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European Journal of Medicinal Chemistry 57 (2012) 112e125

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis and biological activities of polyquinoline derivatives: New Bcl-2 familyprotein modulators

Emmanuelle Saugues a,b, Anne-Laure Debaud c,d,e, Fabrice Anizon a,b, Nathalie Bonnefoy c,d,e,**,1,Pascale Moreau a,b,*,1

aClermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, FrancebCNRS, UMR 6296, ICCF, BP 80026, 63171 Aubière, FrancecUniversité de Lyon, Franced Inserm, U851, 21 avenue Tony Garnier, Lyon F-69007, FranceeUniversité Lyon 1, UMS3444/US8, France

a r t i c l e i n f o

Article history:Received 29 June 2012Received in revised form5 September 2012Accepted 7 September 2012Available online 17 September 2012

Keywords:QuinolineSuzuki couplingBcl-2 family protein interactions modulatorsIn vitro pro-apoptotic activities

* Corresponding author. Clermont Université, UniveChimie de Clermont-Ferrand, BP 10448, 63000 Clerm(0) 4 73 40 79 63; fax: þ33 (0) 4 73 40 77 17.** Corresponding author. Université Lyon 1, UMS34437 28 23 72.

E-mail addresses: [email protected]@univ-bpclermont.fr (P. Moreau).

1 NB and PM equally contributed to this work aauthorship.

0223-5234/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2012.09.012

a b s t r a c t

The synthesis of quinoline derivatives, designed to interact with Bcl-xL, and to inhibit its interaction withpro-apoptotic partners, is described and their biological effects investigated. We showed that 5 out of 28synthetized compounds restored cell death of 3T3 cells overexpressing Bcl-xL following serum starvation.Active compounds were further characterized for their binding capacity to the anti-apoptotic member ofthe Bcl-2 family, Bcl-xL as well as Bcl-2, Bfl-1 and Mcl-1, and for their pro-apoptotic activities towardlymphoid tumor cells and peripheral blood mononuclear cells. Altogether our results indicate thatdimeric, rather than trimeric quinoline derivatives, represent a new attractive class of BH3 mimetics forcancer therapy.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Bcl-2 (B-cell lymphoma 2) family members are importantregulators of apoptosis that can be divided into three groups [1].One group protects against apoptosis and is comprised of fivedifferent proteins: Bcl-2 itself, Mcl-1 (myeloid cell leukemia 1), Bcl-xL (B-cell lymphoma extra long), Bcl-w (B-cell lymphoma w), andBfl-1 (B-cell lymphoma-2-related gene expressed in fetal liver). Thesecond group of proteins is represented by Bax (Bcl-2 associatedprotein X) and Bak (Bcl-2 antagonist/killer). Although structurallyvery similar to the pro-survival members, Bax and Bak are the mainactivators of the apoptosis machinery in response to cellular stressstimuli. The third group is comprised of a heterogeneous group ofproteins e the “Bcl-2 Homology-3 domain (BH3)-only proteins” e

rsité Blaise Pascal, Institut deont-Ferrand, France. Tel.: þ33

4/US8, France. Tel.: þ33 (0) 4

rm.fr (N. Bonnefoy),

nd therefore share the last

son SAS. All rights reserved.

and includes such members as Bid (BH3 interacting death domain),Bim (Bcl-2 interacting mediator), Puma (Bcl-2 binding component3), Bik (Bcl-2 interacting killer), Bad (Bcl-2-associated deathpromoter), and others. The BH3-only proteins act to coupleupstream cellular stress stimuli to responses by Bcl-2 pro-survivalproteins and Bax, Bak death proteins [2,3]. How these three groupsintegrate cell signaling into the decision to live or die is notcompletely understood and the mechanism remains controversial.However, key elements have been elucidated, which have uncov-ered a way to therapeutically modulate their behavior [4]. It isknown, for example, that pro-survival members can bind pro-apoptotic members through the docking of the a-helix of the BH3domain of death members into a deep groove on the surface ofsurvival members [5e7]. If the pro-survival members are in excessin the cell, the pro-apoptotic members cannot trigger apoptosis dueto these proteineprotein interactions. An abnormal apoptosisregulation by overexpression of the Bcl-2 pro-survival membershas been noticed in many cancers. Overcoming altered apoptosisleading to the resistance of tumor cells to conventional chemo-therapy is an attractive approach for anticancer therapy. Thus, thedevelopment of new drugs that can inhibit the action of anti-apoptotic Bcl-2 members is very attractive [8e10]. One strategy isthe synthesis of pro-apoptotic protein BH3 domain mimics that are

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E. Saugues et al. / European Journal of Medicinal Chemistry 57 (2012) 112e125 113

able to interact with the hydrophobic groove of their anti-apoptoticcounterparts. Consequently, we recently developed a researchprogram concerning the synthesis and biological activitiesof quinoline derivatives (Fig. 1) designed to interact with Bcl-xL[11e14].

