DOI: 10.1002/cmdc.201200334

Design and Synthesis of 3-Carbamoylbenzoic AcidDerivatives as Inhibitors of Human Apurinic/ApyrimidinicEndonuclease 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]

Introduction

DNA bases are susceptible to damaging modifications inflictedby various endogenous and exogenous sources, which, whenacted 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 ofthe source of damage, all abasic sites, which must be repairedin order for cells to survive, are repaired by the base-excisionrepair (BER) pathway. Apurinic/apyrimidinic endonuclease(APE1) has the important role of recognizing AP sites in thegenome and incising the DNA backbone immediately 5’ to theabasic sites, producing a 3’-hydroxy and a 5’-abasic deoxyri-bose phosphate, thereby initiating their repair by an ensuingsuccession of BER enzymes. The outstanding majority of abasicsites are repaired by what is known as the short-patch orsingle-nucleotide BER (SP/SN-BER), whereby a single nucleotideis replaced, following APE1 incision, by the actions of DNApolymerase (pol) b and DNA ligase III. However, there is alsoa second, less prevalent arm of BER: long-patch BER (LP-BER)involves replacement of the damaged nucleotide with a stretchof 2–10 newly synthesized nucleotides, carried out by DNApol 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 repairprotein responsible for and capable of processing AP sites inboth the SP/SN and LP arms of BER, thereby enabling DNArepair.[2] While the endonuclease function is the predominantrole for APE1 in cells, it also exhibits weaker 3’-phosphodiester-ase and 3’!5’ exonuclease activities. In addition to its role asa DNA repair enzyme, APE1 also functions as a reduction–oxi-dation factor, owing to an active cysteine residue located ina 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 nuclearfactor 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 BERpathway 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 hasemerged as an attractive therapeutic target for anticancerdrug development, as demonstrated by studies that link itsoverexpression with resistance to radio- and chemotherapy.[6]

Although APE1 is considered to be a ubiquitously expressedprotein with an estimated 350 000–7 000 000 copies per cell, itsexpression in cancer cells has been shown to be even higherthan surrounding tissue.[7] Additionally, siRNA knockdown ofAPE1 in cancer cells, which has the effect of potentiating thecell-killing ability of cytotoxic agents, has further confirmed itsprotective role in cancer against a variety of DNA damagingagents.[8] Therapeutics that target APE1 could decrease onco-genic cell advantage to evade apoptosis and sensitize cancersto both radiation and chemotherapy.[9] For instance, radio-re-sistance in glioma cell lines, which is associated with higher ex-

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 thiswork, we designed and synthesized different classes of small-molecule inhibitors of the catalytic endonuclease function of

APE1 that contain a 3-carbamoylbenzoic acid scaffold. Furtherstructural modifications were made with the aim of increasingthe activity and cytotoxicity of these inhibitors. Several of ourcompounds were shown to inhibit the catalytic endonucleasefunction of APE1 with potencies in the low-micromolar rangein vitro, and therefore represent novel classes of APE1 inhibi-tors worthy of further development.

[a] Dr. F. Aiello, Dr. F. Grande, Prof. A. GarofaloDipartimento di Scienze FarmaceuticheUniversit� della Calabria, 87036 Arcavacata di Rende, CS (Italy)E-mail : francesca.aiello@unical.it

[b] Y. Shabaik, A. Esqueda, T. W. Sanchez, Prof. N. NeamatiDepartment of Pharmacology and Pharmaceutical SciencesSchool of Pharmacy, University of Southern California1985 Zonal Avenue, Los Angeles, CA 90089 (USA)

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pression of APE1, can be reversed following chemical inhibitionof APE1, as can cisplatin resistance in non-small-cell lungcancer.[10] Alterations in APE1 expression and localization arethought to have prognostic and/or predictive significance inseveral cancers, including liver, lung, and ovarian cancer.[11] Pre-dictive cancer-specific survival following radiotherapy has alsobeen attributed to APE1 expression.[11c, 12] Overexpression incancer cells and drug-induced acetylation of APE1 have alsobeen shown to promote expression of the multidrug-resistance(MDR1) gene.[13] The tumor microenvironment is also affectedby 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 centralrole in BER and the fact that many of the classically employedanticancer treatments including alkylating agents and ionizingradiation 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 APE1and that can potentiate the cell-killing effect of DNA-targetedchemotherapeutics. We are aided in our endeavor by the avail-ability of structural information on APE1 in complex with itssubstrate DNA bearing an abasic site.[2a] In a previous study wedeveloped a set of three-dimensional (3D) pharmacophoremodels based on APE1 interactions with the abasic deoxyri-bose 3’- and 5’-phosphate backbone in a co-crystal structureof APE1 in complex with its substrate DNA bearing an abasicsite.[15] APE1 endonuclease activity requires binding of divalentcations in the active site and can be exploited for drugdesign.[16] The pharmacophore perception is a popular tech-nique in drug design to identify small-molecule inhibitors withdiverse chemical scaffolds. It has been successfully used to dis-cover several classes of clinically relevant small molecules thattarget 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 toestablish quantitative structure–activity relationship models forfurther development of clinically relevant APE1 inhibitors. In aneffort to better characterize the mode of action of these com-pounds, we planned a synthetic pathway for the preparationof new classes of 3-carbamoylbenzyl derivatives (Figure 1 a)active against APE1. Several of the synthesized compounds dis-played good APE1 inhibitory activity in preliminary in vitroassays. 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 fashionsimilar to APE1, IN catalytically cleaves the phosphodiesterbackbone of virally encoded DNA 5’ to its recognition site,leaving a 3’-hydroxy group. Furthermore, IN requires a divalentmetal cation for catalysis, making it a suitable counter-screen-ing target for establishing the specificity of our compounds.Structure–activity analysis of synthesized compounds reachedan informative structural platform for the rational optimizationof this class of small molecules, because among the classesstudied, namely 6-benzylcarbamoyl pyridines, 8-hydroxyquino-

line-2-carboxamides, and N-benzylbenzamide derivatives (Fig-ure 1 b), none showed significant activity and selectivity,whereas 3-carbamoylbenzoic acid derivatives showed the mostpotent and selective activity against APE1, in the micromolarconcentration range.

Results and Discussion

Chemistry

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-disubstitutedbenzylamines, using EDC in the presence of a catalytic amountof 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 theappropriate benzylamine. All the methoxy derivatives weresubjected to demethylation by HBr/CH3COOH or LiI/collidine,depending on the sensitivity of the substrate. Compound F327was demethylated by BBr3 in CH2Cl2 (Scheme 1).

Derivatives F-M260 and F-M262 were obtained by theSchotten–Baumann procedure, reacting 2-methoxyisophthalic1 acid with one equivalent of methyl 3-amino-3-(4-chlorophe-

Figure 1. Synthesized compounds with descriptive hydrophobic, negativeionizable, and linker moieties. a) Synthesized 3-carbamoylbenzoic acid deriv-atives. b) Subsequently synthesized 6-benzylcarbamoylpyridine, 8-hydroxy-qunoline-2-carboxamide, and N-benzylbenzamide derivatives.

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nyl)propanoate or 3-amino-3-(4-fluorophenyl)propanoate, re-spectively. Ether and ester functions were hydrolyzed at thesame time by the LiI/collidine system to give derivatives F-260D and F-262D, while derivatives F-M260S and F-M262Swere obtained by alkaline hydrolysis selective for the estergroup (Scheme 2).

The 6-benzylcarbamoyl pyri-dine derivatives F-1, F-2, and thesymmetric di-3-chlorobenzyl de-rivative F-4 were prepared bythe Schotten–Baumann proce-dure, starting from pyridine-2,6-dicarbonyl dichloride 2 and theappropriate amine, as depictedin Scheme 3. The methyl ester F-3 was prepared starting from2,6-pyridine dicarboxylic acid 3first transformed by a two-stepprocedure into the monomethylester, 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 ionin 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 substitutedbenzylamine. The hydroxy derivative F-7 was obtained by BBr3-catalyzed demethylation of F-6 (Scheme 5).

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 moietywith the alternative bioisosteretetrazole 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 keystep.[20] The tetrazole ring wasfirst installed starting frommethyl 3-cyanobenzoate 5 andsodium azide. The intermediateester was then hydrolyzed intothe corresponding acid, whichwas in turn condensed with theappropriate 3-fluorobenzyla-mine.

