Bioorganic & Medicinal Chemistry Letters 27 (2017) 607–611

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/locate /bmcl

Design, synthesis, and biological evaluation of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs in MCF-7and MDA-MB-468 breast cancer cell lines

http://dx.doi.org/10.1016/j.bmcl.2016.12.0060960-894X/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors.E-mail addresses: youngjung@pusan.ac.kr (Y.-S. Jung), jhkwak@ks.ac.kr

(J.-H. Kwak).c These authors have contributed equally to the preparation of this manuscript.

Ye Jin Kim a,c, Jae Sung Pyo a,c, Young-Suk Jung b,⇑, Jae-Hwan Kwak a,⇑aCollege of Pharmacy, Kyungsung University, Busan 48434, Republic of KoreabCollege of Pharmacy, Pusan National University, Busan 46241, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 September 2016Revised 29 November 2016Accepted 2 December 2016Available online 5 December 2016

Keywords:Anti-breast cancer activityPyrazinoindolone scaffoldGefitinib-resistant TNBCInhibition of p-Akt

A series of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs were designedand synthesized for developing pyrazinoindolone scaffolds as anti-breast cancer agents. Compounds1h and 1i, having a furan-2-yl-methylamide and benzylamide group, respectively, exhibited more potentcytotoxicity in MDA-MB-468 triple-negative breast cancer (TNBC) cells than compounds possessing ali-phatic groups. Compounds 2a and 2b, as (R)-enantiomers of 1h and 1i, also had inhibitory activity againstMDA-MB-468 cells. Moreover, analogs (3a–b and 3d–e) bearing a benzyl group at the N-2 positionshowed more potent activity than gefitinib, as a potent EFGR-TK inhibitor. Especially, compound 3aexhibited selective cytotoxic activity against MDA-MB-468 cells; it also had a synergistic effect in com-bination with gefitinib against MDA-MB-468 cells. In addition, we confirmed that compounds 3a and 3dinhibit phosphorylation of Akt in MDA-MB-468 cells using western blot analysis. Therefore, these 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs may be helpful for investigating newanti-TNBC agents.

� 2016 Elsevier Ltd. All rights reserved.

Breast cancer is the most common cancer in women worldwide,and its incidence rate has continuously increased.1 As a heteroge-neous disease, breast cancer is classified into at least three majorsubtypes according to the gene expression states, including lumi-nal A, luminal B, and human epidermal growth factor receptor 2(HER2).2–4 Luminal breast cancers are positive for estrogen or pro-gesterone receptors and can typically be treated with hormonaltherapy (such as tamoxifen or aromatase inhibitors) that lowershormone levels or blocks hormone receptors.5 In contrast, HER2positive tumors, such as HER2 subtype and some luminal B sub-types with HER2+, have amplification and overexpression of theerbB2 oncogene. Therefore, HER2 positive tumors effectivelyrespond to anti-HER2 therapies (such as trastuzumab).3 Triple-negative breast cancer (TNBC) is reported as an aggressive andintractable subtype of breast cancer that is defined by the absenceof estrogen receptors, progesterone receptors, and HER2; approxi-mately 10 to 20% of women with breast cancer have TNBC.6,7

Generally, patients with TNBC respond poorly and progressrapidly, and their prognosis is very poor due to difficulties admin-istering hormone and HER-2 targeted therapies as well as a strongresistance to general anti-cancer agents. Approximately 20% ofTNBC responds to standard chemotherapy.3 Although, there isnot specific approved drug, various clinical trials of targeted ther-apies are in progress for treatment of TNBC.8,9 Therefore, the devel-opment of an effective targeted therapy for TNBC is one of thehighest priorities of current breast cancer research.

