Isoquinoline-1,3-diones as Selective Inhibitors of Tyrosyl DNA Phosphodiesterase II (TDP2)

Article abstract of DOI:10.1021/acs.jmedchem.5b01973

Tyrosyl DNA phosphodiesterase II (TDP2) is a recently discovered enzyme that specifically repairs DNA damages induced by topoisomerase II (Top2) poisons and causes resistance to these drugs. Inhibiting TDP2 is expected to enhance the efficacy of clinically important Top2-targeting anticancer drugs. However, TDP2 as a therapeutic target remains poorly understood. We report herein the discovery of isoquinoline-1,3-dione as a viable chemotype for selectively inhibiting TDP2. The initial hit compound 43 was identified by screening our in-house collection of synthetic compounds. Further structure-activity relationship (SAR) studies identified numerous analogues inhibiting TDP2 in low micromolar range without appreciable inhibition against the homologous TDP1 at the highest testing concentration (111 μM). The best compound 64 inhibited recombinant TDP2 with an IC₅₀ of 1.9 μM. The discovery of this chemotype may provide a platform toward understanding TDP2 as a drug target.

Full text of DOI:10.1021/acs.jmedchem.5b01973

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Article
Isoquinoline-1,3-diones as Selective Inhibitors
of Tyrosyl DNA Phosphodiesterase II (TDP2)
jayakanth kankanala, Christophe Marchand, Monica Abdelmalak,
Hideki Aihara, Yves Pommier, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01973 • Publication Date (Web): 24 Feb 2016
Downloaded from http://pubs.acs.org on February 29, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Isoquinoline-1,3-diones as Selective Inhibitors of Tyrosyl DNA
Phosphodiesterase II (TDP2)
Jayakanth Kankanala a
b
b
c
,
Christophe Marchand , Monica Abdelmalak , Hideki Aihara ,
Yves Pommier and Zhengqiang Wang*a
b
aCenter for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455
bLaboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
c
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455
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Abstract
Tyrosyl DNA phosphodiesterase II (TDP2) is a recently discovered enzyme that
specifically repairs DNA damages induced by topoisomerase II (Top2) poisons and
causes resistance to these drugs. Inhibiting TDP2 is expected to enhance the efficacy of
clinically important Top2-targeting anticancer drugs. However, TDP2 as a therapeutic
target remains poorly understood. We report herein the discovery of isoquinoline-1,3-
dione as a viable chemotype for selectively inhibiting TDP2. The initial hit compound 43
was identified by screening our in-house collection of synthetic compounds. Further
structure-activity-relationship (SAR) studies identified numerous analogues inhibiting
TDP2 in low micromolar range without appreciable inhibition against the homologous
TDP1 at the highest testing concentration (111 M). The best compound 64 inhibited
recombinant TDP2 with an IC50 of 1.9 µM. The discovery of this chemotype may provide
a platform towards understanding TDP2 as a drug target.
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Introduction
Many cancer chemotherapies work by inducing excessive DNA damage that causes cell
death. The survival of damaged cancer cells hinges on the repair of these DNA lesions
through various pathways which ultimately cause therapeutic resistance. Therefore,
targeting DNA repair pathways can enhance the efficacy of DNA-damaging anticancer
1
drugs. Among DNA damaging drugs are Top2 poisons, such as etoposide (ETP),
teniposide, doxorubicin, epirubicin and idarubicin, which are widely used for treating a
2
broad spectrum of cancers. Mechanistically, Top2 (1, Fig. 1) relaxes DNA torsional
strains by cutting DNA substrate (2), using its tyrosine residue to form the covalent
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Top2cc (4) along with the released 3' DNA-end (3) (Fig. 1). The DNA break is normally
resealed when the 3'-end (3) attacks back to resolve Top2cc, which allows the
continuation of functional DNA transcription and replication. However, the dynamic
equilibrium between DNA and Top2cc can shift toward the accumulation of abortive
Top2cc (5) in which DNA breaks fail to rejoin. Clinical Top2 poisons work by this exact
mechanism as they bind to and stabilize the Top2cc to prevent re-ligation (5, Fig. 1).
