Design and Synthesis of 3-Carbamoylbenzoic Acid Derivatives as Inhibitors of Human Apurinic/Apyrimidinic Endonuclease 1 (APE1)

Article abstract of DOI:10.1002/cmdc.201200334

Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifaceted protein with an essential role in the base excision repair (BER) pathway. Its implication in tumor development, progression, and resistance has been confirmed in multiple cancers, making it a viable target for intensive investigation. In this work, we designed and synthesized different classes of small-molecule inhibitors of the catalytic endonuclease function of APE1 that contain a 3-carbamoylbenzoic acid scaffold. Further structural modifications were made with the aim of increasing the activity and cytotoxicity of these inhibitors. Several of our compounds were shown to inhibit the catalytic endonuclease function of APE1 with potencies in the low-micromolar range invitro, and therefore represent novel classes of APE1 inhibitors worthy of further development.

Full text of DOI:10.1002/cmdc.201200334

MED
DOI: 10.1002/cmdc.201200334
Design and Synthesis of 3-Carbamoylbenzoic Acid
Derivatives as Inhibitors of Human Apurinic/Apyrimidinic
Endonuclease 1 (APE1)
Francesca Aiello,*[a] Yumna Shabaik,[b] Adrian Esqueda,[b] Tino W. Sanchez,[b] Fedora Grande,[a]
Antonio Garofalo,[a] and Nouri Neamati[b]
Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifac-
eted protein with an essential role in the base excision repair
(BER) pathway. Its implication in tumor development, progres-
sion, and resistance has been confirmed in multiple cancers,
making it a viable target for intensive investigation. In this
work, we designed and synthesized different classes of small-
molecule inhibitors of the catalytic endonuclease function of
APE1 that contain a 3-carbamoylbenzoic acid scaffold. Further
structural modifications were made with the aim of increasing
the activity and cytotoxicity of these inhibitors. Several of our
compounds were shown to inhibit the catalytic endonuclease
function of APE1 with potencies in the low-micromolar range
in vitro, and therefore represent novel classes of APE1 inhibi-
tors worthy of further development.
Introduction
DNA bases are susceptible to damaging modifications inflicted
by various endogenous and exogenous sources, which, when
acted upon by glycosylases, produce apurinic/apyrimidinic (AP)
sites that are potentially cytotoxic and mutagenic if left unre-
paired. Alternatively, AP sites can be generated by the sponta-
neous hydrolysis of labile N-glycosidic bonds.[1] Regardless of
the source of damage, all abasic sites, which must be repaired
in order for cells to survive, are repaired by the base-excision
repair (BER) pathway. Apurinic/apyrimidinic endonuclease
(APE1) has the important role of recognizing AP sites in the
genome and incising the DNA backbone immediately 5’ to the
abasic sites, producing a 3’-hydroxy and a 5’-abasic deoxyri-
bose phosphate, thereby initiating their repair by an ensuing
succession of BER enzymes. The outstanding majority of abasic
sites are repaired by what is known as the short-patch or
single-nucleotide BER (SP/SN-BER), whereby a single nucleotide
is replaced, following APE1 incision, by the actions of DNA
polymerase (pol) b and DNA ligase III. However, there is also
a second, less prevalent arm of BER: long-patch BER (LP-BER)
involves replacement of the damaged nucleotide with a stretch
of 2–10 newly synthesized nucleotides, carried out by DNA
pol b or d/e, flap endonuclease (FEN1), and finally DNA ligase I.
The importance of APE1 among other DNA repair proteins,
and in contrast to the array of damage-specific DNA glycosy-
lases in BER, is emphasized by APE1 being the only DNA repair
protein responsible for and capable of processing AP sites in
both the SP/SN and LP arms of BER, thereby enabling DNA
repair.[2] While the endonuclease function is the predominant
role for APE1 in cells, it also exhibits weaker 3’-phosphodiester-
ase and 3’!5’ exonuclease activities. In addition to its role as
a DNA repair enzyme, APE1 also functions as a reduction–oxi-
dation factor, owing to an active cysteine residue located in
a domain that is separate from its DNA processing capabili-
ties.[3] By way of this redox domain, APE1 is capable of activat-
ing various transcription factors including, but not limited to,
tumor protein 53 (p53), activator protein 1 (AP-1) and nuclear
factor kappa-light-chain-enhancer of activated B cells (NF-kB),
thereby contributing to various growth-signaling pathways.[4]
Moreover, APE1 has been shown to participate in RNA metabo-
lism, as evidenced by its interactions with nucleophosmin
(NPM1), its ability to cleave abasic RNA, and its regulation of c-
Myc mRNA levels.[5] As the foremost repair pathway responsi-
ble for removal and replacement of damaged bases, the BER
pathway has garnered great interest for pharmacological inhib-
ition. Several of the proteins in this pathway show altered ex-
pression and activation in cancer. In particular, APE1 has
emerged as an attractive therapeutic target for anticancer
drug development, as demonstrated by studies that link its
overexpression with resistance to radio- and chemotherapy.[6]
Although APE1 is considered to be a ubiquitously expressed
protein with an estimated 350000–7000000 copies per cell, its
expression in cancer cells has been shown to be even higher
than surrounding tissue.[7] Additionally, siRNA knockdown of
APE1 in cancer cells, which has the effect of potentiating the
cell-killing ability of cytotoxic agents, has further confirmed its
protective role in cancer against a variety of DNA damaging
agents.[8] Therapeutics that target APE1 could decrease onco-
genic cell advantage to evade apoptosis and sensitize cancers
to both radiation and chemotherapy.[9] For instance, radio-re-
sistance in glioma cell lines, which is associated with higher ex-
[a] Dr. F. Aiello, Dr. F. Grande, Prof. A. Garofalo
Dipartimento di Scienze Farmaceutiche
Universitꢀ della Calabria, 87036 Arcavacata di Rende, CS (Italy)
E-mail: francesca.aiello@unical.it
[b] Y. Shabaik, A. Esqueda, T. W. Sanchez, Prof. N. Neamati
Department of Pharmacology and Pharmaceutical Sciences
School of Pharmacy, University of Southern California
1985 Zonal Avenue, Los Angeles, CA 90089 (USA)
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pression of APE1, can be reversed following chemical inhibition
of APE1, as can cisplatin resistance in non-small-cell lung
cancer.[10] Alterations in APE1 expression and localization are
thought to have prognostic and/or predictive significance in
several cancers, including liver, lung, and ovarian cancer.[11] Pre-
dictive cancer-specific survival following radiotherapy has also
been attributed to APE1 expression.[11c,12] Overexpression in
cancer cells and drug-induced acetylation of APE1 have also
been shown to promote expression of the multidrug-resistance
(MDR1) gene.[13] The tumor microenvironment is also affected
by APE1, whereby inhibition of its redox activity leads to inhib-
ition of release of pro-inflammatory signals from tumor-associ-
ated macrophages.[14] Considering that APE1 plays a central
role in BER and the fact that many of the classically employed
anticancer treatments including alkylating agents and ionizing
radiation produce base damage that requires intact BER func-
tionality to overcome, inhibiting APE1 as a means to interrupt-
ing BER is viewed as a viable strategy to enhance chemothera-
peutic outcomes. Toward that goal, we have devoted our ef-
forts to the design of novel classes of small-molecule com-
pounds that can inhibit the endonuclease function of APE1
and that can potentiate the cell-killing effect of DNA-targeted
chemotherapeutics. We are aided in our endeavor by the avail-
ability of structural information on APE1 in complex with its
substrate DNA bearing an abasic site.[2a] In a previous study we
developed a set of three-dimensional (3D) pharmacophore
models based on APE1 interactions with the abasic deoxyri-
bose 3’- and 5’-phosphate backbone in a co-crystal structure
of APE1 in complex with its substrate DNA bearing an abasic
site.[15] APE1 endonuclease activity requires binding of divalent
cations in the active site and can be exploited for drug
design.[16] The pharmacophore perception is a popular tech-
nique in drug design to identify small-molecule inhibitors with
diverse chemical scaffolds. It has been successfully used to dis-
cover several classes of clinically relevant small molecules that
target a number of pathways, including DNA repair. The struc-
turally diverse set of molecules we have identified are APE1 in-
hibitors and are important and suitable as lead molecules to
establish quantitative structure–activity relationship models for
further development of clinically relevant APE1 inhibitors. In an
Figure 1. Synthesized compounds with descriptive hydrophobic, negative
ionizable, and linker moieties. a) Synthesized 3-carbamoylbenzoic acid deriv-
atives. b) Subsequently synthesized 6-benzylcarbamoylpyridine, 8-hydroxy-
qunoline-2-carboxamide, and N-benzylbenzamide derivatives.
line-2-carboxamides, and N-benzylbenzamide derivatives (Fig-
ure 1b), none showed significant activity and selectivity,
whereas 3-carbamoylbenzoic acid derivatives showed the most
potent and selective activity against APE1, in the micromolar
concentration range.
effort to better characterize the mode of action of these com- Results and Discussion
pounds, we planned a synthetic pathway for the preparation
Chemistry
of new classes of 3-carbamoylbenzyl derivatives (Figure 1a)
active against APE1. Several of the synthesized compounds dis-
played good APE1 inhibitory activity in preliminary in vitro
assays. A similar radiolabeled DNA assay was conducted on re-
combinant HIV-1 integrase (IN) to observe the degree of selec-
tivity of these compounds in inhibiting APE1.[17] In a fashion
similar to APE1, IN catalytically cleaves the phosphodiester
backbone of virally encoded DNA 5’ to its recognition site,
leaving a 3’-hydroxy group. Furthermore, IN requires a divalent
metal cation for catalysis, making it a suitable counter-screen-
ing target for establishing the specificity of our compounds.
Structure–activity analysis of synthesized compounds reached
an informative structural platform for the rational optimization
of this class of small molecules, because among the classes
studied, namely 6-benzylcarbamoyl pyridines, 8-hydroxyquino-
The synthesis of 3-carbamoylbenzoic acids F325–F350 was car-
ried out by condensing commercially available 2-methoxyi-
sophthalic acid 1 with 3- or 4-substituted or 3,4-disubstituted
benzylamines, using EDC in the presence of a catalytic amount
of DMAP as a condensing agent and THF as the solvent.