Finally, we decided to preparethe N-benzylbenzamide series ofcompounds shown in Scheme 7to further investigate the impor-

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 ortrifluoromethyl groups instead of a more polar carboxylic func-tion, giving rise to a two-point complexing moiety with an

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 (1 m in CH2Cl2).

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.

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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overall geometry similar to that of the original triad. Thesecompounds 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 of3-carbamoylbenzoic acid deriv-atives

Inhibition of APE1 endonucleaseactivity was determined with anin vitro enzymatic assay with a ra-diolabeled DNA substrate, sche-matically shown in Figure 2. Op-timal assay conditions forscreening compounds againstour purified APE1 protein werepreviously determined.[15a] Con-comitantly, we assayed our com-pounds for their ability to inhibit

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 ouragents for APE1 over IN are reported. A representative IN gel isshown in Figure 3.

Compounds F325–F327 structurally varied only in thenumber and position of the methoxy group on the phenyl ringand were selectively active for APE1, with IC50 values between18�8 and 21�7 mm. Halogenated compounds F328–F330were generated by the replacement of methoxy groups withchlorine atoms. Interestingly, para-chloro compound F328 didnot show any inhibitory activity, whereas compounds F329and F330 had IC50 values of 16�6 and 18�12 mm, respective-ly. Compound F331, with a fluorine atom in the para position,and meta-fluoro compound F332 inhibited APE1 endonucleaseactivity with IC50 values of 26�2 and 27�16 mm, respectively.However, F333, with two fluorine atoms, did not show inhibi-tion properties. Apart from F350, which weakly inhibited INendonuclease and strand-transfer activity with IC50 values near500 mm, all of the compounds showed no activity against INand displayed selectivity for APE1 greater than 35-fold(Table 1).

Sequentially replacing the methoxy groups in compoundsF326 and F327 with hydroxy groups to generate deprotectedcompounds F326D–F327DD produced a negligible to pro-found decrease in their ability to inhibit APE1. F327DDshowed a decrease in activity from 18�8 mm to >100 mm rela-tive to F327. On the other hand, these modifications mostlyimproved IN inhibition (Table 2) compared with counterparts inTable 1, with the result of decreased selectivity indexes ranging

Scheme 4. Reagents and conditions : a) 1. H2SO4/MeOH, 2. KOH/MeOH; b) 3-chlorobenzylamine, EDC/toluene.

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,H2O; b) 1. trimethylsilyl chloride, py, toluene, 2. SOCl2, 3-fluorobenzylamine,py.

Scheme 7. Reagents and conditions : a) 1. SOCl2, DMF, 2. 3-fluoro- or 3-chloro-benzylamine, Et3N, toluene.

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 sitein the 26mer oligomer was tetrahydrofuran (F).

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from 0.52 to 26.3. Deprotected compounds F328–F333D alsowith methoxy to hydroxy substitutions between the carbonylgroups had a slight to substantial decrease in activities, withthe exception of F328D and F333D, which had increased activ-ity and lower IC50 values of 44�37 and 39�22 mm, respective-ly, relative to their methoxy-containing counterparts listed inTable 1. Following the same trend as the previous compounds,the compounds in Table 2 also had improved IN inhibition, butstill retained modest selectivity for APE1. Compound F327DD,with two hydroxy groups in the meta and para positions ofthe appended phenyl ring, displayed no inhibitory propertiestoward APE1 endonuclease activity, whereas compoundF326DD, with a single hydroxy substituent at the meta posi-tion, produced a weak IC50 value of 40�31 mm. CompoundF350D, containing an additional phenyl group, showed an IC50

value of 33�24 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 theirselectivity, but were still able to retain modest selectivity in-dexes favoring APE1, with the exception of F327DD, whichlacked 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: 25�3 to 66�5 mm) gave similar activities to thatof 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 selectedactive compounds that inhibit APE1 function can be observedin the representative gel image in Figure 4.

Cytotoxicity of 3-carbamoylbenzoic acid derivatives assingle agents and in combination with MMS or 5-FdUrd

The effect of our compounds on the proliferation of the H630colon 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 largelyunaffected in H630 cells at the highest doses tested. It wastherefore not possible to conclude any useful structure–activityrelationship from the activities of these compounds as singleagents in cell culture. We further examined the effect thatthese compounds might have as part of a combination regi-men with methyl methanesulfonate (MMS) on the proliferativecapability of H630 cells. MMS forms alkylated bases that are re-paired by BER and as such, compounds that can inhibit APE1in cells should exhibit a sensitizing effect to the cytotoxicityproduced by MMS treatment. We therefore treated cells witha range of concentrations of MMS alone, or in combinationwith a single dose of five of our compounds: F328, F329,F330, F329D and F332D. Pairs of drugs in each combinationwere added simultaneously to cells and incubated together fora total duration of 72 h, at the end of which the cell viabilitywas determined (Figure 5 a). This potentiation was specific tothe BER-dependent DNA lesions caused by MMS, as the combi-nation of F332D with other genotoxic agents did not producea similar synergistic outcome on cell viability Figure 5 a,b).

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.

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It is known that 5-FU, an anti-cancer drug commonly used totreat colon cancer, gets incorpo-rated into the DNA of treatedcells at significant levels and thatBER glycosylases are capable ofdetecting and removing the mis-incorporated base, so that DNAcan be repaired.[21] Our results in-dicate that F332D may be clini-cally useful as a combinationtherapy with 5-FU. This potentia-tion was specific to the BER-de-pendent DNA lesions caused byMMS, as the combination ofF332D with other genotoxicagents did not produce a similarsynergistic outcome on cell via-bility (Figure 6). Although BERproteins recognize and remove5-FU lesions from DNA, recentreports suggest that intact BERis more important for the repairof DNA damage resulting from5-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 F332Din an attempt to correlate syner-gistic activity with APE1 inhibi-tion. Among these compounds,F330, F328D, and F332Dshowed enhanced potentiation

Table 1. Inhibition of APE1 endonuclease activity by compounds F325–F350.

Compound HIV-1 IN APE1 Cell culture SI[c]

3P[a] ST[b] IC50 [mm] MTT Colony

F325 >1000 >1000 21�7 >30 >30 >47.6

F326 >1000 >1000 19�9 >30 >30 >52.6

F327 >1000 >1000 18�8 >30 >30 >55.6

F328 >1000 >1000 >100 >30 >30 NA

F329 <1000 1000 16�6 >30 >30 <62.5

F330 >1000 >1000 18�12 >30 >30 >55.6

F331 >1000 >1000 26�2 >30 >30 >38.5

F332 >1000 >1000 27�16 >30 >30 >37

F333 1000 >1000 >100 >30 >30 NA

F350 505 460 31�11 >30 >30 16.3

[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1endonuclease 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 is0.05 nm.

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of 5-FdUrd-induced cytotoxicity (Figure 7). While F328D andF330 were among the most active analogues tested in the invitro 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 itsmode of action. The synergistic combinations achieved withour inhibitors support their further development toward com-bination therapies with 5-FdUrd for the treatment of coloncancer.

Conclusions

A new class of APE1 inhibitors based on previously reportedpharmacophore models was identified in this work. Amongthe newly synthesized compounds, derivatives F326, F327,F329, and F330, having a 3-benzylcarbamoyl-2-methoxybenzo-

ic acid structure, resulted in themost active and selective inhibi-tion of APE1, showing IC50 values<20 mm and with no activityagainst HIV1 IN at the highestconcentrations tested. Replace-ment of the methoxy groupwith a hydroxy group at posi-tion 2 (compound F237D) didnot result in a significant changein activity, whereas such an alter-ation considerably affected se-lectivity. Notably, some activityand selectivity was retained byseveral dicarbamoyl derivatives,while the replacement of thecarbocyclic aromatic ring togeth-er with the carboxylic functionfor a pyridine or quinolinesystem proved detrimental toactivity. Thus, the 3-benzylcarba-moyl-2-methoxybenzoic acidstructure, common to the activederivatives, represents a leadscaffold to be further developedwith the aim of finding morepotent and selective APE1 inhibi-tors.