TDP2 is the only known human enzyme that can cleave the unique 5’ phosphotyrosyl
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covalent bond featured in abortive Top2cc, demonstrating that TDP2 plays a critical role
in the repair of Top2-mediated DNA damage and in resistance development to clinical
Top2 poisons. This is supported by observations both in cultured cells and animal models
that lack of TDP2 leads to enhanced sensitivity to Top2-induced DNA breaks caused by
Top2 poisons.3-7 Importantly, up-regulation of TDP2 transcription through a gain-of-
function p53 mutation has indeed been linked to etoposide resistance in human lung
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cancer. These observations provide strong rationales for developing TDP2 inhibitors to
sensitize cancer cells towards clinical Top2 poisons.
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Fig. 1. TDP2 repairs abortive Top2-DNA cleavage complex: 2, DNA substrate; 4,
transient Top2-DNA complex; 5, Top2-DNA complex stabilized by a Top2 poison; cyan,
Top 2; green, TDP2; yellow, Top2 poison.
In addition, recent studies have also linked the unique function of TDP2 to the replication
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of picornaviruses, and particularly hepatitis B virus (HBV). HBV chronically infects
over 350 million people globally. Current antivirals against HBV can not eradicate the
virus to achieve disease cure. This is mainly due to the persistence of viral covalently
closed circular DNA (cccDNA)11, 12 produced from a precursor relaxed circular DNA
(
RC-DNA) during a key step of HBV replication. A recent report10 found that this
conversion is critically dependent on cellular TDP2 for the cleavage of a protein-DNA
adduct similar to the Top2cc. Inhibiting cellular TDP2 thus may lead to the depletion of
HBV reservoir cccDNA and may provide a path towards viral eradication, though the
role of Tdp2 in HBV cccDNA formation in vivo remains debatable.13
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Since the discovery of TDP2 in 2009, efforts in biochemistry and crystallography have
generated critical knowledge to allow basic understanding on TDP2 active site structure,
mode of substrate recognition, enzyme kinetics, and mechanism of catalysis.14-16 These
studies support either a one- or two-metal catalytic mechanism characteristic of the
exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily. Based on these
mechanisms, TDP2 repairs abortive Top2cc through a hydrolytic reaction promoted by
either one or two Mg2+ ion(s) and a few key residues at the active site. Particularly
important to catalysis are residues D262, E152, H351 and N120, which are highly
conserved across species. Knowledge on substrate binding and mechanism of catalysis
could assist inhibitor discovery; particularly the structural and biochemical kinship of
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TDP2 to another DNA repair enzyme, human apurinic/apyrimidinic endonuclease 1
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(
hAPE1), could allow ligand-based inhibitor design. However, full-fledged medicinal
chemistry efforts targeting TDP2 require good lead compounds and the understanding of
their inhibition mechanisms, both of which are still lacking. As a result, TDP2 inhibitors
remain largely unexplored. Toxoflavins (6) and Deazaflavins (7) (Fig. 2a) identified
through a high-throughput screening (HTS) are the only known inhibitor scaffolds of
TDP2.18 While biochemically inhibiting TDP2, these scaffolds are flawed as drug
candidates because 6 poses a redox liability and shows poor in vitro pharmacokinetics
(PK) profiles due to its inhibition of metabolic enzyme CYP450, whereas 7 lacks cell
permeability.18 Therefore, there is a pressing need to identify more drug-like TDP2
inhibitors that can also be used as tool compounds towards understanding the mechanism
of TDP2 inhibition.
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Fig. 2. Chemotypes of interest. (a) Reported inhibitor types of TDP2; (b) in-house
synthetic chemotypes screened against TDP2.
Results and Discussion
Hit Identification. Our efforts began with screening a carefully curated library
comprising our in-house synthetic compounds. The screen used a biochemical assay with
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recombinant human TDP2. The library comprises compounds of various chemotypes
(Fig. 2b), many featuring a metal chelating functionality, such as 8–9, 12–14, 17 and 19b
2
+
(
X = OH). The rationale is that the catalysis of TDP2 requires at least one Mg . From
this initial screening, one analogue of the isoquinoline-1,3-dione chemotype (19a),
namely 6-furanoisoquinoline-1,3-dione (43, Fig. 3a) was found to selectively inhibit
TDP2 (Fig. 3c). Dose-response testing revealed that the inhibitory IC50 for 43 was 10 M
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(
Fig. 3d). This potency was confirmed through a secondary assay using a more robust
assay where endogenous TDP2 from whole cell extracts was used (WCE, IC50 = 12 M,
Fig. 3b). Remarkably, none of any other screened chemotypes showed significant TDP2
inhibition, including the highly homologous 19b where the only structural difference is
the presence of the N-2 OH group. That the 2-hydroxyisoquinoline-1,3-dione (HID)
analogue 46 did not inhibit TDP2 at concentrations up to 111 M was remarkable. It
implies that unlike the inhibition of some other Mg2+ dependent DNA processing
enzymes, TDP2 inhibition may not require a chelating functionality. On the other hand,
counter assays against the homologous TDP1 showed no inhibition by 43 (Fig. 3b),
strongly suggesting that the observed TDP2 inhibition was highly specific. In addition,
the small size (mw = 227) and the lack of apparent PK liabilities render 43 an attractive
hit, which is amenable to further SAR.
