). LCMS found, 468 [M − H]+.
4-(8-Iodo-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-1-yl)benzonitrile (15b). Prepared from intermediate 14b
using General Procedure D. Compound 15b was isolated as a yellow
1
95:5) to give 16b as a pale yellow solid (yield: 43%). H NMR (400
1
MHz, DMSO-d6) δ ppm: 9.08 (s, 1H), 8.90 (d, J = 2.5 Hz, 1H), 8.37
(d, J = 2.3 Hz, 1H), 8.26 (d, J = 8.6 Hz, 2H), 8.21 (m, 1H), 8.12 (dd, J
= 9.1, 2.0 Hz, 1H), 8.03 (m, 2H), 7.97 (m, 2H), 7.79 (m, 1H), 7.67
(m, 1H), 7.35 (d, J = 1.8 Hz, 1H), 3.62 (s, 3H). LCMS found, 428 [M
+ H]+.
solid (yield: 57%). H NMR (400 MHz, DMSO-d6) δ ppm: 9.05 (s,
1H), 8.20 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.81 (m, 2H),
7.30 (m, 1H), 3.58 (s, 3H). LCMS found, 427 [M + H]+.
8-Iodo-3-methyl-1-phenyl-1H-imidazo[4,5-c]quinolin-2(3H)-
one (15c). Prepared from intermediate 14c using General Procedure
D. Compound 15c was isolated as a pale yellow solid (yield: 44%). 1H
NMR (400 MHz, DMSO-d6) δ ppm: 9.01 (s, 1H), 7.77 (m, 2H), 7.69
(m, 2H), 7.60 (m, 2H), 7.27 (m, 2H), 3.58 (s, 3H). LCMS found, 402
[M + H]+.
2-(4-(8-Iodo-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-1-yl)phenyl)acetonitrile (15d). To a solution of 14d (0.4
g, 0.94 mmol) in DMF (25 mL) were successively added K2CO3 (156
mg, 1.13 mmol) and methyl iodide (70 μL, 1.13 mmol). The resultant
mixture was stirred for 4.5 h at rt and then filtered. Volatiles were
evaporated under reduced pressure to give a precipitate that was
3-Methyl-1-phenyl-8-(quinolin-3-yl)-1H-imidazo[4,5-c]-
quinolin-2(3H)-one (16c). Prepared using General Procedure E,
reacting intermediate 15c with 3-quinoline boronic acid. Purification
was carried out by flash column chromatography over silica gel
(eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 16c as a white solid
1
(yield: 38%). H NMR (400 MHz, DMSO-d6) δ ppm: 9.04 (s, 1H),
8.79 (d, J = 2.3 Hz, 1H), 8.37 (d, J = 2.3 Hz, 1H), 8.18 (d, J = 8.8 Hz,
1H), 8.10 (dd, J = 8.8, 2.0 Hz, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.98 (d, J
= 7.3 Hz, 1H), 7.74 (m, 6H), 7.66 (m, 1H), 7.35 (d, J = 1.8 Hz,
1H),3.62 (s, 3H). LCMS found, 403 [M + H]+.
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2-(4-(3-Methyl-2-oxo-8-(pyridin-4-yl)-2,3-dihydro-1H-
imidazo[4,5-c]quinolin-1-yl)phenyl)acetonitrile (16d). Prepared
using General Procedure E, reacting intermediate 15d with pyridine 4-
boronic acid pinacol ester. Purification was carried out by flash column
chromatography over silica gel (eluent: CH2Cl2/MeOH 100:0 to
95:5) to give 16d as a yellow solid (yield: 17%). 1H NMR (400 MHz,
DMSO-d6) δ ppm: 9.05 (s, 1H), 8.60 (d, J = 5.0 Hz, 2H), 8.15 (d, J =
8.6 Hz, 1H), 8.00 (d, J = 8.08 Hz, 1H), 7.73 (m, 4H), 7.30 (m, 3H),
4.28 (s, 2H), 3.61 (s, 3H). LCMS found, 392 [M + H]+.
out by flash column chromatography over silica gel (eluent: CH2Cl2/
MeOH 100:0 to 90:10) to give 16k as a yellow solid (yield: 20%). 1H
NMR (400 MHz, CDCl3) δ ppm: 8.76 (m, 3H), 8.51 (d, J = 1.7 Hz,
1H), 8.28 (d, J = 8.8 Hz, 1H), 7.90 (dd, J = 8.8, 1.7 Hz, 1H), 7.64 (dd,
J = 4.5, 1.6, 2H), 4.03 (s, 3H), 3.66 (s, 3H). LCMS found, 291 [M +
H]+.
