Synthesis of New Quinolinequinone Derivatives and Preliminary Exploration of their Cytotoxic Properties

Article abstract of DOI:10.1021/jm301689x

A series of 7-amino- and 7-acetamidoquinoline-5,8-diones with aryl substituents at the 2-position were synthesized, characterized, and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1) -directed antitumor agents. The synthesis of lavendamycin analogues is illustrated. Metabolism studies demonstrated that 7-amino analogues were generally better substrates for NQO1 than 7-amido analogues, as were compounds with smaller heteroaromatic substituents at the C-2 position. Surprisingly, only two compounds, 7-acetamido-2-(8′-quinolinyl)quinoline-5,8-dione (11) and 7-amino-2-(2-pyridinyl)quinoline-5,8-dione (23), showed selective cytotoxicity toward the NQO1-expressing MDA468-NQ16 breast cancer cells versus the NQO1-null MDA468-WT cells. For all other compounds, NQO1 protected against quinoline-5,8-dione cytotoxicity. Compound 22 showed potent activity against human breast cancer cells expressing or not expressing NQO1, with respective IC₅₀ values of 190 nM and 140 nM and a low NQO1-mediated reduction rate, which suggests that the mode of action of 22 differs from that of lavendamycin and involves an unidentified target(s).

Full text of DOI:10.1021/jm301689x

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Article
Synthesis of New Quinolinequinone Derivatives and
Preliminary Exploration of their Cytotoxic Properties.
Charles Mambo Keyari, Alison K Kearns, Nathan S Duncan, Emily
A Eickholt, Geoffrey Abbott, Howard D. Beall, and Philippe Diaz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301689x • Publication Date (Web): 10 Apr 2013
Downloaded from http://pubs.acs.org on April 15, 2013
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Synthesis of New Quinolinequinone Derivatives and Preliminary Exploration of
their Cytotoxic Properties
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Charles M. Keyari, Alison K. Kearns, Nathan S. Duncan, Emily A. Eickholt, Geoffrey Abbott, Howard D.
Beall* and Philippe Diaz*
Core Laboratory for Neuromolecular Production, Department of Biomedical and Pharmaceutical
Sciences, The University of Montana, Missoula, MT 59812, USA
ABSTRACT
A series of 7ꢀaminoꢀ and 7ꢀacetamidoquinolineꢀ5,8ꢀdiones with aryl substituents at the 2ꢀposition were
synthesized, characterized and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1)ꢀdirected
antitumor agents. The synthesis of lavendamycin analogs is illustrated. Metabolism studies demonstrated
that 7ꢀaminoꢀ analogues were generally better substrates for NQO1 than 7ꢀamidoꢀ analogues as were
compounds with smaller heteroaromatic substituents at the Cꢀ2 position. Surprisingly, only two
compounds, 7ꢀacetamidoꢀ2ꢀ(8’ꢀquinolinyl)quinolineꢀ5,8ꢀdione (11) and 7ꢀaminoꢀ2ꢀ(2ꢀpyridinyl)quinolineꢀ
5,8ꢀdione (23) showed selective cytotoxicity toward the NQO1ꢀexpressing MDA468ꢀNQ16 breast cancer
cells versus the NQO1ꢀnull MDA468ꢀWT cells. For all other compounds, NQO1 protected against
quinolineꢀ5,8ꢀdione cytotoxicity. Compound 22 showed a potent activity against human breast cancer cells
expressing or not expressing NQO1 with IC50 values of respectively 190 nM and 140 nM and a low NQO1
mediated reduction rate, which suggests that the mode of action of 22 differs from lavendamycin and
involves an unidentified target(s).
KEYWORDS: Lavendamycin, Suzuki coupling, microwave irradiation, palladium (0) catalysis,
quinolinequinones, NQO1, antitumor, cytotoxicity
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INTRODUCTION
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Lavendamycin (Figure 1) is a quinolinequinone antibiotic with antitumor activity first isolated from
Streptomyces lavendulae by Balitz et al in 1982.1ꢀ2 It is structurally related to Streptonigrin which was first
isolated from Streptomyces flocculus.3ꢀ4 Streptonigrin is known for its potent cytotoxic properties,
antitumor activity, and in vitro and in vivo antiviral properties and potent, broad spectrum antimicrobial
properties. Although lavendamycin is not suitable for clinical use due to its toxicity, its analogs are less
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Figure 1. Natural quinolinequinone antibiotics
toxic and hence have potential as antitumor agents.5 Recent findings6ꢀ11 suggest that some indolequinones
and quinolinequinones are excellent substrates for the quinone reductase enzyme, NAD(P)H:quinone
oxidoreductase 1 (NQO1), and are selectively cytotoxic to cancer cell lines that overexpress NQO1. NQO1
is a ubiquitous flavoenzyme that catalyzes the 2ꢀelectron reduction of quinones to hydroquinones, and it is
highly expressed in many solid tumors.12 This forms the basis for the synthesis of novel quinolinequinones
structurally related to lavendamycin as potential NQO1ꢀdirected antitumor agents.
Behforouz et al. (1996)13 first demonstrated that 7ꢀaminoquinolineꢀ5,8ꢀdiones can be efficiently
prepared from commercially available 8ꢀhydroxyꢀ2ꢀmethylquinoline. Fryatt and coꢀworkers7 also showed
that by starting with 6ꢀmethoxyquinoline, 6ꢀmethoxyꢀ2ꢀchloroquinolineꢀ5,8ꢀdione was prepared and
subsequent palladium(0) catalyzed reaction with boronic acids gave novel quinoline quinones under reflux
for 24 hours. Further, in 2004, 14 arylboronic acids were shown to be more reactive than their counterparts,
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the arylpinacolboronate esters when reacted with indole bromides in Suzuki couplings under reflux. The
lower reactivity was attributed to steric factors in the arylpinacolboronate esters. Also, 3ꢀarylꢀindazoles
have been synthesized by the reaction of haloindazoles (3ꢀbromoindazole and 3ꢀiodoindazole) with aryl
boronic acids under Pd(0) catalysis in Suzuki type cross couplings. 15 The reaction times ranged from 1 ꢀ18
hours under reflux conditions. In this present study we report a direct more efficient approach to 7ꢀ
aminoquinoline quinones starting from commercially available 7ꢀaminoꢀ8ꢀhydroxyquinoline under
microwave conditions where the reaction times are shorter. Computational, metabolism and cytotoxicity
studies on the quinolineꢀ5,8ꢀdiones are also described.
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CHEMISTRY
The synthesis commenced with the nitration of 5ꢀchloroꢀ8ꢀhydroxyquinoline under HNO3/H2SO4
according to a procedure reported by Musser et al.16 to give the 5ꢀchloroꢀ7ꢀnitroꢀ8ꢀhydroxyquinoline (1) in
good yield (79%). Hydrogenation under Pd/Cꢀcatalysis at 40ꢀ50 psi not only reduced the nitro group to the
free amine but also removed the chloride to provide the desired 7ꢀaminoꢀ8ꢀhydroxyquinoline (2) in
excellent yield (99%). A direct approach to the amino alcohol 2 involves the heating of a mixture of 8ꢀ
hydroxyquinoline and NꢀmethylꢀNꢀphenylhydrazine at 90°C, albeit very low yields were obtained. 17Our
attempt to synthesize the amino alcohol by heating in a microwave between 130ꢀ160°C did not improve the
yield. Acetylation proceeded smoothly where both the amino and hydroxyl groups were protected. The
resulting diacetylated product (3) was hydrolyzed in MeOH/H2O under reflux to form 7ꢀacetamidoꢀ8ꢀ
hydroxyquinoline. Subsequent benzylation of the free hydroxyl was effected by reacting with BnBr/K2CO3
in DMF at 50°C for 24 hrs to give the 7ꢀacetamidoꢀ8ꢀbenzyloxyquinoline (4) in 90% yield. Oxidation using
mCPBA in 1,2ꢀdichloroethane at rt for 48 hrs gave the Nꢀoxide (5) in 82% yield.18 The key intermediate in
the synthesis, the 2ꢀchloroꢀ7ꢀacetamidoꢀ8ꢀbenzyloxyquinoline (6), was obtained in 62% yield by refuxing
the Nꢀoxide with POCl3 in CHCl3.19 The high regioselectivity of the reaction can be rationalized in terms of
sterics as well as formation of an oxyphosphorane adduct anion in a rapid concerted mechanism. 20 We also
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attempted refluxing the Nꢀoxide 5 with SO2Cl2 as reported in literature,9 but only resulted in massive
decomposition of the starting material. Deprotection of the benzyl group was effected with BCl3•SMe2 in
CH2Cl2 and subsequent oxidation using Fremy’s salt (potassium nitrosodisulfonateꢀ(KO3S)2NO) gave the
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7ꢀacetamidoꢀ2ꢀchloroꢀquinolineꢀ5,8ꢀdione (8) in 71% yield. The results are summarized in Scheme 1
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below.