Experimental Section

Chemistry

All reactions were carried outunder 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 weredried over MgSO4 and evaporated

on a rotary evaporator under reduced pressure. Melting pointswere measured using an Electrothermal 8103 apparatus and areuncorrected. IR spectra were recorded as thin films on PerkinElmer398 and FT 1600 spectrophotometers. 1H NMR spectra were re-corded on a Bruker 300 MHz spectrometer with TMS as an internalstandard; chemical shifts (d) are expressed in ppm, and couplingconstants (J) in Hz. MS data were determined by direct insertion at70 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 elementalanalyzer, and the results are within 0.4 % of theoretical values.Yields refer to purified products and are not optimized.

General procedures for the synthesis of carbamoyl derivatives

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 inone portion to a solution of 2-methoxyisopthalic acid (0.200 g,1.020 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-

Table 2. Inhibition of APE1 endonuclease activity by compounds F326D–F350D.

Compound HIV-1 IN APE1 Cell culture SI[c]

3P[a] ST[b] IC50 [mm] MTT Colony

F326D >1000 >1000 38�30 >30 >30 >26.3

F326DD 280 195 40�31 >30 >30 7

F327D 61 99 19�2 >30 >30 3.2

F327DD 52�30 17�9 >100 >30 >30 <0.52

F328D >333 >216 44�37 >30 >30 >7.6

F329D >100 95�8 33�26 >30 >30 >3

F330D >100 82�25 28�19 >30 >30 >3.6

F331D >100 70�14 27�9 >30 >30 >3.7

F332D >1000 >1000 >100 >30 >30 NA

F333D >333 98�3 39�22 >30 >30 >8.5

F350D 95�6 35�14 33�24 >30 >30 2.9

[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1endonuclease catalysis versus IN endonuclease 3’-processing.

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drochloride (EDC; 0.195 g, 1.020 mmol), and 4-dimethylaminopyri-dine (DMAP) (cat.) in THF (5.5 mL). The mixture was stirred for 72 hat 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 aqueouslayer was acidified with 2 n 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-

tallized from petroleum ether (PE);yield 48 % (0.154 g); mp: 173 8C;1H NMR (300 MHz, [D6]acetone):d= 8.10 (dd, J = 6.0, 1.6 Hz, 2 H),7.50 (bs, 1 H), 7.20 (m, 2 H), 6.78(m, 3 H), 4.55 (d, J = 5.5 Hz, 2 H),3.76 (s, 3 H), 3.74 ppm (s, 3 H); IR(KBr): n= 3288, 1649 cm�1; MS (EI70 eV) m/z 315 [M]+ ; Anal. calcdfor C17H17NO5 : C 64.75, H 5.43, N4.44, found: C 65.03, H 5.52, N4.40.

3-(4-Methoxybenzylcarbamoyl)-2-methoxybenzoic acid (F325):white powder; yield 48 % (0.154 g);mp: 189 8C (PE); 1H NMR (300 MHz,[D6]acetone): d= 8.20 (m, 2 H), 7.59(s, 1 H), 7.26 (m, 2 H), 6.85 (m, 3 H),4.55 (d, J = 5.5 Hz, 2 H), 3.76 (s, 3 H),3.74 ppm (s, 3 H), IR (KBr): n= 3288,1649 cm�1; MS (EI 70 eV) m/z 315[M]+ ; Anal. calcd for C17H17NO5 : C64.75, H 5.43, N 4.44, found: C64.98, H 5.52; N 4.04.

3-(3,4-Dimethoxybenzylcarbamo-yl)-2-methoxybenzoic acid (F327):white powder; yield 30 % (0.096 g);mp: 197 8C (PE); 1H NMR (300 MHz,[D6]acetone): d= 11.5 (bs, 1 H), 8.11(bs, 1 H), 7.93 (dd, J = 7.7,3.9 Hz,1 H), 7.88 (dd, J = 5.7, 1.9 Hz,1 H), 6.81–7.39 (m, 4 H), 4.50 (d, J =5.9 Hz, 2 H), 3.89 (s, 3 H), 3.62 ppm(s, 6 H), IR (KBr): n= 3281,1650 cm�1; MS (EI 70 eV) m/z 345[M]+ ; Anal. calcd for C18H19NO6 : C62.60, H 5.55, N 4.06, found: C62.40, H 5.35, N 4.10.

3-(4-Chlorobenzylcarbamoyl)-2-methoxybenzoic acid (F328):white powder; yield 40 % (0.130 g);mp: 172 8C (PE); 1H NMR (300 MHz,CDCl3): d= 8.19 (dd, J = 7.7, 1.9 Hz,1 H), 8.11 (dd, J = 7.8, 1.9 Hz, 1 H),7.52 (s, 1 H), 7.30 (m, 5 H), 4.57 (d,J = 5.8 Hz, 2 H), 3.79 ppm (s, 3 H); IR(KBr): n= 3280, 1641 cm�1; MS (EI70 eV) m/z 319.06 [M]+ ; Anal. calcdfor C16H14ClNO4 : C 60.10, H 4.41, N4.38, found: C 60.23, H 4.62, N4.50.

3-(3-Chlorobenzylcarbamoyl)-2-methoxybenzoic acid (F329):white powder; yield 40 % (0.130 g);

mp: 188 8C (PE); 1H NMR (300 MHz, CDCl3): d= 8.27 (m, 1 H), 8.18(bs, 1 H), 7.79 (m, 1 H), 7.74 (m, 1 H), 7.10–7.45 (m, 4 H), 4.62 (m,2 H), 3.87 ppm (s, 3 H); IR (KBr): n= 3280, 1641 cm�1; MS (EI 70 eV)m/z 319.06 [M]+ ; Anal. calcd for C16H14ClNO4 : C 60.10, H 4.41, N4.38, found: C 60.25, H 4.05, N 4.50.

3-(3,4-Dichlorobenzylcarbamoyl)-2-methoxybenzoic acid (F330):white powder; yield 45 % (0.162 g); mp: 158 8C (PE); 1H NMR

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 Cell culture SI[c]

3P[a] ST[b] IC50 [mm] MTT Colony

F-1 >100 >100 >100 <20 (55 %) >30 NA

F-2 >100 >100 100 20 >30 >1

F-3 >100 >100 100 >20 >30 >1

F-5 >100 >100 >100 >20 >30 NA

F-6 >100 >100 >100 20 >30 NA

F-7 >100 >100 70 <20 (57 %) >30 >1.4

[a] 3’-Processing. [b] Strand transfer. [c] Selectivity index: fold difference in IC50 values of the inhibitors for APE1endonuclease catalysis versus IN endonuclease 3’-processing.

Table 4. Inhibition of APE1 endonuclease activity by compounds F330bis–F-4.

Compound HIV-1 IN APE1 Cell culture SI[c]

3P[a] ST[b] IC50 [mm] MTT Colony

F330bis 666 395 27�5 >30 >30 24.7

F330bisD 195 105�8 66�5 >30 >30 3

F328bisD 150 170 28�4 >30 >30 5.4

F329bisD 140 98�4 41�22 >30 >30 3.4

F350bisD 105 37 25�3 >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 APE1endonuclease catalysis versus IN endonuclease 3’-processing.

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(300 MHz, CDCl3): d= 8.20 (dd, 1 H, J = 7.7, 1.8 Hz), 8.11 (dd, J = 7.8,1.9 Hz, 1 H), 7.77 (s, 1 H), 7.41–7.27 (m, 2 H), 7.18 (m, 2 H), 4.56 (d,J = 5.3 Hz, 2 H), 3.84 ppm (s, 3 H); IR (KBr): n= 3291, 1639 cm�1; MS(EI 70 eV) m/z 353.06 [M]+ ; Anal. calcd for C16H13Cl2NO4 : C 54.26, H3.70, N 3.95, found: C 54.60, H 4.05, N 4.22.

3-(4-Fluorobenzylcarbamoyl)-2-methoxybenzoic acid (F331):white powder; yield 42 % (0.129 g);mp: 156 8C (PE); 1H NMR (300 MHz,CDCl3): d= 8.25 (m, 2 H), 7.50 (bs,1 H), 7.29 (m, 3 H), 6.99 (m, 2 H),4.57 (d, J = 5.0 Hz, 2 H), 3.79 ppm(s, 3 H); IR (KBr): n= 3280, 1638,1350 cm�1; MS (EI 70 eV) m/z303.09 [M]+ ; Anal. calcd forC16H14FNO4 : C 63.36, H 4.65, N4.62, found: C 63.30, H 5.01, N4.60.