N1,N3-Dibenzylamides F328bis–F330bis and F350bis were ob-
tained after the same reaction by using two equivalents of the
appropriate benzylamine. All the methoxy derivatives were
subjected to demethylation by HBr/CH3COOH or LiI/collidine,
depending on the sensitivity of the substrate. Compound F327
was demethylated by BBr3 in CH2Cl2 (Scheme 1).
Derivatives F-M260 and F-M262 were obtained by the
Schotten–Baumann procedure, reacting 2-methoxyisophthalic
1 acid with one equivalent of methyl 3-amino-3-(4-chlorophe-
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It is known that the carboxyl-
ate ion often hinders the pene-
tration of potentially active mol-
ecules inside cells.[19] Better re-
sults could be pursued by re-
placing the carboxylic moiety
with the alternative bioisostere
tetrazole ring. To this aim we de-
signed
compound
T3F
(Scheme 6), which was synthe-
sized by a typical [3+2] cycload-
dition procedure as the key
step.[20] The tetrazole ring was
first installed starting from
methyl 3-cyanobenzoate 5 and
sodium azide. The intermediate
ester was then hydrolyzed into
the corresponding acid, which
was in turn condensed with the
appropriate
mine.
3-fluorobenzyla-
Scheme 1. Reagents and conditions: a) benzylamine (1 equiv), EDC, DMAP, CH2Cl2, RT; b) benzylamine (2 equiv),
EDC, DMAP, CH2Cl2, RT; c) HBr/CH3COOH, 1208, 2 h; d) LiI, collidine, reflux, 2 h; e) BBr3 (1m in CH2Cl2).
Finally, we decided to prepare
the N-benzylbenzamide series of
compounds shown in Scheme 7
to further investigate the impor-
nyl)propanoate or 3-amino-3-(4-fluorophenyl)propanoate, re-
spectively. Ether and ester functions were hydrolyzed at the
same time by the LiI/collidine system to give derivatives F-
260D and F-262D, while derivatives F-M260S and F-M262S
were obtained by alkaline hydrolysis selective for the ester
group (Scheme 2).
tance of the original complexing triad consisting of an elec-
tron-rich functional group between the two carbonyl groups.
Beside removal of the central oxygen or replacement of the ni-
trogen atom, these compounds show the presence of cyano or
trifluoromethyl groups instead of a more polar carboxylic func-
tion, giving rise to a two-point complexing moiety with an
The 6-benzylcarbamoyl pyri-
dine derivatives F-1, F-2, and the
symmetric di-3-chlorobenzyl de-
rivative F-4 were prepared by
the Schotten–Baumann proce-
dure, starting from pyridine-2,6-
dicarbonyl dichloride 2 and the
appropriate amine, as depicted
in Scheme 3. The methyl ester F-
3 was prepared starting from
2,6-pyridine dicarboxylic acid 3
first transformed by a two-step
procedure into the monomethyl
Scheme 2. Reagents and conditions: a) SOCl2, DMF, 2-methyl-3-amino-3-(4-fluoro- or 4-chlorophenyl)propanoate,
THF, reflux, 24 h; b) LiI, collidine, reflux, 1 h; c) LiOH, H2O, THF, MeOH, H2O, RT, 18 h.
ester, which was then con-
densed with 3-chlorobenzyla-
mine by the EDC/DMAP system
(Scheme 4).[18]
With the aim of determining the ideal size of the hydropho-
bic group that supports the triad coordinating the divalent ion
in the enzyme active site, we decided to prepare 8-hydroxyqui-
noline derivatives F-5 and F-6, starting from 8-hydroxyquino-
line-2-carboxylic acid 4, which was transformed into the corre-
sponding acid chloride, to be condensed with a substituted
benzylamine. The hydroxy derivative F-7 was obtained by BBr3-
catalyzed demethylation of F-6 (Scheme 5).
Scheme 3. Reagents and conditions: a) 3-chlorobenzylamine (2 equiv), pyri-
dine/toluene; b) 3-fluoro- or 3-chlorobenzylamine (1 equiv), pyridine/tolu-
ene.
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Scheme 7. Reagents and conditions: a) 1. SOCl2, DMF, 2. 3-fluoro- or 3-chloro-
benzylamine, Et3N, toluene.
Scheme 4. Reagents and conditions: a) 1. H2SO4/MeOH, 2. KOH/MeOH; b) 3-
chlorobenzylamine, EDC/toluene.
HIV-1 IN endonuclease and polynucleotidyl transferase activi-
ties as indicated by 3’-processing and strand-transfer efficien-
cies, respectively. Where possible, the selectivity index of our
agents for APE1 over IN are reported. A representative IN gel is
shown in Figure 3.
Compounds F325–F327 structurally varied only in the
number and position of the methoxy group on the phenyl ring
and were selectively active for APE1, with IC50 values between
18ꢀ8 and 21ꢀ7 mm. Halogenated compounds F328–F330
were generated by the replacement of methoxy groups with
chlorine atoms. Interestingly, para-chloro compound F328 did
not show any inhibitory activity, whereas compounds F329
and F330 had IC50 values of 16ꢀ6 and 18ꢀ12 mm, respective-
ly. Compound F331, with a fluorine atom in the para position,
and meta-fluoro compound F332 inhibited APE1 endonuclease
activity with IC50 values of 26ꢀ2 and 27ꢀ16 mm, respectively.
However, F333, with two fluorine atoms, did not show inhibi-
tion properties. Apart from F350, which weakly inhibited IN
endonuclease and strand-transfer activity with IC50 values near
500 mm, all of the compounds showed no activity against IN
and displayed selectivity for APE1 greater than 35-fold
(Table 1).
Scheme 5. Reagents and conditions: a) SOCl2, 2,3-ethoxybenzylamine or 4-
chlorobenzylamine, iPr2EtN, toluene. b) BBr3, CH2Cl2.
Scheme 6. Reagents and conditions: a) 1. NaN3, NH4Cl, DMF, 2. NaOH, MeOH,
Sequentially replacing the methoxy groups in compounds
F326 and F327 with hydroxy groups to generate deprotected
compounds F326D–F327DD produced a negligible to pro-
found decrease in their ability to inhibit APE1. F327DD
showed a decrease in activity from 18ꢀ8 mm to >100 mm rela-
tive to F327. On the other hand, these modifications mostly
improved IN inhibition (Table 2) compared with counterparts in
Table 1, with the result of decreased selectivity indexes ranging
H2O; b) 1. trimethylsilyl chloride, py, toluene, 2. SOCl2, 3-fluorobenzylamine,
py.
overall geometry similar to that of the original triad. These
compounds were prepared by the simple condensation of a 3-
cyano 6 or 3-trifluoromethylbenzoic acid chloride 7 and a suita-
ble benzylamine, as depicted in Scheme 7.
In vitro APE1 inhibitory activi-
ties and selectivity indexes of
3-carbamoylbenzoic acid deriv-
atives
Inhibition of APE1 endonuclease
activity was determined with an
in vitro enzymatic assay with a ra-
diolabeled DNA substrate, sche-
matically shown in Figure 2. Op-
timal assay conditions for
screening compounds against
our purified APE1 protein were
previously determined.[15a] Con-
Figure 2. Endonuclease activity of APE1. During BER, APE1 cleaves the phosphodiester backbone 5’ to the AP site.
The 26mer oligomer used in this assay was radiolabeled with 32P at the 5’ end containing the AP site. The AP site
comitantly, we assayed our com-
pounds for their ability to inhibit in the 26mer oligomer was tetrahydrofuran (F).
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Figure 3. Representative gel showing inhibition of purified IN by selected compounds. Lane 13: DNA alone; Lane 14: DNA and IN without compounds;
Lanes 1–12 and 15–26: DNA with IN and selected compounds at 100, 33, and 11 mm. Final IN concentration is 200 nm.
from 0.52 to 26.3. Deprotected compounds F328–F333D also
with methoxy to hydroxy substitutions between the carbonyl
groups had a slight to substantial decrease in activities, with
the exception of F328D and F333D, which had increased activ-
ity and lower IC50 values of 44ꢀ37 and 39ꢀ22 mm, respective-
ly, relative to their methoxy-containing counterparts listed in
Table 1. Following the same trend as the previous compounds,
the compounds in Table 2 also had improved IN inhibition, but
still retained modest selectivity for APE1. Compound F327DD,
with two hydroxy groups in the meta and para positions of
the appended phenyl ring, displayed no inhibitory properties
toward APE1 endonuclease activity, whereas compound
F326DD, with a single hydroxy substituent at the meta posi-
tion, produced a weak IC50 value of 40ꢀ31 mm. Compound
F350D, containing an additional phenyl group, showed an IC50
value of 33ꢀ24 mm, similar to that of compound F350
(Table 1). Interestingly, these modifications improved the com-
pounds’ ability to inhibit IN and resulted in a decrease in their
selectivity, but were still able to retain modest selectivity in-
dexes favoring APE1, with the exception of F327DD, which
lacked APE1 inhibition, but was active against IN. Pyridine de-
rivatives F-1, F-2, and ester derivative F-3, designed as ana-
logues of compounds F329 and F332, did not show any activi-
ty. Similarly, compounds F-5, F-6, and F-7, based on an 8-hy-
droxyquinoline scaffold, were devoid of any significant activity
(Table 3). These inactive compounds also did not inhibit IN. In-
terestingly, the symmetric diamide derivatives listed in Table 4
(IC50 range: 25ꢀ3 to 66ꢀ5 mm) gave similar activities to that
of monosubstituted analogues, with a concomitant drop in se-
lectivity of APE1 over IN. Finally, derivatives F-M260, F260D, F-
M260S, F-M262, F262D, F-M262S, CF34Ph, CF33Cl, CF33F,
CN4Ph, and tetrazole derivative T3F (Table 5) were devoid of
activity against both APE1 and IN. The efficacy of selected
active compounds that inhibit APE1 function can be observed
in the representative gel image in Figure 4.