Scheme 1a
Cl
Cl
i
ii
iii
HN
N
H2N
N
O
N
O2N
N
O
OH
OH
OH
2
O
1
3
O
vi
vii
iv-v
HN
N
Cl
HN
N
N
N+
O-
H
O
O
O
O
Bn
O
Bn
Bn
4
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5
O
O
O
O
O
viii
ix
x
HN
N
Cl
N
N
R
HN
N
Cl
H
OH
O
8
R= aryl, heteroaryl
O
9-16
7
aReagents and conditions: (i) HNO3/H2SO4; (ii) H2/PdꢀC, MeOH, 40ꢀ50psi, overnight; (iii) CH3COCl, DIEA,
THF, 2hrs.; (iv) H2OꢀMeOH, reflux, 1hr; (v) BnBr, K2CO3, DMF, 50°C, 24 hrs; (vi) mCPBA, ClCH2CH2Cl,
48hrs; (vii) POCl3, CHCl3, reflux, 2 hrs; (viii) BCl3ꢁSMe2, CH2Cl2, overnight; (ix) Fremy's salt, rt, 1 hr; (x)
RB(OH)2, Pd(PPh3)4, 110ꢀ140 C, W 20ꢀ25 min.
After the successful formation of the quinolinequinone 8, the stage was now set for Suzuki coupling
chemistry. This was accomplished by reaction with different boronic acids under Pd(0) catalysis in a
microwave as illustrated in Table 1 below. Generally, the reactions were complete within 20ꢀ30 minutes in
good yields except for the arylboronate ester where only 27% of the product (16) was obtained. The
mechanistic details of the reaction have been well studied in which case oxidative addition, transmetallation
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and reductive elimination being the most critical steps.21 Interestingly, the 7ꢀaminoꢀ2ꢀ(2ꢀpyridyl)quinolineꢀ
5,8ꢀdione was prepared in 9 steps starting from 3ꢀhydroxybenzoic acid where the key step was a
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Table 1. Suzuki coupling products
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RꢀX
Reaction conditions
R
Yield (%)
OH
OH
70
Pd(PPh3)4, DME/Na2CO3
140 C, 20 min
9
( )
CF3
F3C
B
N
OH
OH
Pd(PPh3)4, DME/Na2CO3
120 C, 20 min
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(10)
(11)
B
N
Pd(PPh3)4, DME/Na2CO3
120 C, 20 min
N
N
B
HO
OH
OH
Pd(PPh3)4, DME/Na2CO3
110 C, 25 min
(12)
67
B
N
N
OH
Boc
Boc
N
*Pd(PPh3)4, p-dioxane
120 C, 20 min
Sn(CH3)3
71
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13
(
)
N
OH
B
(14)
Pd(PPh3)4, DME/Na2CO3
110 C, 25 min
N
N
OH
Boc
Boc
NH
N
OH
B
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HN
N
(
15)
Pd(PPh3)4, DME/Na2CO3
120 C, 20 min
OH
N
PdCl2(dppf), p-dioxane,K3PO4
120 C,30 min
N
O
B
O
NHAc
(16)
AcHN
*
Stille coupling reaction
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Friedlander condensation of 2ꢀacetylpyridine and 2ꢀaminoꢀ3ꢀbenzyloxyꢀ4ꢀbromobenzaldehyde to give the
8ꢀbenzyloxyꢀ7ꢀbromoꢀ2ꢀ(2’ꢀpyridyl)quinoline.22 Although this seems an attractive strategy, the method
lacks the flexibility needed to create a library of lavendamycin analogs.
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The final step in the synthesis involved the removal of the acetate protecting group which was effected
by reaction with H2SO4ꢀMeOH at rt. The Bocꢀprotected derivatives were also subjected to TFA/CH2Cl2 at
rt for 2 hrs to provide the 7ꢀacetamido derivatives.
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ELECTROCHEMISTRY
Eletrochemistry was performed to compare the electrochemical behavior of the quinolinequinones with
their reduction rates by NQO1, and the data are shown in Table 2. Tetrahydrofuran was used as the solvent
for all compounds except 15, which was run in DMSO. The compounds were run against a Ag/AgCl
electrode cathodic and anodic peak potentials, Epc and Epa, respectively, were measured at a potential
sweep rate of 50 mV/sec, and the midpoint of the peak potentials was used to determine E1/2 values, (Epc +
Epa)/2. Unfortunately, many of the analogues did not show reversible electrochemistry, and in some cases,
there were multiple reduction peaks. This makes interpretation of the numbers somewhat difficult, but
some conclusions can be drawn. For instance, most of the acetylated quinolinequinones had a reduction
peak between ꢀ1.08V and ꢀ1.18V, an indication that they are easier to reduce than the nonꢀacetylated
compounds due to the presence of this electronꢀwithdrawing group. This is consistent with what we
reported previously for lavendamycins. However, there was no correlation between reduction potentials
and reduction rates by NQO1, in line with previous publications on this topic.6ꢀ8, 23, 24 This suggests that
steric interactions are more likely to be predictive of substrate efficiency than reduction potentials.
NMR SPECTROSCOPY AND SPECTROPHOTOMETRY
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Complexation of zinc(II) triflate by compounds 19, 22 and 23 was studied using H NMR spectroscopy.
No new peaks were observed in NMR spectra, indicating that free and complexed forms of zinc(II) triflate
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were in a rapid exchange relative to NMR timescale. The aromatic region of the NMR spectrum of
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compound 19 in THFꢀD8 at room temperature is shown below in Figure 2.
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2'
3'
H2N
19
N
O
CF3
Figure 2. Aromatic proton region NMR spectra of 19 upon addition of increasing equivalent of
Zn(SO3CF3)2 in [2H8]THF. Note the change in δ of Hꢀ2’ and Hꢀ3 on addition of Zn2+. Equivalents of Zn2+:
A=0, B=1, C=2, D=3, E=4, F=5 and G=10.
There was a small difference in chemical shifts of Hꢀ2’ (moving upfield) and Hꢀ3 (moving downfield) after
addition of one equivalent zinc(II) triflate to the NMR solution (Table S1 and Figure 2) whereas the
changes in δ of the other protons were barely noticeable. The biggest change in δ of Hꢀ2’ (ꢀ0.04 ppm) and
Hꢀ3’ (+0.07 ppm) occurs after addition of ten equivalents of Zn(SO3CF3)2. This suggests that weak binding
occurs at low Zn2+ concentration.
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Figure 3. Aromatic protons region NMR spectra of 22 upon addition of increasing equivalent of
Zn(SO3CF3)2 in [2H8]THF. Equivalents of Zn2+: A=0, B=1, C=2, D=3, E=4, F=5 and G=10.
In contrast, addition of only one equivalent of Zn(SO3CF3)2 to compound 22 caused larger chemical shift
variations of all the protons (Table S2 and Figure 3) Increasing the amount of Zn2+ (2ꢀ10 equiv.) added to
compound 22 made little or no difference in δ afterwards (>0.01 ppm). This means that the quinoline
derivative binds the Zn2+ more efficiently than compound 19 and only one equivalent of Zn2+ is enough to
cause chemical shift variations.
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Zn(SO3CF3)2 in [2H8]THF. Equivalents of Zn2+: A=0, B=1, C=2, D=3, E=4, F=5 and G=10.
Similar observations were made with compounds 23 (Table S3 and Figure 4) and 13 (Table S4). This is
consistent with the results reported by Long and Harding25 where they demonstrated that the 1:1 bipyridyl
complex of streptonigrin was the major complex formed at room temperature by perfoming an NMR study
in [2H8]THF with addition of Zn2+. Titration of compound 23 with Zn2+ in a mixture of dimethylsulfoxideꢀ
methanol (1:3) was monitored by a spectrophotometer as reported in literature.26 A plot of ꢀλ355 against
Zn2+ concentration gave a moderate affinity constant of 1.41×104 Mꢀ1 for compound 23 binding with Zn2+.