4-(2-Oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-1-yl)benzonitrile (17b). Prepared using General Proce-
dure E, reacting intermediate 14b with 3-quinoline boronic acid.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 17b as a
white solid (yield: 43%). 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.90
(d, J = 2.3 Hz, 1H), 8.81 (s, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.23 (d, J =
8.6 Hz, 2H), 8.18 (d, J = 8.8 Hz, 1H), 8.04 (m, 3H), 7.96 (d, J = 8.6
Hz, 2H), 7.78 (m, 1H), 7.66 (m, 1H), 7.38 (d, J = 2.0 Hz, 1H), 5.95
(s, 1H). LCMS found, 414 [M + H]+.
3-Methyl-8-(pyridin-4-yl)-1-(p-tolyl)-1H-imidazo[4,5-c]-
quinolin-2(3H)-one (16e). Prepared using General Procedure E,
reacting intermediate 15e with pyridine 4-boronic acid pinacol ester.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 90:10) to give 16e as a
1
yellow solid (yield: 54%). H NMR (400 MHz, DMSO-d6) δ ppm:
9.04 (s, 1H), 8.59 (dd, J = 4.5, 1.9 Hz, 2H), 8.14 (d, J = 8.8 Hz, 1H),
7.98 (dd, J = 8.8, 1.9 Hz, 1H), 7.55 (m, 4H), 7.31 (dd, J = 4.5, 1.5 Hz,
2H), 7.25 (d, J = 2.0 Hz, 1H), 3.61 (s, 3H), 3.31 (s, 3H). LCMS
found, 367 [M + H]+.
2-(4-(2-Oxo-8-(pyridin-4-yl)-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-1-yl)phenyl)acetonitrile (17d). Prepared using General
Procedure E, reacting intermediate 14d with 4-pyridine boronic ester.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 17d as a
4-(3-Methyl-2-oxo-8-(pyridin-4-yl)-2,3-dihydro-1H-imidazo-
[4,5-c]quinolin-1-yl)benzonitrile (16f). Prepared using General
Procedure E, reacting intermediate 15b with pyridine 4-boronic acid
pinacol ester. Purification was carried out by flash column
chromatography over silica gel (eluent: CH2Cl2/MeOH 100:0 to
1
yellow solid (yield: 12%). H NMR (400 MHz, DMSO-d6) δ ppm:
8.81 (s, 1H), 8.59 (dd, J = 4.5, 1.7 Hz, 2H), 8.12 (d, J = 8.9 Hz, 1H),
7.99 (dd, J = 8.9, 2.0 Hz, 1H), 7.72 (m, 4H), 7.31 (dd, J = 4.5, 1.7 Hz,
2H), 7.29 (d, J = 2.0 Hz, 1H), 4.28 (s, 2H), 3.33 (brs, 1H). LCMS
found, 378 [M + H]+.
1
95:5) to give 16f as a white solid (yield: 5%). H NMR (400 MHz,
DMSO-d6) δ ppm: 9.08 (s, 1H), 8.59 (d, J = 6.3 Hz, 2H), 8.22 (d, J =
8.6 Hz, 2H), 8.17 (d, J = 9.1 Hz, 1H), 7.98 (dd, J = 9.1, 2.0 Hz, 1H),
7.94 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 6.3 Hz, 2H), 7.29 (d, J = 2.0 Hz,
1H), 3.61 (s, 3H). LCMS found, 428 [M + H]+.