Compound B, inhibited Bcl-xL/Bak, Bcl-xL/Bax and Bcl-xL/Bidinteractions with IC50 values around 25 mM while compound Ainhibited the Bcl-xL/Bid interaction with an IC50 value of 21 mM andcompound I inhibited the Bcl-xL/Bim interaction with an IC50 of29 mM [13], showing that this series could be of potential interestfor the development of new Bcl-2 anti-apoptotic group inhibitors.Thus, to enlarge the structural diversity of the quinoline derivativesstudied, the preparation of various analogs bearing pyrrolidinyl ormorpholinyl groups was undertaken. In addition, in vitro activitiesof these new di- and trimeric quinoline analogs have been evalu-ated toward 3T3 cells that overexpress Bcl-xL (Bcl-xL-3T3) as wellas toward lymphoma and leukemic cell lines known to expressBcl-xL and other Bcl-2 anti-apoptotic proteins such as Bcl-2, Bfl-1and Mcl-1.

2. Results and discussion

As already described [12e14], polymeric quinoline derivativesare easily prepared by Suzuki cross-coupling reactions betweenheteroaromatic boronic acids and bromoheteroaromatic deriva-tives. First, we decided to prepare analogs with the amino group(pyrrolidin-1-yl or morpholin-4-yl) located at the right-end oftrimeric quinoline derivatives (compounds 8e11, Scheme 1).

Thus, the required bromo monomeric moieties 4 and 5 wereprepared in good yields by substitution of the chlorine atom ofcompound 1 [13] using either pyrrolidine or morpholine and thencatalytic hydrogenation of the propenyl double bound. Trimericquinoline derivatives 8e11 were obtained in reasonable yields by

AN

N

N

OEt

OMe

OMe

N OR'

N OR'

R N OR'

R''

C R = H,D R = H,E R = iPr,F R = iPr,G R = iPr,H R = iPr,

R' = Et,R' = Pr,R' = Et,R' = Pr,R' = Et,R' = Pr,

R'' = iPr,R'' = iPr,R'' = H,R'' = H,R'' = iPr,R'' = iPr,

4

3

7'

8'

4'

3'

7''

8''

IC50 Bcl-xL/Bak = 74 µMIC50 Bcl-xL/Bax = 243 µMIC50 Bcl-xL/Bid = 21 µMIC50 Bcl-xL/Bim = 47 µMIC50 Bcl-xL/PUMA = 54 µM

Fig. 1. Structure and biological activities of di- and trimeric q

coupling of bromoquinolines 4 or 5 and boronic acids producedfrom biquinolines 6 [14] or 7 [14] in the presence of lithium dii-sopropylamide and triisopropylborate and subsequent hydrolysis.The Suzuki reactionwas performed under microwave irradiation inthe presence of PdCl2(PPh3)2 and sodium carbonate (Scheme 1).

Then, we prepared compounds 21e26 with the amino group(pyrrolidin-1-yl or morpholin-4-yl) located at the left-end of thetrimeric quinoline derivatives (Scheme 2).

The dimers 15 and 16 were prepared by Suzuki cross-couplingbetween the bromoquinolines 13 and 14 that were alreadydescribed [14] and the 2-chloroquinolin-3-boronic acid 12 obtainedby lithiationeborylation of 2-chloroquinoline [15]. Suzuki couplingwas achieved using palladium acetate and tri-tert-butylphosphine.The introduction of the amino function was performed, asdescribed for the preparation of 2 and 3, by nucleophilic aromaticsubstitution of the chlorine atom using either pyrrolidine or mor-pholine. Finally, trimers 21e26 were prepared using the methoddescribed above for trimers 8e11. Thus, coupling reaction betweenbromoquinolines 13 or 14 and boronic acids produced from biqui-nolines 17e20 led to trimers 21e26 in 43e98% yields (Scheme 2).

Finally, compounds 27e34 presenting amino groups (pyrrolidin-1-yl ormorpholin-4-yl) at bothendsof the trimerswere synthesizedusing the same procedure as the one described for 21e26 in thepresence of bromoquinoline 4/5 instead of 13/14 (Scheme 3).

In vitro biological activities of compounds 8e11, and 17e34werefirst tested by evaluating their capacities to inhibit Bcl-xL pro-survival activity in Bcl-xL-3T3 cells following serum starvation(Table 1).

As shown Table 1, none of the trimeric quinoline derivativetested (compounds 8e11 and 21e34) induced cell death of Bcl-xL-3T3 cells. Only three dimeric analogs (compounds 17, 18 and 20)bearing both alkoxy and amino substituents strongly decreasedviability of Bcl-xL-3T3 cells. Indeed, Bcl-xL-3T3 cells treated with

N

N

N

OMe

OMe

OMe

B

N

N

OMe

OMe

I

IC50 Bcl-xL/Bak > 1 mMIC50 Bcl-xL/Bax > 1 mMIC50 Bcl-xL/Bid > 1 mMIC50 Bcl-xL/Bim = 29 µMIC50 Bcl-xL/PUMA > 1 mM

IC50 Bcl-xL/Bak = 25 µMIC50 Bcl-xL/Bax = 26 µMIC50 Bcl-xL/Bid = 27 µMIC50 Bcl-xL/Bim = 92.5 µMIC50 Bcl-xL/PUMA = 42 µM

uinoline derivatives previously described by our group.