RESULTS AND DISCUSSION
Quinolinequinone metabolism by recombinant human NQO1 was examined using a spectrophotometric
assay that employs cytochrome c as the terminal electron acceptor. Initial rates of reduction (ꢂmol
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cytochrome c reduced/min/mg NQO1) were calculated from the linear portion (0ꢀ30 s) of the reaction
graphs. The 7ꢀacetamidoꢀ2ꢀ(2ꢀpyridinyl) compound 13 displayed the highest reduction rate by NQO1
(Table 2), although it was the only acetylated analogue with a high reduction rate. In all other cases, 7ꢀ
amino compounds had much higher reduction rates than corresponding 7ꢀacetamido compounds with
identical substituents at the quinolineꢀ2ꢀposition. Although unusual, higher rates for acetylated analogues
have been observed in other series.6, 11 With regard to the aromatic substituents at the quinolineꢀ2ꢀposition,
no clear trend in reduction rates was observed except that bulkier groups generally decreased reduction
rates. Oxygen consumption is a measure of the ability of the reduced quinone (hydroquinone) to redox
cycle following reduction by NQO1. This could lead to production of toxic reactive oxygen species and
ultimately to cell death. Oxygen consumption was measured for select quinolinequinones, and the trend, if
not the magnitude, mirrored the reduction rates. (Table 2).
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Table 2. Reduction ratesa and oxygen consumptionb as a result of quinolineꢀ5,8ꢀdione metabolism by
recombinant human NQO1 and electrochemical reduction potentials vs ferrocenec. Reduction rates were
determined by spectrophotometric cytochrome c assay and oxygen uptake by oxygen electrode.
O
R1
N
H
N
R2
O
Reduction rate by
hNQO1
(ꢂmol cyt c reduced (ꢂmol/min/mg
Oxygen
Consumption
Reduction Potential
(E1/2(V) vs Fc)
No.
R1
R2
/min/mg NQO1
4.5 +/ꢀ 0.5
NQO1)
CH3CO
CH3CO
5.2 +/ꢀ 1.0
ꢀ1.93 nrd
9
25 +/ꢀ 4
ꢀ
ꢀ1.17, ꢀ1.92 nr
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CH3CO
CH3CO
7.0 +/ꢀ 0.3
ꢀ
ꢀ1.18, ꢀ1.68, ꢀ1.77
ꢀ1.90 nr
11
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7
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9
N
480 +/ꢀ 200
34 +/ꢀ 3
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CH3CO
CH3CO
16 +/ꢀ 1
ꢀ
ꢀ
ꢀ1.08, ꢀ1.36, ꢀ1.58
ꢀ1.10, ꢀ1.60, ꢀ1.94
15
2.8 +/ꢀ 0.2
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CH3CO
31 +/ꢀ 9
ꢀ
ꢀ
H
H
78 +/ꢀ 7
ꢀ
ꢀ
ꢀ
19
20
170 +/ꢀ 30
ꢀ1.99 nr
H
H
80 +/ꢀ 8
18 +/ꢀ 6
ꢀ
ꢀ
ꢀ1.84 nr
21
22
ꢀ1.53, ꢀ1.65
N
H
H
71 +/ꢀ 13
ꢀ
ꢀ1.85 nr
ꢀ
23
24
120 +/ꢀ 10
8.5 +/ꢀ 1.6
aSpectrophotometric assay using cytochrome c as terminal electron acceptor (550 nm).
bOxygen concentration monitored using an oxygen electrode.
cE1/2 values calculated as (Epc+Epa)/2 are average values from voltammograms recorded potential sweep
rate of 50 mV/sec. Epc = cathodic peak potential; Epa = anodic peak potential.
dnr = nonꢀreversible, anodic peak only.
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Cell survival was measured using the MTT colorimetric assay. In previous work, we demonstrated that IC50
values generated from standard clonogenic assays and MTT assays were positively correlated suggesting
that the MTT assay is a reliable indicator of cytotoxicity. 6 We utilized MDAꢀMBꢀ468 human breast cancer
cells stably transfected with human NQO1 cDNA (MDA468ꢀNQ16) along with the nonꢀtransfected wild
type cells (MDA468) to compare the cytotoxicity of the quinolinequinones (Table 3).27
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12
13
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16
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20
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22
23
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Table 3. Cytotoxicity of quinolineꢀ5,8ꢀdiones toward MDA468ꢀWT (NQO1ꢀdeficient) and MDA468ꢀ
NQ16 (NQO1ꢀrich) human breast cancer cell lines.
O
R1
N
H
N
R2
O
Selectivity Ratio
IC50 (WT)/
IC50 (ꢂM)
IC50 (ꢂM)
No.
R1
R2
MDA468ꢀWT
MDA468ꢀNQ16
IC50 NQ16
CH3CO
1.7 +/ꢀ 0.8
3.3 +/ꢀ 0.1
2.4 +/ꢀ 1.9
6.3 +/ꢀ 0.2
0.73
0.52
9
CH3CO
CH3CO
10
11
N
0.80 +/ꢀ 0.33
0.64 +/ꢀ 0.41
1.2
CH3CO
CH3CO
H
0.53 +/ꢀ 0.27
7.4 +/ꢀ 5.0
5.3 +/ꢀ 0.8
2.2 +/ꢀ 0.5
19.1 +/ꢀ 5.9
17 +/ꢀ 5
0.24
0.39
0.31
13
17
19
N
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H
H
H
5.6 +/ꢀ 1.3
4.8 +/ꢀ 0.9
15 +/ꢀ 2
10 +/ꢀ 1
0.37
0.47
0.75
20
4
5
6
7
8
9
10
11
12
13
14
15
N
21
0.14 +/ꢀ 0.02
0.19 +/ꢀ 0.04
22
N
16
23
17
18
19
20
24
21
H
H
19 +/ꢀ 12
5.3 +/ꢀ 2.1
17 +/ꢀ 2
3.5
N
4.5 +/ꢀ 1.9
0.26
22
23
24
25
26
Quinolinequinone cytotoxicity (IC50) to MDA468 cells was generally in the single digit micromolar range
following 2ꢀh exposures with some in the high nanomolar range (11, 13, 22). Surprisingly, selectivity ratios
[IC50 (MDA468) / IC50 (MDA468ꢀNQ16)] were generally <1 meaning that the quinolinequinones were less
cytotoxic to the NQO1ꢀrich MDA468ꢀNQ16 cells rather than more cytotoxic. This suggests that NQO1 was
protective to the cells rather than functioning as an activating enzyme.27 Only two compounds (11, 23)
were selectively cytotoxic to the MDA468ꢀNQ16 cells. The reason for the general absence of selective
cytotoxicity with this particular series of compounds is unclear, but it is consistent with NQO1’s primary
role as a detoxification enzyme.27
27
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Molecular docking of the quinolinequinones in the NQO1 active site was performed using Sybyl 8.1.1
and GOLD 5.1 for scoring. Three good NQO1 substrates (13, 20, 24) and three poor NQO1 substrates (9,
11, 17) were docked and scored using ChemPLP and ChemScore (Table 4). The highest scores representing
a good fit for the model were found for 20 and 24 consistent with the metabolism data. The exception again
was 13, which scored the lowest, but was the best substrate. Interatomic distances between
quinolinequinone carbonyl groups and FAD N5 and His161 were shortest for 20, but all were within a
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reasonable distance for hydride transfer from FAD when the dynamic effects of the quinoneꢀenzyme
interaction are considered. Figure 5 shows possible docking conformations for 20 and 11 with NQO1. All
quinolinequinones orient with the quinone ring above the FAD isoalloxazine ring as needed for hydride
transfer.
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The mechanism of action of lavendamycin and streptonigrin is not clearly understood. However previous
studies demonstrated that quinone moieties are reduced by NQO1 to the corresponding hydroquinones
which undergo autoꢀoxidation producing activated oxygen species including not only the semiquinone
derivatives but also superoxide and hydroxyl radicals.28 In addition, both streptonigrin and lavendamycin
chelate divalent cationic metal ions. This property might confer to streptonigrin and lavendamycin the
ability to shuttle iron cations into the cells which in turn can catalyze production of reactive oxygen species
through a Fenton reaction. On the other hand, this chelation can result in depletion of intracellular cationic
metals which might result in cell death. 29Generation of the semiquinone radical, after reduction of the
quinone to the hydroquinone followed by autoꢀoxidation, results in a decrease of activity in 9 compounds.
