The identification and optimization of 2,4-diketobutyric acids as flap endonuclease 1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.07.028

Flap endonuclease 1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. In this report, we describe the identification and SAR of a series of 2,4-diketobutyric acid FEN1 inhibi

Full text of DOI:10.1016/j.bmcl.2004.07.028

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4915–4918
The identification and optimization of 2,4-diketobutyric acids
as flap endonuclease 1 inhibitors
L. Nathan Tumey,* Bayard Huck, Elizabeth Gleason, Jianmin Wang,
Daniel Silver, Kurt Brunden, Sherry Boozer, Stephen Rundlett,
Bruce Sherf, Steven Murphy, Andrew Bailey, Tom Dent,
Christina Leventhal, John Harrington and Youssef L. Bennani
Athersys, Inc., 3201 Carnegie Ave., Cleveland, OH 44115, USA
Received 23 April 2004; revised 13 July 2004; accepted 14 July 2004
Available online 4 August 2004
Abstract—There have been several recent reports of chemopotentiation via inhibition of DNA repair processes. Flap endonuclease 1
(FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA
damage. In this report, we describe the identification and SAR of a series of 2,4-diketobutyric acid FEN1 inhibitors.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Flap endonuclease-1 (FEN1) is a 43kDa metal-depend-
ent nuclear enzyme that exhibits both DNA structure-
specific endonuclease activity and 50 exonuclease
activity. Arguably, the most well studied aspect of
FEN1 function is its role in the cleavage of Okazaki
fragments during DNA replication.1 However, FEN1 also
acts to cleave 50 DNA flaps generated during a variety of
other cellular processes including double-strand break
repair,2 homologous recombination,3 and base excision
repair (BER).4 FEN1 is necessary for normal embryonic
development and homozygous mouse knockouts of
FEN1 are lethal.5,6 Interestingly, however, yeast knock-
outs of FEN1 are viable, albeit at a slower growth rate.7
and O6-alkylguanine–DNA alkyltransferase (ATase or
MGMT).12 These inhibitors are reported to potentiate
the activity of various chemotherapeutic agents includ-
ing temozolamide13 and topotecan.11 In light of this re-
cent work, we embarked on a strategy to identify
selective small-molecule inhibitors of FEN1 for use as
chemopotentiating agents.
FEN1 is highly homologous to a related endonuclease,
xeroderma pigmentosum G (XPG).14 XPG is part of a
repair pathway that excises DNA containing pyrimidine
dimers, a common form of damage caused by exposure
to UV light. Defects in XPG are known to cause hyper-
sensitivity to UV light, resulting in light-induced skin
lesions and carcinoma.15 Therefore, selective inhibition
of FEN1 over XPG was a key goal of this program.
BER is an important cellular mechanism for the repair
of DNA damage caused by alkylating agents.8 The role
of FEN1 in BER is clearly exemplified in a recent report
that shows nuclease-defective FEN1 results in increased
cellular sensitivity to methylmethane sulfonate (MMS),
a potent DNA alkylating agent.9 Sensitization to
DNA damaging agents may improve the therapeutic
window of classical chemotherapeutics by lowering the
minimum effective dose.10 Recent reports describe sev-
eral small molecule inhibitors of DNA repair proteins
including poly(ADP-ribose) polymerase-1 (PARP)11
A high-throughput screen was performed with our com-
pound library in order to identify compounds with
inhibitory activity against FEN1. Selected com-
pounds were subsequently screened against XPG.
FEN1 and XPG inhibition assays were performed using
a fluorogenic substrate consisting of a triple-labeled
double-stranded DNA molecule containing an internal
2-nucleotide gap.16 Several classes of compounds were
identified from this screen, the most prominent of which
contained a 2,4-diketobutyric acid functionality (Fig. 1,
compounds 1–3). Generally, these compounds were low
micromolar inhibitors of FEN1 (Table 1).
Keywords: Endonuclease; Cancer; FEN1.
*
Corresponding author. Tel.: +1-216-4319900x498; fax: +1-216-
3619495; e-mail: ntumey@athersys.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.028

