Synthesis and Biological Evaluation of the First Triple Inhibitors of Human Topoisomerase 1, Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), and Tyrosyl-DNA Phosphodiesterase 2 (Tdp2)

Article abstract of DOI:10.1021/acs.jmedchem.6b01565

Tdp1 and Tdp2 are two tyrosyl-DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both Tdp1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, Tdp1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, Tdp1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, Tdp1, and Tdp2 was used to design the inhibitors and facilitate the structure-activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA damage in the cancer cells vs normal cells.

Full text of DOI:10.1021/acs.jmedchem.6b01565

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of the First Triple Inhibitors of
Human Topoisomerase 1, Tyrosyl−DNA Phosphodiesterase 1 (Tdp1),
and Tyrosyl−DNA Phosphodiesterase 2 (Tdp2)
Ping Wang,† Mohamed S. A. Elsayed,† Caroline B. Plescia,‡ Azhar Ravji,‡ Christophe E. Redon,‡
Evgeny Kiselev,‡ Christophe Marchand,‡ Olga Zeleznik,‡ Keli Agama,‡ Yves Pommier,‡
and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the Purdue Center for Cancer
Research, Purdue University, West Lafayette, Indiana 47907, United States
‡Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Institutes of
Health, Bethesda, Frederick, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Tdp1 and Tdp2 are two tyrosyl−DNA phosphodiesterases that can repair damaged DNA resulting from
topoisomerase inhibitors and a variety of other DNA-damaging agents. Both Tdp1 and Tdp2 inhibition could hypothetically
potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of
new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, Tdp1, and Tdp2. Enzyme inhibitory data and cytotoxicity data
from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can
modulate their inhibitory potencies and selectivities vs Top1, Tdp1, and Tdp2. Molecular modeling of selected target compounds
bound to Top1, Tdp1, and Tdp2 was used to design the inhibitors and facilitate the structure−activity relationship analysis. The
monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia
CCRF-CEM cells documented significantly more DNA damage in the cancer cells vs normal cells.
proposed mechanism for Tdp1-mediated hydrolysis involves
two sequential steps.8,10,11 In the first step, a conserved
histidine residue His263 performs a nucleophilic attack on the
phosphate moiety that is linked to Top1 catalytic residue
Tyr723 at the 3′-end of DNA (Figure 1A), releasing tyrosine
and forming a covalent enzyme−DNA complex (Figure 1B),
while in the second step the intermediate phosphohistidine
complex is hydrolyzed via nucleophilic attack by a His493-
activated water molecule (Figure 1C), yielding a DNA 3′-
phosphate and the native enzyme (Figure 1D). Subsequent
processing by polynucleotide kinase phosphatase (PNKP),
DNA polymerases, and DNA ligases finishes DNA restora-
tion.6,7,14 In addition, Tdp1 has also been reported to repair
oxidative damage-induced 3′-phosphoglycolates and alkylation
damage-induced DNA breaks.12,15,16 It is believed that by
INTRODUCTION
■
The inhibition of DNA topoisomerase I (Top1) has proven to
be a successful approach to the design of anticancer agents.
Camptothecins (CPTs) and indenoisoquinolines are two
established classes of Top1 inhibitors.1,2 The CPTs and
indenoisoquinolines act through stabilization of the DNA-
Top1 covalent cleavage complex (Top1cc) by a dual DNA
intercalation and protein binding mechanism that leads to
inhibition of the DNA religation process.1,3−5 Subsequent
collision of the DNA replication fork with drug-stabilized
complexes causes DNA double-stranded breaks. Ultimately, this
leads to tumor cell death. DNA repair after Top1-mediated
DNA cleavage is a complex process that can be initiated by
tyrosyl−DNA phosphodiesterase 1 (Tdp1),6−8 which plays a
critical role in development of drug resistance.9 Tdp1 is a
member of the phospholipase D superfamily10 of enzymes that
catalyze the hydrolysis of the 3′-phosphotyrosyl linker found in
the Top1cc and other 3′-end DNA blocking lesions.10−13 The
Received: October 21, 2016
© XXXX American Chemical Society
A
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Figure 1. Mechanism proposed for Tdp1.17 (A) His263 carries out a nucleophilic attack on the DNA-Top1 phosphodiester link, resulting in Tdp1-
linked DNA (B). (C) Enzyme-catalyzed hydrolysis of the Tdp1−DNA phosphodiester link involving His493 results in a DNA-3′-phosphate and free
enzyme (D).
Figure 2. Proposed reaction mechanism for phosphodiester bond cleavage by Tdp2.23 (A) Top2-derived peptide (trapped topoisomerase) linked to
5′-DNA via a phosphotyrosyl bond. (B) Upon binding of the covalent enzyme−DNA substrate in the Tdp2 active site, two magnesium ions are
coordinated by the Tdp2 catalytic residues to activate nucleophilic attack of the phosphotyrosyl bond (curved arrows). (C) Cleavage products
consisting of Top2 and the liberated DNA with a 5′-phosphate end.15
reducing the repair of Top1−DNA lesions, Tdp1 inhibitors
have the potential to augment the anticancer activities of Top1
inhibitors, which makes Tdp1 a rational anticancer drug
development target.15,17,18
Tyrosyl−DNA phosphodiesterase II (Tdp2) is a member of
the metal-dependent phosphodiesterases that was recently
discovered with repair function linked to topoisomerase II
(Top2)-mediated DNA damage.15,19,20 Tdp2 cleaves Top2−
DNA adducts by catalyzing the hydrolysis of 5′-phosphotyrosyl
bonds and thereby releasing trapped Top2 from 5′-termini
(Figure 2), thus playing a key role in maintaining normal DNA
topology.15 Inhibition of Tdp2 may therefore be a useful
approach to overcome intrinsic or acquired resistance to Top2-
targeted drug therapy.19,20 More recently, it was revealed that
Tdp2 also promotes repair of Top1-mediated DNA damage in
the absence of Tdp1 and that cells lacking both Tdp1 and Tdp2
are more sensitive to Top1 inhibitors than Tdp1-deficient
cells.21,22 The hypothesis that Tdp2 may serve as a potential
therapeutic co-target of Top1 and Tdp1 needs to be tested.15,19
The functional relationships among these enzymes make
Tdp1 and Tdp2 attractive targets for cancer treatment in
combination with Top1 inhibitors, an idea that is also
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supported by the hypersensitivity of Tdp1- and Tdp2-deficient
vertebrate cells to topoisomerase inhibitors.12,14,19,21,24−29 It has
therefore been proposed that combination therapies that target
both Tdp1 and Top1 should improve potency and selectivity
toward cancer cells with preexisting repair and checkpoint
deficiencies.15 A limited number of Tdp1 and Tdp2 inhibitors
have been reported in the literature, but many of them are
hampered by problems of chemical or metabolic instability and
low potency, or by being PAINS (pan-assay interference)
compounds.8,17,18,28−42 Encouragingly, a series of indenoiso-
quinoline dual Top1-Tdp1 inhibitors displaying good activity
on both Top1 and Tdp1 were recently reported (e.g.,
compounds 1−4 in Figure 3),43−45 suggesting that chemical
modifications of the indenoisoquinoline ring system might
allow the generation of dual Top1-Tdp1 inhibitors with
optimized potencies.
investigate the hypothesis that manipulation of the side chain
on the lactam nitrogen in 7-azaindenoisoquinolines could lead
to compounds capable of simultaneously inhibiting Top1,
Tdp1, and Tdp2. Prior reports on the crystal structures of
Top1,4 Tdp1,50 and Tdp251,52 provided the foundation for a
structure-based drug design approach.