In recent studies, TNBC has frequently been shown to highlyexpress epidermal growth factor receptor (EGFR) and to be moresensitive to EGFR inhibition than luminal type breast cancersare.10–12 Thus, overexpressed EGFR is considered an important tar-get for treatment of TNBC. Especially, several EGFR-targetedagents, including monoclonal antibodies such as cetuximab andpanitumumab and small-molecule kinase inhibitors such as gefi-tinib and erlotinib, have been approved for clinical use against var-ious human cancers.13 However, these EGFR inhibitors are oflimited benefit in EGFR overexpressed TNBC because Akt andHER3 signaling pathways act as feedback loops that can give riseto acquired resistance.9 Due to the limitation of EGFR inhibitorsin TNBC, monotherapy using cetuximab results in a lower rate ofresponse than combinational therapy with cetuximab and

NHOH

O∗

NH2 HClOMe

O

HO

6

NH ∗NH

O

OMe

OOH

N ∗NH

O

OMe

O

serine methyl ester 4 (S form) / 5 (R form)

9 (S form) / 10 (R form)

7 (S form) / 8 (R form)

N ∗NH

O

HN

OR

1a-w (S form)2a and 2b (R form)

NNH

O

OH

O11

NNH

O

HN

OR

1a1b1c1d1e1f1g

R= Me-R= Pr-R= i-Pr-R= Hex-R= Cy-R= Allyl-R= Ph-

NNH

O

HN

O

XR1

NNH

O

HN

O

R2

R3

1h1j1k

X= O-X= O-X= S

R1= Me-R1= H

R1= H

1i1l1m1n1o1p

R2= FR2= H

R2= H

NNH

O

HN

OOH

1q1r1s1t1u1v

R2= HR2= OMe

R2= Me

; R3= H; R3= H

; R3= OMe

; R3= Me; R3= H

; R3= H

R2= H

R2= H

R2= HR2= Cl

R2= CF3

; R3= H; R3= NH3

; R3= Cl

; R3= CF3

; R3= H

; R3= H

R2= NH3

NNH

O

HN

O

Ar

2a2b

Ar= 2-furanyl-Ar= Ph-

1w

a b

c d

e

Scheme 1. General methods for preparing pyrazinoindolone analogs. Reagents andconditions: (a) EDC, HOBt, Et3N, CH2Cl2, 18 h, 71–98%; (b) DEAD, PPh3, THF, 24 h,32–54 %; (c) LiOH, H2O, 1 h, 48%; (d) R-NH2, EDC, HOBt, Et3N, DMF, 18 h, 11–13%;(e) R-NH2, MeOH, 15 h, 10–66%.

608 Y.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 607–611

carboplatin (a platinum-based antineoplastic agent) does, in clini-cal trials.9,14 Therefore, we aimed to discover and develop novelagents to overcome the limitations of current EGFR-tyrosine kinase(EGFR-TK) inhibitors by using TNBC cells that overexpress EGFRand are resistant to EGFR-TK inhibitors such as a gefitinib.

In many studies, N-heterocyclic ring systems such as indole andpyrazine moieties serve as pharmacophores in various compoundswith biological and pharmacological activities (Fig. 1a). Forinstance, indolypyrazine analogs have anti-proliferative activityagainst various human tumor cell lines including breast cancercells (GI50 value = 0.29–0.82 lM).15 Further, the natural productma’edamine A and its analogs having a unique pyrazine-2-(1H)-one exhibit cytotoxic activity against breast cancer cell lines.16

Especially, compounds possessing pyrazinoindolone have beenreported to be potent MK2 (MAPKAP-K2; mitogen-activated pro-tein kinase-activated protein kinase 2) inhibitors.17 Based on theirstructures, we designed and synthesized a library of 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs (series 1–3) by hybridizing with a pyrazinone and indole ring. In addition,we attempted to establish structure–activity relationships (SAR)according to the substituents of the amide, stereo–configurationon C-3, and alkyl group on N-2 (Fig. 1b).