4916
L. N. Tumey et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4915–4918
O
O
O
"Method A"
1 (R=Ph)
2 (R=Cl)
"Method B"
O
O
O
OMe
OH
OH
N(Me)2
N
3 (R=OPh)
R
O
R
R
O
R
OH
Figure 1. 2,4-Diketobutyrate hits from high-throughput screening.
O
Scheme 1. Reagents and conditions: Method A: (1) diethyl oxalate,
NaOMe; (2) NaOH. Method B: (1) MeLi, HMPA/THF; (2) HCl.
Compound 1 has been previously reported to inhibit the
cap (m7GpppXm)-dependent endonuclease of influenza
virus. This endonuclease cleaves host-cell RNA in order
to provide primers for viral mRNA synthesis. The
blockage of this viral endonuclease by compounds re-
lated to 1 has been shown to block in vitro viral replica-
tion.17 Similar diketobutyrates have been reported to
inhibit HIV integrase18 and glycolic acid oxidase.19
(4equiv) and HMPA (12equiv). Fewer equivalents of
either generally results in decreased yield and purity.
While the reaction requires somewhat harsh conditions,
it allows the synthesis of 3,4-diketobutyrates from a
wide variety of carboxylic acids that are commercially
available or readily synthesized. This method was read-
ily adapted to a parallel synthesis format using a Boh-
dan mini-block system, which allowed the expeditious
synthesis of a large variety of 2,4-diketobutyrates for
biological evaluation.22
The synthesis of 2,4-diketobutyrates is generally accom-
plished through a base-promoted Claisen condensation
of the appropriate methyl ketone and a dialkyl oxalate
(Scheme 1, Method A). Saponification gives the requi-
site 2,4-diketobutyrate in good overall yield.20 While
this two-step procedure works well with a variety of sub-
strates, the lack of availability of appropriately substi-
tuted methyl ketones greatly limited its usefulness.
Comparison of compounds 1–4 (Table 1) clearly illus-
trate that hydrophobic substitution at the para position
increased activity ꢀ10-fold against both FEN1 and
XPG. Substitution at the ortho position (5–7) slightly in-
creased FEN1 activity, but more importantly decreases
the activity against XPG. In particular, an ortho Cl in-
creased FEN1 selectivity to over 10-fold. meta Substitu-
tion (8–11) resulted in increased activity, but fairly poor
selectivity. meta And para-biaryl substitution resulted in
especially potent compounds (2, 9, 10). The combination
of an ortho-Cl with a hydrophobic group at the meta or
A conceptually more attractive route involves the addi-
tion of the dianion of a pyruvic acid equivalent to an es-
ter.21 The dimethyl hydrazone of pyruvic acid was used
as the nucleophile (Scheme 1, Method B). The hydra-
zone was easily prepared on a 60mmol scale and was
stable for at least 1month at 0ꢁC. Addition of the dian-
ion of the hydrazone to esters required methyl lithium
Table 1. FEN1 and XPG activity for compounds 1–18
O
O
OH
O
R
Compounda
Synthetic method
R
IC50 (lM)
FEN1
XPG
12.7
1.60
1.87
75.4
1
A
A
A
A
A
A
A
A
B
4-Cl
4-Ph
19.3
3.50
1.75
24.9
3.51
14.5
12.9
6.96
3.62
1.36
7.31
37.9
1.82
5.53
17.7
0.41
0.33
0.19
2
3
4-OPh
4
H
2-Cl
5
38.1
78.5
54.5
13.1
6
2-F
2-CH3
7
8
3-Cl
3-Ph
9
3.24
10
11
12
13
14
15
16
17
18
B
3-(4-OMe-Ph)
3-OPh
1.59
5.44
A
A
A
A
A
A
B
2,6-DiCl
2,5-DiCl
2,4-DiCl
2,3-DiCl
2-Cl, 4-(4-Cl-Ph)
2-Cl, 5-Ph
2-Cl, 5-(4-OMe-Ph)
>100
7.00
9.85
22.1
1.77
1.13
0.57
B
a Compound identity and purity were established by 1H NMR and analytical HPLC–MS.