The best NQO1 substrates are less active compounds (13, 20, 24) in NQO1 expressing cells. In contrast,
poor NQO1 substrates such as compound 22 or 11, exhibit the best activity in both cancer cells expressing
NQO1 and not expressing NQO1. According to the NMR experiments, the quinoline derivative 22 and
compound 13 binds the Zn2+ more efficiently than compound 19; and only one equivalent of Zn2+ is
enough to cause important chemical shift variations. Similar observations were made with compound 23,
which was less cytotoxic than compound 22. Even though metal chelation by these compounds is still a
plausible mechanism to explain their activity against breast cancer cells, another mode of action cannot be
discarded. Most active compounds (11, 13 and 22) are potential tridentate ligands for metals. Compound 23
exhibits lower activity than the corresponding acetylated amino analogue 13. It was proposed that metals
can assist tautomeric shift from the active quinone analogues to the quinoid analogue which has an
isoelectronic structure with the biologically inactive azastreptonigrin.24 This tautomeric shift can explain
the decrease of activity of the amino derivative compared to the amido derivative. In our series of aryl
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substituted quinonequinolines the active molecule is the quinone derivatives and not the semiquinone
derivatives. A similar mode of action than the bidentate metal ligands derivatives 8ꢀhydroxyquinone is
currently under investigation. 30, 31
8
9
Table 4. Computational parameters for selected quinolineꢀ5,8ꢀdiones.
10
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O
R1
N
H
N
R2
O
C=O8…
NH5 ((Ǻ)
C=O5…
His161NE2 (Ǻ)
No. R1
R2
ChemPLP
63.2
ChemScore
22.6
CH3CO
CH3CO
3.9
4.7
3.6
3.3
9
63.8
22.6
11
CH3CO
CH3CO
H
57.6
63.8
72.8
67.3
21.4
22.1
26.0
22.7
4.3
4.2
3.6
4.1
3.5
3.3
3.2
3.3
13
17
20
24
N
N
H
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Figure 5. Quinolinequinones docked in NQO1 active site: 20, cyan; 11, magenta; FAD, green.
CONCLUSIONS
A six step synthetic scheme led to good yields for quinolinequinone analogs of lavendamycin projected
as NQO1ꢀdirected antitumor agents. Unexpectedly, ten of eleven analogs demonstrated excellent
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cytotoxicity (IC50 values of single digit micromolar or better) towards MDA468 breast cancer cells, but
only two were selectively cytotoxic to NQO1ꢀexpressing MDA468ꢀNQ16 cells. Compounds 22 and 11 are
poor NQO1 substrates and exhibit the best activity against breast cancer cells. In our novel series of aryl
substituted quinonequinolines the active molecule appears to be the quinone derivatives and not the
semiquinone derivatives resulting from NQO1 reduction, suggesting that the mode of action of this novel
series differs from lavendamycin and involves an unidentified target. Quinolinequinone derivatives 11, 13
and 22 cytotoxicities (IC50) to MDA468 cells were in the high nanomolar range. Our results seem to
indicate that compounds 11, 13 and 22 effects could be also, at least partially, mediated by metal chelation.
These aryl quinonequinoline derivatives represent a novel promising class of cytotoxic agents with a
potential novel therapeutic value.
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EXPERIMENTAL SECTION
Cell Culture. MDAꢀMBꢀ468 (MDA468) human breast cancer cells and stably NQO1ꢀtransfected
MDA468ꢀNQ1632 were a gift from Dr. David Ross (University of ColoradoꢀDenver, Denver, CO).
MDA468 cells had no measurable NQO1 activiy whereas activity in MDA468ꢀNQ16 cells was 1070
nmol/min/mg total cell protein using dichlorophenolindophenol (DCPIP) as the standard electron acceptor.
Cells were grown in RPMI 1640 medium with Lꢀglutamine and penicillin/streptomycin, and supplemented
with 10% fetal bovine serum (FBS). Cell culture medium and supplements were obtained from Invitrogen
(Carlsbad, CA). The cells were incubated at 37 °C under a humidified atmosphere containing 5% CO2.
Spectrophotometric cytochrome c assay. Quinolinequinone reduction was monitored using a
spectrophotometric assay in which the rate of reduction of cytochrome c was quantified at 550 nm. Briefly,
the assay mixture contained cytochrome c (70 ꢂM), NADH (1 mM), recombinant human NQO1 (0.1ꢀ10
ꢂg) (gift from Dr. David Ross, University of ColoradoꢀDenver, Denver, CO) and quinonlinequinones (25
ꢂM) in a final volume of 1 mL TrisꢀHCl (25 mM, pH 7.4) containing 0.7 mg/mL BSA and 0.1% Tweenꢀ20.
Reactions were carried out at room temperature and started by the addition of NADH. Rates of reduction
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were calculated from the initial linear part of the reaction curve (0ꢀ30 s) and results were expressed in terms
of ꢂmol of cytochrome c reduced/min/mg of NQO1 using a molar extinction coefficient of 21.1 mMꢀ1 cmꢀ1
for cytochrome c. All reactions were carried out at least in triplicate.
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9
Oxygen Consumption. Oxygen concentration was monitored using a MIꢀ730 MicroꢀOxygen Electrode
(Microelectrodes, Bedford, NH) with concentrations adjusted for temperature (25 °C). Assay mixtures
contained 25 ꢂM quinonlinequinones, 200 ꢂM NADH and 1 ꢂg/mL NQO1 in a 2 mL TrisꢀHclꢀ
BSA/Tween (0.1%) buffer system. Reactions were started with NADH and measured over 3 minute
intervals at room temperature. All reactions were carried out in triplicate.
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Electrochemistry. Cyclic Voltammagrams were collected for 10 analogues using a BAS CVꢀ50W
electrochemical analyzer using a standard 3 electrode cell. Experiments were performed using an Ag/AgCl
reference electrode, a glossy carbon working electrode and a platinum wire auxiliary electrode. The
reported potentials are referenced by the Ferrocene (0/+) couple in the solvent used, primarily THF, which
occurs at +.60V vs. Ag/AgCl. The compounds were run at concentrations of 1mM in THF, except
compound 15 which was run in DMSO, with a.1M concentration of tetrabutylammonium
hexafluorophosphate as a supporting electrolyte. All samples were purged and run under an Ar atmosphere
during the course of the experiment, and the electrodes were washed and wiped down between each
sample. Each CV was collected at a sweep rate of 50mV/s in the potential range of 0V to ꢀ2V at room
temperature of 21°C.
NMR spectroscopy. Oneꢀdimensional 1H NMR spectra were recorded at room temperature on Bruker
Avance IIITM spectrometer (The Woodlands, Texas) at 400 MHz using a 5ꢀmm probe and a simple pulseꢀ
acquire sequence. Acquisition parameters consisted of spectral width of 4000 Hz with an acquisition time
3.98 s, number of scans of 128, and relaxation delay of 1 s. Complexes were prepared in a mixture of
CDCl3 and THFꢀD8.
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Cell Viability Assay. Growth inhibition was determined using the MTT colorimetric assay. Cells were
plated in 96ꢀwell plates at a density of 10,000 cells/mL and allowed to attach overnight (16 h).
Quinolinequinone solutions were applied in medium for 2 hours, removed and replaced with fresh medium,
and the plates were incubated at 37 °C under a humidified atmosphere containing 5% CO2 for 3ꢀ5 days.
MTT (50 ꢂg) was added and the cells were incubated for another 4 hours. Medium/MTT solutions were
removed carefully by aspiration, the MTT formazan crystals were dissolved in 100 ꢂL DMSO, and
absorbance was determined on a plate reader at 560 nm. IC50 values (concentration at which cell survival
equals 50% of control) were determined from semiꢀlog plots of percent of control vs. concentration.
Selectivity ratios were defined as the IC50 value for the MDA468 cell line divided by the IC50 value for the
MDA468ꢀNQ16 cell line.