For the target (S)- and (R)-configuration of N-substituted-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide (series 1and 2, respectively), the general synthetic procedure is outlinedin Scheme 1. The commercially available starting materials L-serinemethyl ester (4) and D-serine methyl ester (5) were treated withindole-2-carboxylic acid in the presence of HOBt (1-hydroxylben-zotriazole), EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide), and trimethylamine in CH2Cl2 to obtain amides 7 and 8,respectively. To prepare methyl ester 9 and 10 with a cyclizedpyrazinoindolone core, an intramolecular Mitsunobu reaction

N NH

OHN

O

N

SNH

O

N

MK2 inhibitor (Pyrazinoindolone; IC50 = 2 nM)

N N

NH2N

BrAntitumor agent(Indolypyrazine)

GI50 = 0.29 ~ 0.82 μM (against breast cancer)

1-Oxo-1,2,3,4-tetrahydro-pyrazinoindole analogs

NNH

O

Indole5,6-Dihydro

-pyrazin-2-one

hybridizationNHN ∗

N

OR'

HN

OR

1

34

2

b) Design

a) Bioactive Compounds

Ts

N

HNO

OMe

Br

BrO

Br

N

Ma'edamine A (pyrazine-2-(1 H)-one )c-erbB-2 kinase inhibitor IC50 = 6.7μg/mL

N ∗NH

O

HN

OR

NN

O

HN

OR

R'

S form: Series 1 (1a-w )R form: Series 2 (2a-b )

Introduction of substituentsSeries 3 (3a-g )

R'= BnMeAllyl

Fig. 1. (a) Biologically active compounds having an indolypyrazine,15 pyrazine-2-(1H)-one,16 and pyrazinoindolone core17; (b) The design of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs.

was performed from compound 7 and 8 using DEAD (diethyldiazenedicarboxylate) and triphenylphosphine in tetrahydrofuran.Generally, targeted N-substituted-1-oxo-1,2,3,4-tetrahydropy-razino[1,2-a]indole-3-carboxamide analogs were concisely pre-pared through direct transamidation from methyl ester 9 and 10with various primary amines. Hydrolysis of methyl ester 9 fol-lowed by peptide coupling with EDC and HOBt afforded compound1d and 1g, having an N-hexyl and N-phenyl group, respectively(Scheme 1).

Cell viability of the newly synthesized library of (S)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs(series 1) was evaluated in the luminal type breast cancer cell line,MCF-7, and the EGFR over-expressed TNBC cell line,18 MDA-MB-468, using a dose dependent MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Table 1). In particular,MDA-MB-468 cell lines have a gefitinib resistance, due to existingamplification of the Akt pathway and lack of PTEN (phosphataseand tensin homolog), resulting in feedback loops.9,19

As shown in Table 1, compounds in series 1 generally haveweak activity in MCF-7 cells regardless of substituents on theamide nitrogen atom. However, five compounds possessing afuran-2-yl-methylamide (1h: 39.7%), benzylamide (1i: 48.8%), 5-methyl-furan-2-yl-methylamide (1j: 43.0%), thiophen-2-yl-methy-lamide (1k: 39.2%), and 3-chlorobenzylamide (1s: 34.8%) grouphad more potent cytotoxic activity in the MDA-MB-468 cell linethan compounds with other substituted groups did. Especially,(S)-N-(furan-2-yl-methyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide (1h) (GI50: 23.1 lM) exhibited the bestactivity, but was less potent than gefitinib was, which was usedas a reference compound.

In the preliminary SAR study, we determined that compoundsbearing an aryl methyl group on the amide nitrogen atom exhibitmore potent cytotoxicity than compounds with an alkyl (such asmethyl, propyl, isopropyl, hexyl, and cyclohexyl), allyl, phenyl,

Table 1Anticancer activity of 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs (series 1 and 2) against the MCF-7 and MDA-MB-468 cell lines.

Comp. MCF-7 Cell viability at30 lM (% of untreatedcontrol)

MDA-MB-468 Cell viability at 30 lM(% of untreated control) (aGI50 value)

Comp. MCF-7 Cell viability at 30 lM (% ofuntreated control) (aGI50 value)

MDA-MB-468 Cell viability at 30 lM(% of untreated control) (aGI50 value)