3-(3-Fluorobenzylcarbamoyl)-2-methoxybenzoic acid (F332):white powder; yield 50 % (0.154 g);mp: 144 8C (PE); 1H NMR (300 MHz,CDCl3): d= 8.24 (dd, J = 7.7, 1.8 Hz,1 H), 8.09 (dd, J = 7.7, 1.9 Hz, 1 H),7.85 (s, 1 H), 7.33–7.14 (m, 2 H),7.12–6.84 (m, 3 H), 4.61 (d, J =5.7 Hz, 2 H), 3.79 ppm (s, 3 H); IR(KBr): n= 3288, 1638, 1370 cm�1;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, H5.00, N 4.75.

3-(3,4-Difluorobenzylcarbamoyl)-2-methoxybenzoic acid (F333):white powder; yield 30 % (0.098 g);mp: 161 8C (PE); 1H NMR (300 MHz,CDCl3): d= 8.24 (dd, J = 7.7, 1.8 Hz,1 H), 8.09 (dd, J = 7.8, 1.8 Hz, 1 H),7.89 (s, 1 H), 7.30 (m, 1 H), 7.19–7.03 (m, 3 H), 4.56 (d, J = 5.86 Hz,2 H), 3.81 ppm (s, 3 H); IR (KBr): n=3280, 1630, 1344 cm�1; MS (EI70 eV) m/z 321.08 [M]+ ; Anal. calcdfor C16H13F2NO4 : C 59.82, H 4.08, N4.36, found: C 60.02, H 4.31, N4.16.

3-(4-Phenylbenzylcarbamoyl)-2-methoxybenzoic acid (F350):white powder; yield 30 % (0.110 g);mp: 198 8C (n-hexane); 1H NMR(300 MHz, CDCl3): d= 8.26 (dd, J =7.7, 1.9 Hz, 1 H), 8.11 (dd, J = 7.8,1.9 Hz, 1 H), 7.61 (bs, 1 H), 7.58 (m,4 H), 7.47 (m, 6 H), 4.68 (m, 2 H),3.86 ppm (s, 3 H); IR (KBr): n= 3288,1638, 1344 cm�1; MS (EI 70 eV) m/z361.13 [M]+ ; Anal. calcd forC22H19NO4: C 73.12, H 5.30, N 3.88,found: C 73.37, H 5.42, N 4.08.

The following N1,N3-dibenzyla-mides F328bis–F330bis and

F350bis were obtained after the same reaction by using 2 equivreactants with respect to starting 2-methoxyisophthalic acid.

N1,N2-Bis(4-chlorobenzyl)-2-methoxybenzene-1,3-diamide(F328bis): white powder; yield 60 % (0.270 g); mp: 195 8C (PE);1H NMR (300 MHz, [D6]acetone): d= 8.30 (bs, 2 H), 7.87 (m, 5 H),

Table 5. Compounds that exhibit no activity against APE1 endonuclease activity.

Compound HIV-1 IN APE1 Cell culture3P[a] ST[b] IC50 [mm] MTT Colony

F-M260 >100 >100 >100 >30 >30

F260D 410 250 >100 >30 >30

F-M260S 590 560 >100 >30 >30

F-M262 >1000 >1000 >100 >30 >30

F262D 600 510 >100 >30 >30

F-M262S 490 333 NT >30 >30

CF34Ph 1000 1000 >100 >30 >30

CF33Cl >1000 >1000 >100 >30 >30

CF33F >1000 >1000 >100 >30 >30

CN4Ph 1000 1000 >100 >30 >30

T3F >1000 >1000 NT >30 >30

[a] 3’-Processing. [b] Strand transfer.

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MED3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors

7.20 (m, 6 H), 4.41(m, 4 H), 3.79 ppm (s, 3 H); IR (KBr): n= 1690 cm�1;MS (EI 70 eV) m/z 442.09 [M]+ ; Anal. calcd for C23H20Cl2N2O3: C62.31, 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(F329bis): white powder; yield 60 % (0.270 g); mp: 197 8C (n-hexane); 1H NMR (300 MHz, [D6]acetone): d= 8.41 (bs, 2 H), 8.00 (m,3 H), 7.38 (m, 8 H), 4.56 (d, J = 5.8 Hz, 4 H), 3.87 ppm (s, 3 H); IR(KBr): n= 1690 cm�1; MS (EI 70 eV) m/z 442.09 [M]+ ; Anal. calcd forC23H20Cl2N2O3 : C 62.31, H 4.55, N 6.32, found: C 62.51, H 4.60, N6.52.

N1,N2-Bis(3,4-dichlorobenzyl)-2-methoxybenzene-1,3-diamide(F330bis): white powder; yield 30 % (0.156 g); mp: 187 8C (PE); IR1H NMR (300 MHz, [D6]acetone): d= 8.51 (bs, 2 H), 8.03 (m, 4 H),7.68–7.22 (m, 5 H), 4.56 (d, J = 5.4 Hz, 4 H), 3.79 ppm (s, 3 H); (KBr):n= 1690 cm�1; MS (EI 70 eV) m/z 510.01 [M]+ ; Anal. calcd forC23H18Cl4N2O3 : C 53.93, H 3.54, N 5.47, found: C 53.92, H 3.68, N5.27.

N1,N2-Bis(4-phenylbenzyl)-2-methoxybenzene-1,3-diamide(F350bis): brown solid; yield 20 % (0.104 g); mp: 193 8C (PE);1H NMR (300 MHz, CD3OD): d= 8.00(bs, 2 H), 7.79–7.35 (m, 21 H),4.99 (s, 3 H), 4.15 ppm (m, 4 H); IR (KBr): n= 1690 cm�1; MS (EI70 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: 117 8C (cyclohexane);1H NMR (300 MHz, [D6]acetone): d= 9.30 (bs, 1 H), 8.50–8.00 (m,3 H), 7.50–6.80 (m, 4 H), 4.50 (s, 2 H), 2.00 ppm (s, 3 H); IR (KBr): n=1735, 1650 cm�1; MS (EI 70 eV) m/z 304 [M]+ ; Anal. calcd forC15H13ClN2O: 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 solutionof 3-cyanobenzoic acid (0.150 g, 1.020 mmol) in SOCl2 (1.5 mL) andDMF (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 (seeSchemes 3 and 5)] in dry toluene (3 mL) was added, and the wholewas stirred at reflux overnight. The reaction was quenched withH2O (1 mL) and concentrated, the aqueous phase was extractedwith Et2O, and the organic layer was dried and concentrated togive a residue which was purified by flash chromatography (PE/EtOAc, 2:1 as eluent); yellow solid; yield 53 % (0.137 g); mp: 101–103 8C (cyclohexane); 1H NMR (300 MHz CDCl3): d= 8.14–8.01 (m,2 H), 7.79 (dt, J = 7.7, 1.2 Hz, 1 H), 7.58 (1 H, m), 7.40–7.25 (m, 1 H),6.94–7.17 (m, 3 H), 6.86 (bs, 1 H), 4.63 ppm (d, J = 5.7 Hz, 2 H); IR(KBr): n= 2220, 1640, 1367 cm�1; 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; H4.30, N 11.32.

N-(3-Chlorobenzyl)-3-cyanobenzamide (CN3Cl): beige powder;yield 20 % (0.055 g); mp: 109 8C (cyclohexane); 1H NMR (300 MHzCDCl3): d= 8.06 (m, 2 H), 7.73 (d, J = 7.7 Hz, 1 H), 7.51 (t, J = 7.7 Hz,1 H), 7.28 (m, 4 H), 6.65 (s, 1 H), 4.54 ppm (d, J = 5.7 Hz, 2 H); IR(KBr): n= 2236, 1639 cm�1; MS (EI 70 eV) m/z 270 [M]+ ; Anal. calcdfor C15H11ClN2O: C 66.55, H 4.10, N 10.35, found: C 65.75, H 4.16, N10.09.

N-(4-Phenylbenzyl)-3-cyanobenzamide (CN4Ph): light-yellowpowder; yield 19 % (0.060 g); mp: 164 8C (cyclohexane); 1H NMR(300 MHz CDCl3): d= 8.40 (s, 1 H), 7.97 (dd, J = 7.9, 1.0 Hz, 1 H), 7.72(dd, J = 7.7, 1.0 Hz, 1 H), 7.60–7.33 (m, 10 H), 6.53 (s, 1 H), 4.63 ppm(d, J = 5.5 Hz, 2 H); IR (KBr): n= 2229, 1640 cm�1; MS (EI 70 eV) m/z312 [M]+ ; Anal. calcd for C21H16N2O: C 80.75, H 5.16, N 8.97, found:C 80.97, H 5.50, N 9.09.