Cytotoxicity of 3-carbamoylbenzoic acid derivatives as
single agents and in combination with MMS or 5-FdUrd
The effect of our compounds on the proliferation of the H630
colon cancer cell line was examined with viability and colony-
formation assays. Although many of the represented 3-carba-
moylbenzoic acid derivatives inhibited APE1 catalytic endonu-
clease function effectively in vitro, cell proliferation was largely
unaffected in H630 cells at the highest doses tested. It was
therefore not possible to conclude any useful structure–activity
relationship from the activities of these compounds as single
agents in cell culture. We further examined the effect that
these compounds might have as part of a combination regi-
men with methyl methanesulfonate (MMS) on the proliferative
capability of H630 cells. MMS forms alkylated bases that are re-
paired by BER and as such, compounds that can inhibit APE1
in cells should exhibit a sensitizing effect to the cytotoxicity
produced by MMS treatment. We therefore treated cells with
a range of concentrations of MMS alone, or in combination
with a single dose of five of our compounds: F328, F329,
F330, F329D and F332D. Pairs of drugs in each combination
were added simultaneously to cells and incubated together for
a total duration of 72 h, at the end of which the cell viability
was determined (Figure 5a). This potentiation was specific to
the BER-dependent DNA lesions caused by MMS, as the combi-
nation of F332D with other genotoxic agents did not produce
a similar synergistic outcome on cell viability Figure 5a,b).
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It is known that 5-FU, an anti-
cancer drug commonly used to
treat colon cancer, gets incorpo-
rated into the DNA of treated
cells at significant levels and that
BER glycosylases are capable of
detecting and removing the mis-
incorporated base, so that DNA
can be repaired.[21] Our results in-
dicate that F332D may be clini-
cally useful as a combination
therapy with 5-FU. This potentia-
tion was specific to the BER-de-
pendent DNA lesions caused by
MMS, as the combination of
F332D with other genotoxic
agents did not produce a similar
synergistic outcome on cell via-
bility (Figure 6). Although BER
proteins recognize and remove
5-FU lesions from DNA, recent
reports suggest that intact BER
is more important for the repair
of DNA damage resulting from
5-fluorodeoxyuridine (5-FdUrd)
treatment.[22] Therefore, we ex-
amined the effect of the com-
bined treatment of our APE1 in-
hibitors with 5-FdUrd on the sur-
vival of HT-29 colon cancer cells.
We tested F328, F329, F330,
F332, F328D, F329D, and F332D
in an attempt to correlate syner-
gistic activity with APE1 inhibi-
tion. Among these compounds,
F330, F328D, and F332D
showed enhanced potentiation
Table 1. Inhibition of APE1 endonuclease activity by compounds F325–F350.
Compound
HIV-1 IN
3P[a]
APE1
IC50 [mm]
Cell culture
SI[c]
ST[b]
MTT
Colony
F325
F326
F327
F328
F329
F330
F331
F332
F333
>1000
>1000
>1000
>1000
<1000
>1000
>1000
>1000
1000
>1000
21ꢀ7
19ꢀ9
18ꢀ8
>100
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>47.6
>52.6
>55.6
NA
>1000
>1000
>1000
1000
>30
>30
>30
>30
>30
>30
>30
>30
16ꢀ6
18ꢀ12
26ꢀ2
27ꢀ16
>100
<62.5
>55.6
>38.5
>37
>1000
>1000
>1000
>1000
NA
F350
505
460
31ꢀ11
>30
>30
16.3
[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1
endonuclease catalysis versus IN endonuclease 3’-processing.
Figure 4. Representative gel showing inhibition of purified APE1 by selected compounds. Lane 17: DNA alone; Lanes 18 and 19: DNA and APE1 without com-
pounds; Lanes 1–16 and 20–27: DNA with APE1 and selected compounds by varying concentrations 100, 33, 11, and 3.7 mm. Final APE1 concentration is
0.05 nm.
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3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors
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ic acid structure, resulted in the
most active and selective inhibi-
tion of APE1, showing IC50 values
<20 mm and with no activity
against HIV1 IN at the highest
concentrations tested. Replace-
ment of the methoxy group
with a hydroxy group at posi-
tion 2 (compound F237D) did
not result in a significant change
in activity, whereas such an alter-
ation considerably affected se-
lectivity. Notably, some activity
and selectivity was retained by
several dicarbamoyl derivatives,
while the replacement of the
carbocyclic aromatic ring togeth-
er with the carboxylic function
Table 2. Inhibition of APE1 endonuclease activity by compounds F326D–F350D.
Compound
HIV-1 IN
APE1
IC50 [mm]
Cell culture
MTT Colony
SI[c]
3P[a]
ST[b]
F326D >1000
>1000
38ꢀ30 >30
>30
>30
>26.3
F326DD
F327D
280
61
195
40ꢀ31 >30
7
99
19ꢀ2
>30
>30
>30
3.2
F327DD
F328D
F329D
F330D
F331D
52ꢀ30
17ꢀ9
>100
>30
>30
>30
>30
>30
>30
>30
<0.52
>7.6
>3
>333
>100
>100
>100
>216
44ꢀ37 >30
33ꢀ26 >30
28ꢀ19 >30
for
a
pyridine or quinoline
95ꢀ8
system proved detrimental to
activity. Thus, the 3-benzylcarba-
moyl-2-methoxybenzoic
structure, common to the active
derivatives, represents lead
acid
82ꢀ25
70ꢀ14
>3.6
>3.7
NA
a
27ꢀ9
>30
>30
scaffold to be further developed
with the aim of finding more
potent and selective APE1 inhibi-
tors.
F332D >1000
>1000
>100
F333D
F350D
>333
98ꢀ3
39ꢀ22 >30
33ꢀ24 >30
>8.5
Experimental Section
Chemistry
95ꢀ6
35ꢀ14
>30
2.9
All reactions were carried out
under
a
nitrogen atmosphere.
Progress of the reaction was moni-
tored by TLC on silica gel plates
(Merck, 60, 230–400 mesh, 0.040–
0.063 mm). Organic solutions were
dried over MgSO4 and evaporated
[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1
endonuclease catalysis versus IN endonuclease 3’-processing.
on a rotary evaporator under reduced pressure. Melting points
were measured using an Electrothermal 8103 apparatus and are
uncorrected. IR spectra were recorded as thin films on PerkinElmer
398 and FT 1600 spectrophotometers. 1H NMR spectra were re-
corded on a Bruker 300 MHz spectrometer with TMS as an internal
standard; chemical shifts (d) are expressed in ppm, and coupling
constants (J) in Hz. MS data were determined by direct insertion at
70 eV with a VG70 spectrometer. Merck silica gel (Kieselgel 60,
230–400 mesh) was used for flash chromatography columns. Ele-
mental analyses were performed on a PerkinElmer 240C elemental
analyzer, and the results are within 0.4% of theoretical values.
Yields refer to purified products and are not optimized.
of 5-FdUrd-induced cytotoxicity (Figure 7). While F328D and
F330 were among the most active analogues tested in the in
vitro APE1 assay, F332D exhibited very weak activity. However,
F332D was able to specifically potentiate the cytotoxicity pro-
duced by both MMS and 5-FdUrd which are repaired by BER.
Because it is not presently clear how F332D achieves this po-
tentiation, future studies are required to better understand its
mode of action. The synergistic combinations achieved with
our inhibitors support their further development toward com-
bination therapies with 5-FdUrd for the treatment of colon
cancer.
General procedures for the synthesis of carbamoyl derivatives
Conclusions
Method A. The preparation of 3-(3-methoxybenzylcarbamoyl)-2-
methoxybenzoic acid (F326) is described as a representative exam-
ple. 4-Methoxybenzylamine (132 mL, 1.020 mmol) was added in
one portion to a solution of 2-methoxyisopthalic acid (0.200 g,
1.020 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
A new class of APE1 inhibitors based on previously reported
pharmacophore models was identified in this work. Among
the newly synthesized compounds, derivatives F326, F327,
F329, and F330, having a 3-benzylcarbamoyl-2-methoxybenzo-
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tallized from petroleum ether (PE);
yield 48% (0.154 g); mp: 1738C;
1H NMR (300 MHz, [D6]acetone):
d=8.10 (dd, J=6.0, 1.6 Hz, 2H),
7.50 (bs, 1H), 7.20 (m, 2H), 6.78
(m, 3H), 4.55 (d, J=5.5 Hz, 2H),
3.76 (s, 3H), 3.74 ppm (s, 3H); IR
(KBr): n˜ =3288, 1649 cmꢁ1; MS (EI
70 eV) m/z 315 [M]+; Anal. calcd
for C17H17NO5: C 64.75, H 5.43, N
4.44, found: C 65.03, H 5.52, N
4.40.
Table 3. Inhibition of APE1 endonuclease activity by compounds F-1, F-2, F-3, F-5, F-6 and F-7.
Compound
HIV-1 IN
APE1
IC50 [mm]
Cell culture
SI[c]
3P[a]
ST[b]
MTT
Colony
F-1
F-2
F-3
>100
>100
>100
>100
>100
100
<20(55%)
20
>30
NA
>1
>1
>100
>100
>100
>30
>30
>30
100
>20
3-(4-Methoxybenzylcarbamoyl)-2-
methoxybenzoic acid (F325):
white powder; yield 48% (0.154 g);
F-5
>100
>100
>20
NA
1
mp: 1898C (PE); H NMR (300 MHz,
[D6]acetone): d=8.20 (m, 2H), 7.59
(s, 1H), 7.26 (m, 2H), 6.85 (m, 3H),
4.55 (d, J=5.5 Hz, 2H), 3.76 (s, 3H),
3.74 ppm (s, 3H), IR (KBr): n˜ =3288,
1649 cmꢁ1; MS (EI 70 eV) m/z 315
[M]+; Anal. calcd for C17H17NO5: C
64.75, H 5.43, N 4.44, found: C
64.98, H 5.52; N 4.04.
F-6
F-7
>100
>100
>100
>100
>100
20
>30
>30
NA
70
<20(57%)
>1.4
[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1
endonuclease catalysis versus IN endonuclease 3’-processing.