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Rec IC50 (M)
WCE IC50 (M)
Cpd
Tdp2
Tdp1
Tdp2
Tdp1
(a)
(b)
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>111
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Compound 43
(c)
(d)
(c)
Fig. 3. SAR and selective TDP2 inhibition of the isoquinolinedione chemotype: (a)
structure of isoquinoline-1,3-dione 43 and HID 46; (b) inhibition profile: 43 selectively
inhibits TDP2 in assays using either recombinant (Rec) or endogenous TDP2 from whole
cell extracts (WCE), while 46 was inactive in either condition; (c) gel images of dose
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response testing of 43 in Rec. TDP2 biochemical assays. Conc. (μM):1.4, 4.1, 12.3, 37,
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11; (d) dose response curve for 43 in Rec. TDP2 assay expressed as mean ± SD from at
least three independent experiments.
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Chemistry. The SAR of compound 43 concerns primarily three distinct series of
compounds: 1) isoquinoline-1,3-diones with a simple functional group (Br, I, CO
2
H, CF ,
3
NO ) around the left ring (33–42, Table 1); 2) isoquinoline-1,3-diones with an aromatic
2
ring substituted around the left ring (43–88, Table 1); and 3) scaffold analogues (89–96,
Table 1). Synthetically, all isoquinoline-1,3-dione analogues of the first two series (43–
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8) were accessed by either of the two routes delineated in Schemes 1–2. The first
approach (Scheme 1) was adapted based on our previously reported route for the
1
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synthesis of HID. This approach features key iodo homophthalic acid intermediates (20)
19
which were synthesized using a Hurtley reaction. The structural diversity for analogues
of series two was subsequently introduced via a Suzuki coupling reaction to afford
intermediates 21, which were condensed with urea in 1,2-dichlorobenzene to yield
desired isoquinoline-1,3-dione analogues (Scheme 1).20
Scheme 1a.
a
Reagents and conditions: a) arylboronic acid, K
2
CO
3
, Pd(PPh
3
)
4
, EtOH/H O (1:1), 150
2
°C, 30 min, MW, 60-85%; b) urea, 1,2-dichlorobenzene, 170 °C, 45 min, MW, 55-80%.
To limit the usage of 1,2-dichlorobenzene and to produce a range of substituted
isoquinoline-1,3-diones, an alternative method was employed (Scheme 2). This method
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began with the S
N
Ar reaction of substituted-2-fluorobenzonitrile (22) with methyl
cyanoacetate at 90 °C to produce the ester intermediate (23) which upon heating in a
mixture of DMSO/H O (9:1) at 120 °C resulted in the smooth decarboxylation to furnish
2
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the halogen (I or Br) substituted dinitrile compounds (24). At this stage structural
diversity was introduced via either a Suzuki coupling to afford various aryl substituted
dinitrile intermediates (25) or a palladium assisted arylation and carbonyl insertion to
produce benzoyl substituted dinitrile compounds (26). Treatment of dinitrile
intermediates 24–26 with con. HCl at 70 °C afforded the desired cyclized products (33-
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8, Scheme 2a). The sulfonamide (62–64) or acrylamide (65–66) analogues were
synthesized by derivatizing the aniline analogues (59–61) with sulfonyl or acryloyl
chloride (Scheme 2b). The benzothiazole derivative (84) was synthesized using a T3P
mediated coupling of 39 with 2-aminothiophenol (Scheme 2c).
Scheme 2a.
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a
Reagents and conditions: a) Methyl cyanoacetate, NaH, DMSO, 90 °C; b) DMSO/H O
2
(
9:1), 120 °C, 16 h, 70-85%; c) arylboronic acid, K
2
CO
, K
0 min, MW, 50-62%; e) Con HCl, 70 °C, 4 h, 60-90%; f) sulfonyl or acryloyl chloride,
3
, Pd(PPh
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)
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, DME/H O (4:1),
2
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pyridine, dioxane, r.t; 65-79%; g) 2-aminothiophenol, T3P (50% in EtOAc), DIPEA, 100
C, 30 min, MW, 69%.