RESULTS AND DISCUSSION
■
Design. Compound 5 was docked in the active sites of the
three enzymes Top1 (PDB ID: 1K4T),4 Tdp1 (PDB ID:
1NOP),50 and Tdp2 (PDB ID: 5J3S)52 to investigate the
binding modes and the possibilities for modification of the side
chain. As expected, the azaindenoisoquinoline scaffold stacked
between the base pairs of DNA in the Top1−DNA covalent
complex with a hydrogen bond between the ketone carbonyl of
the drug and a side chain nitrogen of Arg364 (Figure 4, top).
The side chain was directed toward a flexible site in the major
groove of DNA that can accommodate a wide range of
substituents.
The substrate binding site of Tdp1 is quite large in order to
accommodate DNA and a Top1-derived peptide (Figure 4,
middle, and Figure 5). The DNA part of the substrate binds to
a phenylalanine Phe259 via π stacking while maintaining a
network of hydrogen bonds with Arg535, Lys469, and Ser403,
and the peptide part of the complex is directed to the catalytic
residues deep in the active site (Figure 5). Compound 5 was
calculated to adopt a similar binding mode as the substrate in
which the 7-azaindenoisoquinoline scaffold stacked with the
phenylalanine residue and the side chain occupies the DNA
binding pocket pointing toward the catalytic site residues
(Figure 4, middle), which are basic and hydrophilic, suggesting
the exploration of azaindenoisoquinolines having hydrophilic
side chains that incorporate hydrogen bonding groups.
The predicted binding mode of the lead compound 5 in the
active site of Tdp2 (Figure 4, bottom) supports the idea to add
hydrophilic side chains since the amino group is calculated to
be pointed toward a solvent-exposed area. Based on the
molecular modeling studies, the present series of 7-
azaindenoisoquinolines with various hydrophilic side chains
were designed and synthesized to explore the effect of side
chain variation on the activity of this class against Top1, Tdp1,
and Tdp2.
Chemistry. Preparation of compound 7 was first explored
through Mitsunobu reaction by treating 646 with 3-
bromopropanol, Ph3P, and DIAD in THF (Scheme 1), but
only a moderate yield (39%) was obtained, and tedious
chromatography was required to get pure product. An SN2
substitution with 1,3-dibromopropane was then investigated to
install the 3-bromopropyl side chain. After thorough
optimization of the reaction conditions, it was determined
that when compound 6 was treated with sodium hydride in
anhydrous DMF, followed by addition of sodium iodide (0.1
equiv) and 1,3-dibromopropane at −10 to 0 °C for 30 h,
compound 7 could be obtained in 72% yield. This improve-
Figure 3. Representative dual Top1-Tdp1 inhibitors.
Compared to indenoisoquinolines, azaindenoisoquinolines
offer the possibility of improved drug−Top1−DNA ternary
complex stabilization through enhanced charge transfer
interactions involving the donation of electron density to the
drug from the flanking DNA base pairs.46−48 Previous studies of
azaindenoisoquinolines in which a nitrogen atom was system-
atically rotated to eight different locations in the two aromatic
rings established the 7-position (e.g., in compound 5) as the
one that conferred the greatest potency vs Top1.46,47 The
addition of a methoxy group at the 9 position and a nitro group
at the 3 position further improved the potencies of the
azaindenoisoquinolines, resulting in compounds with potent
Top1 inhibition and cytotoxicity.48 Encouraged by these
reports and others,21,43,44,49 the present study was initiated to
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Figure 5. Binding of DNA (magenta) and a Top1-derived, Tyr723-
containing octapeptide (cyan) in the active site of Tdp150 (PDB ID:
1NOP) (tan). The catalytic residues of the enzyme are displayed in
green.
a
Scheme 1
a
Reagents and conditions: (a) 3-bromopropanol, triphenylphosphine,
DIAD, THF, 23 °C, 60 h, 39%; (b) (i) NaH, DMF, 0 to 23 °C, 3 h;
(ii) 1,3-dibromopropane, 0.1 equiv NaI, −10 to 0 °C, 30 h, 72%.
a
Scheme 2
Figure 4. Hypothetical binding modes of 7-azaindenoisoquinoline 5
with Top14 (PDB ID: 1K4T) (top), Tdp150 (PDB ID: 1NOP)
(middle), and Tdp252 (PDB ID: 5J3S) (bottom).
ment in yield over the previously reported reaction conditions
resulted from the use of NaI.48 The practical preparation of
compound 7 on a gram scale made it possible to synthesize 7-
azaindenoisoquinoline analogues with maximum side chain
variation, thereby facilitating the structure−activity relationship
(SAR) study.
a
The SAR study was restricted to varying the lactam γ-
aminopropyl side chain. As illustrated in Scheme 2, compounds
8, 9, 10, 12, 13, 16, and 17 were prepared in 50−77% yields by
heating compound 7 with the corresponding amines at reflux in
1,4-dioxane in the presence of K2CO3 and NaI, while
compounds 11, 14, and 15 were obtained by treatment of 7
with methylamine, isopropylamine, and ethylamine in DMF at
room temperature in the presence of triethylamine and 0.1
equiv of NaI in 68%, 80%, and 73% yields, respectively.
Reagents and conditions: (a) amine (pyrrolidine for 8, 1-
methylpiperazine for 9, piperazine for 10, 2-amino-2-thiazoline for
12, and 4-(2-aminoethyl)morpholine for 13, 1-(2-hydroxyethyl)-
piperazine for 16, and 4-aminopiperidine for 17), NaI, K2CO3, 1,4-
dioxane, reflux, 6 h (8, 68%; 9, 77%; 10, 66%; 12, 57%; 13, 50%; 16,
53%, 17, 63%); (b) amine (methylamine for 11, isopropylamine for
14, ethylamine for 15), NaI, Et3N, DMF, room temperature, 12 h (11,
68%; 14, 80%; 15, 73%).
D
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Table 1. Top1, Tdp1, and Tdp2 Inhibitory Activities and Cytotoxicities of 7-Azaindenoisoquinolines
a
cytotoxicity (GI50, μM)
enzyme inhibitory activity
c
d
d
compd lung HOP-62 CNS SF-539 melanoma UACC-62 ovarian OVCAR-3 renal SN12C breast MCF-7 MGM
Top1
Tdp1
Tdp2
7
0.058
<0.01
0.081
0.065
0.030
0.014
0.150
0.085
0.107
2.76
0.279
0.031
0.191
0.355
0.051
0.036
0.311
0.051
0.070
1.70
0.082
0.015
0.183
0.175
0.020
<0.01
0.044
0.019
0.027
1.22
0.248
0.059
0.171
1.190
0.174
0.137
0.315
0.082
0.062
1.46
0.269
0.036
0.214
0.230
0.046
<0.01
0.080
0.039
0.053
0.046
<0.01
0.016
0.036
1.06
0.112
0.045
0.562
0.316
0.741
0.058
0.177
0.058
0.079
1.66
++
++
++
++
+++
++
+
8
+++
++
+
9
++
+
10
11
12
13
14
15
16
17
18
19
++
+++
+++
++
+
<0.01
0.032
0.012
0.019
0.142
NS
++++
+++
++
+
++++
++++
+++
++
+
+++
0
+
0
b
NS
NS
NS
NS
NS
NS
++
+++
0
+++
0
<0.01
0.02
<0.01
0.04
<0.01
0.03
0.22
0.020
<0.01
0.013
<0.01
0.040
0.21
++++
++++
0.50
+/++
+++
a
b
The cytotoxicity GI50 values listed are the concentrations corresponding to 50% growth inhibition and are the result of single determinations. Not
c
selected for 5-concentration testing due to low cytotoxicity in the one-concentration preliminary testing. Compound-induced DNA cleavage
resulting from Top1 inhibition is graded by the following semiquantitative scale relative to 1 μM MJ-III-65 (19) and 1 μM camptothecin (18): 0, no
detectable activity; +, weak activity; ++, activity less than that of MJ-III-65 (19); +++, activity equal to that of 19; ++++, activity equipotent with 18.
d
Tdp1 and Tdp2 IC50 values were determined in duplicate using a semiquantitative scale: 0, IC50 > 111 μM; +, IC50 between 37 and 111 μM; ++,
IC50 between 12 and 37 μM; +++, IC50 between 1 and 12 μM; ++++, IC50 < 1 μM.