N ClBr

N OR

N OR

N NR1R2Br

N OR

N OR

N NR1R2

N NR1R2Br

6, R = Et7, R = Pr

8, R = Et, NR1R2 = pyrrolidin-1-yl, 79%9, R = Pr, NR1R2 = pyrrolidin-1-yl, 49%10, R = Et, NR1R2 = morpholin-4-yl, 77%11, R = Pr, NR1R2 = morpholin-4-yl, 73%

a

1

2, NR1R2 = pyrrolidin-1-yl, 94%3, NR1R2 = morpholin-4-yl, 91%

b

4, NR1R2 = pyrrolidin-1-yl, 77%5, NR1R2 = morpholin-4-yl, 73%

c, d, e

Reagents and conditions: (a) Pyrrolidine or morpholine; (b) H2, PtO2, EtOAc; (c) LDA, B(OiPr)3, THF; (d) aq. NH4Cl; (e) 4 or 5, PdCl2(PPh3)2, 2 M aq. Na2CO3, THF, w.µ

Scheme 1. Synthesis of compounds 8e11.

E. Saugues et al. / European Journal of Medicinal Chemistry 57 (2012) 112e125114

5 mM solution of compounds 17,18 and 20 have shown residual cellviability of 0.4%, 37.9%, 32.7%, respectively, suggesting that the threedimeric quinolines inhibited pro-survival activity of Bcl-xL inserum-starved 3T3 cells.

N Cl

B(OH)2

N ORBr

N NR1R2

N OR

N O

N

15, R16, R

21, NR1R2 = pyrrolidin-1-yl, R = Et, R' = Et, 70%22, NR1R2 = pyrrolidin-1-yl, R = Pr, R' = Pr, 98%23, NR1R2 = pyrrolidin-1-yl, R = Et, R' = Pr, 43%24, NR1R2 = pyrrolidin-1-yl, R = Pr, R' = Et, 82%25, NR1R2 = morpholin-4-yl, R = Pr, R' = Pr, 72%26, NR1R2 = morpholin-4-yl, R = Pr, R' = Et, 68%

a

c, d, e

12

+

13 R = Et14 R = Pr

Reagents and conditions: (a) Pd(OAc)2, P(tBu)3, dTHF; (d) aq. NH4Cl; (e) 13 or 14, PdCl2(PPh3)2, 2 M

Scheme 2. Synthesis of

As dimeric species were found to be more active than thetrimeric ones (except for compound 19 bearing pyrrolidinyl andpropoxy groups at the 2- and 20-positions, respectively) weprepared additional dimers (Scheme 4).

R'

Cl

N OR b

N NR1R2

N OR

= Et, 57%= Pr, 56%

17, NR1R2 = pyrrolidin-1-yl, R = Et, 68%18, NR1R2 = morpholin-4-yl, R = Et, 65%19, NR1R2 = pyrrolidin-1-yl, R = Pr, 92%20, NR1R2 = morpholin-4-yl, R = Pr, 75%

ioxane/H2O; (b) NHR1R2; (c) LDA, B(OiPr)3, aq. Na2CO3, THF, w.µ

compounds 17e26.

N NR1R2

N OR

N NR1R2

N OR

N NR3R4

17 NR1R2 = pyrrolidin-1-yl, R = Et18 NR1R2 = morpholin-4-yl, R = Et19 NR1R2 = pyrrolidin-1-yl, R = Pr20 NR1R2 = morpholin-4-yl, R = Pr

27, NR1R2 = NR3R4 = pyrrolidin-1-yl, R = Et, 61%28, NR1R2 = NR3R4 = pyrrolidin-1-yl, R = Pr, 51%29, NR1R2 = NR3R4 = morpholin-1-yl, R = Et, 96%30, NR1R2 = NR3R4 = morpholin-4-yl, R = Pr, 40%31, NR1R2 = pyrrolidin-1-yl, R = Et, NR3R4 = morpholin-4-yl, 72%32, NR1R2 = pyrrolidin-1-yl, R = Pr, NR3R4 = morpholin-4-yl, 82%33, NR1R2 = morpholin-4-yl, R = Et, NR3R4 = pyrrolidin-1-yl, 48%34, NR1R2 = morpholin-4-yl, R = Pr, NR3R4 = pyrrolidin-1-yl, 57%

a, b, c

Reagents and conditions: (a) LDA, B(OiPr)3, THF; (b) aq. NH4Cl; (c) 4 or 5, LDA, PdCl2(PPh3)2, 2 M aq. Na2CO3, THF, µw.

Scheme 3. Synthesis of compounds 27e34.


Compound 35 prepared in 88% yield by hydrogenation of 1 inthe presence of PtO2 was engaged in a Suzuki cross-couplingreaction with 3-boronic acid derivatives of 36 and 37 [14] to give38 and 39 in reasonable yields. Subsequent substitution of thechlorine atom by pyrrolidine or morpholine led to the corre-sponding alkoxy amino dimeric quinoline derivatives 40e43.

Additionally, in order to enlarge the structural diversity of thedimers tested, compounds 45 and 46 bearing two amino groupswere easily prepared from 44 yielded by Suzuki cross-couplingbetween bromoquinoline 35 and 2-chloroquinolin-3-boronicacid 12.