1-(4-(Dimethylamino)phenyl)-8-(pyridin-4-yl)-1H-imidazo-
[4,5-c]quinolin-2(3H)-one (17h). Prepared using General Procedure
E, reacting intermediate 14h with pyridine 4-boronic acid pinacol
ester. Purification was carried out by flash column chromatography
over silica gel (eluent: CH2Cl2/MeOH 100:0 to 90:10) to give 17h as
a salmon solid (yield: 9%, purity: 94%). 1H NMR (400 MHz, DMSO-
d6) δ ppm: 8.76 (s, 1H), 8.56 (d, J = 5.8 Hz, 2H), 8.09 (d, J = 8.8 Hz,
1H), 7.97 (dd, J = 8.8, 2.0 Hz, 1H), 7.38 (m, 6H), 6.99 (d, J = 9.1 Hz,
2H), 3.06 (s, 6H). LCMS found, 382 [M + H]+. Also isolated from
this reaction was 1-(4-(dimethylamino)phenyl)-1H-imidazo[4,5-c]-
1-(4-Methoxyphenyl)-3-methyl-8-(pyridin-4-yl)-1H-imidazo-
[4,5-c]quinolin-2(3H)-one (16g). Prepared using General Procedure
E, reacting intermediate 15g with pyridine 4-boronic acid pinacol ester.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 16g as a tan
1
solid (yield: 32%). H NMR (400 MHz, DMSO-d6) δ ppm: 9.02 (s,
1H), 8.57 (m, 2H), 8.13 (d, J = 8.8 Hz, 1H), 7.96 (dd, J = 8.8, 2.0 Hz,
1H), 7.58 (d, J = 8.8 Hz, 2H), 7.34 (m, 2H), 7.30 (d, J = 2.0 Hz, 1H),
7.26 (m, 2H), 3.92 (s, 3H), 3.60 (s, 3H). LCMS found, 383 [M + H]+.
1-(4-(Dimethylamino)phenyl)-3-methyl-8-(pyridin-4-yl)-1H-
imidazo[4,5-c]quinolin-2(3H)-one (16h). Prepared using General
Procedure E, reacting intermediate 15h with pyridine 4-boronic acid
pinacol ester. Purification was carried out by flash column
chromatography over silica gel (eluent: CH2Cl2/MeOH 100:0 to
1
quinolin-2(3H)-one M1009/84/4. (18) (yield: 5%). H NMR (400
MHz, DMSO-d6) δ ppm: 8.72 (s, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.47
(m, 2H), 7.31 (m, 3H), 7.15 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 8.8 Hz,
2H), 3.02 (s, 6H). LCMS found, 305 [M + H]+.
1-(4-(Dimethylamino)phenyl)-3-methyl-1H-imidazo[4,5-c]-
quinolin-2(3H)-one (19). Prepared using the General Procedure E,
reacting intermediate 15h in the absence of boronic acid. Purification
was carried out by flash column chromatography over silica gel
(eluent: CH2Cl2/MeOH 100:0 to 95:5) to give 19 as a salmon solid.
1
90:10) to give 16h as a salmon solid (yield: 16%). H NMR (400
MHz, DMSO-d6) δ ppm: 9.00 (s, 1H), 8.56 (d, J = 5.8 Hz, 2H), 8.12
(d, J = 8.8 Hz, 1H), 7.98 (dd, J = 8.4, 1.78 Hz, 1H), 7.38 (m, 5H), 6.99
(d, J = 8.8 Hz, 2H), 3.60 (s, 3H), 3.06 (s, 6H). LCMS found, 396 [M
+ H]+.
3-Methyl-1-(4-morpholinophenyl)-8-(pyridin-4-yl)-1H-
imidazo[4,5-c]quinolin-2(3H)-one (16i). Prepared using General
Procedure E, reacting intermediate 15i with pyridine 4-boronic acid
pinacol ester. Purification was carried out by flash column
chromatography over silica gel (eluent: CH2Cl2/MeOH 100:0 to
90:10) to give 16i as a salmon solid (yield: 28%). 1H NMR (400 MHz,
DMSO-d6) δ ppm: 9.02 (s, 1H), 8.58 (d, J = 5.8 Hz, 2H), 8.13 (d, J =
8.8 Hz, 1H), 7.99 (dd, J = 8.8, 1.9 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H),
7.37 (m, 3H), 7.25 (d, J = 8.8 Hz, 2H), 3.83 (m, 4H), 3.61 (s, 3H),
3.24 (m, 4H). LCMS found, 438 [M + H]+.
1-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-8-(pyridin-4-
yl)-1H-imidazo[4,5-c]quinolin-2(3H)-one (16j). Prepared using
General Procedure E, reacting intermediate 14j with pyridine 4-
boronic acid pinacol ester. Purification was carried out by flash column
chromatography over amino-silica gel (eluent: CH2Cl2/MeOH 100:0
to 95:5) to give 16j as a white solid (yield: 23%). 1H NMR (400 MHz,
DMSO-d6) δ ppm: 8.78 (s, 1H), 8.54 (m, 2H), 8.10 (d, J = 9.1 Hz,
1H), 7.91 (dd, J = 8.8, 1.9 Hz, 1H), 7.60 (m, 3H), 7.52 (m, 2H), 7.36
(d, J = 1.9 Hz, 1H), 7.31 (dd, J = 4.4, 1.6 Hz, 2H), 3.65 (s, 2H), 2.45
(m, 4H), 2.33 (m, 4H), 2.17 (s, 3H). LCMS found, 451 [M + H]+.