N-(3-Fluorobenzyl)-3-(trifluoromethyl)benzamide (CF33F): brownoil ; yield 50 % (0.151 g); 1H NMR (300 MHz CDCl3): d= 7.96 (s, 1 H),7.86 (d, J = 7.8 Hz, 1 H), 7.64 (d, J = 7.8 Hz, 1 H), 7.41 (t, J = 7.8 Hz,1 H), 7.27 (bs, 1 H), 7.17 (s, 1 H), 7.00–6.80 (m, 3 H), 4.46 ppm (d, J =5.8 Hz, 2 H); IR (KBr): n= 1642, 1332 cm�1; MS (EI 70 eV) m/z 297[M]+ ; Anal. calcd for C15H11F4NO: C 60.61, H 3.73, N 4.71, found: C60.87, H 4.03, N 5.01.

N-(3-Chlorobenzyl)-3-(trifluoromethyl)benzamide (CF33Cl): yellowoil ; yield 70 % (0.223 g); 1H NMR (300 MHz CDCl3): d= 8.06 (s, 1 H);7.94 (d, J = 7.8 Hz, 1 H), 7.83 (m, 1 H), 7.70 (d, J = 7.8 Hz, 1 H), 7.45 (t,J = 7.8 Hz, 1 H), 7.26–7.08 (m, 4 H), 4.48 ppm (d, J = 5.8 Hz, 2 H); IR(KBr): n= 1641, 1334 cm�1; MS (EI 70 eV) m/z 313 [M]+ ; Anal. calcdfor C15H11 ClF3NO: C 57.43, H 3.53, N 4.46, found: C 57.68, H 3.35, N4.59.

Figure 5. Effect of combination treatment of a) F332D with MMS, andb) 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 thepresence or absence of a minimally effective concentration of F332D drug(50 mm) for 72 h. Values are the average of four independent experimentsfor MMS combinations and only one experiment for 5-FU combinations. Allconcentration combinations were tested in triplicate for each experiment.Two-tailed, paired student’s t-test was used to calculate the p values for thedifference in mean survival values between the combination treatmentgroup and MMS/5-FU only or F332D only treatment groups; *p<0.05 inboth cases.

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N-(4-Phenylbenzyl)-3-(trifluoromethyl)benzamide (CF34Ph): whitepowder; yield 63 % (0.201 g); mp: 117 8C (cyclohexane); 1H NMR(300 MHz CDCl3): d= 8.05 (s, 1 H), 7.93 (d, J = 7.7 Hz, 1 H), 7.65 (d,J = 7.4 Hz, 1 H), 7.60–7.15 (m, 10 H), 6.63 (bs, 1 H), 4.60 ppm (d, J =5.4 Hz, 2 H); IR (KBr): n= 1640, 1330 cm�1; MS (EI 70 eV) m/z 313[M]+ ; Anal. calcd for C21H16F3NO: C 70.98, H 4.54, N 3.94, found: C70.73, 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, 1 H),8.06 (d, J = 7.7 Hz, 1 H), 7.30 (m, 5 H), 5.63 (m, 1 H), 3.80–3.60 (m,6 H), 3.05–2.85 ppm (m, 2 H); IR (KBr): n= 3400, 1687 cm�1; MS (EI70 eV) m/z 391 [M]+ ; Anal. calcd for C19H18ClNO6 : C 58.24, H 4.63, N3.57, found: C 58.36, H 4.40, N 3.37.

Figure 6. a) Effect of combination treatment of F332D with a) MMS, b) doxorubicin, c) oxaliplatin, and d) camptothecin. DNA-damage-inducing agents’ effectson 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 differencein 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 ofindicated inhibitor (50 mm) for an additional 24 h. Drug-containing medium was then replaced with fresh media, and colonies were allowed to form. Percentsurvival indicates fraction of surviving colonies relative to untreated control.

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MED3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors

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, 1 H), 8.06 (d, J =7.7 Hz, 1 H), 7.35 (m, 2 H), 7.27 (t, J = 7.7 Hz, 1 H), 7.03 (m, 2 H), 5.64(dd, J = 14.0, 6.1 Hz, 1 H), 3.80–3.60 (m, 6 H), 3.80–2.87 ppm (m,2 H); IR (KBr): n= 3450, 1691 cm�1; 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.

N-(3-Fluorobenzyl)-3-(1H-tetrazol-5-yl)benzamide (T3F): amor-phous solid; yield 3 % (0.009 g); 1H NMR (300 MHz CDCl3): d= 8.65(bs, 1 H), 8.25 (bs, 1 H), 8.00 (m, 1 H), 7.70 (t, J = 7.7 Hz, 1 H), 7.59 (t,J = 7.9 Hz, 1 H), 7.35 (m, 1 H), 7.20–6.95 (m, 2 H), 6.80–6.75 (m, 2 H),4.65 ppm (d, J = 5.6 Hz, 2 H); IR (KBr): n= 1693 cm�1; 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.

6-(3-Chlorobenzylcarbamoyl)pyridine-2-carboxylic acid (F-1):brown solid; yield 42 % (0.124 g); mp: 290 8C (dec.) (Et2O); 1H NMR(300 MHz DMSO): d= 11.00 (bs, 2 H), 8.30–7.70 (m, 3 H), 7.60–7.20(m, 4 H), 4.50 ppm (s, 2 H); IR (KBr): n= 1710, 1690 cm�1; MS (EI70 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: 156 8C (dec.) (MeOH/Et2O),1H NMR (300 MHz CDCl3): d= 9.00 (bs, 1 H), 8.90 (s, 1 H), 8.50 (m,1 H), 8.10 (m, 1 H), 7.70 (t, J = 7.7 Hz, 1 H), 7.50–7.00 (m, 4 H),4.70 ppm (m, 2 H); IR (KBr): n= 1710, 1650 cm�1; MS (EI 70 eV) m/z290 [M]+ ; Anal. calcd for C14H11FN2O3 : C 57.84, H 3.81;N, 9.64,found: C 58.00, H 3.71, N 9.65.

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, 2 H), 7.70–7.60 (d, J = 7.6 Hz, 2 H), 7.53–7.40 (t, J =7.0 Hz, 1 H), 6.90–6.40 (m, 8 H), 4.30 ppm (s, 4 H); IR (KBr): n=1690 cm�1; MS (EI 70 eV) m/z 414 [M]+ ; Anal. calcd forC21H17Cl2N3O2 : C 60.88, H 4.14, N 10.14, found: C 61.08, H 4.21, N9.96.

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, 1 H), 8.43 (bs, 1 H), 7.85 (d, J = 8.6 Hz, 1 H), 7.70 (d, J =

8.5 Hz, 1 H), 7.60–7.20 (m, 7 H), 4.80 ppm (s, 2 H), IR (KBr): n=1710 cm�1; MS (EI 70 eV) m/z 312 [M]+ ; Anal. calcd forC17H13ClN2O2 : C 65.29, H 4.19, N 8.96, found: C 65.19, H 4.39, N8.77.

N-(3-Methoxybenzyl)-8-hydroxyquinoline-2-carboxamide (F-6):brown solid; yield 30 % (0.126 g); mp: 122 8C (EtOAc); 1H NMR(300 MHz CDCl3): d= 8.99 (bs, 1 H), 8.50 (bs, 1 H), 8.20 (d, J = 8.5 Hz,1 H), 8.10 (d, J = 8.5 Hz, 1 H), 7.45 (t, J = 8.2 Hz, 1 H), 7.28 (d, J =8.2 Hz, 1 H), 7.08 (d, J = 7.6 Hz, 1 H), 6.99 (t, J = 8.0 Hz, 1 H), 6.75 (m,2 H), 6.55 (d, J = 8.2 Hz, 1 H), 4.50 (s, 2 H), 3.60 ppm (s, 3 H), IR (KBr):n= 1710 cm�1; MS (EI 70 eV) m/z 414 [M]+ ; Anal. calcd forC18H16N2O3 : C 70.12, H 5.23, N 9.09, found: C 69.98, H 4.99, N 9.07.