Biological Results. All of the synthesized compounds 7−
17 were tested for their inhibitory activities against Top1,
Tdp1, and Tdp2. Their antiproliferative activities were also
evaluated against the NCI-60 panel of human cancer cell lines.
Top1 inhibition was recorded as the ability of the drug to
induce enzyme-linked DNA breaks in the Top1-mediated DNA
cleavage assay.53 The results of this assay are designated relative
to the Top1 inhibitory activities of camptothecin (18) and MJ-
III-65 (19, also known as NSC 706744 and LMP744)54,55 and
are expressed in semiquantitative fashion: 0, no detectable
activity; +, weak activity; ++, less activity than that of
compound 19; +++, similar activity to that of 19; ++++,
equipotent to 18. As shown in Table 1 and Figure 6, all of the
compounds 7−17 exhibited moderate to potent Top1
inhibitory activity. Compound 7 expressed Top1 inhibitory
activity at the ++ potency level. Further evaluation of analogues
with various amino groups in the side chain (pyrrolidine, 8; 1-
methylpiperazine, 9; and piperazine, 10) revealed similar Top1
inhibitory activities at the ++ level. Interestingly, compound 11,
in which a methylamine was introduced at the end of propyl
chain, displayed improved Top1 inhibitory activity relative to 7
at the +++ level. Notably, the isopropylamine 14 and
ethylamine 15 compounds demonstrated excellent Top1
inhibitory activity at the ++++ level. The 4-(2-aminoethyl)-
morpholine compound 13 and 2-amino-2-thiazoline compound
12 displayed improved Top1 inhibitory activity relative to 7 at
the +++ and ++++ levels, respectively. The improved potencies
of compounds 11−15 compared to those of 8−10 indicate that
a secondary amine in side chain of 7-azaindenoisoquinolines is
better than a tertiary amine for the Top1 inhibitory activity.
Top1 inhibitors can be classified as being poisons or
suppressors. The Top1 poisons inhibit the DNA religation
reaction in the drug−DNA−Top1 ternary complex, while
suppressors inhibit the DNA cleavage reaction in which the
enzyme forms a covalent bond with the DNA through a
phosphodiester linkage. It is apparent from the gel data
depicted in Figure 6 that the azaindenoisoquinoline 12 acts as a
poison in the 0.1−10 μM range, but it acts as a suppressor at a
concentration of 100 μM. This behavior has been routinely
observed in both the indenoisoquinoline43,49 and azaindenoi-
soquinoline46,48 series, and most likely results from the binding
of the drug to DNA at high drug concentrations, thus making
the DNA a poorer substrate. It can be modulated by altering
the substituted side chain that is connected to the lactam.
Figure 7 shows the binding mode of compound 12 between
the DNA base pairs in the active site of Top1. The 7-
azaindenoisoquinoline scaffold is stacked between the base
pairs of DNA with a hydrogen bond between the ketone
carbonyl and a side chain nitrogen of Arg364. The nitro group
hydrogen bonds to the side chain hydroxyl group of Thr718 as
well as a side chain N−H of Asn722. The 2-amino-2-thiazoline
side chain is directed to a solvent-exposed area in the major
groove. The hydrophilic nature of the side chain facilitates the
Figure 6. Representative gel showing Top1-mediated DNA cleavage
induced by indenoisoquinolines 12−15. From left to right: lane 1,
DNA alone; lane 2, DNA + Top1; lane 3, 1 μM camptothecin (CPT,
18) + DNA + Top 1; lane 4, 1 μM MJ-III-65 (19) + DNA + Top1;
Lanes 5−20, compounds 12−15 (each at 0.1, 1.0, 10, and 100 μM).
Numbers and arrows on the left indicate arbitrary cleavage site
positions. CPT (18) and the indenoisoquinoline MJ-III-65 (19) are
positive controls.
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Figure 7. Hypothetical binding mode of compound 12 in ternary complex with DNA and Top14 (PDB ID: 1K4T). The stereoview is programmed
for walleyed (relaxed) viewing.
binding and stabilization of the 7-azaindenoisoquinoline system
between the DNA base pairs.
(N14Y), thus preventing the generation of an oligonucleotide
with a free 3′-phosphate (N14P).32 Therefore, the disappear-
ance of the gel band for N14P indicates Tdp1 inhibition
(Figure 8). The Tdp1 inhibitory activities of the 7-
azaindenoisoquinolines are summarized in Table 1, and
representative gels demonstrating dose-dependent Tdp1
inhibition are depicted in Figure 8. Tdp1 inhibitory potencies
were determined in duplicate using a semiquantitative scale: 0,
IC50 > 111 μM; +, IC50 between 37 and 111 μM; ++, IC50
between 12 and 37 μM; +++, IC50 between 1 and 12 μM; and
++++, IC50 < 1 μM.44 Compounds 11 and 15, with
methylamine and ethylamine at the end of the side chain,
respectively, display good Tdp1 inhibitory activities with +++
potency. Compounds 8 and 12, with five-membered cyclic
amine substituents at the end of their side chains, also exhibit
The Tdp1 inhibitory activities of the 7-azaindenoisoquino-
lines were determined by measuring their abilities to inhibit the
hydrolysis of the phosphodiester linkage between the tyrosine
residue and the 3′-end of a DNA oligonucleotide substrate
Figure 8. Representative gels showing concentration-dependent Tdp1 inhibition by 7-azaindenoisoquinolines 7−17. From left to right (upper): lane
1, DNA alone; lane 2, DNA + Tdp1; lanes 3−32, DNA + Tdp1 + compounds 7−11 (each at 1.4, 4.1, 12.3, 37, and 111 μM). From left to right
(middle): lane 1, DNA alone; lane 2, DNA + Tdp1; lanes 3−32, DNA + Tdp1 + compounds AM-8−3 (2) and 12−15 (each at 1.4, 4.1, 12.3, 37, and
111 μM). From left to right (lower): lane 1, DNA alone; lane 2, DNA + Tdp1; lanes 3−20, DNA + Tdp1 + compounds 2, 17, and 16 (each at 1.4,
4.1, 12.3, 37, and 111 μM). Compound 2 is the positive control.
F
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Figure 9. Hypothetical binding mode of compound 12 with Tdp150 (PDB ID: 1NOP). The stereoview is programmed for walleyed (relaxed)
viewing.