°
The methods for synthesizing scaffold analogues are depicted in Scheme 3. The synthesis
of quinazoline-2,4-dione (90) involved a Suzuki coupling reaction of the starting 4-
bromomethyl anthranilate 27. The resulting intermediate 28 was then converted to the
desired cyclized product 90 via the treatment with potassium cyanate and the subsequent
2
1
saponification (Scheme 3a). The 1,8-naphthalimide derivatives (91–92) were
synthesized by the condensation of 1,8-naphthalic anhydrides (29–30) with ammonium
2
2
hydroxide (Scheme 3b). The 5,6-fused heterocyclic core was obtained by treating 2-
amino-1-propene-1,1,3-tricarbonitrile (31) with various substituted hydrazine in refluxing
ethanol to yield dinitrile compounds (32), which were then cyclized with the aid of acid
to furnish compounds 93–95. Compound 95 was acetylated to produce 96 (Scheme 3c).
Scheme 3a.
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a
Reagents and conditions: a) phenylboronic acid, K
10 °C, 45 min, MW, 50%; b) i) KOCN, toluene, r.t; ii) NaOH, EtOH, reflux, 68%; c)
OH, 40 °C, 4 h, 70-90%; d) 2-(tributylstannyl)furan, Pd(PPh , toluene, 150 °C, 30
min, 72%; e) R-NHNH , EtOH, reflux, 70-95%; f) Con HCl, 70 °C, 4 h, 65-85%; g)
2
CO
3
, Pd(PPh
3
)
4
, DME/H
2
O (4:1),
1
NH
4
)
3 4
2
acetyl chloride, AcOH, 70 °C, 62%.
Biology. All final compounds 33–96 were evaluated biochemically for inhibition of
human TDP2. The primary assay used recombinant TDP2 and all compounds were tested
in dose response fashion at concentrations up to 111 M. A counter assay was also run
with each compound against the homologous TDP1 and none of the tested compounds
showed significant inhibition at the highest concentration (111M). The testing results
are summarized in Table 1. The most striking SAR trend was that while the vast majority
of the quinolone-1,3-dione analogues (33-89) potently inhibited recombinant human
TDP2 in the low micromolar range, none of the scaffold analogues (90-96) showed
considerable TDP2 inhibition in the same assay. This observation strongly validates
quinolone-1,3-dione as an important TDP2 inhibitor type. In addition, the dramatic OH
1
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effect at the N-2 position initially observed with compound 46 was confirmed with an
additional pair of analogues (44 and 47). Consistent with the SAR trend, the 2-NH
analogue 44 inhibited TDP2 with an IC50 of 13 M whereas the 2-NOH analogue 47 was
completely inactive (IC50 > 111M). This confirmed SAR trend suggests that compounds
with a chleting triad, such as the HID chemotype, should not be considered as favorable
TDP2 inhibitor types. The third major SAR observation concerns the substitution position
on the left ring of the isoquinoline-1,3-dione core. Notably, most of the C6 or C7
substituted analogues potently inhibited recombinant TDP2, whereas C8 analogues (53
and 57) showed substantially reduced inhibitory activity (compared to 51-52 and 55-56),
and more dramatically, C5 substituted analogues (34, 45 and 58, Table 1) were all
inactive regardless of the nature of the substituent.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Biochemical inhibition of synthetic isoquinolinedione analogues against
recombinant hTDP2.