3-(3,4-Dimethoxybenzylcarbamo-
yl)-2-methoxybenzoic acid (F327):
white powder; yield 30% (0.096 g);
1
Table 4. Inhibition of APE1 endonuclease activity by compounds F330bis–F-4.
mp: 1978C (PE); H NMR (300 MHz,
[D6]acetone): d=11.5 (bs, 1H), 8.11
(bs, 1H), 7.93 (dd, J=7.7,
3.9 Hz,1H), 7.88 (dd, J=5.7, 1.9 Hz,
1H), 6.81–7.39 (m, 4H), 4.50 (d, J=
5.9 Hz, 2H), 3.89 (s, 3H), 3.62 ppm
(s, 6H), IR (KBr): n˜ =3281,
1650 cmꢁ1; MS (EI 70 eV) m/z 345
[M]+; Anal. calcd for C18H19NO6: C
62.60, H 5.55, N 4.06, found: C
62.40, H 5.35, N 4.10.
Compound
HIV-1 IN
3P[a] ST[b]
APE1
IC50 [mm] MTT
Cell culture
Colony
SI[c]
F330bis
666
395
27ꢀ5 >30 >30
24.7
F330bisD
F328bisD
F329bisD
195
150
140
105ꢀ8
170
66ꢀ5 >30 >30
28ꢀ4 >30 >30
41ꢀ22 >30 >30
3
5.4
3.4
3-(4-Chlorobenzylcarbamoyl)-2-
methoxybenzoic acid (F328):
white powder; yield 40% (0.130 g);
1
98ꢀ4
mp: 1728C (PE); H NMR (300 MHz,
CDCl3): d=8.19 (dd, J=7.7, 1.9 Hz,
1H), 8.11 (dd, J=7.8, 1.9 Hz, 1H),
7.52 (s, 1H), 7.30 (m, 5H), 4.57 (d,
J=5.8 Hz, 2H), 3.79 ppm (s, 3H); IR
(KBr): n˜ =3280, 1641 cmꢁ1; MS (EI
70 eV) m/z 319.06 [M]+; Anal. calcd
for C16H14ClNO4: C 60.10, H 4.41, N
4.38, found: C 60.23, H 4.62, N
4.50.
F350bisD
105
37
25ꢀ3 >30 >30
4.2
F-4 >100 >100
>100
>20 <30(53%) NA
[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1
endonuclease catalysis versus IN endonuclease 3’-processing.
3-(3-Chlorobenzylcarbamoyl)-2-
methoxybenzoic acid (F329):
white powder; yield 40% (0.130 g);
drochloride (EDC; 0.195 g, 1.020 mmol), and 4-dimethylaminopyri-
mp: 1888C (PE); 1H NMR (300 MHz, CDCl3): d=8.27 (m, 1H), 8.18
(bs, 1H), 7.79 (m, 1H), 7.74 (m, 1H), 7.10–7.45 (m, 4H), 4.62 (m,
2H), 3.87 ppm (s, 3H); IR (KBr): n˜ =3280, 1641 cmꢁ1; MS (EI 70 eV)
m/z 319.06 [M]+; Anal. calcd for C16H14ClNO4: C 60.10, H 4.41, N
4.38, found: C 60.25, H 4.05, N 4.50.
dine (DMAP) (cat.) in THF (5.5 mL). The mixture was stirred for 72 h
at room temperature. After this time, the solvent was removed,
and the crude oil made alkaline (pH 8) by adding a saturated solu-
tion of aqueous NaHCO3, and extracted with Et2O. The aqueous
layer was acidified with 2n HCl to pH 2 and extracted with EtOAc.
The organic phase was dried over anhydrous Na2SO4 and concen-
trated to give compound F326 as a white solid, which was recrys-
3-(3,4-Dichlorobenzylcarbamoyl)-2-methoxybenzoic acid (F330):
white powder; yield 45% (0.162 g); mp: 1588C (PE); 1H NMR
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3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors
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3-(4-Fluorobenzylcarbamoyl)-2-
methoxybenzoic acid (F331):
white powder; yield 42% (0.129 g);
Table 5. Compounds that exhibit no activity against APE1 endonuclease activity.
Compound
HIV-1 IN
APE1
Cell culture
1
3P[a]
ST[b]
IC50 [mm]
MTT
Colony
mp: 1568C (PE); H NMR (300 MHz,
CDCl3): d=8.25 (m, 2H), 7.50 (bs,
1H), 7.29 (m, 3H), 6.99 (m, 2H),
4.57 (d, J=5.0 Hz, 2H), 3.79 ppm
(s, 3H); IR (KBr): n˜ =3280, 1638,
F-M260
F260D
>100
>100
>100
>100
>100
>100
>100
NT
>30
>30
>30
>30
>30
>30
>30
1350 cmꢁ1
; MS (EI 70 eV) m/z
303.09 [M]+; Anal. calcd for
C16H14FNO4: C 63.36, H 4.65, N
4.62, found: C 63.30, H 5.01, N
4.60.
410
250
560
>30
>30
>30
>30
>30
3-(3-Fluorobenzylcarbamoyl)-2-
methoxybenzoic acid (F332):
white powder; yield 50% (0.154 g);
1
mp: 1448C (PE); H NMR (300 MHz,
F-M260S
F-M262
F262D
590
CDCl3): d=8.24 (dd, J=7.7, 1.8 Hz,
1H), 8.09 (dd, J=7.7, 1.9 Hz, 1H),
7.85 (s, 1H), 7.33–7.14 (m, 2H),
7.12–6.84 (m, 3H), 4.61 (d, J=
5.7 Hz, 2H), 3.79 ppm (s, 3H); IR
(KBr): n˜ =3288, 1638, 1370 cmꢁ1
;
>1000
>1000
MS (EI 70 eV) m/z 303.09 [M]+;
Anal. calcd for C16H14FNO4: C 63.36,
H 4.65, N 4.62, found: C 63.33, H
5.00, N 4.75.
600
510
3-(3,4-Difluorobenzylcarbamoyl)-
2-methoxybenzoic acid (F333):
white powder; yield 30% (0.098 g);
1
mp: 1618C (PE); H NMR (300 MHz,
CDCl3): d=8.24 (dd, J=7.7, 1.8 Hz,
1H), 8.09 (dd, J=7.8, 1.8 Hz, 1H),
7.89 (s, 1H), 7.30 (m, 1H), 7.19–
7.03 (m, 3H), 4.56 (d, J=5.86 Hz,
2H), 3.81 ppm (s, 3H); IR (KBr): n˜ =
F-M262S
490
333
3280, 1630, 1344 cmꢁ1
; MS (EI
CF34Ph
CF33Cl
CF33F
1000
>1000
>1000
1000
>1000
>1000
>100
>100
>100
>30
>30
>30
>30
>30
>30
70 eV) m/z 321.08 [M]+; Anal. calcd
for C16H13F2NO4: C 59.82, H 4.08, N
4.36, found: C 60.02, H 4.31, N
4.16.
3-(4-Phenylbenzylcarbamoyl)-2-
methoxybenzoic acid (F350):
white powder; yield 30% (0.110 g);
mp: 1988C (n-hexane); 1H NMR
(300 MHz, CDCl3): d=8.26 (dd, J=
7.7, 1.9 Hz, 1H), 8.11 (dd, J=7.8,
1.9 Hz, 1H), 7.61 (bs, 1H), 7.58 (m,
4H), 7.47 (m, 6H), 4.68 (m, 2H),
3.86 ppm (s, 3H); IR (KBr): n˜ =3288,
1638, 1344 cmꢁ1; MS (EI 70 eV) m/z
361.13 [M]+; Anal. calcd for
C22H19NO4: C 73.12, H 5.30, N 3.88,
found: C 73.37, H 5.42, N 4.08.
CN4Ph
T3F
1000
1000
>100
>30
>30
>30
>30
>1000
>1000
NT
[a] 3’-Processing. [b] Strand transfer.
The following N1,N3-dibenzyla-
mides
F328bis–F330bis
and
F350bis were obtained after the same reaction by using 2 equiv
reactants with respect to starting 2-methoxyisophthalic acid.
(300 MHz, CDCl3): d=8.20 (dd, 1H, J=7.7, 1.8 Hz), 8.11 (dd, J=7.8,
1.9 Hz, 1H), 7.77 (s, 1H), 7.41–7.27 (m, 2H), 7.18 (m, 2H), 4.56 (d,
J=5.3 Hz, 2H), 3.84 ppm (s, 3H); IR (KBr): n˜ =3291, 1639 cmꢁ1; MS
(EI 70 eV) m/z 353.06 [M]+; Anal. calcd for C16H13Cl2NO4: C 54.26, H
3.70, N 3.95, found: C 54.60, H 4.05, N 4.22.
N1,N2-Bis(4-chlorobenzyl)-2-methoxybenzene-1,3-diamide
(F328bis): white powder; yield 60% (0.270 g); mp: 1958C (PE);
1H NMR (300 MHz, [D6]acetone): d=8.30 (bs, 2H), 7.87 (m, 5H),
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N1,N2-Bis(4-phenylbenzyl)-2-methoxybenzene-1,3-diamide
(F350bis): brown solid; yield 20% (0.104 g); mp: 1938C (PE);
1H NMR (300 MHz, CD3OD): d=8.00(bs, 2H), 7.79–7.35 (m, 21H),
4.99 (s, 3H), 4.15 ppm (m, 4H); IR (KBr): n˜ =1690 cmꢁ1; MS (EI
70 eV) m/z 510.01 [M]+; Anal. calcd for C35H30N2O3: C 79.82, H 5.74,
N 5.32, found: C 80.02, H 5.94, N 5.32.
Methyl 6-(3-chlorobenzylcarbamoyl)pyridine-2-carboxylate (F-3):
yellow solid; yield 61% (0.102 g); mp: 1178C (cyclohexane);
1H NMR (300 MHz, [D6]acetone): d=9.30 (bs, 1H), 8.50–8.00 (m,
3H), 7.50–6.80 (m, 4H), 4.50 (s, 2H), 2.00 ppm (s, 3H); IR (KBr): n˜ =
1735, 1650 cmꢁ1; MS (EI 70 eV) m/z 304 [M]+; Anal. calcd for
C15H13ClN2O: C 59.12, H 4.30, N 9.19, found: C,59.32, H 4.61, N 8.89.