Figure 10. Representative gels showing concentration-dependent Tdp2 inhibition by 7-azaindenoisoquinolines 7−17: From left to right (upper):
lane 1, DNA alone; lane 2, DNA + Tdp2; lanes 3−32, DNA + Tdp2 + compounds 7−11 (each at 0.46, 1.4, 4.1, 12.3, 37, and 111 μM). From left to
right (middle): lane 1, DNA alone; lane 2, DNA + Tdp2; lanes 3−32, DNA + Tdp2 + compound 20 (at 0.017, 0.05, 0.15, 0.46, 1.4, and 4.1 μM) and
12−15 (each at 0.46, 1.4, 4.1, 12.3, 37, and 111 μM). From left to right (lower): lane 1, DNA alone; lane 2, DNA + Tdp2; lanes 3−20, DNA + Tdp2
+ compound 20 (at 0.017, 0.05, 0.15, 0.46, 1.4, 4.1 μM), 17 and 16 (each at 0.46, 1.4, 4.1, 12.3, 37, and 111 μM). Compound 20 is the positive
control.
good inhibition of Tdp1 at the +++ potency level. However,
when six-membered cyclic amine or isopropylamine substitu-
ents were introduced to the side chain, only moderate Tdp1
inhibitory activities at the ++ level were found for the
corresponding compounds 9, 10, 13, and 14.
enzyme. Finally, the 2-amino-2-thiazoline side chain of the
compound is directed to the deep region of the enzyme (green)
where the catalytic residues reside. The hydrophilic nature of
this side chain is important for the binding as the catalytic
residues are hydrophilic in nature.
Figure 9 illustrates the hypothetical binding mode of
compound 12 in the active site of Tdp1. The 7-
azaindenoisoquinoline scaffold binds to the Phe259 benzene
ring via π stacking, while the nitro group hydrogen bonds with
Arg535. The lactam carbonyl oxygen of the scaffold is involved
in hydrogen bonding with Ser518, which is an important
residue for the binding of the DNA−peptide substrate to the
The inhibitory effects of these 7-azaindenoisoquinolines on
Tdp2 were examined in a similar fashion as Tdp1. As shown in
Table 1 and Figure 10, all of the compounds displayed some
degree of inhibitory activity vs Tdp2 with potencies varying
from the + to the +++ levels. The previously described inhibitor
SV-5−153 (20, Figure 10) was included as a positive
control.28,38 Compound 17 is the best one with Tdp2
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inhibitory activity at the +++ level, the highest in this series.
Fortunately, the crystal structure of the human Tdp2 (PDB:
5J3S) has just been released and was used for docking studies
involving 17.52 As demonstrated in Figure 11, the side chain is
calculated to be directed toward a solvent exposed area, and the
lactam carbonyl oxygen hydrogen bonds to the Arg276 side
chain.
against 60 human cancer cell lines (the “NCI-60”).56 The GI50
values obtained with selected cell lines, along with the mean
graph midpoint (MGM) values based on all 60 cell lines, are
summarized in Table 1. The MGM is based on a calculation of
the average GI50 values for all of the cell lines tested in which
GI50 values below and above the test range (0.01 to 100 μM)
are taken as the minimum (0.01 μM) and maximum (100 μM)
drug concentrations used in the screening test. Many of these
new compounds displayed significant potencies against various
cell lines with GI50 MGMs in the low micromolar to
submicromolar range (0.045−1.66 μM). The Top1 ++++
compounds 12, 14, and 15 were among the most cytotoxic,
with MGM values of 0.058, 0.058, and 0.079 μM, respectively.
The differences in cytotoxicity among these three compounds
are low despite clear differences in Tdp1 and Tdp2 inhibition,
suggesting that Tdp1 and Tdp2 inhibition do not contribute
significantly to the cytotoxicities of these compounds. The
Top1 +++ compounds are 11 (MGM 0.714 μM), 13 (MGM
0.177 μM), and 16 (MGM 1.650 μM). On the basis of the
Tdp1 and Tdp2 inhibitory potencies, 16 is expected to be the
least potent, which it clearly is. However, one would expect 11
to be the most cytotoxic, and in fact, 13 is the most cytotoxic of
the three compounds. Among the Top1 ++ compounds, 7
(MGM 0.112 μM), 8 (MGM 0.045 μM), 9 (MGM 0.562 μM),
10 (MGM 0.316 μM), and 17 (not tested due to low
cytotoxicity in the one-concentration preliminary testing), 17 is
expected to be the most cytotoxic on the basis of Tdp1 and
Tdp2 inhibition, but it is actually the least cytotoxic. Based on
their performance against Top1, Tdp1, and Tdp2, compound 7
or 10 should be the least cytotoxic, but actually 17 is. However,
compound 8 is surprisingly the most cytotoxic compound in
the whole series despite having relatively moderate Top1
inhibitory activity. Overall, these triple inhibitors are very
cytotoxic anticancer agents, but their relative cytotoxicities
cannot be rationalized simply on the basis of their inhibitory
activities vs the three enzymes. Other factors that could
possibly contribute to the lack of agreement between the
enzyme inhibitory activities and cytotoxicities of these 7-
azaindenoisoquinoline include differences in cellular uptake,
distribution within the cell, metabolism, efflux from the cell, off-
target effects, and lack of sufficient potency vs Tdp1 and Tdp2
to exert a significant synergistic effect.
In order to contrast the biological activities of the
compounds in cancer cells versus normal cells, human
lymphoblastic leukemia CCRF-CEM cells were compared to
normal lymphocytes (PBMCs) with respect to their sensitivity
to DNA damage induced by five 7-azaindenoisoquinolines at
two different concentrations. DNA damage was monitored by
testing the ability of the drugs to induce the phosphorylation of
histone H2AX on serine 139 (γ-H2AX), a well-established
marker.57 As documented in Figure 12, each of the compounds
12, 14, 15, 17, and 18 (camptothecin) produced a significantly
more intense γ-H2AX response in the cancer cells as opposed
to normal cells after a 2 h exposure, an effect that was evident at
both 1 μM and 100 nM. The results indicate that the new
compounds elicit markedly more DNA damage in cancer cells
versus normal cells.
Figure 11. Upper: hypothetical binding mode of compound 17 with
Tdp2 (PDB code: 5J3S).28,38,52 Lower: the structure of 17 (orange) is
overlapped with that of 20 (green), which is present in the crystal
structure of the enzyme complex with 20.
CONCLUSIONS
■
In summary, a series of 7-azaindenoisoquinolines were
rationally designed through a structure-based approach and
synthesized. Subsequent biological evaluation revealed that all
of the compounds except 16 have inhibitory activity vs all three
To investigate their potential as anticancer agents, the 7-
azaindenoisoquinolines 7−17 were evaluated in the National
Cancer Institute’s Developmental Therapeutics Program screen
H
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Article
added to a suspension of 7-aza-5,6-dihydro-9-methoxy-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]-isoquinoline (6, 1.5 g, 4.64 mmol) in dry
DMF (230 mL) at 0 °C. After the reaction mixture had been warmed
to room temperature and stirred for 3 h, a dark solution formed. The
solution was cooled to −10−0 °C in an ice-salt-water bath, and 1,3-
dibromopropane (4.686 g, 23.2 mmol) was added. The solution was
stirred for 30 h and the reaction quenched with water (200 mL). The
product was extracted with ethyl acetate (5 × 200 mL). The combined
extracts were washed with water (6 × 150 mL) and brine (3 × 100
mL), dried with sodium sulfate, and evaporated to dryness under
reduced pressure. The residue was triturated with ether, filtered, and
washed with ether to provide compound 7 as a red solid (1.47 g,
1
72%): mp 170−172 °C. H NMR (300 MHz, CDCl3) δ 9.19 (d, J =
2.4 Hz, 1 H), 8.74 (d, J = 8.9 Hz, 1 H), 8.48 (dd, J = 9.0, 2.3 Hz, 1 H),
8.23 (d, J = 2.6 Hz, 1 H), 7.44 (d, J = 2.7 Hz, 1 H), 5.16−5.05 (m, 2
H), 3.98 (s, 3 H), 3.55 (t, J = 6.8 Hz, 2 H), 2.52−2.37 (m, 2 H);
ESIMS m/z (rel intensity) 444.0 (MH+, 100).