IC50
M)
Compd
Structure
a
(
3
3
25±8.2
>37
34
3
5
11±8.3
1
2
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Journal of Medicinal Chemistry
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7
8
9
3
3
6
7
7.6±4.1
9.8±3.7
7.5±3.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
38
39
>111
4
0
3.0±1.0
7.0±1.7
19±9.5
41
4
4
4
2
3
4
10±2.5
13, 26b
1
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
5
6
>111
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>111
>111
47
48
23, 71b
4
5
5
5
9
0
1
2
16±5.0
20±6.0
13±3.0
15±9.5
1
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Journal of Medicinal Chemistry
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5
6
7
8
9
5
3
26±5.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
5
5
4
5
6
14±9.2
10±2.0
11±4.5
5
7
19±4.3
58
>37
5
6
9
0
42±2.0
>111
1
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5
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
61
36±3.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
6
2
3
18±8.0
4.5±2.8
64
1.9±0.90
10±7.3
6
6
5
6
11±3.0
>111
67
68
30±5.0
1
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Journal of Medicinal Chemistry
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8
9
69
21±8.0
16±5.8
5.5±2.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
7
0
1
7
7
7
2
3
4
5.6±2.6
1.7±0.30
6.4±4.2
75
76
8.0±5.1
7.1±5.3
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
77
>37
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
7
8
8
8
8
8
9
0
1
2
3
16±6.9
3.0±2.3
4.1±1.9
4.5±1.5
3.2±2.5
>111
84
5.1±2.0
1
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Journal of Medicinal Chemistry
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9
85
86
9.3, 6.5b
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>111
8
8
7
8
11±2.0
5.4±1.8
8
9
9
9
9
0
1
2
14±5.3
>111
>111
>111
>111
93
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
9
4
5
>111
>111
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
6
>111
a
IC50: concentration of a compound producing 50% inhibition, expressed as
mean±standard deviation from at least three independent experiments.
b
Value from a second independent experiment.
To confirm the observed potency with C6 or C7 analogues, selected compounds were
also tested in a secondary assay using the whole cell extracts of the TDP2 expressing
cells. As shown in Table 2, with the exception of 48 and 85, all compounds tested
retained activity in this assay with IC50 values consistent with those obtained from the
assays using recombinant TDP2. These data strongly support a genuine and selective
TDP2 inhibitory profile of the C6 or C7 substituted isoqunioline-1,3-dione chemotype. In
addition, a few compounds (36, 40, 43 and 84) were also tested for sensitivity against
DT40 knockout cells complemented with human hTDP2 using a previously reported
6
assay. Although synergy to Top2 inhibitor ETP was not observed with any of these
compounds, three of them (36, 43 and 84) exhibited potent cytotoxicity at low
micromolar concentrations (CC90 = 2.5—12 M). It is likely that further improved TDP2
inhibitors will produce the desired synergism.
2
0
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Journal of Medicinal Chemistry
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8
9
10
11
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13
14
15
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Inhibitory activity of selected compounds against hTDP2 whole cell extract
(
WCE).
Rec.
WCE
CC90
Compd TDP2 IC50 IC50
c
(M)
a
b
(M)
(M)
36d
38
7.6±4.1
7.5±3.0
3.0±1.0
7.0±1.7
8.5
9.3
9.5
--
2.6
5.2
3.5
25
4
0d
>50
--
4
1
4
3d
2.4
--
4
4
8
9
13
50
4
23
>111
23
--
4
16±5.0
13±3.0
10±2.0
42±2.0
36±3.0
--
51
55
35
--
25
--
5
6
9
1
55
--
40
--
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
62
64
69
18±8.0
1.9±0.90
21±8.0
5.5±2.0
5.6±2.6
1.7±0.30
6.4±4.2
8.0±5.1
3.0±2.3
4.1±1.9
4.5±1.5
3.2±2.5
5.1±2.0
9.3
25
--
2.2
42
--
--
--
--
--
--
--
--
--
--
--
12
--
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
7
7
7
7
7
1
2
3
4
5
9
15
3.3
3.2
4.5
6.5
4.3
5.3
5.5
2.7
11
80
81
8
2
8
4d
8
5
>111
a
IC50: concentration of a compound producing 50% inhibition, expressed as
mean±standard deviation from at least three independent experiments.
b
IC50: concentration of a compound producing 50% inhibition.
c
CC90: concentration of a compound killing 90% of the cells. Assay was done in the
absence of EPT against hTDP2 DT40 cells.6
d
Synergy was not observed when tested in combination with different concentrations of
EPT.6
2
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9
Molecular Modeling. With the lack of a TDP2 / inhibitor co-crystal structure,
understanding on the inhibitor binding mode and mechanism of inhibition remains
elusive. With reported inhibitors 6 and 7, it is unclear exactly how they inhibit TDP2 as
molecular modeling revealed little about their binding. To provide a predicted binding
mode of our new inhibitors, we have constructed a homology model of hTDP2 based on
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
the available crystal structure of mouse TDP2 (mTDP2; PDB code:4GZ1 ) which shares
a high degree of sequence identity (65%) with hTDP2. With this model, docking analysis
was performed using Glide XP (v.6.4).23, 24 The predicted binding mode of compound 56
in the active site of hTDP2 suggests an interaction between the carbonyl at position 1 of
isoquinoline-1,3-dione core and the residues, H351 and the backbone NH of N264 of
TDP2 (Fig. 4a). The NH at position 2 and carbonyl group at position 3 of 56 can
2+
coordinate to the magnesium ion (Mg ). The carbonyl group at position 7 of 56 forms a
salt bridge with the R266 within the active site of hTDP2. Whereas in compound 40, the
carbonyl group at position 1 forms an H-bond interaction with the backbone NH of N264.