Method B. The preparation of N-(3-fluorobenzyl)-3-cyanobenza-
mide (CN3F) is described as a representative example. A solution
of 3-cyanobenzoic acid (0.150 g, 1.020 mmol) in SOCl2 (1.5 mL) and
DMF (60 mL) was stirred at reflux for 2 h. After this time, the SOCl2
was distilled off, and the crude solid was dissolved in dry toluene
(1.5 mL). To this solution,
a mixture of 3-fluorobenzylamine
(0.153 g, 1.224 mmol) and Et3N (0.105 g, 1.02 mmol) [in some in-
stances pyridine or diisopropylethylamine were used (see
Schemes 3 and 5)] in dry toluene (3 mL) was added, and the whole
was stirred at reflux overnight. The reaction was quenched with
H2O (1 mL) and concentrated, the aqueous phase was extracted
with Et2O, and the organic layer was dried and concentrated to
give a residue which was purified by flash chromatography (PE/
EtOAc, 2:1 as eluent); yellow solid; yield 53% (0.137 g); mp: 101–
1038C (cyclohexane); 1H NMR (300 MHz CDCl3): d=8.14–8.01 (m,
2H), 7.79 (dt, J=7.7, 1.2 Hz, 1H), 7.58 (1H, m), 7.40–7.25 (m, 1H),
6.94–7.17 (m, 3H), 6.86 (bs, 1H), 4.63 ppm (d, J=5.7 Hz, 2H); IR
(KBr): n˜ =2220, 1640, 1367 cmꢁ1; MS (EI 70 eV) m/z 254 [M]+; Anal.
calcd for C15H11FN2O C 70.86, H 4.36, N 11.02, found: C 71.00; H
4.30, N 11.32.
Figure 5. Effect of combination treatment of a) F332D with MMS, and
b) F332D with 5-FU. Cell survival was measured by MTT in the H630 cell line.
Cells were treated with a range of concentrations of MMS or 5-FU in the
presence or absence of a minimally effective concentration of F332D drug
(50 mm) for 72 h. Values are the average of four independent experiments
for MMS combinations and only one experiment for 5-FU combinations. All
concentration combinations were tested in triplicate for each experiment.
Two-tailed, paired student’s t-test was used to calculate the p values for the
difference in mean survival values between the combination treatment
group and MMS/5-FU only or F332D only treatment groups; *p<0.05 in
both cases.
N-(3-Chlorobenzyl)-3-cyanobenzamide (CN3Cl): beige powder;
yield 20% (0.055 g); mp: 1098C (cyclohexane); 1H NMR (300 MHz
CDCl3): d=8.06 (m, 2H), 7.73 (d, J=7.7 Hz, 1H), 7.51 (t, J=7.7 Hz,
1H), 7.28 (m, 4H), 6.65 (s, 1H), 4.54 ppm (d, J=5.7 Hz, 2H); IR
(KBr): n˜ =2236, 1639 cmꢁ1; MS (EI 70 eV) m/z 270 [M]+; Anal. calcd
for C15H11ClN2O: C 66.55, H 4.10, N 10.35, found: C 65.75, H 4.16, N
10.09.
N-(4-Phenylbenzyl)-3-cyanobenzamide (CN4Ph): light-yellow
powder; yield 19% (0.060 g); mp: 1648C (cyclohexane); 1H NMR
(300 MHz CDCl3): d=8.40 (s, 1H), 7.97 (dd, J=7.9, 1.0 Hz, 1H), 7.72
(dd, J=7.7, 1.0 Hz, 1H), 7.60–7.33 (m, 10H), 6.53 (s, 1H), 4.63 ppm
(d, J=5.5 Hz, 2H); IR (KBr): n˜ =2229, 1640 cmꢁ1; MS (EI 70 eV) m/z
312 [M]+; Anal. calcd for C21H16N2O: C 80.75, H 5.16, N 8.97, found:
C 80.97, H 5.50, N 9.09.
7.20 (m, 6H), 4.41(m, 4H), 3.79 ppm (s, 3H); IR (KBr): n˜ =1690 cmꢁ1
;
MS (EI 70 eV) m/z 442.09 [M]+; Anal. calcd for C23H20Cl2N2O3: C
62.31, H 4.55, N 6.32, found: C 61.99, H 4.60, N 6.52.
N1,N2-Bis(3-chlorobenzyl)-2-methoxybenzene-1,3-diamide
N-(3-Fluorobenzyl)-3-(trifluoromethyl)benzamide (CF33F): brown
oil; yield 50% (0.151 g); H NMR (300 MHz CDCl3): d=7.96 (s, 1H),
(F329bis): white powder; yield 60% (0.270 g); mp: 1978C (n-
1
1
hexane); H NMR (300 MHz, [D6]acetone): d=8.41 (bs, 2H), 8.00 (m,
7.86 (d, J=7.8 Hz, 1H), 7.64 (d, J=7.8 Hz, 1H), 7.41 (t, J=7.8 Hz,
1H), 7.27 (bs, 1H), 7.17 (s, 1H), 7.00–6.80 (m, 3H), 4.46 ppm (d, J=
5.8 Hz, 2H); IR (KBr): n˜ =1642, 1332 cmꢁ1; MS (EI 70 eV) m/z 297
[M]+; Anal. calcd for C15H11F4NO: C 60.61, H 3.73, N 4.71, found: C
60.87, H 4.03, N 5.01.
3H), 7.38 (m, 8H), 4.56 (d, J=5.8 Hz, 4H), 3.87 ppm (s, 3H); IR
(KBr): n˜ =1690 cmꢁ1; MS (EI 70 eV) m/z 442.09 [M]+; Anal. calcd for
C23H20Cl2N2O3: C 62.31, H 4.55, N 6.32, found: C 62.51, H 4.60, N
6.52.
N1,N2-Bis(3,4-dichlorobenzyl)-2-methoxybenzene-1,3-diamide
(F330bis): white powder; yield 30% (0.156 g); mp: 1878C (PE); IR
1H NMR (300 MHz, [D6]acetone): d=8.51 (bs, 2H), 8.03 (m, 4H),
7.68–7.22 (m, 5H), 4.56 (d, J=5.4 Hz, 4H), 3.79 ppm (s, 3H); (KBr):
n˜ =1690 cmꢁ1; MS (EI 70 eV) m/z 510.01 [M]+; Anal. calcd for
C23H18Cl4N2O3: C 53.93, H 3.54, N 5.47, found: C 53.92, H 3.68, N
5.27.
N-(3-Chlorobenzyl)-3-(trifluoromethyl)benzamide (CF33Cl): yellow
oil; yield 70% (0.223 g); H NMR (300 MHz CDCl3): d=8.06 (s, 1H);
1
7.94 (d, J=7.8 Hz, 1H), 7.83 (m, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.45 (t,
J=7.8 Hz, 1H), 7.26–7.08 (m, 4H), 4.48 ppm (d, J=5.8 Hz, 2H); IR
(KBr): n˜ =1641, 1334 cmꢁ1; MS (EI 70 eV) m/z 313 [M]+; Anal. calcd
for C15H11 ClF3NO: C 57.43, H 3.53, N 4.46, found: C 57.68, H 3.35, N
4.59.
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3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors
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Figure 6. a) Effect of combination treatment of F332D with a) MMS, b) doxorubicin, c) oxaliplatin, and d) camptothecin. DNA-damage-inducing agents’ effects
on cell survival was measured by MTT in the H630 cell line. Cells were treated with a range of concentrations of MMS (or DNA damaging drug) in the pres-
ence or absence of a minimally effective concentration of F332D (50 mm) for 72 h. Values are the average of four independent experiments for MMS. All con-
centration combinations were tested in triplicate for each experiment. Two-tailed, paired student’s t-test was used to calculate the p values for the difference
in mean survival values between the combination treatment group and MMS only or F332D only treatment groups; *p<0.05 in both cases.
Figure 7. Effect of combination treatment of a) F328D with 5-FdUrd, b) F330 with 5-FdUrd, and c) F332D with 5-FdUrd. Cell survival was measured by colony-
formation assay in the HT-29 colon cancer cell line. Cells were pretreated with a single concentration of 5-FdUrd (0.1 mm) for 24 h followed by the addition of
indicated inhibitor (50 mm) for an additional 24 h. Drug-containing medium was then replaced with fresh media, and colonies were allowed to form. Percent
survival indicates fraction of surviving colonies relative to untreated control.
N-(4-Phenylbenzyl)-3-(trifluoromethyl)benzamide (CF34Ph): white
powder; yield 63% (0.201 g); mp: 1178C (cyclohexane); 1H NMR
(300 MHz CDCl3): d=8.05 (s, 1H), 7.93 (d, J=7.7 Hz, 1H), 7.65 (d,
J=7.4 Hz, 1H), 7.60–7.15 (m, 10H), 6.63 (bs, 1H), 4.60 ppm (d, J=
5.4 Hz, 2H); IR (KBr): n˜ =1640, 1330 cmꢁ1; MS (EI 70 eV) m/z 313
[M]+; Anal. calcd for C21H16F3NO: C 70.98, H 4.54, N 3.94, found: C
70.73, H 4.39, N 3.89.
3-{[1-(4-Chlorophenyl)-3-methoxy-3-oxopropyl]carbamoyl}-2-me-
thoxybenzoic acid (F-M260): light-yellow oil; yield 23%
(0.091 g>); 1H NMR (300 MHz CDCl3): d=8.39 (d, J=8.0 Hz, 1H),
8.06 (d, J=7.7 Hz, 1H), 7.30 (m, 5H), 5.63 (m, 1H), 3.80–3.60 (m,
6H), 3.05–2.85 ppm (m, 2H); IR (KBr): n˜ =3400, 1687 cmꢁ1; MS (EI
70 eV) m/z 391 [M]+; Anal. calcd for C19H18ClNO6: C 58.24, H 4.63, N
3.57, found: C 58.36, H 4.40, N 3.37.
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F. Aiello et al.