L. N. Tumey et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4915–4918
Table 2. FEN1 and XPG activity for compounds 19–29
4917
1.) Suzukib
O
O
O
2.) TMS-CHN2
3.) Method B
O
OH
OH
Method A
O
X
R
X
R
X
Br
Compounda
Synthetic method
X
R
IC50 (lM)
FEN1
XPG
41.0
1.81
19
20
21
22
23
24
25
26
27
28
29
A
B
B
B
B
B
A
B
B
B
B
O
O
O
O
O
O
S
H
Ph
64.2
1.66
13.5
10.8
11.3
12.2
10.1
7.65
1.52
0.57
0.44
4-t-Bu-Ph
4-Cl-Ph
4-OEt-Ph
2-Cl-Ph
H
3.13
0.98
1.40
1.85
38.7
S
Ph
3-CF3
1.85
2.32
0.90
0.31
S
S
4-Cl, 3-F-Ph
4-OCF3-Ph
S
a Compound identity and purity were established by 1H NMR and analytical HPLC–MS.
bArB(OH)2, 2M Na2CO3, Pd(PPh3)4, NMP, heat.
para position resulted in several sub-micromolar com-
pounds (16–18). Interestingly, comparisons of com-
pounds 9 and 10 to compounds 17 and 18 show that
the ortho chloro consistently gave a 3- to 5-fold improve-
ment in selectivity against XPG.
N
O
N
S
a
Ph
Ph
HO
S
30
O
Heterocyclic diketobutyrates were also explored (Table
2). These compounds were generally made through a
Suzuki coupling to the heterocyclic carboxylic acid as
O
O
O
OTBS
OMe
b
MeO
HO
shown in Table 2.23 In general, unsubstituted heterocy-
cles showed poor activity (19, 25). 5-Substituted furans
were generally more potent but were quite selective for
O
N
O
N
31
OPh
O
O
R
O
PMB
XPG (21–24). 5-Substituted thiophenes were quite
potent against FEN1, but showed little of the desired
selectivity (26–29).
N
c
N
N
N
N
N
Ph
N
32 (R = PMB)
33 (R = H)
N
d
It has been reported that 2,4-diketobutyrates suffer from
metabolic and toxicity problems due to the electrophilic
nature of the a-keto acid.24 In response, several groups
have reported alternatives including 8-hydroxy-[1,6]
naphthyridines24 and 5-keto-tetrazoles.25 Based on this
literature, we designed several molecules to mimic the
2,4-diketobutyric acid moiety (Scheme 2). Interestingly,
the N-substituted tetrazole (32, Table 3) proved to be
one of the most active isosteres. Unsubstituted tetrazoles
were largely inactive (33–34). The 8-hydroxy-[1,6] naph-
thyridine (36) also showed low micromolar activity.
Unfortunately, none of the isosteres were sufficiently
active to warrant further investigation.
O
O
H
N
N
N
N
34
OPh
OPh
CH2
O
O
O
O
f
O
HO
O
35
OH
O
OH
O
N
g
N
OMe
N
N
OPh
In conclusion, we have identified a sub-micromolar
series of 2,4-diketobutyrates as inhibitors of the DNA
repair protein FEN1. These inhibitors are highly
related to a series of HIV integrase inhibitors that
have shown efficacy in cell culture. Several compounds
showed greater than 10-fold selectivity over the highly
related endonuclease, XPG. Further studies have been
36
Scheme 2. Reagents and conditions: (a) NaHMDS, diethyl oxalate; (b)
(1) LiHMDS, THF, 4-phenoxy acetophenone; (2) TFA; (c) 4-phenyl
acetophenone, diethyl oxalate, NaOMe; (d) TFA; (e) 4-phenoxy
acetophenone, diethyl oxalate, NaOMe; (f) AlCl3, DCE; (g) 4-phenoxy
phenyl lithium, THF.

4918
L. N. Tumey et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4915–4918
Table 3. FEN1 and XPG activity for compounds 30–36 (Scheme 2)
gagcaa-Black Hole Quencher; Integrated DNA Technol-
ogies). High-throughput enzyme inhibition assays were
performed using 60lL reaction mixes (50mM Tris–HCl;
pH8.0, 10mM MgCl2, 0.5mM 2-ME, 6lg BSA, 2.5lg
circular plasmid, 180U FEN1 or 50ng XPG, 25lM test
compound, and 5pmol BVT substrate) contained in black
96-well plates. Reactions were incubated at room temper-
ature for 90min, stopped through the addition of 40lL
stop buffer (0.025% SDS, 12.5mM EDTA) and fluores-
cence was measured using a Fluoroscan plate-reading
fluorometer fitted with 485nm excitation/538nm emission
filters.
Compound
IC50 (lM)
FEN1
XPG
31.7
>100
52.4
30
31
32
33
34
35
36
46.4
>100
34.3
>100
63.2
>100
97
>100
29.0
>100
>100
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Lewis, J.; McClements, W.; Ponticello, G.; Radzilowski,
E.; Smith, G.; Tebben, A.; Wolfe, A. Antimicrob. Agents
Chemother. 1994, 38, 2827–2837.
undertaken to determine the clinical usefulness of com-
pounds in this series as chemopotentiating agents.
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16. This molecule, referred to as BVT substrate, was prepared
by annealing oligonucleotide VT (50-VIC-ccctccgcc-
gtcgcgttt-TAMRA; Applied BioSystems) with oligonucleo-
tide B (50-aaacgcgacggcggagggtcttgctcagtgtc gtctccgacact-