General procedure for the demethylation reaction

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-methoxybenzoicacid (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 180 8C.3 n 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

30 min. The yellow solid formed was filtered, washed with H2O,and purified by ion-exchange chromatography (DOWEX� 200), togive a light-yellow solid; yield 60 % (0.011 g); mp: 196 8C (EtOAc/PE); 1H NMR (300 MHz [D6]acetone): d= 8.66 (bs, 1 H), 8.34 (dd, J =7.8, 1.8 Hz, 1 H), 8.12 (dd, J = 6.0, 1.8 Hz, 1 H), 7.26 (t, J = 7.6 Hz, 1 H),7.11 (t, J = 7.8 Hz, 1 H), 6.90 (m, 3 H), 4.66 (d, J = 5.8 Hz, 2 H),3.78 ppm (s, 3 H); IR (KBr): n= 3280, 1644 cm�1; MS (EI 70 eV) m/z302 [M]+ ; Anal. calcd for C16H15NO5 : C 63.78, H 5.02, N, 4.65, found:C 63.68, H 5.12, N, 4.90.

3-(3,4-Dihydroxybenzylcarbamoyl)-2-hydroxybenzoic acid(F327D): light-yellow solid; yield 60 % (0.012 g); mp: 205 8C(EtOAc); 1H NMR (300 MHz [D6]acetone): d= 9.10 (bs, 1 H), 8.51 (s,1 H), 8.26 (d, J = 7.7 Hz, 1 H), 8.08 (m, 1 H), 7.95 (d, J = 7.7 Hz, 1 H),7.30 (m, 1 H),6.92 (m, 2 H), 4.55 (s, 2 H), 3.91 ppm (s, 6 H); IR (KBr):n= 3288, 1639 cm�1; MS (EI 70 eV) m/z 331 [M]+ ; Anal. calcd forC17H17NO6: C 61.63, H 5.17, N 4.23, found: C 61.45, H 5.25, N 4.37.

3-{[2-Carboxy-1-(4-chlorophenyl)ethyl]carbamoyl}-2-hydroxyben-zoic acid (F260D): orange powder; yield 98 % (0.022 g); mp: 122–125 8C (EtOAc); 1H NMR (300 MHz CD3OD): d= 8.10 (d, J = 7.8 Hz,2 H), 7.43 (m, 4 H), 7.05 (t, J = 7.8 Hz, 1 H), 5.62 (t, J = 7.1 Hz, 1 H),2.90 ppm (m, 2 H); IR (KBr): n= 3710, 1678 cm�1; MS (EI 70 eV) m/z363 [M]+ ; Anal. calcd for C17H14ClNO6: C 58.24, H 4.63, N 3.57,found: C 58.54, H 4.99, N 3.41.

3-{[2-Carboxy-1-(4-fluorophenyl)ethyl]carbamoyl}-2-hydroxyben-zoic acid (F262D): brown powder; yield 61 % (0.013 g); mp: 98–103 8C (dichloromethane); 1H NMR (300 MHz CD3OD): d= 7.92 (d,J = 7.8 Hz, 2 H), 7.35 (m, 3 H), 7.05 (t, J = 7.8 Hz, 1 H), 6.90 (m, 2 H),5.55 (t, J = 6.9 Hz, 1 H), 2.80 ppm (m, 2 H); IR (KBr): n= 3700,1688 cm�1; 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.

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 aceticacid (0.7 mL). The mixture was stirred at 120 8C for 2 h; H2O(1.5 mL) was then added, and the mixture was left to cool to roomtemperature to give a grey solid, which was filtered and thorough-ly washed with H2O; yield 30 % (0.029 g); mp: 225 8C (MeOH);1H NMR (300 MHz DMSO): d= 11.01 (bs, 1 H), 9.08 (bs, 1 H), 8.00(dd, J = 16.9, 7.3 Hz, 2 H), 7.39 (m, 2 H), 7.22 (s, 1 H), 7.02 (t, J =7.5 Hz, 1 H), 4.50 ppm (d, J = 5.1 Hz, 2 H); IR (KBr): n= 3290,1641 cm�1; 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.

3-(4-Chlorobenzylcarbamoyl)-2-hydroxybenzoic acid (F328D):grey powder; yield 42 % (0.038 g); mp: 248 8C (MeOH); 1H NMR(300 MHz DMSO): d= 8.95 (bs, 1 H), 7.95 (m, 2 H), 7.33 (m, 4 H), 6.95(t, J = 7.8 Hz, 1 H), 4,45 ppm (d, J = 5.6 Hz, 2 H); IR (KBr): n= 3291,1649 cm�1; 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.

3-(4-Phenylbenzylcarbamoyl)-2-hydroxybenzoic acid (F350D):grey powder; yield 30 % (0.031 g>) ; mp: 260 8C (EtOH); 1H NMR(300 MHz DMSO): d= 9.11 (s, 1 H), 8.10–7.91 (m, 2 H), 7.68–7.35 (m,9 H), 7.11 (s, 1 H), 4.59 ppm (s, 2 H); IR (KBr): n= 3299, 1641 cm�1;MS (EI 70 eV) m/z 347 [M]+ ; Anal. calcd for C21H17NO4 : C 72.61, H4.93, 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: 215 8C (MeCN);1H NMR (300 MHz [D6]acetone): d= 13,10 (bs, 1 H), 8.71 (s, 2 H), 8.29(dd, J = 7.7, 1.7 Hz, 1 H), 8.12 (m, 5 H), 7.99 (d, J = 7.7 Hz, 1 H), 7.35(m, 2 H), 7.10 (m, 2 H), 4.60 ppm (m, 4 H); IR (KBr): n= 3450,

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1645 cm�1; MS (EI 70 eV) m/z 429 [M]+ ; Anal. calcd forC22H18Cl2N2O3 : C 61.55, H 4.23, N 6.53, found: C 61.29, H 4.54, N6.23.

N1,N2-Bis(3-chlorobenzyl)-2-hydroxybenzene-1,3-diamide(F329bisD): grey powder; yield 33 % (0.042 g>) ; mp: 222 8C (n-hexane); 1H NMR (300 MHz [D6]acetone): d= 13.01 (bs, 1 H), 8.63 (s,2 H), 8.21 (d, J = 7.7 Hz, 2 H), 7.95 (m, 5 H), 7.20 (m, 2 H), 6.97 (m,2 H), 4.58 ppm (s, 4 H); IR (KBr): n= 3455, 1675 cm�1; 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: 244 8C (EtOH);1H NMR (300 MHz [D6]acetone): d= 13,01 (bs, 1 H), 8.90 (bs, 2 H),8.71 (s, 1 H), 8.30 (m, 2 H), 7.90–7.00 (m, 6 H), 4.55 ppm (m, 4 H); IR(KBr): n= 3475, 1657 cm�1; MS (EI 70 eV) m/z 497 [M]+ ; Anal. calcdfor C22H16Cl4N2O3 : C 53.04, H 3.24, N 5.62, found: C 53.05, H 3.44, N6.00.

N1,N2-Bis(4-phenylbenzyl)-2-hydroxybenzene-1,3-diamide(F350bisD): brown solid; yield 40 % (0.061 g); mp: 228 8C (MeCN);1H NMR (300 MHz [D6]acetone): d= 13.21 (s, 1 H), 8.71 (s, 2 H), 8.21–6.99 (m, 21 H), 5.01–4.35 ppm (m, 4 H); IR (KBr): n= 3761,1699 cm�1; 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.

Method E. The preparation of N-(3-hydroxybenzyl)-8-hydroxyqui-noline-2-carboxamide (F-7) is described as a representative exam-ple. 1 n 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 0 8C. The mixturewas stirred at room temperature for 24 h under nitrogen and thendiluted with H2O (5 mL) and extracted with EtOAc (3 � 10 mL). Thedried organic phases were concentrated, and the crude residuewas 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, 2 H), 9.03(bs, 1 H), 8.43 (d, J = 8.5 Hz, 1 H), 8.24 (m, 1 H), 7.45 (m, 2 H), 7.10(m, 2 H), 6.75 (m, 2 H), 6.63 (m, 1 H), 4.54 ppm (d, J = 6.4 Hz, 2 H); IR(KBr): n= 3600, 1710 cm�1; MS (EI 70 eV) m/z 295 [M]+ ; Anal. calcdfor C17H14N2O3 : C 69.38, H 4.79, N 9.52, found: C 69.28, H 4.68, N9.82.