Then, the activity of new dimeric quinoline derivatives 40e43,45 and 46 was evaluated for their capacity to restore cell death ofBcl-xL-3T3 following serum starvation (Table 2). As reported inTable 2, with the exception of compounds 40 and 42, all the newdimers tested were again more active than the trimeric derivativespresented above. More particularly, 5 mM solutions of compounds41 and 45 efficiently promote cell death of Bcl-xL-3T3 cells withrespectively, 55.5% and 4.37% of residual cell viability at 96 h. As thebest active compounds of this series were found to be 17 and 45, thepreferred scaffold identified was a dimeric quinoline structuresubstituted at the 50-position by an isopropyl group, at the 2-position by a pyrrolidinyl moiety, and either by an ethoxy (17) oranother pyrrolidinyl group (45) at the 20-position.

In order to further characterize biological activity of the dimericquinoline derivatives 17, 18, 20, 41 and 45, we next tested theirability to induce apoptosis of the diffused large B-cell lymphoma

Table 1Cell death induction by compounds 8e11 and 17e34. % Of residual Bcl-xL-3T3 cellsviability with compounds tested at 5 mM.

Cpds % Of residualBcl-xL-3T3 viability


8 92.3 24 94.59 94.6 25 92.310 93.6 26 95.211 92.9 27 95.117 0.42 28 90.518 37.9 29 96.519 84.4 30 81.620 32.7 31 91.021 95.3 32 96.522 92.8 33 92.223 96.4 34 93.4

cell line BP3, the B lymphoblastoid cell line IM9 and the leukemiccell line RS4.11. We observed that compound 17 very efficientlyinduced apoptosis of IM9 and RS4.11 cell lines at 10 mM concen-tration (Fig. 2), while it showed a lower pro-apoptotic effect towardBP3 cells and showed very low toxicity toward normal peripheralblood mononuclear cells (PBMC) at the same concentration(Fig. 3A). With the exception of compound 20 that also slightlyreduced viability of IM9 cells, other compounds did not signifi-cantly induced apoptosis of lymphoid tumor cell lines (Fig. 2).

We then compared the activity of compound 17 toward BP3, IM9and RS4.11 cell lines with three other known inhibitors of Bcl-2anti-apoptotic molecules, such as the ABT-737 molecule fromAbbott laboratories that binds with high affinity (Ki < 1 nM) to theanti-apoptotic Bcl-2 family proteins Bcl-xL, Bcl-2 and Bcl-w, butdisplays a poor affinity towardMcl-1 and Bfl-1 [16,17], as well as thenatural product gambogic acid (GA) and the GeminX’s lead mole-cule called Obatoclax (GX015-070), two pan-Bcl-2 inhibitorsmolecules [18,19]. While GA is highly toxic for all cell lines tested,we observed that both compound 17 and ABT-737 induced veryefficient cell death of IM9 and RS4.11 cell lines, but have an inter-mediary pro-apoptotic activity toward BP3 cells (Fig. 3B). Obatoclaxalso efficiently promoted cell death of IM9 cells, while it has anintermediary pro-apoptotic activity toward BP3 and RS4.11 celllines (Fig. 3B).

Anti-apoptotic Bcl-2 suppress apoptosis by inhibiting Bax andBak pro-apoptotic Bcl-2 members. Therefore cells lacking both Baxand Bak proteins are resistant to apoptotic stimuli that act throughdisruption of mitochondrial function, such as apoptosis induced bystaurosporine, ultraviolet radiation or growth factor deprivation[20]. We observed here that mouse embryonic fibroblasts that areBax/Bak double knockout (MEF DKO) were not killed by compound17 (Fig. 3C), ABT-737 or Obatoclax (Fig. 3D), while MEF DKO werekilled by GA (Fig. 3D), illustrating, as previously reported, that evenif GA was described as a pan-Bcl-2 inhibitor its cytotoxic effect isonly partly dependent on Bcl-2 proteins [18]. In contrast withstaurosporine that efficiently killed wild type MEF, we observedthat neither compound 17 nor ABT-737 induced death of thesecells, while the effect of Obatoclax was minor (Fig. 3C and D),suggesting that compound 17, as ABT-737 or Obatoclax, is unable toefficiently counteract activity of all anti-apoptotic Bcl-2 members.

As di- and trimeric quinoline analogs were initially designed tointeract with Bcl-xL and to inhibit interactionwith its pro-apoptoticpartners, we assessed the capacity of compounds 17, 18, 20, 41 and

N OR'

R

N ClBr

a

N Cl

N Cl

N Cl

B(OH)2g

N OR'

N Cl

R

N NR1R2

N NR1R2

N OR'

N NR1R2

R

38, R = H,R' = Pr, 58%39, R = iPr, R' = Et, 59%

40, R = H, R' = Pr, NR1R2 = pyrrolidin-1-yl, 94%41, R = H, R' = Pr, NR1R2 = morpholin-4-yl, 86%42, R = iPr, R' = Et, NR1R2 = pyrrolidin-1-yl, 85%43, R = iPr, R' = Et, NR1R2 = morpholin-4-yl, 46%

36, R = H, R' = Pr37, R = iPr, R' = Et

1 35, 88%

b, c, d

44, 43% 45, NR1R2 = pyrrolidin-1-yl, 93%46, NR1R2 = morpholin-4-yl, 90%

f

12

N ClBr

e

Reagents and conditions: (a) PtO2/H2, EtOAc; (b) LDA, B(OiPr)3, THF; (c) aq. NH4Cl; (d) 35, PEPPSI-iPr, tBuOK, iPrOH/THF; (e) NHR1R2; (f) 35, PEPPSI-iPr, tBuOK, iPrOH/THF; (g) NHR1R2.