The NH at position 2 and carbonyl group at position 3 within 40 coordinate to the E152
2
+
and magnesium ion (Mg ) respectively (Fig. 4b).
2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Fig. 4. Predicted binding mode of a) 56 and b) 40 within active site of hTDP2
(
numbering was based on hTDP2 sequence). The active site residues are shown as green
sticks and metal ions as magenta sphere. The H-bond interactions are depicted as black
dotted lines and the distance between bonded atoms are represented in red. Homology
1
5
model (hTDP2) was built using the crystal structure of mTDP2 (PDB code: 4GZ1 ).
Conclusions
By screening our in-house collection of synthetic small molecules, isoquinoline-1,3-dione
was identified as a novel inhibitor type of TDP2. The selective and potent inhibitory
profile of this chemotype against TDP2 was further established through the chemical
synthesis of 63 functional and scaffold analogues, and validated via a secondary assay
2
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9
using WCE as well as a counter assay against TDP1, which shares some substrates with
25
TDP2. Analogue synthesis identified compound 64 as the best of the series with an IC50
of 1.9 µM against recombinant TDP2 and 2.2 µM against TDP2 in WCE assay. The main
SAR observations also suggest that the C6 or C7 substitution is strongly preferred and
that the OH substitution at the N2 position resulting in a CO-NOH-CO chelating triad is
undesired. While efforts towards obtaining the TDP2 / inhibitor co-crystal structures are
still underway, homology modeling did reveal a few specific interactions within the
TDP2 active site that may contribute to understanding the molecular mechanism of TDP2
inhibition. Identifying this novel and selective TDP2 inhibitor type may provide an
important path towards employing TDP2 as a drug target and developing TDP2 inhibitors
as molecular probes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental
Chemistry
General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Flash chromatography was performed on a Teledyne Combiflash RF-200 with
RediSep columns (silica) and indicated mobile phase. All moisture sensitive reactions
were performed under an inert atmosphere of ultra-pure argon with oven-dried glassware.
1
13
H and C NMR spectra were recorded on a Varian 600 MHz spectrometer. Mass data
were acquired on an Agilent TOF II TOS/MS spectrometer capable of ESI and APCI ion
sources. Analysis of sample purity was performed on a Varian Prepstar SD-1 HPLC
system with a Phenomenex Gemini, 5 micron C18 column (250mm x 4.6 mm). HPLC
conditions: solvent A = H O, solvent B = MeCN; flow rate = 1.0 mL/min; compounds
2
2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were eluted with a gradient of 10% MeCN/H
2
O for 0-5 min and to 80% MeCN/ H O
2
from 5-30 min followed by 100% MeCN from 30-35 min and then 10% MeCN/H O from
2
3
5-40 min. Purity was determined by total absorbance at 254 nm. All tested compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
have a purity ≥ 98%.
General Procedure for Suzuki Coupling (21, 25). The mixture of compound 20 or 24
(
(
1.0 eq), aryl boronic acid (1.6 eq), Pd(PPh
3
)
4
(0.065 eq), K
2
CO
3
(3.6 eq) in EtOH/H O
2
1:1) was irradiated at 150 °C for 30 min under microwave conditions. The black residue
formed was filtered through celite and the solvent was concentrated in vacuo. The
resulting aqueous solution was acidified (pH = 3) using 2N HCl. A white precipitate was
obtained via filtration, which was washed with water and dried under vacuum to furnish
desired compounds (21, 25) as colorless solid.
General Procedure for the S Ar Reaction (23). To a suspension of NaH (32 mmol, 2.0
N
eq, 60% dispersion in oil) in DMSO (10 mL) at 0 °C was added methyl cyanoacetate (32
mmol, 2.0 eq) slowly and the mixture was stirred at r.t for 30 min before 2-fluoro-4-
iodobenzonitrile (22, 16 mmol, 1.0 eq) in DMSO (16 mL) was added. The resulting
solution was stirred at 90 °C for 8 h and quenched by adding 2N HCl (20 mL). After
extraction with the EtOAc (2 x 30 mL), the combined organics were washed with
NaHCO
3
(2 x 20 mL), brine (30 mL), dried over Na
2
SO and concentrated in vacuo to
4
leave brown oil. The crude material was used for next step without further purification.