1-Methyl-8-(pyridin-4-yl)-1H-imidazo[4,5-c]quinolin-2(3H)-
one (16k). Prepared using General Procedure E, reacting intermediate
15k with pyridine 4-boronic acid pinacol ester. Purification was carried
1
(yield: 13%, purity: 91%). H NMR (400 MHz, DMSO-d6) δ ppm:
8.96 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.56 (m, 1H), 7.31 (m, 3H),
7.16 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 9.10 Hz, 2H), 3.57 (s, 3H), 3.03
(s, 6H). LCMS found, 319 [M + H]+.
2-(4-((3′-Amino-[3,6′-biquinolin]-4′-yl)amino)phenyl)-2-
methylpropanenitrile (20). Prepared using General Procedure E,
reacting compound 13 and 3-quinoline boronic acid. Purification was
carried out by flash column chromatography over silica gel (eluent:
CH2Cl2/MeOH 100:0 to 95:5) to give 20 as a pale yellow solid (yield:
1
2%). H NMR (400 MHz, DMSO-d6) δ ppm: 9.23 (d, J = 2.3 Hz,
1H), 8.63 (s, 2H), 8.16 (m, 1H), 8.09 (s, 1H), 8.02 (m, 3H), 7.87 (dd,
J = 8.8, 2.0 Hz, 1H), 7.76 (m, 1H), 7.64 (m, 1H), 7.28 (d, 8.8 Hz, 2H),
6.63 (d, J = 8.8 Hz, 2H), 5.30 (brs, 2H), 1.60 (s, 6H). LCMS found,
430 [M + H]+.
4-(3-Methyl-2-oxo-1-(p-tolyl)-2,3-dihydro-1H-imidazo[4,5-c]-
quinolin-8-yl)benzoic Acid (21). Prepared using General Procedure
E, reacting intermediate 15e with 4-carboxyphenyl boronic acid.
Purification was carried out by flash column chromatography over
silica gel (eluent: CH2Cl2/MeOH 100:0 to 90:10) to give 21 as a
1
yellow solid (yield: 27%). H NMR (400 MHz, DMSO-d6) δ ppm:
9.00 (s, 1H), 8.10 (d, J = 9.1 Hz, 1H), 7.93 (m, 3H), 7.54 (m, 4H),
7.39 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 1.3 Hz, 1H), 3.61 (s, 3H), 3.31
(s, 3H). LCMS found, 410 [M + H]+.
3,8-Dimethyl-1-phenyl-1H-imidazo[4,5-c]quinolin-2(3H)-one
(22). Prepared from intermediate 15c using General Procedure G.
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1
Compound 22 was obtained as a white solid (yield: 17%). H NMR
(400 MHz, DMSO-d6) δ ppm: 8.92 (s, 1H), 7.92 (d, J = 8.8 Hz 1H),
7.67 (m, 3H), 7.59 (m, 2H), 7.38 (dd, J = 8.8, 1.8 Hz, 1H), 6.70 (s,
1H), 3.58 (s, 3H), 2.16 (s, 3H). LCMS found, 290 [M + H]+.
3-Methyl-1-phenyl-1H-imidazo[4,5-c]quinolin-2(3H)-one
(23). Prepared using General Procedure E, reacting intermediate 15c
with trimethylboroxin. Purification was carried out by flash column
chromatography over silica gel (eluent: CH2Cl2/MeOH 100:0 to
Cytotoxicity Assay. This assay was used as a selectivity assay, and
compounds were tested at dose−response concentrations against
HepG2 in order to identify the level of cytotoxicity. Dose− response
starting at 10 mM, unless indicated, with 3-fold dilutions for 11 points
was made in master plates (1536). Fifty nanoliters per well was
stamped in final assay plates.