Scheme 4. Synthesis of compounds 40e43, 45 and 46.


45 to bind Bcl-xL and to inhibit its interaction with Bim pro-apoptotic protein. We thus measured the capacity of compounds17, 18, 20, 41 and 45 to bind Bcl-xL using an ELISA assay incompetition with biotinylated Bim peptide. We observed that allthe five compounds bound Bcl-xL and disrupted Bcl-xL/Bim inter-action, compound 17 being the most efficient with an EC50

calculated z 6 mM (Fig. 4A and Table 3). Compared to 17,compounds 18, 20, 41 and 45 were less active toward Bcl-xL/Biminteractions but showed Mcl-1/Bim, Bcl-2/Bim and Bfl-1/Bim EC50values in the same range as the one measured for 17 (Fig. 4BeD,Table 3).

However EC50 from compound 17 was z7 and z1000 foldhigher than Obatoclax and ABT-737 respectively (Table 3). Inter-estingly, we observed that compound 17 also efficiently disruptedMcl-1/Bim (EC50 ¼ 2.1 mM), Bcl-2/Bim (EC50 ¼ 3.5 mM) and, toa lesser extend, Bfl-1/Bim (EC50 ¼ 25.5 mM) interactions. As ex-pected from previous reports, ABT-737 very efficiently disruptedBim interactions with Bcl-xL and Bcl-2 but not with Bfl-1 andMcl-1,

Table 2Cell death induction by compounds 40e43, 45 and 46. % Of residual Bcl-xL-3T3 cellsviability with compounds tested at 5 mM.



40 86.8 43 70.341 55.5 45 4.3742 91.6 46 67.9

whereas the pan-inhibitor Obatoclax disrupted all the interactions(Fig. 4AeD).

In conclusion, we have described the synthesis of new di- andtrimeric alkoxyalkylaminoquinoline derivatives. The results ob-tained in this structureeactivity relationship study have pointedout that in this series, the most active compound is dimer 17 thathas demonstrated significant capacity to promote cell death of Bcl-xL-3T3 cells but also apoptosis of lymphoid tumor cells known toexpress Bcl-xL, as well as other anti-apoptotic proteins [21], while itshowed very low toxic effect toward PBMC, when tested at thesame concentration. Specificity of compound 17 toward Bcl-2apoptotic pathway was revealed by its absence of toxic effect onBax/Bak double deficient cells.

Moreover, dimeric quinoline derivatives were shown to beefficient inhibitors of Bim interaction with Bcl-xL but also with Bcl-2, Bfl-1 and Mcl-1 and thus represent leads of a new attractive classof BH3 mimetics for cancer therapy. Co-crystallization assaysbetween 17 and Bcl-xL will be undertaken to investigate their modeof interaction and thus provide valuable information for furtherdevelopment of this series as Bcl-2 anti-apoptotic group inhibitors.

3. Experimental section

3.1. Chemistry

Starting materials were obtained from commercial suppliersand used without further purification. IR spectra were recorded on

Fig. 2. BP3, IM9 and RS4.11 tumor cells were cultured for 24 h in the presence of compounds 17, 18, 20, 41 and 45 at the indicated concentrations. Percentages of viable cells weredetermined by flow cytometry after Annexin V and propidium iodide labeling, and expressed as percentages of specific viable cells calculated according to the formula: (% viabletreated cells � 100)/% viable non-treated cells. Results are expressed as mean � SEM of 3 experiments.


a Shimadzu FTIR-8400S spectrometer (n in cm�1). NMR spectra,performed on a Bruker AVANCE 400 (1H: 400 MHz, 13C: 100 MHz),are reported in ppm using the solvent residual peak as an internalstandard (1H: DMSO-d6, 2.50 ppm, CDCl3, 7.26 ppm or acetone-d6,2.05 ppm; 13C: DMSO-d6, 39.52 ppm, CDCl3, 77.16 ppm or acetone-d6, 29.84 ppm); the following abbreviations are used: singlet (s),

Fig. 3. A, PBMC were treated for 24 h with compound 17 used at the indicated concentrcompounds 17 (10 mM), ABT-737 (10 mM), GA (5 mM) and Obatoclax (10 mM). Percentages ocultured in medium alone (Med) or in the presence of staurosporine (STS) (1 mM), ABT-737 (1the indicated concentrations (C). A, B, C and D, Cell viability was determined by flow cytom

doublet (d), triplet (t), quartet (q), sextet (sex), septet (sep), doubletof doublet (dd), doublet of doublet of doublet (ddd), multiplet (m),broad signal (br s). High resolution mass spectra (ESIþ) weredetermined on a high-resolution Micro Q-Tof apparatus (CRMP,Université Blaise Pascal, Clermont-Ferrand, France). Chromato-graphic purifications were performed by flash silica gel Geduran SI

ations. B, BP3, IM9 and RS4.11 tumor cells were cultured for 24 h in the presence off viable cells were calculated as indicated in Fig. 2. C and D, WT and DKO MEFs were0 mM), GA (1 mM) and Obatoclax (10 mM) (D) or in the presence of compound 17 used atetry, and results are expressed as mean � SEM of 3 independent experiments.