Mitsunobu Approach. 7-Aza-5,6-dihydro-9-methoxy-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]-isoquinoline (6, 97 mg, 0.3 mmol) and
triphenylphosphine (236 mg, 0.9 mmol) were diluted in THF (30
mL). 3-Bromopropanol (129 mg, 0.9 mmol) was added, followed by
DIAD (182 mg, 0.9 mmol). The solution was stirred at room
temperature for 60 h, and the reaction mixture was concentrated to
dryness. The solid was purified by flash column chromatography,
eluting with hexane−ethyl acetate (2:1) to provide the product 7 as a
red solid (50 mg, 39%).
Figure 12. γ-H2AX foci (green) formation in human PBMCs
(lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells
treated with indenoisoquinolines 12, 14, 15, 17, or camptothecin (18)
at 100 nM (lower panels) and 1 μM (upper panels) for 2 h.
Representative confocal microscopy images are shown. DNA was
stained with DAPI (blue).
7-Aza-5,6-dihydro-6-[3-(4-pyrrolidine-1-yl)propyl]-9-me-
thoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (8). 7-
Aza-6-(3-bromopropyl)-5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline (7, 133 mg, 0.3 mmol), pyrrolidine
(214 mg, 3 mmol), NaI (10 mg, 0.03 mmol), and potassium carbonate
(208 mg, 1.5 mmol) were diluted with 1,4-dioxane (20 mL). The
resulting mixture was heated at reflux for 6 h. The solvent was
evaporated under reduced pressure, and the residue was redissolved in
chloroform (100 mL). The chloroform solution was washed with
water (3 × 10 mL) and brine (20 mL), dried with sodium sulfate, and
evaporated to dryness. The solid residue was subjected to column
chromatography (silica gel), eluting with 5−20% methanol in
dichloromethane to yield the red solid product 8 (88 mg, 68%): mp
248−250 °C. IR (film) 3412, 1613, 1483, 1335, 1300 cm−1; 1H NMR
(300 MHz, CDCl3) δ 9.17 (d, J = 2.1 Hz, 1 H), 8.78 (d, J = 8.7 Hz, 1
H), 8.52 (dd, J = 9.0, 2.7 Hz, 1 H), 8.27 (d, J = 2.6 Hz, 1 H), 7.45 (d, J
= 2.7 Hz, 1 H), 5.07 (t, J = 6.9 Hz, 2 H), 3.99 (s, 3 H), 3.87−3.83 (m,
2 H), 3.28−3.23 (m, 2 H), 2.85−2.80 (m, 2 H), 2.55−2.49 (m, 2 H),
2.28−2.23 (m, 2 H), 2.10−2.0 7 (m, 2 H); ESIMS m/z (rel intensity)
435.1 (MH+, 100). HPLC purity: 99.42% (C18 reverse phase, MeOH-
H2O, 85:15); HRMS-ESI m/z: MH+ calcd for C23H23N4O5, 435.1669;
found, 435.1656.
Mitsunobu Approach. 7-Aza-5,6-dihydro-9-methoxy-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]-isoquinoline (6, 97 mg, 0.3 mmol) and
triphenylphosphine (236 mg, 0.9 mmol) were diluted in THF (30
mL). 3-(1-Pyrrolidinyl)-1-propanol (117 mg, 0.9 mmol) was added,
followed by DIAD (182 mg, 0.9 mmol). The solution was stirred at
room temperature for 72 h. The reaction mixture was concentrated to
dryness. The solid was purified by flash column chromatography,
eluting with 5−20% methanol in dichloromethane to yield the red
solid product 8 (52 mg, 40%).
enzymes. Of the tested compounds, the dihydrothiazole
derivative 12 displayed the best overall inhibition of Top1,
Tdp1, and Tdp2, while the aminopiperidine analogue 17 was
found to be the most potent inhibitor of Tdp2. Enzyme
inhibitory data and cytotoxicity data from human cancer cell
cultures establish that modification of the lactam side chain of
the 7-azaindenoisoquinolines can modulate the cytotoxicity and
inhibitory potency vs Top1, Tdp1, and Tdp2, and γ-H2AX foci
detection experiments indicate greater DNA damage in cancer
vs normal cells. The present report proves that it is actually
possible to simultaneously inhibit Top1, Tdp1, and Tdp2, and
this fact may serve as a springboard to facilitate the elaboration
of more potent 7-azaindenoisoquinoline triple inhibitors as
potential anticancer agents.
EXPERIMENTAL SECTION
■
General. NMR spectra were obtained at 300 or 500 (1H) and 75 or
125 (13C) MHz using Bruker ARX300 or Bruker DX-2 500 [QNP
probe or multinuclear broadband observe (BBO) probe, respectively]
spectrometers. Column chromatography was performed with 230−400
mesh silica gel. The melting points were determined using capillary
tubes with a Mel-Temp apparatus and are uncorrected. IR spectra were
obtained using a PerkinElmer 1600 series FTIR spectrometer on salt
plates or as KBr pellets. ESIMS analyses were recorded on a
FinniganMAT LCQ Classic mass spectrometer. APCI−MS analyses
were performed using an Agilent 6320 ion trap mass spectrometer. EI/
CIMS analyses were obtained with a Hewlett-Packard Engine mass
spectrometer. All mass spectral analyses were performed at the
Campus-Wide Mass Spectrometry Center of Purdue University.
HPLC analyses were carried out on a Waters 1525 binary HPLC
pump/Waters 2487 dual λ absorbance detector system using a 5 μm
C18 reverse phase column. All reported yields refer to pure isolated
compounds. Chemicals and solvents were of reagent grade and used as
obtained from commercial sources without further purification. The
purities of all of the biologically tested compounds were ≥95% as
estimated by HPLC or determined by elemental analysis. For HPLC,
the peak area of the major product was ≥95% of the combined total
peak areas when monitored by a UV detector at 254 nm.
3-(1-Pyrrolidinyl)-1-propanol. Potassium carbonate (1.4 g, 0.94
mol) and pyrrolidine (0.87 mL, 1.8 mol) were added to a stirred
solution of 3-bromopropanol (1 g, 0.635 mol) in 30 mL of THF at 0
°C, and the resulting mixture was stirred at room temperature for 15 h.
The resulting mixture was diluted with ethyl acetate (200 mL) and
filtered through Celite. The filtrate was concentrated, and the residue
was subjected to column chromatography (silica gel), eluting with 50%
1
methanol in dichloromethane, to yield a yellow oil (0.57 g, 70%). H
NMR (300 MHz, CDCl3) δ 3.79 (t, J = 5.1 Hz, 2 H), 2.71 (t, J = 5.6
Hz, 2 H), 2.57−2.51 (m, 4 H), 1.75−1.66 (m, 6 H); 13C NMR (125
MHz, CDCl3) δ 64.18, 56.02, 54.09, 29.24, 23.27.