General Procedure for the Synthesis of Dinitriles (24). A solution of compound 23 in
DMSO/H
water. The aqueous solution was extracted using EtOAc (2 x 30 mL). The combined
organics were washed with water (2 x 20 mL), brine (30 mL), dried over Na SO , and
2
O (9:1) mixture was stirred at 120 °C for 16 h before being quenched with
2
4
2
6
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concentrated in vacuo. The crude mixture was purified using CombiFlash with 0-20%
EtOAc in hexane as an eluent to yield the desired products (24) as yellow solid. Yield:
7
0-85% over 2 steps.
General Procedure for the Synthesis of Arylketone (26). A mixture of compound 24
1.0 eq), arylboronic acid (2.5 eq), Pd(OAC) (0.1 eq), Mo(CO) (1.5 eq), K CO (3.0 eq)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
2
6
2
3
in anisole (0.2M, 5 mL) was irradiated at 140 °C for 30 min under microwave conditions.
The black residue formed was filtered through celite and the solvent was concentrated in
vacuo. The crude mixture was purified using CombiFlash with 0-50% EtOAc in hexane
as an eluent to yield the desired product 26 as colorless solid. Yield: 50-62%.
General Procedure for Isoquinoline-1,3-dione (33-88). Method A: To a suspension of
compound 21 (1.0 eq) in 1,2-dichlorobenzene was added urea (2.0 eq) and the resulting
mixture was irradiated at 175 °C for 45 min under microwave conditions. The solvent
was removed in vacuo to produce the crude product as brown solid which was purified
using CombiFlash with 0-50% EtOAc in hexane as an eluent to furnish titled compounds
(
33-88) as yellow solid. Method B: A solution of compound 24 / 25 / 26 (0.45 mmol) in
Conc. HCl (2 mL) was stirred at 70 °C for 4 h. After the solvent was removed in vacuo
the residue was purified using CombiFlash with 0-30% EtOAc in hexane as an eluent to
yield desired products (33-88) as colorless solid.
1
Isoquinoline-1,3(2H, 4H)-dione (33). H NMR (600 MHz, DMSO-d
6
) δ 11.28 (s, 1H),
8.01 (dd, J = 7.8, 1.4 Hz, 1H), 7.64 (td, J = 7.5, 1.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.38
13
(
d, J = 7.6 Hz, 1H), 4.03 (s, 2H). C NMR (150 MHz, DMSO-d
6
) δ 171.1, 165.5, 136.8,
NO ,
1
33.6, 127.9, 127.7, 127.5, 125.1, 36.1. HRMS-ESI (−) m/z calculated for C
9
H
6
2
160.0404 [M-H]-; found: 160.0400.
2
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5
6
1
5
-Bromoisoquinoline-1,3(2H, 4H)-dione (34). H NMR (600 MHz, DMSO-d
6
) δ 11.48
(
(
s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 3.87
13
s, 2H). C NMR (150 MHz, DMSO-d
26.7, 122.2, 37.1. HRMS-ESI (−) m/z calculated for C
found: 237.9506.
-Bromoisoquinoline-1,3(2H, 4H)-dione (35). H NMR (600 MHz, DMSO-d
6
) δ 169.2, 164.1, 136.6, 135.7, 128.5, 126.9,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
H
5
BrNO , 237.9509 [M-H]-;
2
1
6
6
) δ 11.37
s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.67-7.65 (m, 2H), 4.04 (s, 2H). C NMR (150 MHz,
DMSO-d ) δ 174.0, 162.4, 156.9, 137.1, 133.6, 129.9, 128.6, 127.6, 35.9. HRMS-ESI (−)
m/z calculated for C
1
3
(
6
9
H
5
BrNO
2
, 237.9509 [M-H]-; found: 237.9515.
1
6
1
d
-Iodoisoquinoline-1,3(2H, 4H)-dione (36). H NMR (600 MHz, DMSO-d
6
) δ 11.35 (s,
1
3
H), 7.83 (m, 2H), 7.74 (d, J = 8.7 Hz, 1H), 4.01 (s, 2H). C NMR (150 MHz, DMSO-
) δ 170.9, 165.5, 139.1, 136.9, 136.8, 136.4, 125.0, 102.5, 35.9. HRMS-ESI (−) m/z
6
calculated for C
9
H
5
INO
2
, 285.9370 [M-H]-; found: 285.9376.