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Molecular Modeling. For docking purposes, the crystallographic coordinates of the human NQO1
complex with 3ꢀ(hydroxymethyl)ꢀ5ꢀ(2ꢀmethylaziridinꢀ1ꢀyl)ꢀ1ꢀmethylꢀ2ꢀphenylindoleꢀ4,7ꢀdione (25) were
obtained from the Brookhaven Database (PDB code 1H6933 and resolution 1.86Ǻ) and was edited
accordingly to provide a monomer of the protein. The protein complex was then minimized within Sybyl
7.3 (Tripos Ltd., St Louis) while holding all heavy atoms stationary. The ligand was then removed to leave
the receptor complex which was used for the subsequent docking studies. For preparation of ligand
structures, fragments from Sybyl 8.1.1 were used to construct the compounds and all symmetric
compounds were prepared as monoanionic ligands. Ligands were subject to 1000 iterations of energy
minimization using the Powell method with MMFF94s force field standard method. For computational
docking, the GOLD 5.1 software was used in combination with the ChemPLP34 scoring function (rescoring
with ChemScore.35 The active site was defined as being any volume within 8Ǻ of the quinone scaffold of
25 in its crystal pose in 1H69. Each GA run comprised using the default parameters of: 100000 genetic
operations on an initial population of 100 members divided into five subpopulations with weights for
crossover, mutation, and migration being set to 95, 95, and 10, respectively. GOLD allows a userꢀdefinable
number of GA runs per ligand, each of which starts from a different orientation. For these experiments, the
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number of GA runs was set to 10, and scoring of the docked poses was performed with the ChemPLP
scoring function using ChemScore rescore. Each GOLD run was saved and the strongest scoring binding
pose of each ligand (subject to a rmsd default distance threshold of 1.5Ǻ) was compared to that of the
reference ligand position observed in the crystal structure. The best output pose (orientations) of the ligands
generated were analyzed based on its ChemPLP/ChemScore score, feasibility of hydride transfer process
and Hꢀbonding to the enzyme. The best pose(s) were visualized using PyMOL Molecular Graphics System
version 1.3.
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Chemistry. All moisture sensitive reactions were performed in an inert, dry atmosphere of argon in flame
dried glassware. Air sensitive liquids were transferred via syringe or cannula through rubber septa. Reagent
grade solvents were used for extraction and flash chromatography. THF was distilled from
Na/benzophenone under argon; dichloromethane (CH2Cl2) and chloroform (CHCl3) were distilled from
CaH2 under argon. All other reagents and solvents which were purchased from commercial sources, were
used directly without further purification. The progress of reactions was checked by analytical thinꢀlayer
chromatography (Sorbent Technologies, Silica G TLC plates w/UV 254). The plates were visualized first
with UV illumination followed by charring with ninhydrin (0.3% ninhydrin (w/v), 97:3 EtOHꢀAcOH).
Flash column chromatography was performed using prepacked Biotage SNAP cartridges on a Biotage
Isolera One instrument. Microwave reactions were performed using a Biotage Initiator instrument. The
solvent compositions reported for all chromatographic separations are on a volume/volume (v/v) basis.
1HNMR spectra were recorded at 400 or 500 MHz and are reported in parts per million (ppm) on the δ
scale relative to tetramethylsilane as an internal standard. 13CNMR spectra were recorded at 100 or 125
MHz and are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 77.00). Melting points
were determined on a Stuart melting point apparatus from Bibby Scientific Limited and are uncorrected.
High Resolution mass spectrometry (HRMS) was performed on a Waters/Micromass LCTꢀTOF instrument.
All compounds were more than 95% pure.
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5-chloro-8-hydroxy-7-nitroquinoline (1). This compound was prepared according to the literature
procedure to yield a yellow solid, 4.40 g (79%). M.p. 198ꢀ200°C, [lit.12, m.p. 192ꢀ194°C]; H NMR (500
1
5
6
7
MHz, DMSO) δ 9.09 (dd, J = 4.2, 0.5 Hz, 1H), 8.58 (dd, J = 8.5, 0.8 Hz, 1H), 8.18 (s, 1H), 7.94 (dd, J =
8
9
13
8.5, 4.3 Hz, 1H). C NMR (126 MHz, DMSO) δ 150.5, 150.1, 139.9, 133.6, 132.3, 128.5, 125.9, 122.0,
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+
117.9. HRMS (TOF MS ES+) for C9H6ClN2O3 (MH+) calcd. 225.0067, found 225.0055.
7-Amino-8-hydroxyquinoline (2). Compound 1 (2.4 g, 10.69 mmol) was placed in a hydrogenation
apparatus equipped with a magnetic stir bar and methanol added. Pd/C (150 mg) in a small amount of
MeOH (60 mL) was added and stirring commenced. H2 gas was introduced at a pressure of 40ꢀ50 psi and
reacted at rt overnight. TLC showed full conversion. The black solution was filtered using a celite pad and
1
concentrated under reduced pressure to yield 2 as a black oil, 99% yield. H NMR (500 MHz, CDCl3) δ
8.66 (dd, J = 4.4, 1.6 Hz, 1H), 8.03 (dd, J = 8.2, 1.6 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 8.2, 4.4
Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 148.0, 137.9, 136.6, 136.1, 132.1, 122.4,
119.3, 118.5, 117.7. HRMS (TOF MS ES+) for C9H9N2O+ (MH+) calcd. 161.0715, found 161.0707.
7-acetamido-8-acetyloxyquinoline (3). Compound 2 (330 mg, 2.06 mmol) was dissolved in dried THF (10
mL) and DIEA added with stirring. AcCl (176 ꢁL) in 1mL THF was added drop wise while stirring and
reacted at rt for 2 hrs. Then concentrated under reduced pressure followed by redissolving in CH2Cl2 (20
mL) and water (10 mL). The two layers were allowed to partition and extracted 2x 20 mL CH2Cl2. The
combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. Then
purified on a Biotage SNAP cartridge (25 g) at a flow rate of 25 mL/min to yield an orange solid, 382 mg
(76%); m.p. 151ꢀ153°C; 1H NMR (500 MHz, CDCl3) δ 8.85 (dd, J = 4.1, 1.3 Hz, 1H), 8.49 (d, J = 9.1 Hz,
1H), 8.13 (dd, J = 8.3, 1.5 Hz, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.67 (s, 1H), 7.36 (dd, J = 8.2, 4.2 Hz, 1H),
2.56 (s, 1H), 2.04 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 169.7, 168.5, 150.6, 140.7, 135.8, 134.9, 130.8,
+
125.8, 125.6, 121.3, 120.6, 24.5, 21.0; HRMS (TOF MS ES+) for C13H13N2O3 (MH+) calcd. 245.0926,
found 245.0923.
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7-acetamido-8-benzyloxyquinoline (4). To a solution of 3 (1.2 g, 4.91 mmol) in MeOH (100 mL) was
added water (10 mL) and the reaction stirred under reflux for 1 hr. The black solution was concentrated and
in vacuo and flash chromatographed on a KPꢀSil 100 g Biotage SNAP cartridge using MeOH: DCM as the
solvent (0ꢀ5% MeOH). A white solid (0.9 g) obtained and used for the next step directly. Rf= 0.11 (5%
MeOH:CH2Cl2).
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To a solution of 7ꢀacetamidoꢀ8ꢀhydroxyquinoline (2.27 g, 11.23 mmol) in 40 mL DMF was added K2CO3
(2.33 g, 16.80 mmol) and BnBr (2 mL, 16.80 mmol) respectively. The reaction was stirred at 50°C for 24
hrs after which TLC showed almost all the starting material was consumed. The reaction mixture was
diluted with 30 mL CH2Cl2, filtered with a pad of celite and concentrated under reduced pressure. The
residue was loaded onto a 100 g Biotage SNAP cartridge by dissolving in a small amount of CH2Cl2 and
eluted with EtOAc:heptane gradient (0ꢀ50%). Yield 2.95 g (90%) of a yellow oil was obtained. Rf= 0.50
(60% EtOAc:heptane). 1H NMR (500 MHz, CDCl3) δ 8.95 (dd, J = 4.2, 1.7 Hz, 1H), 8.58 (d, J = 9.0 Hz,
1H), 8.14 (dd, J = 8.3, 1.7 Hz, 1H), 7.77 (s, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.40 – 7.35 (m, 6H), 5.49 (s, 2H),
1.93 (s, 4H). 13C NMR (126 MHz, CDCl3) δ 168.3, 150.0, 142.0, 141.0, 137.4, 136.2, 132.0, 128.9, 128.8,
+
128.8, 126.0, 124.0, 120.0, 120.0, 77.3, 24.6. HRMS (TOF MS ES+) for C18H17N2O2 (MH+) calcd.