7-Aza-5,6-dihydro-6-[3-(3H-methylpiperazine-1-yl)propyl]-
9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline
7-Aza-6-(3-bromopropyl)-5,6-dihydro-9-methoxy-3-nitro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (7).48 Sodium hydride
(234 mg, 9.28 mmol) and sodium iodide (70 mg, 0.464 mmol) were
I
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Article
(9). Compound 7 (133 mg, 0.3 mmol), 1-methylpiperazine (300 mg, 3
mmol), NaI (10 mg, 0.03 mmol), and potassium carbonate (208 mg,
1.5 mmol) were diluted with 1,4-dioxane (20 mL). The resulting
mixture was heated at reflux for 6 h. The solvent was evaporated under
reduced pressure, and the residue was redissolved in chloroform (100
mL). The chloroform solution was washed with water (3 × 10 mL)
and brine (20 mL), dried with sodium sulfate, and evaporated to
dryness. The solid residue was subjected to column chromatography
(silica gel), eluting with 5−20% methanol in dichloromethane, to yield
the orange solid product (107 mg, 77%): mp 211−213 °C. IR (film)
dichloromethane, to yield the red solid product (63 mg, 57%): mp
175−177 °C. IR (film) 3430, 1641, 1613, 1504, 1482, 1335 cm−1; 1H
NMR (300 MHz, CDCl3) δ 9.19 (d, J = 2.2 Hz, 1 H), 8.75 (d, J = 8.9
Hz, 1 H), 8.49 (dd, J = 8.9, 2.1 Hz, 1 H), 8.28 (d, J = 2.7 Hz, 1 H),
7.43 (d, J = 2.8 Hz, 1 H), 5.05 (t, J = 5.2 Hz, 2 H), 3.97 (s, 3 H), 3.76
(t, J = 6.7 Hz, 2 H), 3.64 (t, J = 6.5 Hz, 2 H), 3.42 (t, J = 7.5 Hz, 1 H),
3.24 (t, J = 6.7 Hz, 2 H), 2.20−2.15 (m, 2 H); ESIMS m/z (rel
intensity) 466.1 (MH+, 100); HRMS-ESI m/z MH+ calcd for
C22H19N5O5S, 466.1185; found, 466.1182. HPLC purity: 97.50%
(C18 reverse phase, MeOH/H2O, 90:10).
1
3429, 1666, 1500, 1334, 1286 cm−1; H NMR (300 MHz, CDCl3) δ
7-Aza-5,6-dihydro-9-methoxy-6-[3-((2-morpholinoethyl)-
amino)propyl]-3-nitro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (13). Compound 7 (106 mg, 0.24 mmol), 4-(2-
aminoethyl)-morpholine (314 mg, 2.4 mmol), NaI (9 mg, 0.024
mmol), and potassium carbonate (165 mg, 1.2 mmol) were diluted
with 1,4-dioxane (30 mL). The resulting mixture was heated at reflux
for 6 h. The solvent was evaporated under reduced pressure, and the
residue was redissolved in chloroform (100 mL). The chloroform
solution was washed with water (3 × 10 mL) and brine (20 mL), dried
with sodium sulfate, and evaporated to dryness. The solid residue was
subjected to column chromatography (silica gel), eluting with 5−20%
methanol in dichloromethane, to yield the red solid product (58 mg,
9.20 (d, J = 2.3 Hz, 1 H), 8.77 (d, J = 8.9 Hz, 1 H), 8.49 (dd, J = 8.7,
2.6 Hz, 1 H), 8.22 (d, J = 2.6 Hz, 1 H), 7.46 (d, J = 2.7 Hz, 1 H), 5.07
(t, J = 6.9 Hz, 2 H), 3.98 (s, 3 H), 2.60−2.55 (m, 2 H), 2.46−2.28 (m,
8 H), 2.26 (s, 3 H), 2.06−2.01 (m, 2 H); ESIMS m/z (rel intensity)
464.1 (MH+, 100); HRMS-ESI m/z MH+ calcd for C24H26N5O5,
464.1934; found, 464.1928. HPLC purity: 98.34% (C18 reverse phase,
MeOH/H2O, 90:10).
7-Aza-5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-6-[3-(pi-
perazine-1-yl)propyl]-11H-indeno[1,2-c]isoquinoline (10).
Compound 7 (133 mg, 0.3 mmol), piperazine (258 mg, 3 mmol),
NaI (10 mg, 0.03 mmol), and potassium carbonate (208 mg, 1.5
mmol) were diluted with 1,4-dioxane (20 mL). The resulting mixture
was heated at reflux for 6 h. The solvent was evaporated under reduced
pressure, and the residue was redissolved in chloroform (100 mL).
The chloroform solution was washed with water (3 × 10 mL) and
brine (20 mL), dried with sodium sulfate, and evaporated to dryness.
The solid residue was subjected to column chromatography (silica
gel), eluting with 5−20% methanol in dichloromethane, to yield the
red solid product (88 mg, 66%): mp 134−135 °C. IR (film) 3418,
1
50%): mp 214−216 °C. IR (film) 2770, 1437, 1332, 1219 cm−1; H
NMR (300 MHz, CDCl3) δ 9.04 (d, J = 2.2 Hz, 1 H), 8.80 (d, J = 8.9
Hz, 1 H), 8.51 (dd, J = 8.9, 2.1 Hz, 1 H), 8.26 (d, J = 2.7 Hz, 1 H),
7.47 (d, J = 2.8 Hz, 1 H), 4.97 (t, J = 5.3 Hz, 2 H), 3.99 (s, 3 H),
3.94−3.89 (m, 4 H), 3.78 (t, J = 6.5 Hz, 2 H), 3.20−3.15 (m, 2 H),
3.08−3.02 (m, 2 H), 2.80−2.75 (m, 3 H), 2.65 (m, 2 H), 2.22−2.18
(m, 2 H); ESIMS m/z (rel intensity) 494.2 (MH+, 100); HRMS-ESI
m/z MH+ calcd for C25H27N5O6, 494.2040; found, 494.2038. HPLC
purity: 98.44% (C18 reverse phase, MeOH/H2O, 90:10).
1
2925, 1671, 1613, 1484, 1336 cm−1; H NMR (300 MHz, CDCl3) δ
7-Aza-5,6-dihydro-6-[3-(isopropylamino)propyl]-9-me-
thoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (14).
Compound 7 (133 mg, 0.3 mmol), NaI (10 mg, 0.03 mmol),
isopropylamine (177 mg, 6 mmol), and triethylamine (0.125 mL, 0.9
mmol) were diluted with anhydrous DMF (40 mL). The resulting
mixture was stirred for 12 h at room temperature and the reaction
quenched with water (5 mL). The products were extracted with
chloroform (5 × 40 mL). The combined extracts were washed with
water (6 × 20 mL) and brine (3 × 20 mL), dried with sodium sulfate,
and evaporated to dryness under reduced pressure. The solid residue
was subjected to column chromatography (silica gel), eluting with 5−
20% methanol in dichloromethane containing 1% trimethylamine, to
yield the red solid product (100 mg, 80%): mp 278−280 °C. IR (film)
9.18 (d, J = 2.4 Hz, 1 H), 8.78 (d, J = 8.8 Hz, 1 H), 8.46 (dd, J = 8.9,
2.6 Hz, 1 H), 8.20 (d, J = 2.5 Hz, 1 H), 7.44 (d, J = 2.8 Hz, 1 H), 5.06
(t, J = 6.8 Hz, 2 H), 3.95 (s, 3 H), 2.58−2.54 (m, 2 H), 2.43−2.25 (m,
8 H), 2.04−1.98 (m, 2 H); 1.91 (brs, 1 H); ESIMS m/z (rel intensity)
450.1 (MH+, 100); HRMS-ESI m/z MH+ calcd for C23H23N5O5,
450.1778; found, 450.1779. HPLC purity: 100% (C18 reverse phase,
MeOH/H2O, 80:20).