3-(3-Hydroxybenzylcarbamoyl)-2-hydroxybenzoic acid (F326DD):grey powder; yield 60 % (0.010 g); mp: 207 8C (EtOH); 1H NMR(300 MHz [D6]acetone): d= 13.21 (bs, 2 H), 9.01 (bs, 1 H), 8.78 (s,1 H), 8.45–8.11 (m, 2 H), 7.62–6.69 (m, 5 H), 4.88–4.51 ppm (m, 2 H);IR (KBr): n= 3287, 1640 cm�1; 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, 3 H), 10.02 (bs, 1 H), 8.99 (bs, 1 H), 8.05(m, 1 H), 7.00 (m, 1 H), 6.82 (m, 1 H), 6.70 (m, 3 H), 4.90 ppm (s, 2 H);IR (KBr): n= 3280, 1629 cm�1; 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-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, thenthe volatiles were removed, and the crude residue was partitionedbetween Et2O and H2O. The inorganic layer was made acidic with3 n HCl (pH 2) and extracted twice with Et2O. The organic layer

was concentrated to give a white solid: yield 52 % (0.013 g>) ; mp102 8C (EtOAc); 1H NMR (300 MHz CD3OD): d= 7.90 (m, 2 H), 7.50(m, 2 H), 7.05 (m, 1 H), 7.00 (m, 3 H), 5.75 (m, 1 H), 4.90 (bs, 2 H), 3.85(s, 3 H), 3.25–2.90 ppm (m, 2 H); IR (KBr): n= 3840, 1698 cm�1; MS(EI 70 eV) m/z 391 [M]+ ; Anal. calcd for C17H16ClNO6 : C 58.24, H4.63, N 3.57, found: C 57.94, H 4.86, N 3.89.

3-{[2-Carboxy-1-(4-fluorophenyl)ethyl]carbamoyl}-2-methoxy-benzoic acid (F-M262S): following the same procedure describedfor compound F-M260S, compound F-M262S was obtained asa white solid; yield 46 % (0.011 g); mp 108 8C (EtOAc); 1H NMR(300 MHz CD3OD): d= 8.00 (m, 1 H), 7.58 (d, 1 H, J = 7.6 Hz), 7.30 (m,2 H), 7.10 (t, 1 H, J = 7.7 Hz), 6.95 (t, 2 H, J = 8.5 Hz), 5.44 (t, J =6.9 Hz, 1 H), 3.85 (s, 3 H), 3.52 (s, 3 H), 2.80 ppm (m, 2 H); IR (KBr):n= 3850, 1690 cm�1; MS (EI 70 eV) m/z 375 [M]+ ; Anal. calcd forC17H16FNO6 : C 60.80, H 4.83, N 3.73, found: C 60.98, H 5.00, N 3.89.

Biology

Preparation of oligonucleotide substrates : APE1 substrate oligonu-cleotides in the forward direction labeled as THF top with the se-quence 5’-ATT TCA CCG GTA CG(F) TCT AGA ATC CG-3’ comple-mented the THF bot reverse strand, 3’-TA AAG TGG CCA TGC (C)AGATC TTA GGC-5’. HIV-1 IN substrate oligonucleotides in the forwarddirection labeled as 21 top with the sequence 5’-GTG TGG AAAATC TCT AGC AGT-3’ complemented the 21 bot 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 shadowingon polyacrylamide gel. To analyze the extent of APE1 and IN enzy-matic activity, both top strands were radioactively labeled at the5’-end using T4 polynucleotide kinase (Epicentre, Madison, WI) andg-[32P]ATP (ARC Inc.). The kinase was heat-inactivated, and 21 botwas added in 1.5-molar excess. The mixture was heated at 95 8C,and then allowed to cool slowly to room temperature. The newlyannealed oligonucleotides were purified through a spin 25 mini-column (USA Scientific) to separate annealed double-stranded oli-gonucleotide from unincorporated material.

In vitro APE1 assay : The in vitro APE1 assay was performed as previ-ously described.[15a] The extent of endonuclease activity of APE1was determined by diluting the test compounds in DMSO and in-cubating the diluted compounds with recombinant APE1 at a finalconcentration 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 mg mL�1 bovine serum albumin (BSA), 10 mm

b-mercaptoethanol, 10 % DMSO, and 25 mm MOPS, pH 7.2) at 37 8Cfor 30 min. Thereafter, 5’-end 32P-labeled linear oligonucleotidesubstrate 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 % deionizedformamide, 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 ina PhosphorImager cassette and analyzed using a Typhoon 8610Variable Mode Imager (Amersham Biosciences) and quantified withImageQuant 5.2 software.

In vitro HIV-1 IN assay : To determine the extent of 3’-processingand strand transfer, wild-type recombinant IN was pre-incubated ata 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 mg mL�1 BSA, 10 mm b-mercaptoethanol, 10 % DMSO, and 25 mm MOPS, pH 7.2) at 30 8Cfor 30 min. The 5’-end 32P-labeled linear oligonucleotide substrate

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MED3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors

(20 nm) was then added, and incubation was continued for an ad-ditional 1 h. Reactions were then quenched by the addition of anequal volume (16 mL) of loading dye (98 % deionized formamide,10 mm EDTA, 0.025 % xylene cyanol, and 0.025 % bromophenolblue). An aliquot (5–10 mL) was electrophoresed on denaturing20 % polyacrylamide gels (0.09 m Tris-borate pH 8.3, 2 mm EDTA,20 % acrylamide, 8 m urea). Gels were dried, exposed in a Phosphor-Imager cassette, and analyzed using a Typhoon 8610 VariableMode Imager (Amersham Biosciences) and quantified using Image-Quant 5.2 software.

Quantification of APE1 and IN inhibition : Percent inhibition (% I) wascalculated with the equation: % I = 100 � [1�(D�C)/(N�C)] , forwhich C, N, and D are the fractions of 26mer substrate cleaved to13mer products by APE1 for DNA alone, DNA plus APE1, and DNAplus APE1 plus the test compound, respectively. For IN inhibition,C, N, and D are the fractions of 21mer substrate converted into19mer (3’-processing product) or strand-transfer products for DNAalone, DNA plus IN, and IN plus drug, respectively. The IC50 valueswere determined by plotting the logarithm of drug concentrationversus percent inhibition to obtain the concentration that pro-duced 50 % inhibition.

Cell culture : H630 and HT-29 colon cancer cells were obtained fromthe National Cancer Institute (Bethesda, MD, USA) and were main-tained as adherent monolayer cultures in Dulbecco’s modifiedEagle’s medium (DMEM) and RPMI-1640, respectively, supplement-ed with 10 % fetal bovine serum (FBS; Gemini-Bioproducts, WestSacramento, CA, USA). Cells were grown at 37 8C in a humidifiedatmosphere of 5 % CO2. For all experiments, cells in exponentialgrowth phase were washed with PBS, briefly trypsinized in a smallvolume of 0.25 % trypsin–EDTA solution (Sigma–Aldrich, St. Louis,MO, USA), resuspended in culture media, and centrifuged at1200 rpm (240 g) for 5 min. Pelleted cells were resuspended incomplete growth medium, counted and plated in sterile platesand allowed to adhere overnight before treating.

Purification of recombinant, His-tagged APE1: His-tagged APE1 waspurified 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 4 8C 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 witha Qiagen pQE30 plasmid containing the APE1 gene sequence andampicillin resistance. The APE1 plasmid was expressed in the M15expression strain after induction by IPTG (1 mm) at an absorbanceof 0.6–0.8 optical density at 595 nm. The culture was allowed togrow for an additional 3–4 h at 37 8C. 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 lysisbuffer (20 mm HEPES, pH 7.5, 5 mm imidazole, 100 mm NaCl) andpassed twice through a French press (Thermo Spectronic, Madison,WI, USA). The lysate was centrifuged at 31000 g, and the pellet wassolubilized in a buffer containing 20 mm HEPES, pH 7.5, 5 mm imi-dazole, and 10 mm CHAPS. Recombinant APE1 protein was purifiedusing Ni-affinity chromatography with Swell-gel Nickel-chelateddiscs (Pierce, Rockford, IL, USA). The protein was eluted from thecolumn with increasing concentrations of imidazole from 40 mm to1 m. An aliquot of each concentration post-elution was run on anSDS-PAGE gel, and fractions containing protein were dialyzed inSpectra/Por molecular porous membrane tubing, MWCO: 12–14 kDa (Spectrum Laboratories Inc. , Houston, TX, USA). The protein

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 �80 8C 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 aspreviously described.[22] Cells were seeded in 96-well tissue culturetreated dishes at a density of 4000 cells per well and allowed toattach overnight. Cells were subsequently treated with continuousexposure to a range of concentrations of drugs for 72 h. At theend of exposure, MTT solution was added to each well at a finalconcentration of 0.3 mg mL�1 MTT and further incubated for 3–4 hat 37 8C. After removal of the supernatant, DMSO was added, andthe absorbance of the solubilized dye was read at l 570 nm. Per-cent cytotoxicity for each drug concentration was determinedusing the following formula: cytotoxicity = 100�(100�[Abs(drug)/Ab-s(control)]). Where possible, IC50 values were determined from the plotof percent cytotoxicity versus the logarithm of drug concentration.