1
7
-Iodoisoquinoline-1,3(2H, 4H)-dione (37). H NMR (600 MHz, DMSO-d
6
) δ 11.38 (s,
1H), 8.25 (d, J = 1.8 Hz, 1H), 7.97 (dd, J = 8.1, 1.8 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H),
13
3
1
2
.96 (s, 2H). C NMR (150 MHz, DMSO-d
6
) δ 170.4, 163.9, 141.5, 136.1, 135.3, 129.9,
26.8, 92.1, 35.5. HRMS-ESI (−) m/z calculated for C
9
H
5
INO , 285.9370 [M-H]-; found:
2
85.9367.
1
8-Iodoisoquinoline-1,3(2H, 4H)-dione (38). H NMR (600 MHz, DMSO-d
6
) δ 11.28 (s,
1
2
9
2
H), 8.06 (d, J = 7.7 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.26 (t, J = 7.7 Hz, 1H), 4.05 (s,
13
H). C NMR (150 MHz, DMSO-d ) δ 169.7, 163.6, 141.8, 134.4, 133.4, 128.2, 124.6,
6
4.9, 37.5. HRMS-ESI (−) m/z calculated for C
9
H
5
INO , 285.9370 [M-H]-; found:
2
85.9372.
2
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,3-Dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (39). H NMR (600 MHz,
DMSO-d ) δ 13.37 (s, 1H), 11.40 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz,
6
1
3
1
H), 7.91 (s, 1H), 4.08 (s, 2H). C NMR (150 MHz, DMSO-d
36.8, 134.6, 128.6, 128.1, 127.6, 127.4, 35.8. HRMS-ESI (−) m/z calculated for
NO , 204.0302 [M-H]-; found: 204.0301.
,3-Dioxo-1,2,3,4-tetrahydroisoquinoline-7-carboxylic acid (40). H NMR (600 MHz,
DMSO-d ) δ 13.26 (s, 1H), 11.42 (s, 1H), 8.53 (d, J = 1.8 Hz, 1H), 8.14 (dd, J = 8.1, 1.8
Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 4.11 (s, 2H). C NMR (150 MHz, DMSO-d
165.9, 162.8, 135.9, 135.2, 134.1, 130.2, 128.7, 127.2, 36.1. HRMS-ESI (−) m/z
calculated for C10 NO , 204.0302 [M-H]-; 204.0308.
-(Trifluoromethyl)isoquinoline-1,3(2H, 4H)-dione (41). H NMR (600 MHz, DMSO-
) δ 11.50 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 7.82 (s, 1H), 7.80 (d, J = 8.3 Hz, 1H), 4.12
6
) δ 170.6, 166.3, 164.6,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
1
C
10
H
6
4
1
1
6
13
6
) δ 175.0,
H
6
4
1
6
d
6
13
(s, 2H). C NMR (150 MHz, DMSO-d
24.6, 123.7, 122.7, 36.0. HRMS-ESI (−) m/z calculated for C10
H]-; found: 228.0285.
6
) δ 170.5, 164.4, 138.0, 132.7, 128.5, 124.9,
1
H
5
F
3
NO , 228.0278 [M-
2
1
6
-Nitroisoquinoline-1,3(2H, 4H)-dione (42). H NMR (600 MHz, DMSO-d
H), 8.29 (s, 1H), 8.25-8.25 (m, 2H), 4.17 (s, 2H). HRMS-ESI (−) m/z calculated for
, 205.0255 [M-H]-; found: 205.0252.
6-(Furan-2-yl)isoquinoline-1,3 (2H, 4H)-dione (43). H NMR (600 MHz, DMSO-d
6
) δ 11.59 (s,
1
C
9
H
5
N
2
O
4
1
6
) δ
1
1
1.28 (s, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.86 (s, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.70 (s,
1
3
H), 7.18 (d, J = 3.3 Hz, 1H), 6.67 (dd, J = 3.3, 1.7 Hz, 1H), 4.07 (s, 2H); C NMR (150
MHz, DMSO-d
6
) δ 175.4, 163.0, 150.9, 145.2, 135.0, 133.2, 129.3, 128.9, 128.0, 120.57,
2
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