7-Aza-5,6-dihydro-9-methoxy-6-[3-(methylaminopropyl]-3-
nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (11). Com-
pound 7 (133 mg, 0.3 mmol), NaI (10 mg, 0.03 mmol), methylamine
(2 M in THF) (3 mL, 6 mmol), and triethylamine (0.125 mL, 0.9
mmol) were diluted with anhydrous DMF (40 mL). The resulting
mixture was stirred for 12 h at room temperature and the reaction
quenched with water (5 mL). The products were extracted with
chloroform (5 × 40 mL). The combined extracts were washed with
water (6 × 20 mL) and brine (3 × 20 mL), dried with sodium sulfate,
and evaporated to dryness under reduced pressure. The solid residue
was subjected to column chromatography (silica gel), eluting with 5−
20% methanol in dichloromethane containing 1% trimethylamine, to
yield a red solid product (80 mg, 68%): mp 259−262 °C. IR (film)
3435, 2964, 2782, 1682, 1555, 1481, 1336, 1302 cm−1; 1H NMR (300
MHz, CDCl3) δ 9.18 (d, J = 2.4 Hz, 1 H), 8.75 (d, J = 8.9 Hz, 1 H),
8.48 (dd, J = 8.70, 2.7 Hz, 1 H), 8.23 (d, J = 2.7 Hz, 1 H), 7.43 (d, J =
2.4 Hz, 1 H), 5.03 (t, J = 7.5 Hz, 2 H), 3.97 (s, 3 H), 2.77−2.72 (m, 2
H), 2.45 (s, 3 H), 2.09−1.96 (m, 3 H); ESIMS m/z (rel intensity)
395.1 (MH+, 100); HRMS-ESI m/z MH+ calcd for C20H18N4O5,
395.1356; found, 395.1357. HPLC purity: 96.98% (C18 reverse phase,
MeOH/H2O, 85:15).
7-Aza-5,6-dihydro-6-[3-((4,5-dihydrothiazol-2-yl)amino)-
propyl]-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (12). Compound 7 (106 mg, 0.24 mmol), 2-amino-2-
thiazoline (333 mg, 2.4 mmol), NaI (9 mg, 0.024 mmol), and
potassium carbonate (165 mg, 1.2 mmol) were diluted with 1,4-
dioxane (30 mL). The resulting mixture was heated at reflux for 6 h.
The solvent was evaporated under reduced pressure, and the residue
was redissolved in chloroform (100 mL). The chloroform solution was
washed with water (3 × 10 mL) and brine (20 mL), dried with sodium
sulfate, and evaporated to dryness. The solid residue was subjected to
column chromatography (silica gel), eluting with 5−20% methanol in
1
2961, 2805, 1677, 1610, 1337, 1288, 854 cm−1; H NMR (300 MHz,
DMSO) δ 8.90 (d, J = 2.1 Hz, 1 H), 8.67 (d, J = 8.9 Hz, 1 H), 8.61
(dd, J = 8.8, 2.3 Hz, 1 H), 8.37 (d, J = 2.4 Hz, 1 H), 7.70 (d, J = 2.7
Hz, 1 H), 4.89 (t, J = 7.5 Hz, 2 H), 3.98 (s, 3 H), 3.28−3.21 (m, 1 H),
3.09−3.01 (m, 2 H), 2.16−2.10 (m, 2 H), 1.20 (d, J = 6.5 Hz, 6 H);
ESIMS m/z (rel intensity) 423.2 (MH+, 100); HRMS-ESI m/z MH+
calcd for C22H22N4O5, 423.1669; found, 423.1668. HPLC purity:
98.73% (C18 reverse phase, MeOH/H2O, 80:20).
7-Aza-6-[3-(ethylamino)propyl]-5,6-dihydro-9-methoxy-3-
nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (15). A mixture
of compound 7 (133 mg, 0.3 mmol), anhydrous DMF (35 mL), NaI
(10 mg, 0.03 mmol), ethylamine (3 mL of a 2 M solution in THF, 6
mmol), and triethylamine (0.125 mL, 0.9 mmol) was stirred for 12 h
at room temperature, and the reaction was then quenched with water
(5 mL). The product was extracted with chloroform (6 × 40 mL). The
combined extracts were washed with water (5 × 30 mL) and brine (3
× 20 mL), dried with sodium sulfate, and evaporated to dryness under
reduced pressure. The solid residue was subjected to column
chromatography (silica gel), eluting with 5−20% methanol in
dichloromethane containing 1% trimethylamine, to yield the red
solid product (89 mg, 73%): mp 266−268 °C. IR (film) 3583, 2946,
1
1682, 1571, 1482, 1336, 1300 cm−1; H NMR (300 MHz, DMSO) δ
8.89 (d, J = 2.1 Hz, 1 H), 8.65−8.59 (m, 2 H), 8.36 (d, J = 2.6 Hz, 1
H), 7.69 (d, J = 2.7 Hz, 1 H), 4.89 (t, J = 7.5 Hz, 2 H), 3.98 (s, 3 H),
3.08−3.01 (m, 2 H), 2.97−2.89 (m, 2 H), 2.15−2.09 (m, 2 H), 1.17 (t,
J = 6.5 Hz, 3 H); MALDI: m/z 409 (MH+); HRMS-ESI m/z MH+
J
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Journal of Medicinal Chemistry
Article
calcd for C21H20N4O5, 409.1512; found, 409.1516. HPLC purity:
98.32% (C18 reverse phase, MeOH/H2O, 90:10).
sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue).
Aliquots of each reaction mixture were subjected to 20% denaturing
PAGE. Gels were dried and visualized by using a phosphoimager and
ImageQuant software (Molecular Dynamics). Cleavage sites are
numbered to reflect actual sites on the 117 bp oligonucleotide.30
Recombinant Tdp1 Assay.33,45 A 5′-[32P]-labeled single-
stranded DNA oligonucleotide containing a 3′-phosphotyrosine
(N14Y) was incubated at 1 nM with 10 pM recombinant Tdp1 in
the absence or presence of inhibitor for 15 min at room temperature in
the LMP1 assay buffer containing 50 mM Tris HCl, pH 7.5, 80 mM
KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-
20. Reactions were terminated by the addition of 1 volume of gel
loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v)
xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were
subjected to a 16% denaturing PAGE with multiple loadings at 12 min
intervals. Gels were dried and exposed to a PhosphorImager screen
(GE Healthcare). Gel images were scanned using a Typhoon 8600
(GE Healthcare), and densitometry analyses were performed using the
ImageQuant software (GE Healthcare).
7-Aza-5,6-dihydro-6-[3-(4-(2-hydroxyethyl)piperazin-1-yl)-
propyl]-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (16). Compound 7 (133 mg, 0.3 mmol), 1-(2-
hydroxyethyl)piperazine (270 mg, 6 mmol), NaI (9 mg, 0.024
mmol), and potassium carbonate (165 mg, 1.2 mmol) were diluted
with 1,4-dioxane (30 mL). The resulting mixture was heated at reflux
for 6 h. The solvent was evaporated under reduced pressure, and the
residue was redissolved in chloroform (120 mL). The chloroform
solution was washed with water (3 × 10 mL) and brine (20 mL), dried
with sodium sulfate, and evaporated to dryness. The solid residue was
subjected to column chromatography (silica gel), eluting with 5−20%
methanol in dichloromethane to yield the red solid product (78 mg,
53%): mp 214−216 °C. IR (film) 3439, 1636, 1507, 1483, 1256 cm−1;
1H NMR (300 MHz, CDCl3) δ 9.20 (d, J = 2.3 Hz, 1 H), 8.76 (d, J =
8.9 Hz, 1 H), 8.50 (dd, J = 8.7, 2.7 Hz, 1 H), 8.21 (d, J = 2.8 Hz, 1 H),
7.44 (d, J = 2.7 Hz, 1 H), 4.90 (t, J = 7.5 Hz, 2 H), 3.98 (s, 3 H),
3.59−3.55 (m, 2 H), 2.97 (t, J = 2.5 Hz, 1 H), 2.59−2.44 (m, 12 H),
2.08−2.03 (m, 2 H); ESIMS m/z (rel intensity) 494.2 (MH+, 100);
HRMS-ESI m/z MH+ calcd for C25H27N5O6, 494.2040; found,
494.2041. HPLC purity: 96.97% (C18 reverse phase, MeOH/H2O,
90:10).