Colony formation assay : The colony-forming capacity of H630 cellsin response to drug treatment was determined by plating cells ata density of 100 cells per well in 96-well plates or 300 cells perwell in 12-well plates. Cells were allowed to attach overnight, fol-lowing which drug was added to the media in each well and thenincubated for 7–14 days. At the end of treatment, cells werewashed once with PBS, stained with crystal violet for 30 min(0.25 % crystal violet, 3.7 % formaldehyde, 80 % MeOH), washedwith deionized water, and left to dry overnight. Plates werescanned in the Odyssey Infrared Imaging System (Licor, Lincoln,NE, USA) using the 700 channel laser. Cytotoxicities were calculatedas a ratio of control as described above.

Keywords: anticancer agents · APE1 · carbamoylbenzoicacids · inhibitors · synthesis

[1] T. Lindahl, Nature 1993, 362, 709 – 715.[2] a) J. A. Tainer, C. D. Mol, T. Izumi, S. Mitra, Nature 2000, 403, 451 – 456;

b) A. C. Jacobs, C. R. Kreller, M. M. Greenberg, Biochemistry 2011, 50,136 – 143.

[3] L. J. Walker, C. N. Robson, E. Black, D. Gillespie, I. D. Hickson, Mol. Cell.Biol. 1993, 13, 5370 – 5376.

[4] a) G. Tell, F. Quadrifoglio, C. Tiribelli, M. R. Kelley, Antioxid. Redox Signal-ing 2009, 11, 601 – 620; b) M. R. Kelley, M. M. Georgiadis, M. L. Fishel,Curr. Mol. Pharmacol. 2012, 5, 36 – 53.

[5] a) C. Vascotto, D. Fantini, M. Romanello, L. Cesaratto, M. Deganuto, A.Leonardi, J. P. Radicella, M. R. Kelley, C. D’Ambrosio, A. Scaloni, F. Quadri-foglio, G. Tell, Mol. Cell. Biol. 2009, 29, 1834 – 1854; b) T. Barnes, W. C.Kim, A. K. Mantha, S. E. Kim, T. Izumi, S. Mitra, C. H. Lee, Nucleic AcidsRes. 2009, 37, 3946 – 3958.

[6] a) A. Bapat, M. L. Fishel, M. R. Kelley, Antioxid. Redox Signaling 2009, 11,651 – 668; b) A. Al-Attar, L. Gossage, K. R. Fareed, M. Shehata, M. Mo-hammed, A. M. Zaitoun, I. Soomro, D. N. Lobo, R. Abbotts, S. Chan, S.Madhusudan, Br. J. Cancer 2010, 102, 704 – 709.

[7] B. Demple, T. Herman, D. S. Chen, Proc. Natl. Acad. Sci. USA 1991, 88,11450 – 11454.

[8] a) M. L. Fishel, Y. He, A. M. Reed, H. Chin-Sinex, G. D. Hutchins, M. S.Mendonca, M. R. Kelley, DNA Repair 2008, 7, 177 – 186; b) Y. Jiang, S.Zhou, G. E. Sandusky, M. R. Kelley, M. L. Fishel, Cancer Invest. 2010, 28,885 – 895.

[9] a) H. Fung, B. Demple, J. Biol. Chem. 2011, 286, 4968 – 4977; b) D. R. Mc-Neill, W. Lam, T. L. DeWeese, Y. C. Cheng, D. M. Wilson, Mol. Cancer Res.2009, 7, 897 – 906.

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�� These are not the final page numbers!

MED F. Aiello et al.

[10] a) D. Wang, D. B. Xiang, X. Q. Yang, L. S. Chen, M. X. Li, Z. Y. Zhong, Y. S.Zhang, Lung Cancer 2009, 66, 298 – 304; b) M. D. Naidu, J. M. Mason,R. V. Pica, H. Fung, L. A. Pena, J. Radiat. Res. 2010, 51, 393 – 404.

[11] a) V. Di Maso, C. Avellini, L. S. Croce, N. Rosso, F. Quadrifoglio, L. Cesarat-to, E. Codarin, G. Bedogni, C. A. Beltrami, G. Tell, C. Tiribelli, Mol. Med.2007, 13, 89 – 96; b) F. Puglisi, G. Aprile, A. M. Minisini, F. Barbone, P. Cat-aldi, G. Tell, M. R. Kelley, G. Damante, C. A. Beltrami, C. Di Loreto, Anti-cancer Res. 2001, 21, 4041 – 4049; c) Q. Sheng, Y. Zhang, R. Wang, J.Zhang, B. Chen, J. Wang, W. Zhang, X. Xin, Med. Oncol. 2012, 29, 1265 –1271.

[12] F. Puglisi, F. Barbone, G. Tell, G. Aprile, B. Pertoldi, C. Raiti, M. R. Kelley, G.Damante, A. Sobrero, C. A. Beltrami, C. Di Loreto, Oncol. Rep. 2002, 9,11 – 17.

[13] S. Sengupta, A. K. Mantha, S. Mitra, K. K. Bhakat, Oncogene 2011, 30,482 – 493.

[14] A. Jedinak, S. Dudhgaonkar, M. R. Kelley, D. Sliva, Anticancer Res. 2011,31, 379 – 385.

[15] a) Z. Zawahir, R. Dayam, J. Deng, C. Pereira, N. Neamati, J. Med. Chem.2009, 52, 20 – 32; b) R. I. Al-Safi, S. Odde, Y. Shabaik, N. Neamati, Curr.Mol. Pharmacol. 2012, 5, 14 – 35.

[16] P. T. Beernink, B. W. Segelke, M. Z. Hadi, J. P. Erzberger, D. M. Wilson III, B.Rupp, J. Mol. Biol. 2001, 307, 1023 – 1034.

[17] X. Fan, F. H. Zhang, R. I. Al-Safi, L. F. Zeng, Y. Shabaik, B. Debnath, T. W.Sanchez, S. Odde, N. Neamati, Y. Q. Long, Bioorg. Med. Chem. 2011, 19,4935 – 4952.

[18] C. Schmuck, U. Machon, Chem. Eur. J. 2005, 11, 1109 – 1118.[19] R. J. Herr, Bioorg. Med. Chem. 2002, 10, 3379 – 3393.[20] M. F. M. Esper�n, M. L. Fascio, N. B. D’Accorso, J. Heterocycl. Chem. 2002,

39, 221 – 224.[21] F. Fischer, K. Baerenfaller, J. Jiricny, Gastroenterology 2007, 133, 1858 –

1868.[22] M. Millard, D. Pathania, Y. Shabaik, L. Taheri, J. Deng, N. Neamati, PLoS

One 2010, 5, e13131.

Received: July 4, 2012

Published online on && &&, 0000

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MED3-Carbamoylbenzoic Acid Derivatives as APE1 Inhibitors

FULL PAPERS

F. Aiello,* Y. Shabaik, A. Esqueda,T. W. Sanchez, F. Grande, A. Garofalo,N. Neamati

&& –&&

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

Cancer in disrepair: The base-excisionrepair (BER) pathway is the foremostpathway responsible for removal and re-placement of damaged DNA bases. Sev-eral BER enzymes have altered expres-sion and activation in cancer cells, andamong them APE1 has emerged asa particularly attractive target for anti-cancer drug development, as demon-strated by studies that link its overex-pression with resistance to radio- andchemotherapy.

&16& www.chemmedchem.org � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 16

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