Ctrl 100.0 ± 4.7 100.0 ± 1.5 1m 68.6 ± 6.0 70.5 ± 2.69 85.1 ± 7.6 101.5 ± 2.0 1n 68.8 ± 4.9 74.7 ± 3.911 99.7 ± 10.2 76.2 ± 19.6 1o 78.1 ± 4.1 66.3 ± 2.71a 77.5 ± 12.0 82.6 ± 6.0 1p 68.0 ± 3.4 61.5 ± 2.91b 83.9 ± 9.2 88.0 ± 5.3 1q 70.5 ± 5.4 67.7 ± 2.21c 84.4 ± 5.8 88.1 ± 6.2 1r 76.0 ± 4.1 65.4 ± 2.21d 71.2 ± 5.0 61.8 ± 5.1 1s 61.7 ± 4.1 34.8 ± 0.7 (23.4 lM)1e 80.7 ± 12.9 73.1 ± 11.6 1t 66.1 ± 9.3 60.3 ± 1.91f 104.5 ± 7.3 94.7 ± 4.5 1u 59.1 ± 2.8 62.4 ± 2.91g 87.3 ± 11.6 74.5 ± 4.9 1v 60.2 ± 1.6 70.5 ± 4.11h 62.1 ± 7.7 39.7 ± 2.5 (23.1 lM) 1w 85.5 ± 5.5 63.1 ± 2.21i 71.4 ± 5.1 48.8 ± 5.4 (28.9 lM) 10 79.7 ± 3.9 82.4 ± 2.51j 60.2 ± 3.0 43.0 ± 1.3 2a 82.5 ± 1.3 21.2 ± 1.7 (15.9 lM)1k 61.7 ± 1.6 39.2 ± 1.0 2b 61.8 ± 6.9 45.0 ± 3.0 (24.0 lM)1l 59.7 ± 14.2 63.1 ± 2.5 Gefitinib 16.2 ± 1.3 (20.8 lM) 6.7 ± 0.1 (9.7 lM)

a GI50 values are reported as the mean of four experiments and correspond to the agent’s concentration that causes a 50% decrease in net cell growth.

N ∗

NH

O

OMe

O9 (S form)

NN

O

HN

O

XR1

NN

O

HN

O

R2

R3

3a3c3d3f3g

X= O;-X= O;-;X= S;X= O;-;X= O;-;

R1= Me-R1= H

R1= H

3b

3e

R2= H

R2= H

; R3= H

; R3= Cl

NN

OR'

OMe

O(R'= Bn) (R'= Me) (R'= Allyl)

NN

OBn

HN

OR

3a-g

R'

Bn

a b

121314

R'= Bn;-R'= Bn;R'= Bn;R'= Me;R'= Allyl;

R1= HR1= H

Scheme 2. Synthetic methods to prepare benzylated pyrazinoindolone analogs(series 3). Reagents and conditions: (a) alkyl halide (BnBr, MeI or allyl-Br), K2CO3,DMF, 5 h, 27–38%; (b) R-NH2, MeOH, 15 h, 35–78%.

Table 2Cytotoxic activity of (S)-2-alkyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-car-boxamide analogs (series 3) against the MCF-7 and MDA-MB-468 cell lines.

Comp. MCF-7 MDA-MB-468

Cell viability at 10 lM (% ofuntreated control) (aGI50 value)

Cell viability at 10 lM (% ofuntreated control) (aGI50 value)

Ctrl 100.0 ± 4.7 100.0 ± 1.512 92.6 ± 6.3 98.8 ± 4.63a 58.5 ± 4.3 (19.3 lM) 23.3 ± 3.6 (5.2 lM)3b 47.1 ± 2.7 (9.6 lM) 28.0 ± 0.8 (7.6 lM)3c 68.7 ± 6.3 63.1 ± 3.53d 35.8 ± 2.3 (4.6 lM) 18.5 ± 1.8 (3.8 lM)3e 38.2 ± 2.1 (8.3 lM) 46.7 ± 3.5 (9.5 lM)3f 54.1 ± 3.0 56.1 ± 2.3 (15.0 lM)3g 98.2 ± 10.9 58.4 ± 0.8 (18.5 lM)Gefitinib 89.7 ± 7.6 (20.8 lM) 49.1 ± 3.6 (9.7 lM)

a GI50 values are reported as the mean of four experiments and correspond to theagent’s concentration that causes a 50% decrease in net cell growth.