Log-phase HepG2 cells were removed from a T-175 TC flask using
cell dispersion medium and dispersed by repeated pipetting. Cell
density was adjusted to 60 000 cells/mL as the working concentration
in prewarmed Eagle’s MEM. The seeding density was checked to
ensure that new monolayers were not more than ∼50% confluent at
the time of seeding (typically 3000 cells per well), before completing
preparation of the plates. Five microliters of culture was dispensed in
compound-stamped TC treated, Greiner white 1536-well plates using
a Multidrop Combi Reagent Dispenser (Thermo Scientific) to a final
concentration of 1% DMSO. Cells were incubated for 48 h at 37 °C
and 5% CO2. Viability was determined by CellTiter-Glo kit (Promega)
according to the manufacturer’s instructions. Briefly, reconstituted
CellTiter Buffer was equilibrated to room temperature prior use, and 5
μL per well was dispensed with a Multidrop Combi Reagent
Dispenser. Contents were mixed on a plate orbital shaker and
incubated for 10 min at room temperature to allow the signal to
stabilize before luminescence reading on ViewLux Plate Reader
(PerkinElmer).
1
95:5) to give 23 as an off-white solid (yield: 33%, purity: 94%). H
NMR (400 MHz, DMSO-d6) δ ppm: 9.01 (s, 1H), 8.03 (d, J = 7.8 Hz,
1H), 7.6 (m, 6H), 7.29 (m, 1H), 6.99 (d, J = 8.6 Hz, 1H), 3.59 (s,
3H). LCMS found, 276 [M + H]+.
Cellular Activity Assays. Trypanosome Cell Culture and Cell
Growth Assays. Bloodstream Trypanosoma brucei brucei Lister 427 was
the selected strain to perform the HTS experiments and the profiling
assay. The cell strain was cultured in Hirumi’s modified Iscove’s
medium (HMI-9)29 supplemented with 10% heat-inactivated FBS at
37 °C and 5% CO2 in T-25 vented flasks (Corning). We evaluated the
anti-trypanosomal activity of the newly prepared compounds in
Trypanosoma brucei brucei culture according to resazurin viability test.
Dose−Response Assay. For dose−response experiments, serial
dilutions of compounds were plotted against compound concen-
tration. Dose−response starting at 10 mM, unless indicated, with 3-
fold dilutions for 11 points were made in masterplates. Two hundred
nanoliters per well from those masterplates was stamped in final assay
plates. Controls of 0% response (control 1, 0.2 μL of 100% DMSO)
and 100% response (control 2, 0.2 μL of 1 mM Pentamidine) were
included in each assay plate in columns 6 and 18, respectively. Plates
containing 0.2 μL of 100% DMSO were included in the assay to assess
quality through the entire process. To detect growth inhibition,
parasites in log phase growth were diluted to a working concentration
of 2500 cells/mL in prewarmed HMI-9 medium and gently stirred
until dispensation. Fifty microliters of culture was dispensed in
compound-stamped black, clear-bottom, 384-well Greiner microplates
using a Multidrop Combi Reagent Dispenser (Thermo Scientific) to
give a final solvent concentration of 0.4% DMSO. Plates were covered
with a lid, and cells were incubated for 70 h at 37 °C and 5% CO2.
After this period, 10 μL of 200 μM resazurin solution in prewarmed
HMI-9 was added to each well, and plates were allowed to incubate 2
h more prior to fluorescence reading in a Wallac EnVision multilabel
plate reader (PerkinElmer). Raw fluorescent data from the Envision
plate reader were uploaded into GSK HTS database (ActivityBase).
Activity of each well was normalized as a percentage of inhibition on a
per-plate basis using the following equation
Raw luminescence data from the ViewLux reader were uploaded
into GSK HTS database (ActivityBase). Activity of each well was
normalized as a percentage of inhibition on a per-plate basis using the
following equation
test well − median CTRL1
% inhibition = 100 − 100 ×
median CTRL1 − median CTRL2
where control 1 represents wells from the same plate containing 1%
DMSO (0% inhibition, 100% grown control, n = 128) and control 2
represents wells from the same plate treated with digitoxin (100%
inhibition, 0% grown, n = 128). A Z′ value greater than 0.4 was
required for plate validation during the quality control process.
As previously described for the primary assay, a four-parameter
equation describing a sigmoidal dose−response curve was then fitted
with an adjustable baseline using ActvityBase XE Runner software.
Phospholipidomics Analyses. Lipid Extraction. Total lipids
from mid log phase cells were extracted by the method of Bligh and
Dyer.30 Briefly, mid log phase cells were collected by centrifugation
(800g, 10 min), washed with PBS, resuspended in 100 μL of TDB-
glucose, transferred to a glass tube containing 375 μL of 1:2 (v/v)
CHCl3/MeOH, and vortexed. The sample was agitated vigorously for
a further 10−15 min. The sample was made biphasic by the addition of
125 μL of CHCl3 and vortexed, and then 125 μL of H2O was added.