Fig. 4. Capacity of compounds 17, 18, 20, 41 and 45 to bind Bcl-xL, Bcl-2, Bfl-1 and Mcl-1 were measured using an ELISA assay in competition with biotinylated Bim peptide incomparison with Obatoclax, ABT-737 and GA. Results from one experiment representative of two (for Bcl-2, Bfl-1 and Mcl-1) or three (for Bcl-xL) are shown.


60 (Merck) 0.040e0.063 mm column chromatography. Reactionswere monitored by TLC using fluorescent silica gel plates (60 F254from Merck). Melting points were measured on a Reichert micro-scope and are uncorrected. Experiments under microwave irradi-ation were performed using a CEM Discover Benchmate apparatus.

3.1.1. 7-Bromo-5-(propen-2-yl)-2-(pyrrolidin-1-yl)quinoline 2A mixture of compound 1 (100 mg, 0.35 mmol) and pyrrolidine

(1 mL) was refluxed for 2 h. After cooling, water was added and theproduct was extracted with EtOAc. The assembled organic fractionswere dried over MgSO4 and evaporated. The residue was purifiedby column chromatography (cyclohexane/EtOAc, 95:5) to give 2(105 mg, 0.33 mmol, 94%) as a white solid. Mp ¼ 103e106 �C; IR(ATR): 1610, 1518, 1453, 1409 cm�1; 1H NMR (400 MHz, DMSO-d6):1.94e2.01 (4H, br s), 2.12 (3H, s), 3.49e3.56 (4H, br s), 4.97 (1H, s),5.43 (1H, s), 6.90 (1H, d, J ¼ 9.5 Hz), 7.11 (1H, s), 7.60 (1H, s), 8.00(1H, d, J ¼ 9.5 Hz); 13C NMR (100 MHz, DMSO-d6): 24.6 (CH3), 24.9(2C), 46.5 (2C) (CH2), 111.0, 121.95, 126.5, 134.3 (CHarom), 117.1, 118.1,121.91, 142.1, 143.6, 149.3, 155.4 (]CH2, Carom); HRMS (ESþ) calcdfor C16H18

79BrN2 (M þ H)þ 317.0653, found 317.0647.

3.1.2. 7-Bromo-2-morpholin-4-yl-5-(propen-2-yl)quinoline 3Same procedure as described above in the preparation of

compound 2: compound 1 (200 mg, 0.71 mmol), morpholine(1 mL), 1 h. Column chromatography (cyclohexane/EtOAc, 95:5 to8:2) provided compound 3 (214 mg, 0.64 mmol, 91%) as a whitesolid. Mp ¼ 73e76 �C; IR (ATR): 1609, 1509, 1242, 1121 cm�1; 1H

Table 3Binding activity of compounds 17, 18, 20, 41 and 45 against Bcl-2 family members.EC50 values were determined by competitive ELISA assay, using GraphPad Prismsoftware. nd, not determined.

EC50 (mM)

Obatoclax ABT-737 GA 17 18 20 41 45

Bcl-xL 0.9 <0.01 nd 6.2 14.6 >100 >100 >100Bcl-2 0.3 <0.03 nd 3.5 2.7 9.2 6.6 1.4Mcl-1 0.5 nd nd 2.1 3.5 7.9 11.6 2.3Bfl-1 0.6 nd nd 25.5 24.0 8.2 17.8 21.5

NMR (400 MHz, DMSO-d6): 2.13 (3H, s), 3.62e3.76 (8H, m), 4.96e5.01 (1H, m), 5.42e5.47 (1H, m), 7.21 (1H, d, J ¼ 2 Hz), 7.26 (1H,d, J ¼ 9.5 Hz), 7.66 (1H, d, J ¼ 1.5 Hz), 8.07 (1H, d, J ¼ 9.5 Hz); 13CNMR (100 MHz, DMSO-d6): 24.6 (CH3), 44.8 (2C), 66.0 (2C) (CH2),110.2, 123.1, 126.9, 135.1 (CHarom), 117.4, 118.8, 122.1, 141.9, 143.5,148.5, 157.3 (]CH2, Carom); HRMS (ESþ) calcd for C16H18

79BrN2O(M þ H)þ 333.0602, found 333.0586.