Y.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 607–611 609

and homobenzyl group do. Especially, three compounds (1h, 1j,and 1k) that included an aromatic five membered heterocycle sys-tem such as a furan and thiophene ring had moderate activity.With the exception of the meta-chloro group (1s), m- or p-sub-stituent on phenyl ring (R2 or R3) decreased the activity in 12 ana-logs, 1i–v. Thus, only compound 1i and 1s exhibited valuablecytotoxic activity.

To explore the SAR of stereo-conformation in order to deter-mine cytotoxic activity in EGFR overexpressed TNBC cells, wedesigned and prepared compounds 2a ((R)-N-(furan-2-ylmethyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide)and 2b ((R)-N-benzyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]in-dole-3-carboxamide) in the (R) form on the C-3 position. Com-pounds 2a and 2b showed low biological activity against theMCF-7 cell line. In contrast, GI50 values of compounds 2a and 2bwere 15.9 and 24.0 lM in the MDA-MB-468 cell line, respectively,and were as potent as the (S)-enantiomers 1h (GI50: 23.1 lM) and1i (GI50: 28.9 lM). In brief, the cytotoxic activity of compounds 2aand 2b was maintained in the MDA-MB-468 cell line regardless ofstereo-configuration on C-3 position.

Based on preliminary SAR studies of series 1 and 2, we preparedcompounds that introduced a substituent at N-2 position of com-pound 1h–k and 1s. Benzylated (3a–e), methylated (3f), and ally-lated analogs (3g) at N-2 position of (S)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamides were preparedfrom compound 9 through alkylation with benzylbromide, iodo-methane, or allylbromide and transamidation using aryl methy-lamine (Scheme 2).

In sequence, cell viability at a concentration of 10 lM and GI50values of the prepared seven compounds (3a–g) were evaluated inthe MCF-7 and MDA-MB-468 cell lines (Table 2). Benzylated com-pounds (3a–b and 3d–e) were more cytotoxic in MCF-7 cells thangefitinib, compounds 1h–k and 1s (without a benzyl group at theN-2 position) and compounds 3f and 3g (with a methyl and allylgroup at the N-2 position, respectively) were, with the exceptionof (S)-2-benzyl-N-((5-methylfuran-2-yl)methyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide (3c: 68.7% cellviability at 10 lM). Especially, the inhibitory activity of compound3d (GI50: 4.6 lM) was approximately 4.5-fold stronger than that ofgefitinib (GI50: 20.8 lM). Similarly, all compounds with the excep-tion of 3c, exhibited potent cytotoxic activity against MDA-MB-468cells. Compounds 3a, 3b, and 3d had GI50 values of 5.2, 7.6, and3.8 lM, respectively, and more potent activity than that of theEGFR-TK inhibitor, gefitinib (GI50: 9.7 lM). However, compounds3f (GI50: 15.0 lM) and 3g (GI50: 18.5 lM) exhibited less potent

activity than gefitinib. Compound 3d possessed the best cytotoxicactivity against both MCF-7 (GI50: 4.6 lM) andMDA-MB-468 (GI50:3.8 lM) cell lines.

In the evaluation of their cytotoxic activity, we confirmed thatintroducing a benzyl group at the N-2 position increased anti-breast cancer activity in all compounds except 3c, which bears amethylated furan-2-yl-methylamide group. In addition,

Fig. 2. (a) Combination treatment of gefitinib with or without compound 3a (1 lM) in the MCF-7 and MDA-MB-468 cell lines; (b) Dose matrix of combinational treatmentwith compound 3a and gefitinib in the MDA-MB-468 cell line; (c) Isobologram analysis based on data in the dose matrix; (d) Western blot analysis of phosphorylated Akt andphosphorylated ERK as downstream of the EGFR signaling pathway.

610 Y.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 607–611

compounds 3g and 3f having methyl and allyl group, respectively,also exhibited more potent cytotoxic activity than compound 1hwithout substituent at the N-2 position only in MDA-MB-468 cells.Interestingly, the cytotoxic activity of compound 3a was 3.7-foldmore potent against MDA-MB-468 cells (GI50: 5.2 lM) than againstMCF-7 cells (GI50: 19.3 lM).