293.1290, found 293.1264.
7-acetamido-8-(benzyloxy)quinoline-1-oxide (5). The starting material (4) (428 mg, 1.46 mmol) was
dissolved in 4.3 mL 1,2ꢀdichloroethane with stirring. The mCPBA (340 mg, 1.76 mmol) was added (0.5 M)
and the reaction stirred at rt for 48 hrs. TLC showed almost all the starting material was consumed. The
precipitated mCPBA was filtered and washed with 5 mL 1, 2ꢀdichloroethane. The filtrate was concentrated
under reduced pressure and flash chromatographed on a KPꢀsil 100 g Biotage SNAP cartridge using a 5%
MeOH: DCM gradient at a flow rate of 25 mL/min to yield a yellow solid, 373 mg (82%). M.p. 145ꢀ147°C;
Rf 0.24 (5%MeOH:DCM). 1H NMR (500 MHz, DMSO) δ 9.45 (s, 1H), 8.46 (d, J = 6.1 Hz, 1H), 8.20 (d, J
= 8.9 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.58 – 7.50 (2H), 7.40 – 7.30 (aromatic,
13
4H). C NMR (126 MHz, DMSO) δ 168.9, 139.8, 138.1, 137.1, 136.4, 133.3, 129.8, 129.1, 128.1, 128.0,
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124.7, 124.4, 120.8, 77.7, 23.8. HRMS (TOF MS ES+) for C18H17N2O3 (MH+) calcd. 309.1239, found
4
5
309.1227.
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8
9
7-acetamido-8-benzyloxy-2-chloroquinoline (6). Phosphoryl chloride (280 ꢁL, 3.0 mmol) in CHCl3 (1.0
mL) was added to a stirred solution of the oxide 5 (770 mg, 2.50 mmol) in 21 mL CHCl3 and stirred for 15
min. The mixture was then refluxed for 2 hrs, cooled and poured into ice (50 g) and the pH adjusted to 12
with NaOH (aq.). The aq. layer was extracted with 2 x 50 mL CH2Cl2, washed with 2 x 20 mL H2O, dried
over MgSO4, filtered and concentrated under reduced pressure to yield a brown oil. Then purified on a HPꢀ
Sil 25 g Biotage SNAP cartridge using EtOAc:heptane gradient (0ꢀ50%) as the solvent. Yield 504 mg
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(62%) of an offꢀwhite solid was obtained. Rf= 0.58 (60% EtOAc:heptane); M.P. 92ꢀ94°C; H NMR (500
MHz, CDCl3) δ 8.60 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.81 (s, 1H), 7.54 (d, J = 9.0 Hz, 1H),
7.45 – 7.35 (m, 1H), 7.32 (d, J = 8.5 Hz, 1H), 5.48 (s, 1H), 1.96 (s, 1H). 13C NMR (126 MHz, CDCl3) δ
168.4, 150.5, 141.4, 140.3, 139.0, 137.2, 133.0, 128.9, 128.8, 128.8, 124.3, 123.3, 121.1, 120.1, 77.4, 24.7.
+
HRMS (TOF MS ES+) for C18H16ClN2O2 (MH+) calcd. 327.0900, found 327.0936.
7-acetamido-2-chloro-8-hydroxyquinoline (7). To a solution of 6 (330 mg, 1.01 mmol) in CH2Cl2 (10.1
mL) under an Ar atmosphere was added BCl3•SMe2 (10.1 mL) via a syringe and stirred at rt overnight.
TLC showed the reaction was complete. The reaction was then quenched with saturated NaHCO3(aq.) and
extracted with 2x20 mL CH2Cl2. The organic layers were combined, dried over MgSO4, filtered and
concentrated under reduced pressure. The residue was purified on 50 g KPꢀSil Biotage SNAP cartridge
using a MeOH: CH2Cl2 gradient (0ꢀ5% MeOH) at a flow rate of 25 mL/minute to give a yellow solid, 198
mg (82%). M.P. 176ꢀ178°C; Rf= 0.50 (5% MeOH:CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 9.0
Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.82 (brs, 1H), 7.72 (s, 1H), 7.35 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.5 Hz,
13
1H), 2.29 (s, 3H). C NMR (101 MHz, CDCl3) δ 168.6, 149.7, 138.9, 138.2, 137.1, 124.4, 123.3, 121.5,
+
121.3, 118.0, 24.9. HRMS (TOF MS ES+) for C11H10ClN2O2 (MH+) calcd. 237.0431, found 237.0424.
7-Acetamido-2-chloroquinoline-5,8-dione (8). To a solution of 7 (300 mg, 1.27 mmol) in acetone (30 mL)
was added a solution of Fremy’s salt in NaH2PO4 buffer (0.3 M, 30 mL) and the mixture stirred at rt for 1
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hr. A further solution of Fremy’s salt in the buffer (0.3M, 30 mL) was added and stirring continued for 2
hrs. The acetone was removed under reduced pressure and the residue extracted with 2 x 50 mL CH2Cl2.
The CH2Cl2 phases were combined, dried over MgSO4 and concentrated under reduced pressure. The
residue was purified on a 25 g HPꢀSil Biotage SNAp cartridge using EtOAc:heptanes gradient (0ꢀ60%) to
obtain a yellow solid, 225 mg (71% over 2 steps); m.p. 224ꢀ226°C (decomposes into a black mass), Rf=
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0.49 (60% EtOAc:heptane). H NMR (500 MHz, CDCl3) δ 8.41 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.97 (s,
1H), 7.74 (d, J = 8.2 Hz, 1H), 2.34 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 183.4, 178.1, 169.5, 156.7,
+
145.9, 140.4, 137.2, 129.9, 128.0, 116.3, 25.1. HRMS (TOF MS ES+) for C11H8ClN2O3 (MH+) calcd.
251.0223, found 250.0203.
General procedure for Suzuki coupling under microwave conditions. The 7ꢀacetamidoꢀ2ꢀchloroquinolineꢀ
5,8ꢀdione 8 (21 mg, 0.08 mmol) was dissolved in 4 mL dimethoxyethane (DME) and degassed under
reduced pressure. The palladium (0) catalyst, Pd(PPh3)4 (10 mg, 0084 mmol) was added and the solution
degassed further. The mixture was stirred under Ar atmosphere for 10 minutes. Na2CO3 solution (0.2 mL,
2.0 M) was added followed by the boronic acid (0.126 mmol). The mixture was then heated using a Biotage
microwave initiator at 110ꢀ140°C for 20 minutes. After cooling, TLC showed all the starting material was
consumed. The reaction mixture was poured into DCM and washed with 2 x 10 mL water. Then dried over
MgSO4, filtered and concentrated under reduced pressure. The residue was purified on HPꢀSil 25 g Biotage
SNAP cartridge using EtOAc:heptane gradient (0ꢀ50%) at a flow rate of 20 mL/min. For very polar
products, MeOH:CH2Cl2 (0ꢀ10%MeOH) was used as solvent for purification.
7-acetamido-2-(4-(trifluoromethyl)phenyl)quinoline-5,8-dione (9). Yield 21 mg (70%) of a yellow solid
1
was obtained. Rf= 0.47 (50% EtOAc:heptane); m.p. 250°C(decomposes); H NMR (500 MHz, CDCl3) δ
8.53 (d, J = 8.2 Hz, 1H), 8.45 (s, 1H), 8.27 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 8.2 Hz, 1H), 7.99 (s, 1H), 7.80
(d, J = 8.2 Hz, 2H), 2.35 (s, 4H).13C NMR (126 MHz, CDCl3) δ 184.1, 179.1, 169.5, 160.1, 146.1, 140.6,
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135.7, 128.3, 128.0, 126.0, 126.0, 126.0, 125.3, 116.5, 25.2; HRMS (TOF MS ES+) for
+
C18H12F3N2O3 (MH+) calcd. 361.0800, found 361.0834.