3.1.3. 7-Bromo-5-isopropyl-2-(pyrrolidin-1-yl)quinoline 4A mixture of compound 2 (240 mg, 0.76 mmol) and PtO2

(17.2 mg, 0.076 mmol) in EtOAc (10 mL) was hydrogenated(balloon) at room temperature for 2 h. After filtration throughCelite, the filtrate was evaporated under reduced pressure. Theresidue was purified by column chromatography (cyclohexane/EtOAc, 7:3) to give 4 (187 mg, 0.59 mmol, 77%) as a white solid.Mp ¼ 66e70 �C; IR (ATR): 1612, 1520, 1387 cm�1; 1H NMR(400 MHz, DMSO-d6): 1.27 (6H, d, J ¼ 6.5 Hz), 1.93e2.01 (4H, br s),3.48e3.56 (4H, br s), 3.59 (1H, sep, J ¼ 6.5 Hz), 6.89 (1H, d,J¼ 9.5 Hz), 7.13 (1H, s), 7.54 (1H, s), 8.22 (1H, d, J¼ 9.5 Hz); 13C NMR(100 MHz, DMSO-d6): 23.3 (2C) (CH3), 25.0 (2C), 46.4 (2C) (CH2),27.7 (CH), 110.7, 119.9, 125.7, 132.7 (CHarom), 118.6, 122.6, 147.3,149.5, 155.2 (Carom); HRMS (ESþ) calcd for C16H20

79BrN2 (M þ H)þ

319.0810, found 319.0815.

3.1.4. 7-Bromo-5-isopropyl-2-morpholin-4-ylquinoline 5Same procedure as described above in the preparation of

compound 4: compound 3 (200 mg, 0.60 mmol), PtO2 (13.6 mg,0.060 mmol). Column chromatography provided 5 (146 mg,0.44 mmol, 73%) as a white solid. Mp ¼ 64e65 �C; IR (ATR): 1607,1509, 1240, 1233, 1122, 1115 cm�1; 1H NMR (400 MHz, DMSO-d6):1.28 (6H, d, J ¼ 7 Hz), 3.56e3.69 (5H, m), 3.69e3.75 (4H, m), 7.22(1H, d, J¼ 2 Hz), 7.26 (1H, d, J¼ 9.5 Hz), 7.59 (1H, d, J¼ 1.5 Hz), 8.29(1H, d, J ¼ 9.5 Hz); 13C NMR (100 MHz, DMSO-d6): 23.2 (2C) (CH3),44.8 (2C), 66.0 (2C) (CH2), 27.7 (CH), 109.9, 121.1, 126.1, 133.5(CHarom), 119.3, 122.8, 147.3, 148.5*, 157.1 (Carom), *chemical shiftmeasured from a 1He13C HMBC experiment; HRMS (ESþ) calcd forC16H20

79BrN2O (M þ H)þ 335.0759, found 335.0764.


3.1.5. 2,20-Diethoxy-50,500-diisopropyl-200-pyrrolidin-1-yl-3,70:3,700-terquinoline 8

Step A: a solution of diisopropylamine (146 mL, 1.03 mmol, 4equiv) in THF (1 mL) was cooled to �5 �C before dropwise additionof n-BuLi (2.2 M in hexane, 0.44 mL, 0.97 mmol, 3.7 equiv). Themixture was stirred at �5 �C for 1 h and then added dropwise toa solution, cooled to �78 �C, of compound 6 (100 mg, 0.26 mmol)and triisopropylborate (238 mL, 1.03 mmol, 4 equiv) in THF (1 mL).The mixture was stirred at �78 �C for 4 h and was allowed to reachroom temperature and stirred for 15 h. After addition of a saturatedaqueous NH4Cl solution, the mixture was extracted with EtOAc andthe assembled organic fractions were dried over MgSO4 andevaporated to give the intermediate boronic acid. Step B: to a solu-tion of 4 (41 mg, 0.128 mmol) in THF (1.1 mL) were addedPdCl2(PPh3)2 (4.5 mg, 6.4 mmol), a 2 M aqueous Na2CO3 solution(0.32 mL, 0.64 mmol) and boronic acid from step A. The mixturewas stirred under microwave irradiation (65 �C, Pmax ¼ 50 W, Patm)for 20 min, poured into water and extracted with EtOAc. Theassembled organic fractions were dried over MgSO4, evaporated,and the residue was purified by column chromatography (cyclo-hexane/EtOAc, 99.8:0.2 to 99:1, then to 95:5) to give 8 (63 mg,0.101 mmol, 79%) as a pale yellow solid. Mp ¼ 72e76 �C; IR (ATR):1609, 1450, 1254 cm�1; 1H NMR (400 MHz, acetone-d6): 1.40e1.50(18H, m), 2.02e2.08 (4H under the solvent signal), 3.60e3.67 (4H,m), 3.72 (1H, sep, J ¼ 7 Hz), 3.94 (1H, sep, J ¼ 7 Hz), 4.63 (4H, q,J ¼ 7 Hz), 6.91 (1H, d, J ¼ 9.5 Hz), 7.47 (1H, t, J ¼ 7.5 Hz), 7.57 (1H, s),7.69 (1H, t, J ¼ 7.5 Hz), 7.78 (1H, s), 7.82 (1H, s), 7.86 (1H, d,J ¼ 8.5 Hz), 7.97e8.01 (2H, m), 8.31 (1H, d, J ¼ 9.5 Hz), 8.40 (1H, s),8.55 (1H, s); 13C NMR (100 MHz, acetone-d6): 14.9, 15.0, 24.07 (2C),24.14 (2C) (CH3), 26.2 (2C), 47.4 (2C), 62.5, 62.7 (CH2), 29.0, 29.2(CH), 110.8, 120.1, 123.0, 125.1, 125.9, 126.2, 127.6, 128.8, 130.4, 133.1,134.6, 139.2 (CHarom), 120.6, 123.6, 126.6, 127.2, 127.4, 138.7, 138.8,144.9, 145.6, 147.0, 147.5, 150.2, 156.4, 160.1, 160.2 (Carom); HRMS(ESþ) calcd for C41H45N4O2 (M þ H)þ 625.3543, found 625.3538.