Recently, combinational treatment with an EGFR–TK inhibitorand regulator of EGFR downstream signaling, such as the PI3K/Akt and Ras/ERK pathways, resulted in a synergistic effect againstTNBC.20,21 Thus, we confirmed the combinational effects of com-pound 3a and gefitinib. Compound 3a (1 lM) in combination withgefitinib (1 lM, 3 lM, and 10 lM) resulted in an antagonistic effectin the MCF-7 cell line. However, the combinational treatmentincreased cytotoxicity in the MDA-MB-468 cell line over that ofgefitinib alone (Fig. 2a). For verification of the synergistic effectof compound 3a with gefitinib, we prepared a dose matrix in theMDA-MB-468 cell line using multiple concentrations (0 lM,1 lM, 3 lM, and 10 lM) of compound 3a and gefitinib (Fig. 2b).In sequence, we converted the dose-response matrix to a two-dimensional isobologram22,23 of GI50 values (Fig. 2c). Through anal-ysis of the isobologram graph, we confirmed that compound 3aexhibits synergistic effects with gefitinib.

Finally, western blot analysis was performed in MDA-MB-468cells treated with compounds 3a, 3d, and gefitinib in order to con-firm the role of EGFR overexpressed cancer cells in the synergisticeffects. Specifically, we evaluated the phosphorylation of Akt (p-Akt) and ERK (p-ERK), as downstream of the EGFR signaling path-way, after treatment with compounds 3a, 3d, and gefitinib. After6 h, gefitinib decreased p-Akt and dramatically inhibited p-ERKin the MDA-MB-468 cell line. Compounds 3a and 3d only inhibitedp-Akt. After 18 h, gefitinib also exhibited a potent inhibitory effecton p-ERK, and compounds 3a and 3d continuously reduced p-Akt.However, p-Akt was increased regardless of gefitinib treatment.These results confirm that the signaling pathway downstreamfrom EGFR, Akt in particular, was suppressed. Further, the syner-gistic effects resulted from dual inhibition of EGFR-TK and thepathway associated with phosphorylation of Akt.

In conclusion, a series of 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamides (series 1 and 2) and (S)-2-alkyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamides (series

3) was designed and synthesized from commercially availablestarting materials via the well-known Mitsunobu reaction andamidation reactions using EDC. Compounds 1h and 1i exhibitedmoderate cytotoxic activity and selectivity against MDA-MB-468cells. Compound 2a and 2b, as (R)-enantiomers of 1h and 1i,respectively, also showed inhibitory activity against MDA-MB-468 cells. These (R)-enantiomers had slightly improved anti-TNBCeffect as compared with (S)-enantiomers. In the SAR studies, com-pounds bearing an aryl methyl group on the amide nitrogen atomexhibited more potent cytotoxic activity than compounds with analkyl (such as methyl, propyl, isopropyl, hexyl, and cyclohexyl),allyl, phenyl, and homobenzyl group did. Especially, the furan-2-yl-methyl, benzyl, 5-methyl-furan-2-yl-methyl, thiophen-2-yl-methyl, and 3-chlorobenzyl groups enhanced the cytotoxicityagainst MDA-MB-468 cells. In addition, benzylated 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs havingfuran-2-yl-methylamide (3a; GI50: 5.2 lM), benzylamide (3b;GI50: 7.6 lM), and thiophen-2-yl-methylamide (3d; GI50: 3.8 lM)showed more potent activity than gefitinib (GI50: 9.7 lM), anEGFR–TK inhibitor, did. Interestingly, compound 3a exhibited moreselective cytotoxicity against MDA-MB-468 cells than againstMCF-7 cells. In addition, 3a showed synergism with gefitinib inMDA-MB-468 cells. Using western blot analysis, we confirmed thatcompounds 3a and 3d inhibit phosphorylation of Akt in the MDA-MB-468 cell line. Our studies have encouraged us to investigateadditional novel lead scaffolds exhibiting potent and selectiveanticancer activity against EGFR overexpressed TNBC; this workis in progress.

Acknowledgments

We are grateful to the Basic Science Research Program throughNational Research Foundation of Korea (NRF) funded by the Min-istry of Science, ICT & Future Planning (NRF-2015R1C1A1A01056060).

A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12.006.

Y.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 607–611 611

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