MED
3-{[1-(4-Fluorophenyl)-3-methoxy-3-oxopropyl]carbamoyl}-2-me-
thoxybenzoic acid (F-M262): colorless oil; yield 44% (0.168 g);
1H NMR (300 MHz CDCl3): d=8.35 (d, J=8.1 Hz, 1H), 8.06 (d, J=
7.7 Hz, 1H), 7.35 (m, 2H), 7.27 (t, J=7.7 Hz, 1H), 7.03 (m, 2H), 5.64
(dd, J=14.0, 6.1 Hz, 1H), 3.80–3.60 (m, 6H), 3.80–2.87 ppm (m,
2H); IR (KBr): n˜ =3450, 1691 cmꢁ1; MS (EI 70 eV) m/z 375 [M]+;
Anal. calcd for C19H18FNO6: C 60.80, H 4.83, N 3.73, found: C 61.00,
H 4.88, N 3.56.
30 min. The yellow solid formed was filtered, washed with H2O,
and purified by ion-exchange chromatography (DOWEXꢁ 200), to
give a light-yellow solid; yield 60% (0.011 g); mp: 1968C (EtOAc/
1
PE); H NMR (300 MHz [D6]acetone): d=8.66 (bs, 1H), 8.34 (dd, J=
7.8, 1.8 Hz, 1H), 8.12 (dd, J=6.0, 1.8 Hz, 1H), 7.26 (t, J=7.6 Hz, 1H),
7.11 (t, J=7.8 Hz, 1H), 6.90 (m, 3H), 4.66 (d, J=5.8 Hz, 2H),
3.78 ppm (s, 3H); IR (KBr): n˜ =3280, 1644 cmꢁ1; MS (EI 70 eV) m/z
302 [M]+; Anal. calcd for C16H15NO5: C 63.78, H 5.02, N, 4.65, found:
C 63.68, H 5.12, N, 4.90.
N-(3-Fluorobenzyl)-3-(1H-tetrazol-5-yl)benzamide (T3F): amor-
1
phous solid; yield 3% (0.009 g); H NMR (300 MHz CDCl3): d=8.65
3-(3,4-Dihydroxybenzylcarbamoyl)-2-hydroxybenzoic
acid
(bs, 1H), 8.25 (bs, 1H), 8.00 (m, 1H), 7.70 (t, J=7.7 Hz, 1H), 7.59 (t,
J=7.9 Hz, 1H), 7.35 (m, 1H), 7.20–6.95 (m, 2H), 6.80–6.75 (m, 2H),
4.65 ppm (d, J=5.6 Hz, 2H); IR (KBr): n˜ =1693 cmꢁ1; MS (EI 70 eV)
m/z 297 [M]+; Anal. calcd for C15H12FN5O: C 60.60, H 4.07, N 23.56,
found: C 60.78, H 4.02, N 23.32.
(F327D): light-yellow solid; yield 60% (0.012 g); mp: 2058C
(EtOAc); H NMR (300 MHz [D6]acetone): d=9.10 (bs, 1H), 8.51 (s,
1H), 8.26 (d, J=7.7 Hz, 1H), 8.08 (m, 1H), 7.95 (d, J=7.7 Hz, 1H),
7.30 (m, 1H),6.92 (m, 2H), 4.55 (s, 2H), 3.91 ppm (s, 6H); IR (KBr):
n˜ =3288, 1639 cmꢁ1; MS (EI 70 eV) m/z 331 [M]+; Anal. calcd for
C17H17NO6: C 61.63, H 5.17, N 4.23, found: C 61.45, H 5.25, N 4.37.
1
6-(3-Chlorobenzylcarbamoyl)pyridine-2-carboxylic acid (F-1):
1
brown solid; yield 42% (0.124 g); mp: 2908C (dec.) (Et2O); H NMR
3-{[2-Carboxy-1-(4-chlorophenyl)ethyl]carbamoyl}-2-hydroxyben-
zoic acid (F260D): orange powder; yield 98% (0.022 g); mp: 122–
1258C (EtOAc); 1H NMR (300 MHz CD3OD): d=8.10 (d, J=7.8 Hz,
2H), 7.43 (m, 4H), 7.05 (t, J=7.8 Hz, 1H), 5.62 (t, J=7.1 Hz, 1H),
2.90 ppm (m, 2H); IR (KBr): n˜ =3710, 1678 cmꢁ1; MS (EI 70 eV) m/z
363 [M]+; Anal. calcd for C17H14ClNO6: C 58.24, H 4.63, N 3.57,
found: C 58.54, H 4.99, N 3.41.
(300 MHz DMSO): d=11.00 (bs, 2H), 8.30–7.70 (m, 3H), 7.60–7.20
(m, 4H), 4.50 ppm (s, 2H); IR (KBr): n˜ =1710, 1690 cmꢁ1; MS (EI
70 eV) m/z 290 [M]+; Anal. calcd for C14H11ClN2O3: C 57.84, H 3.81,
N 9.64, found: C 57.56, H 3.83, N 10.01.
6-(3-Fluorobenzylcarbamoyl)pyridine-2-carboxylic acid (F-2):
white solid; yield 22% (0.065 g); mp: 1568C (dec.) (MeOH/Et2O),
1H NMR (300 MHz CDCl3): d=9.00 (bs, 1H), 8.90 (s, 1H), 8.50 (m,
1H), 8.10 (m, 1H), 7.70 (t, J=7.7 Hz, 1H), 7.50–7.00 (m, 4H),
4.70 ppm (m, 2H); IR (KBr): n˜ =1710, 1650 cmꢁ1; MS (EI 70 eV) m/z
290 [M]+; Anal. calcd for C14H11FN2O3: C 57.84, H 3.81;N, 9.64,
found: C 58.00, H 3.71, N 9.65.
3-{[2-Carboxy-1-(4-fluorophenyl)ethyl]carbamoyl}-2-hydroxyben-
zoic acid (F262D): brown powder; yield 61% (0.013 g); mp: 98–
1038C (dichloromethane); 1H NMR (300 MHz CD3OD): d=7.92 (d,
J=7.8 Hz, 2H), 7.35 (m, 3H), 7.05 (t, J=7.8 Hz, 1H), 6.90 (m, 2H),
5.55 (t, J=6.9 Hz, 1H), 2.80 ppm (m, 2H); IR (KBr): n˜ =3700,
1688 cmꢁ1; MS (EI 70 eV) m/z 363 [M]+; Anal. calcd for C17H14FNO6:
C 60.80, H 4.83, N 3.73;, found: C 60.89, H 4.63, N 3.91.
N1,N2-Bis(3-chlorobenzylcarbamoyl)pyridine-2-carboxylic
acid
(F-4): brown oil, yield 16% (0.067 g); 1H NMR (300 MHz CD3OD):
d=9.03 (bs, 2H), 7.70–7.60 (d, J=7.6 Hz, 2H), 7.53–7.40 (t, J=
7.0 Hz, 1H), 6.90–6.40 (m, 8H), 4.30 ppm (s, 4H); IR (KBr): n˜ =
Method D. The preparation of 3-(3,4-difluorobenzylcarbamoyl)-2-
hydroxybenzoic acid (F333D) is described as a representative ex-
ample. HBr (1 mL) was added to a solution of 3-(3,4-difluorobenzyl-
carbamoyl)-2-methoxybenzoic acid (0.090 g, 0.300 mmol) in acetic
acid (0.7 mL). The mixture was stirred at 1208C for 2 h; H2O
(1.5 mL) was then added, and the mixture was left to cool to room
temperature to give a grey solid, which was filtered and thorough-
ly washed with H2O; yield 30% (0.029 g); mp: 2258C (MeOH);
1H NMR (300 MHz DMSO): d=11.01 (bs, 1H), 9.08 (bs, 1H), 8.00
(dd, J=16.9, 7.3 Hz, 2H), 7.39 (m, 2H), 7.22 (s, 1H), 7.02 (t, J=
7.5 Hz, 1H), 4.50 ppm (d, J=5.1 Hz, 2H); IR (KBr): n˜ =3290,
1641 cmꢁ1; MS (EI 70 eV) m/z 322 [M]+; Anal. calcd for C15H11F2NO4:
C 58.64, H 3.61, N 4.56, found: C 58.46; H 3.55, N, 4.72.
1690 cmꢁ1
;
MS (EI 70 eV) m/z 414 [M]+; Anal. calcd for
C21H17Cl2N3O2: C 60.88, H 4.14, N 10.14, found: C 61.08, H 4.21, N
9.96.
N-(4-Chlorobenzyl)-8-hydroxyquinoline-2-carboxamide
(F-5):
brown oil; yield 3% (0.004 g); 1H NMR (300 MHz CDCl3): d=
10.01(bs, 1H), 8.43 (bs, 1H), 7.85 (d, J=8.6 Hz, 1H), 7.70 (d, J=
8.5 Hz, 1H), 7.60–7.20 (m, 7H), 4.80 ppm (s, 2H), IR (KBr): n˜ =
1710 cmꢁ1
;
MS (EI 70 eV) m/z 312 [M]+; Anal. calcd for
C17H13ClN2O2: C 65.29, H 4.19, N 8.96, found: C 65.19, H 4.39, N
8.77.
N-(3-Methoxybenzyl)-8-hydroxyquinoline-2-carboxamide (F-6):
brown solid; yield 30% (0.126 g); mp: 1228C (EtOAc); 1H NMR
(300 MHz CDCl3): d=8.99 (bs, 1H), 8.50 (bs, 1H), 8.20 (d, J=8.5 Hz,
1H), 8.10 (d, J=8.5 Hz, 1H), 7.45 (t, J=8.2 Hz, 1H), 7.28 (d, J=
8.2 Hz, 1H), 7.08 (d, J=7.6 Hz, 1H), 6.99 (t, J=8.0 Hz, 1H), 6.75 (m,
2H), 6.55 (d, J=8.2 Hz, 1H), 4.50 (s, 2H), 3.60 ppm (s, 3H), IR (KBr):
3-(4-Chlorobenzylcarbamoyl)-2-hydroxybenzoic acid (F328D):
grey powder; yield 42% (0.038 g); mp: 2488C (MeOH); 1H NMR
(300 MHz DMSO): d=8.95 (bs, 1H), 7.95 (m, 2H), 7.33 (m, 4H), 6.95
(t, J=7.8 Hz, 1H), 4,45 ppm (d, J=5.6 Hz, 2H); IR (KBr): n˜ =3291,
1649 cmꢁ1; MS (EI 70 eV) m/z 305 [M]+; Anal. calcd for C15H12ClNO4:
C 58.93, H 3.96, N 4.58, found: C 59.03, H 3.71, N 4.45.
n˜ =1710 cmꢁ1 MS (EI 70 eV) m/z 414 [M]+; Anal. calcd for
;
C18H16N2O3: C 70.12, H 5.23, N 9.09, found: C 69.98, H 4.99, N 9.07.