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8
9
7-acetamido-2-(3-pyridinyl))quinoline-5,8-dione (10). Yield 21 mg (41%) of a yellow solid obtained, Rf=
0.19 (5% MeOH:DCM); m.p. >300°C(decomposes); 1H NMR (500 MHz, CDCl3) δ 9.29 (s, 1H), 8.72 (d, J
= 3.9 Hz, 1H), 8.56 (d, J = 8.2 Hz, 1H),8.55 (m, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.00 (s, 1H), 7.56 (dd, J =
8.0, 4.9 Hz, 1H), 2.35 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 184.2, 179.0, 170.4, 158.9, 150.7, 148.1,
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146.1, 140.9, 135.7, 135.7, 128.1, 125.3, 116.6, 24.6 ; HRMS (TOF MS ES+) for C16H12N3O3 MH+)
calcd. 294.0879, found 294.0914. 7ꢀaminoꢀ2ꢀ(3ꢀpyridinyl)quinolineꢀ5,8ꢀdione: 6 mg (12%) of a red solid
1
was obtained. Rf= 0.13 (5% MeOH:DCM); m.p. 195ꢀ197°C (decomposes, turns black); H NMR (500
MHz, CDCl3) δ 9.29 (d, J = 1.7 Hz, 1H), 8.67 (dd, J = 4.9, 1.4 Hz, 1H), 8.58 (ddd, J = 8.0, 2.2, 1.7 Hz,
1H), 8.52 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 7.59 (ddd, J = 8.0, 4.9, 0.7 Hz, 1H), 6.07 (s, 1H).
13C NMR (126 MHz, CDCl3) δ 181.9, 179.8, 157.0, 150.4, 149.6, 147.6, 146.3, 135.4, 135.0, 133.5, 129.4,
+
124.7, 123.8, 102.1. HRMS (TOF MS ES+) for C14H10N3O2 MH+) calcd. 252.0773, found 252.0795.
7-acetamido-2-(8’-quinolinyl)quinoline-5,8-dione (11). Yield 31 mg (51%) of a yellow solid was obtained.
1
Rf= 0.25 (70% EtOAc:heptane), crystallized from MeOH/CH2Cl2; m.p. 295°C (decomposes); H NMR
(500 MHz, CDCl3) δ 8.93 (dd, J = 4.2, 1.8 Hz, 1H), 8.53 (d, J = 8.1 Hz, 1H), 8.48 (d, J = 8.1 Hz, 1H), 8.34
(dd, J = 8.3, 1.8 Hz, 1H), 8.22 (dd, J = 7.2, 1.4 Hz, 1H), 8.04 (dd, J = 8.2, 1.4 Hz, 1H), 8.00 (s, 1H), 7.76
(dd, J = 8.1, 7.3 Hz, 1H), 7.54 (dd, J = 8.3, 4.2 Hz, 1H), 2.34 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 184.8,
179.1, 170.9, 161.9, 150.5, 145.7, 145.1, 140.9, 136.7, 136.5, 133.3, 131.9, 131.7, 130.2, 128.4, 127.6,
+
126.3, 121.4, 116.5, 24.2. HRMS (TOF MS ES+) C20H14N3O3 (MH+) calcd. 344.1035, found 344.1022.
7-acetamido-2-(2-(1-tert-butoxycarbonylindolyl))quinoline-5,8-dione (12). Yield 63mg (67%) of an orange
was obtained. Rf= 0.40 (50% EtOAc:heptane); m.p. 191ꢀ193°C (decomposes); 1H NMR (500 MHz, CDCl3)
δ 8.47 (s, 1H), 8.45 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.97 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H),
7.60 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 9.1, 5.9 Hz, 1H), 6.98 (s, 1H), 2.33 (s, 3H),
1.41 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 184.2, 179.0, 169.6, 157.4, 149.7, 145.3, 140.4, 138.1, 137.5,
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134.2, 128.6, 127.9, 127.6, 126.0, 123.3, 121.5, 116.5, 115.2, 114.0, 84.2, 27.8, 25.1. HRMS (TOF MS
+
ES+) C24H22N3O5 (MH+) calcd. 432.1559, found 432.1568.
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7-acetamido-2-(2-pyridinyl)quinoline-5,8-dione (13). Yield 37 mg (71%) of a yellow solid was obtained.
Rf= 0.19 (5% MeOH:CH2Cl2), crystallized from MeOH/CH2Cl2; m.p. 255ꢀ258°C (decomposes); 1H NMR
(500 MHz, DMSO) δ 10.08 (s, 1H), 8.78 (ddd, J = 4.8, 1.6, 0.8 Hz, 1H), 8.53 (d, J = 7.9 Hz, 1H), 8.46 (d, J
= 8.2 Hz, 1H), 8.08 (td, J = 7.7, 1.8 Hz, 1H), 7.77 (s, 1H), 7.58 (ddd, J = 7.5, 4.7, 1.1 Hz, 1H), 2.28 (s, 3H).
13C NMR (126 MHz, DMSO) δ 184.6, 178.4, 171.5, 158.6, 153.6, 149.8, 146.4, 142.5, 137.8, 135.0, 128.5,
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+
125.5, 124.6, 121.7, 115.3, 24.7. HRMS (TOF MS ES+) C16H12N3O3 (MH+) calcd. 294.0879, found
294.0914.
7-acetamido-2-(2-(1-tert-butoxycarbonylpyrrolyl))quinoline-5,8-dione (14). Yield 36 mg (53%) of a yellow
solid was obtained. Rf= 0.30 (50% EtOAc:heptane); m.p. 191ꢀ193°C (decomposes), recrystallized from
1
methanol; H NMR (500 MHz, CDCl3) δ 8.42 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.95 (s, 1H), 7.79 (d, J =
8.2 Hz, 1H), 7.42 (dd, J = 3.2, 1.7 Hz, 1H), 6.64 (dd, J = 3.4, 1.7 Hz, 1H), 6.29 (t, J = 3.3 Hz, 1H), 2.32 (s,
13
3H), 1.43 (s, 9H). C NMR (126 MHz, CDCl3) δ 184.3, 179.2, 169.5, 156.9, 148.8, 145.3, 140.3, 134.0,
+
132.5, 128.0, 127.3, 125.5, 118.6, 116.4, 111.2, 84.4, 27.7, 25.1. HRMS (TOF MS ES+) C20H20N3O5
(MH+) calcd. 382.1403, found 382.1381.
7-acetamido-2-(4-pyrazolyl))quinoline-5,8-dione (15). Yield 31 mg (42%) of a brown solid was obtained.
1
Rf= 0.33 (5% MeOH:CH2Cl2); m.p. 270°C (decomposes), recrystallized from methanol; H NMR (500
MHz, DMSO) δ 13.34 (s, 1H), 9.97 (s, 1H), 8.55 (s, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.21 (s, 1H), 8.11 (d, J =
8.2 Hz, 1H), 7.69 (s, 1H), 2.26 (s, 3H). 13C NMR (126 MHz, DMSO) δ 184.6, 178.6, 171.4, 156.1, 146.6,
+
142.1, 134.2, 126.1, 123.8, 121.2, 115.1, 24.6. HRMS (TOF MS ES+) C14H11N4O3 (MH+) calcd.
283.0831, found 283.0846.
7-acetamido-2-(3-(2-acetamido-pyridinyl))quinoline-5,8-dione (16). The quinone 8 (71 mg, 0.28 mmol)
was dissolved in 2 mL 1,4ꢀdioxane and degassed under reduced pressure. PdCl2(dppf) (20 mg), K3PO4 (238
mg) and the boronate were added and the solution degassed further. The mixture was stirred under Ar
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atmosphere for 10 minutes. The mixture was then heated heated using a Biotage microwave initiator at
120°C for 30 minutes. After cooling, the reaction mixture was poured into CH2Cl2 and washed with 2 x 10
mL water and extracted 2x 30 mL DCM. The combined organic phases were dried over MgSO4, filtered
and concentrated under reduced pressure. The residue was purified on a HPꢀSil 25 g Biotage SNAP
cartridge using MeOH:CH2Cl2 gradient (0ꢀ5%) at a flow rate of 20 mL/min. Yield 23mg (23%) of a brown
solid was obtained. Rf= 0.32 (5% MeOH:CH2Cl2); m.p. 249°C (decomposes); 1H NMR (500 MHz, DMSO)
δ 10.82 (s, 1H), 10.04 (s, 1H), 9.17 (d, J = 2.5 Hz, 1H), 8.60 (dd, J = 8.8, 2.5 Hz, 1H), 8.45 (d, J = 8.3 Hz,
1H), 8.38 (d, J = 8.2 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 7.75 (s, 1H), 2.28 (s, 3H), 2.14 (s, 3H). 13C NMR
(126 MHz, DMSO) δ 184.6, 178.5, 171.5, 169.7, 157.4, 153.6, 147.3, 146.6, 142.4, 137.0, 134.8, 128.0,
9
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+
127.2, 124.1, 115.3, 113.0, 24.7, 24.0. HRMS (TOF MS ES+) C18H15N4O4 (MH+) calcd. 351.1093, found
351.1064.