The sample was vortexed again and centrifuged at 3000g at rt for 10
min. The lower (organic) phase was transferred into a new glass vial,
and the aqueous phase was re-extracted with the fresh lower phase.
The resultant lower phase lipid extract was dried under a stream of
nitrogen and stored at 4 °C.
100 × test well − median CTRL1
% inhibition = 100 −
median CTRL1 − median CTRL2
where control 1 represents wells from the same plate containing 0.4%
DMSO (0% inhibition, 100% grown control, n = 16) and control 2
represents wells from the same plate treated with pentamidine (100%
inhibition, 0% grown, n = 16). A Z′ value greater than 0.4 was required
for plate validation during the quality control process.
Electrospray Mass Spectrometry Analysis. Lipid extracts were
dissolved in 15 μL of CHCl3/MeOH (1:2) and 15 μL of acetonitrile/
isopropanol/water (6:7:2) and analyzed with a triple quadrupole mass
spectrometer (Absceix 4000 QTrap) equipped with a nanoelectrospray
source. Samples were delivered into the spectrometer using either
thin-walled nanoflow capillary tips or a Nanomate interface in direct
infusion mode (∼125 nL/min). The lipid extracts were analyzed in
both positive and negative ion modes using a capillary voltage of 1.25
kV. MS/MS scanning (daughter, precursor, and neutral loss scans)
were performed using nitrogen as the collision gas, with collision
energies between 35 and 90 V. Each spectrum encompasses at least 50
repetitive scans. Tandem mass spectra (MS/MS) were obtained with
collision energies as follows: 35−45 V, PC/SM in positive ion mode,
parent-ion scanning of m/z 184; 35−55 V, PI in negative ion mode,
parent-ion scanning of m/z 241; 35−65 V, PE in negative ion mode,
parent-ion scanning of m/z 196; 20−35 V, PS in negative ion mode,
neutral loss scanning of m/z 87; and 40−90 V, for all
glycerophospholipids (including PA, PG, and cardiolipin) detected
A four-parameter equation describing a sigmoidal dose−response
curve was then fitted with an adjustable baseline using ActivityBase XE
Runner software. Fitting of dose−response curves and EC50
determination were normalized as percentage of inhibition based on
controls. The curve-fit model was based on a four-parameter logistic
equation
max − min
Y = min +
slope factor
log cmpd conc(M)
⎛
⎜
⎞
⎟
10
10
1 +
EC
50
10log
10
⎝
⎠
HepG2 Cell Culture and Growth Assays. The HepG2 cell line
(human liver hepatocellular carcinoma cell line, ATCC) was cultured
in Eagle’s MEM supplemented with L-glutamine, Earle’s salts, 10%
heat-inactivated FBS, and 1% non-essential amino acids (NEAA) at 37
°C and 5% CO2 in T-175 vented flasks (Corning). The human
biological samples were sourced ethically, and their research use was in
accord with the terms of informed consent.
4846
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by precursor scanning for m/z 153 in negative ion mode. MS/MS
daughter ion scanning was performed with collision energies between
35 and 90 V.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional mass spectrometry data; data tables from this article
annotated with NEU registry numbers; and biochemical and
physicochemical assay details. This material is available free of
charge via the Internet at http://pubs.acs.org. All of the data
included in this work has also been made available as a
publically available data set on www.collaborativedrug.com.
AUTHOR INFORMATION
■
Corresponding Authors
*(M.N.) Phone: +34 958 181651; E-mail: miguel.navarro@ipb.
csic.es.
*(M.P.P.) Phone: 617-373-2703; E-mail: m.pollastri@neu.edu.
Notes
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ACKNOWLEDGMENTS
■
This work was supported in part by the Tres Cantos Open Lab
Foundation (M.P.P., M.N., R.D., S.L.-A., and J.D.S.), NIH
7R01AI082577 (M.P.P.), and Wellcome Trust grant 093228
(T.K.S.). M.S., D.R., and M.N. are supported by grants from
the Spanish MICINN (SAF2012-40029), Junta de Andalucia
(CTS-5841), and RICET (RD12/0018).
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HAT, human African trypanosomiasis; SAR, structure−activity
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