3-(4-Phenylbenzylcarbamoyl)-2-hydroxybenzoic acid (F350D):
grey powder; yield 30% (0.031 g>); mp: 2608C (EtOH); 1H NMR
(300 MHz DMSO): d=9.11 (s, 1H), 8.10–7.91 (m, 2H), 7.68–7.35 (m,
9H), 7.11 (s, 1H), 4.59 ppm (s, 2H); IR (KBr): n˜ =3299, 1641 cmꢁ1
;
General procedure for the demethylation reaction
MS (EI 70 eV) m/z 347 [M]+; Anal. calcd for C21H17NO4: C 72.61, H
Method C. The preparation of 3-(3-methoxybenzylcarbamoyl)-2-hy-
droxybenzoic acid (F326D) is described as a representative exam-
ple. A mixture of 3-(3-methoxybenzylcarbamoyl)-2-methoxybenzoic
acid (0.020 g, 0.063 mmol), 2,4,6-thrimethylpyridine (collidine;
0.193 mL), and LiI (0.030 g, 0.224 mmol) was stirred 2 h at 1808C.
3n HCl (1.48 mL) and H2O (1 mL) were added to the ice-cooled so-
lution, which was then left at room temperature for an additional
4.93, N 4.03, found: C 72.85, H 5.00, N 4.27.
N1,N2-Bis(4-chlorobenzyl)-2-hydroxybenzene-1,3-diamide
(F328bisD): grey powder; yield 30% (0.038 g); mp: 2158C (MeCN);
1H NMR (300 MHz [D6]acetone): d=13,10 (bs, 1H), 8.71 (s, 2H), 8.29
(dd, J=7.7, 1.7 Hz, 1H), 8.12 (m, 5H), 7.99 (d, J=7.7 Hz, 1H), 7.35
(m, 2H), 7.10 (m, 2H), 4.60 ppm (m, 4H); IR (KBr): n˜ =3450,
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3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors
1645 cmꢁ1 MS (EI 70 eV) m/z 429 [M]+; Anal. calcd for
MED
;
was concentrated to give a white solid: yield 52% (0.013 g>); mp
1028C (EtOAc); 1H NMR (300 MHz CD3OD): d=7.90 (m, 2H), 7.50
(m, 2H), 7.05 (m, 1H), 7.00 (m, 3H), 5.75 (m, 1H), 4.90 (bs, 2H), 3.85
(s, 3H), 3.25–2.90 ppm (m, 2H); IR (KBr): n˜ =3840, 1698 cmꢁ1; MS
(EI 70 eV) m/z 391 [M]+; Anal. calcd for C17H16ClNO6: C 58.24, H
4.63, N 3.57, found: C 57.94, H 4.86, N 3.89.
C22H18Cl2N2O3: C 61.55, H 4.23, N 6.53, found: C 61.29, H 4.54, N
6.23.
N1,N2-Bis(3-chlorobenzyl)-2-hydroxybenzene-1,3-diamide
(F329bisD): grey powder; yield 33% (0.042 g>); mp: 2228C (n-
1
hexane); H NMR (300 MHz [D6]acetone): d=13.01 (bs, 1H), 8.63 (s,
3-{[2-Carboxy-1-(4-fluorophenyl)ethyl]carbamoyl}-2-methoxy-
benzoic acid (F-M262S): following the same procedure described
for compound F-M260S, compound F-M262S was obtained as
a white solid; yield 46% (0.011 g); mp 1088C (EtOAc); 1H NMR
(300 MHz CD3OD): d=8.00 (m, 1H), 7.58 (d, 1H, J=7.6 Hz), 7.30 (m,
2H), 7.10 (t, 1H, J=7.7 Hz), 6.95 (t, 2H, J=8.5 Hz), 5.44 (t, J=
6.9 Hz, 1H), 3.85 (s, 3H), 3.52 (s, 3H), 2.80 ppm (m, 2H); IR (KBr):
n˜ =3850, 1690 cmꢁ1; MS (EI 70 eV) m/z 375 [M]+; Anal. calcd for
C17H16FNO6: C 60.80, H 4.83, N 3.73, found: C 60.98, H 5.00, N 3.89.
2H), 8.21 (d, J=7.7 Hz, 2H), 7.95 (m, 5H), 7.20 (m, 2H), 6.97 (m,
2H), 4.58 ppm (s, 4H); IR (KBr): n˜ =3455, 1675 cmꢁ1; MS (EI 70 eV)
m/z 429 [M]+; Anal. calcd for C22H18Cl2N2O3: C 61.55, H 4.23, N 6.53,
found: C 61.29, H 4.54, N 6.23.
N1,N2-Bis(3,4-dichlorobenzyl)-2-hydroxybenzene-1,3-diamide
(F330bisD): grey powder; yield 40% (0.059 g); mp: 2448C (EtOH);
1H NMR (300 MHz [D6]acetone): d=13,01 (bs, 1H), 8.90 (bs, 2H),
8.71 (s, 1H), 8.30 (m, 2H), 7.90–7.00 (m, 6H), 4.55 ppm (m, 4H); IR
(KBr): n˜ =3475, 1657 cmꢁ1; MS (EI 70 eV) m/z 497 [M]+; Anal. calcd
for C22H16Cl4N2O3: C 53.04, H 3.24, N 5.62, found: C 53.05, H 3.44, N
6.00.
Biology
N1,N2-Bis(4-phenylbenzyl)-2-hydroxybenzene-1,3-diamide
(F350bisD): brown solid; yield 40% (0.061 g); mp: 2288C (MeCN);
1H NMR (300 MHz [D6]acetone): d=13.21 (s, 1H), 8.71 (s, 2H), 8.21–
6.99 (m, 21H), 5.01–4.35 ppm (m, 4H); IR (KBr): n˜ =3761,
1699 cmꢁ1; MS (EI 70 eV) m/z 512 [M]+; Anal. calcd for C34H28N2O3:
C 79.67, H 5.51, N 5.46, found: C 80.00, H 5.78, N 5.26.
Preparation of oligonucleotide substrates: APE1 substrate oligonu-
cleotides in the forward direction labeled as THFtop with the se-
quence 5’-ATT TCA CCG GTA CG(F) TCT AGA ATC CG-3’ comple-
mented the THFbot reverse strand, 3’-TA AAG TGG CCA TGC (C)AG
ATC TTA GGC-5’. HIV-1 IN substrate oligonucleotides in the forward
direction labeled as 21top with the sequence 5’-GTG TGG AAA
ATC TCT AGC AGT-3’ complemented the 21bot reverse strand, 5’-
ACT GCT AGA GAT TTT CCA CAC-3’. All oligonucleotides were pur-
chased from Norris Cancer Center Microsequencing Core Facility
(University of Southern California) and purified by UV shadowing
on polyacrylamide gel. To analyze the extent of APE1 and IN enzy-
matic activity, both top strands were radioactively labeled at the
5’-end using T4 polynucleotide kinase (Epicentre, Madison, WI) and
g-[32P]ATP (ARC Inc.). The kinase was heat-inactivated, and 21bot
was added in 1.5-molar excess. The mixture was heated at 958C,
and then allowed to cool slowly to room temperature. The newly
annealed oligonucleotides were purified through a spin 25 mini-
column (USA Scientific) to separate annealed double-stranded oli-
gonucleotide from unincorporated material.
Method E. The preparation of N-(3-hydroxybenzyl)-8-hydroxyqui-
noline-2-carboxamide (F-7) is described as a representative exam-
ple. 1n BBr3 (0.260 mL, 0.260 mmol) was added to a solution of N-
(3-methoxybenzyl)-8-hydroxyquinoline-2-carboxamide
(0.025 g,
0.086 mmol) in dry CH2Cl2 (0.17 mL) cooled to 08C. The mixture
was stirred at room temperature for 24 h under nitrogen and then
diluted with H2O (5 mL) and extracted with EtOAc (3ꢂ10 mL). The
dried organic phases were concentrated, and the crude residue
was purified by flash chromatography using n-hexane/EtOAc
(2:1.5) as eluent, to give a yellow amorphous solid; yield 18%
(0.004 g); 1H NMR (300 MHz [D6]acetone): d=9.51 (bs, 2H), 9.03
(bs, 1H), 8.43 (d, J=8.5 Hz, 1H), 8.24 (m, 1H), 7.45 (m, 2H), 7.10
(m, 2H), 6.75 (m, 2H), 6.63 (m, 1H), 4.54 ppm (d, J=6.4 Hz, 2H); IR
(KBr): n˜ =3600, 1710 cmꢁ1; MS (EI 70 eV) m/z 295 [M]+; Anal. calcd
for C17H14N2O3: C 69.38, H 4.79, N 9.52, found: C 69.28, H 4.68, N
9.82.
In vitro APE1 assay: The in vitro APE1 assay was performed as previ-
ously described.[15a] The extent of endonuclease activity of APE1
was determined by diluting the test compounds in DMSO and in-
cubating the diluted compounds with recombinant APE1 at a final
concentration of 0.05 nm in reaction buffer (50 mm NaCl, 1 mm
HEPES, pH 7.5, 50 mm EDTA, 50 mm dithiothreitol, 10% glycerol (w/
v), 7.5 mm MgCl2, 0.1 mgmLꢁ1 bovine serum albumin (BSA), 10 mm
b-mercaptoethanol, 10% DMSO, and 25 mm MOPS, pH 7.2) at 378C
for 30 min. Thereafter, 5’-end 32P-labeled linear oligonucleotide
substrate containing the AP site (200 nm) was added and incubat-
ed for an additional 15 min. Reactions were quenched with the ad-
dition of an equal volume (8 mL) of loading dye (98% deionized
formamide, 10 mm EDTA, 0.025% xylene cyanol, and 0.025% bro-
mophenol blue). A 10 mL aliquot was electrophoresed on a denatur-
ing 20% polyacrylamide gel. The gels were dried and exposed in
a PhosphorImager cassette and analyzed using a Typhoon 8610
Variable Mode Imager (Amersham Biosciences) and quantified with
ImageQuant 5.2 software.