7-acetamido-2-(2-indolyl)quinoline-5,8-dione (17). The starting material 12 (39 mg, 0.09 mmol) was
dissolved in 2.5 mL CH2Cl2 and cooled to 0°C using an ice bath. Trifluoroacetic acid (140 ꢁL) was the
added dropwise and reacted at rt for 2 hrs. TLC showed full conversion. Then quenched with sat. NaHCO3
(10 mL) and extracted 2x20 mL CH2Cl2. The organic layers were combined, dried over MgSO4, filtered
and concentrated under reduced pressure. The residue was purified on a HPꢀSil 25 g Biotage SNAP
cartridge using EtOAc:heptane gradient (0ꢀ70%) at a flow rate of 20 mL/min. Yield 17 mg (59%) of a red
solid was obtained after recrystallization from MeOH. M.p. 185°C, decomposes; Rf= 0.38 (70%
EtOAc:heptane). 1H NMR (500 MHz, CDCl3) δ 8.35 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 7.92 (s,
1H), 7.67 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.23 (s, 1H), 7.13 (t, J = 7.4
Hz, 1H), 2.34 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 184.2, 180.4, 170.5, 154.7, 145.4, 140.3, 137.8,
134.7, 134.4, 128.3, 126.6, 124.4, 124.4, 121.5, 120.2, 117.0, 111.7, 104.4, 24.4. HRMS (TOF MS ES+)
+
C19H14N3O3 (MH+) calcd. 332.1035, found 332.1030.
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7-acetamido-2-(2-(pyrrolyl))quinoline-5,8-dione (18). The starting material 14 (30 mg, 0.08 mmol) was
dissolved in 3 mL CH2Cl2 and cooled to 0°C using an ice bath. Trifluoroacetic acid (150 ꢁL) was the added
dropwise and reacted at rt for 2 hrs. TLC showed full conversion. Then quenched with sat. NaHCO3 (10
mL) and extracted 2x20 mL CH2Cl2. The organic layers were combined, dried over MgSO4, filtered and
concentrated under reduced pressure. The residue was purified on a HPꢀSil 25 g Biotage SNAP cartridge
using EtOAc:heptane gradient (0ꢀ50%) at a flow rate of 20 mL/min. Yield 21 mg (93%) of a red solid was
obtained after recrystallization from MeOH. M.P. 255°C, decomposes. Rf= 0.11 (50% EtOAc:heptane). 1H
NMR (500 MHz, DMSO) δ 11.65 (s, 1H), 9.95 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H),
7.69 (s, 1H), 7.07 – 7.04 (m, 2H), 6.28 – 6.22 (m, 1H), 2.27 (s, 3H). 13C NMR (126 MHz, DMSO) δ 184.6,
178.7, 171.4, 154.0, 146.6, 141.9, 133.9, 130.1, 125.3, 123.8, 121.8, 115.2, 111.9, 110.4, 24.7. HRMS
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+
(TOF MS ES+) C15H12N3O3 (MH+) calcd. 282.0879, found 282.0909.
General procedure for removal of the acetate group with MeOH-H2SO4. To the starting material (0.1
mmol) in a 20 mL vial was added 175ꢁL of H2SO4 in 3.0 mL MeOH and stirred at rt for 3 hrs. The red
solution was then neutralized with 5 mL 5% NaHCO3 (aq.) and extracted with 5 X 10 mL CH2Cl2. The
combined organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure. Then
purified on a HPꢀSil 25 g Biotage SNAP cartridge using EtOAc:heptanes (0ꢀ70%) or MeOH:CH2Cl2
gradient (0ꢀ5%) at a flow rate of 20 mL/min.
7-Amino-2-(4-(trifluoromethyl)phenyl)quinoline-5,8-dione (19). The general procedure was used to obtain
6.0 mg (67%) of a red solid; Rf= 0.38 (60% EtOAc:heptane); m.p. 151ꢀ153°C (decomposes, turns black);
1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.2 Hz, 1H),
7.58 (d, J = 8.3 Hz, 2H), 5.84 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 182.2, 180.1, 158.7, 150.3, 146.3,
+
140.6, 135.1, 129.4, 127.7, 125.5, 125.5, 125.1, 105.8, 102.4. HRMS (TOF MS ES+) C16H10F3N2O2
(MH+) calcd. 319.0694, found 319.0666.
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Page 29 of 36
Journal of Medicinal Chemistry
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7-amino-2-(3-pyridinyl)quinoline-5,8-dione (20). The general procedure was used to obtain 10 mg (83%)
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of a red solid. Rf= 0.16 (5% MeOH:CH2Cl2); m.p. 195ꢀ197°C (decomposes, turns black). H NMR (500
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MHz, CDCl3) δ 9.29 (d, J = 1.7 Hz, 1H), 8.67 (dd, J = 4.9, 1.4 Hz, 1H), 8.58 (ddd, J = 8.0, 2.2, 1.7 Hz,
1H), 8.52 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 7.59 (ddd, J = 8.0, 4.9, 0.7 Hz, 1H), 6.07 (s, 1H).
13C NMR (126 MHz, CDCl3) δ 181.9, 179.8, 157.0, 150.4, 149.6, 147.6, 146.3, 135.4, 135.0, 133.5, 129.4,
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124.7, 123.8, 102.1. HRMS (TOF MS ES+) for C14H10N3O2 MH+) calcd. 252.0773, found 252.0795.
7-amino-2-(2-indolyl)quinoline-5,8-dione (21). The general procedure was used to obtain 19 mg (63%) of a
darkꢀbrown solid. Rf= 0.22 (70% EtOAc:heptane); m.p. 235°C decomposes. 1H NMR (500 MHz, CDCl3) δ
8.33 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.39
(s, 1H), 7.27 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.19 (s, 1H), 7.12 (td, J = 7.5, 0.8 Hz, 1H), 6.01 (s, 1H). 13C
NMR (126 MHz, CDCl3) δ 182.6, 181.6, 153.6, 149.5, 145.9, 137.6, 135.0, 134.2, 128.3, 128.0, 124.1,
+
124.0, 121.3, 120.0, 111.7, 103.5, 102.9. HRMS (TOF MS ES+) for C17H12N3O2 (MH+) calcd. 290.0930,
found 290.0900.
7-amino-2-(8-quinolinyl)quinoline-5,8-dione (22). The general procedure was used to obtain 55 mg (71%)
1
of a brown solid. Rf= 0.29 (5% MeOH:CH2Cl2); m.p. 243ꢀ245°C, recrystallized from MeOH. H NMR
(500 MHz, CD3OD) δ 8.92 (dd, J = 4.2, 1.8 Hz, 1H), 8.49 (d, J = 8.1 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H),
8.33 (dd, J = 8.3, 1.8 Hz, 1H), 8.21 (dd, J = 7.2, 1.5 Hz, 1H), 8.02 (dd, J = 8.2, 1.4 Hz, 1H), 7.75 (dd, J =
8.1, 7.3 Hz, 1H), 7.53 (dd, J = 8.3, 4.2 Hz, 1H), 6.06 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 182.8, 180.1,
160.5, 150.3, 150.2, 146.2, 145.2, 136.7, 136.7, 133.2, 131.6, 131.5, 129.8, 129.1, 128.4, 126.3, 121.2,
+
102.4. HRMS (TOF MS ES+) for C18H12N3O2 (MH+) calcd. 302.0930, found 302.0939.
7-amino-2-(2-pyridinyl)quinoline-5,8-dione (23). The general procedure was used to obtain 16 mg (76%)
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of a red solid. Rf= 0.25 (20% MeOH:CH2Cl2), recrystallized from MeOH. H NMR (500 MHz, DMSO) δ
8.75 (d, J = 4.1 Hz, 1H), 8.72 (d, J = 8.2 Hz, 1H), 8.50 (d, J = 7.9 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H), 8.05 (t,
+
J = 7.7 Hz, 1H), 7.58 – 7.53 (m, 1H), 5.89 (s, 1H). HRMS (TOF MS ES+) for C14H10N3O2 (MH+) calcd.
252.0773 found 252.0749.
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