6-[3-(4-Aminopiperidin-1-yl)propyl]-7-aza-5,6-dihydro-9-
methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline
(17). Compound 7 (133 mg, 0.3 mmol), 4-aminopiperidine (600 mg,
6 mmol), NaI (9 mg, 0.024 mmol), and potassium carbonate (165 mg,
1.2 mmol) were diluted with 1,4-dioxane (30 mL). The resulting
mixture was heated at reflux for 6 h. The solvent was evaporated under
reduced pressure, and the residue was redissolved in chloroform (140
mL). The chloroform solution was washed with water (3 × 10 mL)
and brine (20 mL), dried with sodium sulfate, and evaporated to
dryness. The solid residue was subjected to column chromatography
(silica gel), eluting with 5−15% methanol in dichloromethane
containing 1% trimethylamine to yield the red solid product (87 mg,
63%): mp 258−260 °C. IR (film) 3440, 1641, 1436, 1335, 1301, 1084
cm−1; 1H NMR (300 MHz, DMSO) δ 8.87 (d, J = 2.2 Hz, 1 H), 8.65−
8.57 (m, 3 H), 8.36 (d, J = 2.6 Hz, 1 H), 8.24 (d, J = 2.3 Hz, 1 H), 7.67
(d, J = 2.6 Hz, 1 H), 4.87 (t, J = 7.4 Hz, 2 H), 3.98 (s, 3 H), 2.50−2.39
(m, 7 H), 2.04−1.90 (m, 6 H); ESIMS m/z (rel intensity) 464.1;
HRMS-ESI m/z MH+ calcd for C24H26N5O5, 464.1934; found,
464.1919. HPLC purity: 95.10% (C18 reverse phase, MeOH/H2O,
80:20).
Molecular Modeling. The PDB files for the X-ray crystal
structures of Top1, Tdp1, and Tdp2 were obtained using the protein
data bank codes 1K4T, 1NOP, and 5J3S, respectively. The protein
structures were cleaned, inspected for errors and missing residues,
hydrogens were added, and the water molecules were deleted. The 7-
azaindenoisoquinolines were constructed and optimized using
ChemDraw and saved in SDF file formats and were corrected using
Accelry’s Discovery studio 2.5 software. GOLD 4.1 was used for
docking with the default parameters except that the iterations were
increased to 300 000, and the early termination option was disabled.
The centroids of the binding sites were defined by the ligands in the
cocrystal structures. The top 10 docking poses per ligand were
inspected visually following the docking runs. Energy minimizations
were performed for selected ligand poses. The CHARMM force field
was utilized within the Accelrys Discovery Studio 2.5 for energy
minimization.
Recombinant Tdp2 Assay.23 Tdp2 reactions were carried out as
described previously with the following modifications. The 18-mer
single-stranded oligonucleotide DNA substrate (TY18, α32P-cordyce-
pin-3′-labeled) was incubated at 1 nM with 25 pM recombinant
human Tdp2 in the absence or presence of inhibitor for 15 min at
room temperature in the LMP2 assay buffer containing 50 mM Tris-
HCl, pH 7.5, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT,
40 μg/mL BSA, and 0.01% Tween 20. Reactions were terminated and
treated similarly to recombinant Tdp1 reactions (see above).
γ-H2AX Detection. Purified lymphocytes were obtained from the
NIH blood bank from paid healthy volunteers who gave written
informed consent to participate in an IRB-approved study for the
collection of blood samples for in vitro research use. The protocol is
designed to protect subjects from research risks as defined in 45CFR46
and to abide by all internal NIH guidelines for human subjects
research (protocol number 99-CC-0168). The lymphocytes were
separated from residual red blood cells using Ficoll gradient (GE
Healthcare, Pittsburgh, PA, USA) following the manufacturer’s
instructions. Cells were maintained in RPMI medium (Thermo
Fischer Scientific, Waltham, MA USA) supplemented with 10% fetal
bovine serum (Thermo Fischer Scientific, Waltham, MA USA) and
100 U/mL penicillin, 100 mg/mL streptomycin (Thermo Fischer
Scientific, Waltham, MA USA) at 37 °C and 5% CO2.
γ-H2AX staining was performed as described.58 Briefly, lymphocytes
(peripheral mononuclear blood cells/PMBCs) or human acute
lymphoblastic leukemia CCRF-CEM cells (2 mil/mL in RPMI 1640
medium supplemented with 10% fetal bovine serum and 100 units/mL
penicillin and 100 μg/mL streptomycin) were treated with drugs at
100 nM and 1 μM concentrations for 2 h at 37 °C. After incubation
cells were fixed in 2% paraformaldehyde in phosphate-buffered saline
(PBS) for 20 min at 23 °C, washed three times with PBS, and
resuspended in PBS at 2 mil/mL. Cells (200 μL aliquots at 2 mil/mL)
were attached to the slide using Cytospin (800 rpm/4 min) and
blocked with 5% bovine serum albumin in PBS-TT (PBS containing
0.5% Tween 20 and 0.1% Triton X-100). Blocked cells were treated
with γ-H2AX antibody (abcam cat.# 05-636; dilution 1:500) in 1%
BSA (bovine serum albumin) in PBS-TT for 2 h in humid chamber,
followed by three 5 min washes with PBS. Cells were then incubated
with secondary antibody (goat anti mouse conjugated to Alexa Fluor
488 dye: dilution 1:500, Life Technologies A11029) in 1% BSA in
PBS-TT, followed by followed by two 5 min washes with PBS. Slides
were mounted with mounting medium containing DAPI and viewed
with confocal microscope. The projections were saved as TIFF files.
Topoisomerase I-Mediated DNA Cleavage Reactions. A 3′-
[32P]-labeled 117-bp DNA oligonucleotide was prepared as previously
described.30 The oligonucleotide contains previously identified Top1
cleavage sites in 161-bp pBluescript SK(−) phagemid DNA.
Approximately 2 nM radiolabeled DNA substrate was incubated
with recombinant Top1 in 20 μL of reaction buffer [10 mM Tris-HCl
(pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 μg/mL
BSA] at 25 °C for 20 min in the presence of various concentrations of
test compounds. The reactions were terminated by adding SDS (0.5%
final concentration) followed by the addition of two volumes of
loading dye (80% formamide, 10 mM sodium hydroxide, 1 mM
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01565.
SMILES molecular formula strings (CSV)
K
DOI: 10.1021/acs.jmedchem.6b01565
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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PDB files of 5 with 1K4T (PDB), 1NOP (PDB), and
5J3S (PDB)
PDB file of DNA and a Top1-derived, Tyr723-containing
octapeptide with 1NOP (PDB)
PDB file of 12 in ternary complex with DNA and 1K4T
(PDB)
PDB file of 12 with 1NOP (PDB)
PDB file of 17 with 5J3S (PDB)
PDB file of 17 and 20 with 5J3S (PDB)
AUTHOR INFORMATION
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6970. E-mail: cushman@
purdue.edu.
■
ORCID
Mark Cushman: 0000-0002-0152-5891
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was made possible by the National Institutes of
Health (NIH) through support with Research Grants
U01CA089566 and P30CACA023168. This research was also
supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research. In
vitro cytotoxicity testing was performed by the Developmental
Therapeutics Program at the National Cancer Institute, under
contract NO1-CO-56000.
ABBREVIATIONS USED
■
CPT, camptothecin; Top1, topoisomerase IB; Tdp1, tyrosyl−
DNA phosphodiesterase 1; Tdp2, tyrosyl−DNA phosphodies-
terase 2
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