3-(3-Hydroxybenzylcarbamoyl)-2-hydroxybenzoic acid (F326DD):
grey powder; yield 60% (0.010 g); mp: 2078C (EtOH); 1H NMR
(300 MHz [D6]acetone): d=13.21 (bs, 2H), 9.01 (bs, 1H), 8.78 (s,
1H), 8.45–8.11 (m, 2H), 7.62–6.69 (m, 5H), 4.88–4.51 ppm (m, 2H);
IR (KBr): n˜ =3287, 1640 cmꢁ1; MS (EI 70 eV) m/z 287 [M]+; Anal.
calcd for C15H13NO5: C 62.72, H 4.56, N 4.88, found: C 62.92, H 4.37,
N 4.98.
3-(3,4-Dihydroxybenzylcarbamoyl)-2-hydroxybenzoic
acid
(F327DD): brown oil; yield 45% (0.008 g); 1H NMR (300 MHz
[D6]acetone): d=13.00 (bs, 3H), 10.02 (bs, 1H), 8.99 (bs, 1H), 8.05
(m, 1H), 7.00 (m, 1H), 6.82 (m, 1H), 6.70 (m, 3H), 4.90 ppm (s, 2H);
IR (KBr): n˜ =3280, 1629 cmꢁ1; MS (EI 70 eV) m/z 303 [M]+; Anal.
calcd for C15H13NO6: C 59.41, H 4.32, N 4.62, found: C 59.04, H 4.22,
N 4.75.
3-{[2-Carboxy-1-(4-chlorophenyl)ethyl]carbamoyl}-2-methoxy-
In vitro HIV-1 IN assay: To determine the extent of 3’-processing
and strand transfer, wild-type recombinant IN was pre-incubated at
a final concentration of 200 nm with the inhibitor in reaction buffer
(50 mm NaCl, 1 mm HEPES, pH 7.5, 50 mm EDTA, 50 mm dithiothrei-
tol, 10% glycerol (w/v), 7.5 mm MnCl2, 0.1 mgmLꢁ1 BSA, 10 mm b-
mercaptoethanol, 10% DMSO, and 25 mm MOPS, pH 7.2) at 308C
for 30 min. The 5’-end 32P-labeled linear oligonucleotide substrate
benzoic acid (F-M260S):
a mixture of LiOH·H2O (0.032 g,
0.075 mmol) and compound F-M260 (0.024 g, 0.063 mmol) in THF/
MeOH/H2O (2:1:1), was stirred at room temperature for 18 h, then
the volatiles were removed, and the crude residue was partitioned
between Et2O and H2O. The inorganic layer was made acidic with
3n HCl (pH 2) and extracted twice with Et2O. The organic layer
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F. Aiello et al.
MED
(20 nm) was then added, and incubation was continued for an ad-
ditional 1 h. Reactions were then quenched by the addition of an
equal volume (16 mL) of loading dye (98% deionized formamide,
10 mm EDTA, 0.025% xylene cyanol, and 0.025% bromophenol
blue). An aliquot (5–10 mL) was electrophoresed on denaturing
20% polyacrylamide gels (0.09m Tris-borate pH 8.3, 2 mm EDTA,
20% acrylamide, 8m urea). Gels were dried, exposed in a Phosphor-
Imager cassette, and analyzed using a Typhoon 8610 Variable
Mode Imager (Amersham Biosciences) and quantified using Image-
Quant 5.2 software.
was dialyzed in buffer containing 20 mm HEPES, pH 7.5, 500 mm
NaCl, 40% glycerol, 0.2 mm EDTA, and 1 mm dithiothreitol (DTT).
After dialysis, the purified enzyme solution contained 50 mm NaCl,
1 mm HEPES, pH 7.5, 50 mm EDTA, 50 mm DTT, and 10% glycerol
(w/v). Aliquots of the protein were then stored at ꢁ808C and re-
moved when necessary for use.
Cell viability assay: Cell viability was assessed by a 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as
previously described.[22] Cells were seeded in 96-well tissue culture
treated dishes at a density of 4000 cells per well and allowed to
attach overnight. Cells were subsequently treated with continuous
exposure to a range of concentrations of drugs for 72 h. At the
end of exposure, MTT solution was added to each well at a final
concentration of 0.3 mgmLꢁ1 MTT and further incubated for 3–4 h
at 378C. After removal of the supernatant, DMSO was added, and
the absorbance of the solubilized dye was read at l 570 nm. Per-
cent cytotoxicity for each drug concentration was determined
using the following formula: cytotoxicity=100ꢁ(100ꢂ[Abs(drug)/Ab-
s(control)]). Where possible, IC50 values were determined from the plot
of percent cytotoxicity versus the logarithm of drug concentration.
Quantification of APE1 and IN inhibition: Percent inhibition (%I) was
calculated with the equation: %I=100ꢂ[1ꢁ(DꢁC)/(NꢁC)], for
which C, N, and D are the fractions of 26mer substrate cleaved to
13mer products by APE1 for DNA alone, DNA plus APE1, and DNA
plus APE1 plus the test compound, respectively. For IN inhibition,
C, N, and D are the fractions of 21mer substrate converted into
19mer (3’-processing product) or strand-transfer products for DNA
alone, DNA plus IN, and IN plus drug, respectively. The IC50 values
were determined by plotting the logarithm of drug concentration
versus percent inhibition to obtain the concentration that pro-
duced 50% inhibition.
Colony formation assay: The colony-forming capacity of H630 cells
in response to drug treatment was determined by plating cells at
a density of 100 cells per well in 96-well plates or 300 cells per
well in 12-well plates. Cells were allowed to attach overnight, fol-
lowing which drug was added to the media in each well and then
incubated for 7–14 days. At the end of treatment, cells were
washed once with PBS, stained with crystal violet for 30 min
(0.25% crystal violet, 3.7% formaldehyde, 80% MeOH), washed
with deionized water, and left to dry overnight. Plates were
scanned in the Odyssey Infrared Imaging System (Licor, Lincoln,
NE, USA) using the 700 channel laser. Cytotoxicities were calculated
as a ratio of control as described above.
Cell culture: H630 and HT-29 colon cancer cells were obtained from
the National Cancer Institute (Bethesda, MD, USA) and were main-
tained as adherent monolayer cultures in Dulbecco’s modified
Eagle’s medium (DMEM) and RPMI-1640, respectively, supplement-
ed with 10% fetal bovine serum (FBS; Gemini-Bioproducts, West
Sacramento, CA, USA). Cells were grown at 378C in a humidified
atmosphere of 5% CO2. For all experiments, cells in exponential
growth phase were washed with PBS, briefly trypsinized in a small
volume of 0.25% trypsin–EDTA solution (Sigma–Aldrich, St. Louis,
MO, USA), resuspended in culture media, and centrifuged at
1200 rpm (240 g) for 5 min. Pelleted cells were resuspended in
complete growth medium, counted and plated in sterile plates
and allowed to adhere overnight before treating.
Keywords: anticancer agents
acids · inhibitors · synthesis
· APE1 · carbamoylbenzoic
Purification of recombinant, His-tagged APE1: His-tagged APE1 was
purified from E. coli M15 cells (Qiagen, Valencia, CA, USA) as previ-
ously described.[15a] Briefly, a pellet of bacterial cells from an over-
night culture was resuspended in TFB1 buffer (100 mm RbCl,
50 mm MnCl2, 30 mm potassium acetate, 10 mm CaCl2, 15% glycer-
ol) and incubated on ice for 90 min. The cells were then recollect-
ed by centrifugation at 4000 g for 5 min at 48C and then resus-
pended in TFB2 buffer (10 mm MOPS, 10 mm RbCl, 75 mm CaCl2,
15% glycerol). An aliquot of these cells were transformed with
a Qiagen pQE30 plasmid containing the APE1 gene sequence and
ampicillin resistance. The APE1 plasmid was expressed in the M15
expression strain after induction by IPTG (1 mm) at an absorbance
of 0.6–0.8 optical density at 595 nm. The culture was allowed to
grow for an additional 3–4 h at 378C. This was followed by centri-
fugation of the cells at 3000 rpm in a bucket rotor centrifuge
(Beckman) for 20 min. Pelleted cells were resuspended in lysis
buffer (20 mm HEPES, pH 7.5, 5 mm imidazole, 100 mm NaCl) and
passed twice through a French press (Thermo Spectronic, Madison,
WI, USA). The lysate was centrifuged at 31000 g, and the pellet was
solubilized in a buffer containing 20 mm HEPES, pH 7.5, 5 mm imi-
dazole, and 10 mm CHAPS. Recombinant APE1 protein was purified
using Ni-affinity chromatography with Swell-gel Nickel-chelated
discs (Pierce, Rockford, IL, USA). The protein was eluted from the
column with increasing concentrations of imidazole from 40 mm to
1m. An aliquot of each concentration post-elution was run on an
SDS-PAGE gel, and fractions containing protein were dialyzed in
Spectra/Por molecular porous membrane tubing, MWCO: 12–
14 kDa (Spectrum Laboratories Inc., Houston, TX, USA). The protein
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Received: July 4, 2012
Published online on && &&, 0000
ChemMedChem 0000, 00, 1 – 16
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www.chemmedchem.org
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FULL PAPERS
F. Aiello,* Y. Shabaik, A. Esqueda,
T. W. Sanchez, F. Grande, A. Garofalo,
N. Neamati
Cancer in disrepair: The base-excision
repair (BER) pathway is the foremost
pathway responsible for removal and re-
placement of damaged DNA bases. Sev-
eral BER enzymes have altered expres-
sion and activation in cancer cells, and
among them APE1 has emerged as
a particularly attractive target for anti-
cancer drug development, as demon-
strated by studies that link its overex-
pression with resistance to radio- and
chemotherapy.
&& – &&
Design and Synthesis of 3-
Carbamoylbenzoic Acid Derivatives as
Inhibitors of Human Apurinic/
Apyrimidinic Endonuclease 1 (APE1)
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 16
ÝÝ
These are not the final page numbers!