3.1.6. 50,500-Diisopropyl-2,20-dipropoxy-200-pyrrolidin-1-yl-3,70:3,700-terquinoline 9

Same procedure as described above in the preparation ofcompound 8, from compound 7 as boronic acid precursor andcompound 4 (37 mg, 0.116 mmol). Column chromatography(cyclohexane/EtOAc, 95:5 to 90:10) provided 9 (37mg, 0.057mmol,49%) as a pale yellow solid. Mp ¼ 70e76 �C; IR (ATR): 1609, 1451,1257 cm�1; 1H NMR (400 MHz, acetone-d6): 1.07 (3H, t, J ¼ 7.5 Hz),1.08 (3H, t, J ¼ 7.5 Hz), 1.42 (6H, d, J ¼ 7 Hz), 1.48 (6H, d, J ¼ 7 Hz),1.81e1.93 (4H, m), 2.03e2.07 (4H under the solvent signal), 3.61e3.66 (4H, m), 3.72 (1H, sep, J ¼ 7 Hz), 3.93 (1H, sep, J ¼ 7 Hz),4.53 (2H, t, J¼ 6.5 Hz), 4.54 (2H, t, J¼ 6.5 Hz), 6.91 (1H, d, J¼ 9.5 Hz),7.47 (1H, ddd, J1 ¼8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.55 (1H, d, J ¼ 1.5 Hz),7.69 (1H, ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.78 (1H, d,J ¼ 1 Hz), 7.80 (1H, d, J ¼ 1.5 Hz), 7.86 (1H, d, J ¼ 8.5 Hz), 7.97e8.01(2H, m), 8.31 (1H, d, J ¼ 9.5 Hz), 8.40 (1H, s), 8.55 (1H, s); 13C NMR(100 MHz, acetone-d6): 11.26, 11.27, 24.0 (2C), 24.1 (2C) (CH3),23.06, 23.07, 26.2 (2C), 47.4 (2C), 68.4, 68.5 (CH2), 29.1, 29.3 (CH),110.8, 120.0,122.8,125.1, 126.1, 126.3,127.6, 128.8,130.4,133.1, 134.7,139.3 (CHarom),120.6,123.6,126.6,127.2,127.5,138.75,138.80,144.9,145.6, 147.0, 147.5, 150.2, 156.4, 160.2, 160.3 (Carom); HRMS (ESþ)calcd for C43H49N4O2 (M þ H)þ 653.3856, found 653.3846.

3.1.7. 2,20-Diethoxy-50,500-diisopropyl-200-morpholin-4-yl-3,70:3,700-terquinoline 10

Same procedure as described above in the preparation ofcompound 8, from compound 6 as boronic acid precursor andcompound 5 (22.5 mg, 0.067 mmol). Column chromatography(cyclohexane/EtOAc, 99.5:0.5 to 90:10) provided 10 (33 mg,0.051mmol, 77%) as a pale yellow solid. Mp¼ 113e116 �C; IR (ATR):

1607, 1438, 1255, 1244, 1237 cm�1; 1H NMR (400 MHz, acetone-d6):1.40e1.51 (18H, m), 3.70e3.78 (5H, m), 3.78e3.83 (4H, m), 3.94 (1H,sep, J ¼ 6.5 Hz), 4.63 (4H, q, J ¼ 7 Hz), 7.26 (1H, d, J ¼ 9.5 Hz), 7.48(1H, t, J ¼ 7.5 Hz), 7.66 (1H, s), 7.70 (1H, t, J ¼ 7.5 Hz), 7.82 (2H, s),7.86 (1H, d, J ¼ 8.5 Hz), 7.97e8.01 (2H, m), 8.37e8.42 (2H, m), 8.57(1H, s); 13C NMR (100 MHz, acetone-d6): 14.9, 15.0, 24.0 (2C), 24.1(2C) (CH3), 23.1 (2C), 46.4 (2C), 62.5, 62.7, 67.4 (2C) (CH2), 29.0, 29.2(CH), 110.2, 121.4, 123.0, 125.1, 126.2, 126.3, 127.6, 128.8, 130.4, 133.9,134.7, 139.2 (CHarom), 121.4, 123.6, 126.6, 127.15, 127.18, 138.8, 139.1,144.9, 145.7, 147.0, 147.5, 149.3, 158.3, 160.0, 160.2 (Carom); HRMS(ESþ) calcd for C41H45N4O3 (M þ H)þ 641.3492, found 641.3502.

3.1.8. 50,500-Diisopropyl-200-morpholin-4-yl-2,20-dipropoxy-3,70:3,700-terquinoline 11

Same procedure as described above in the preparation ofcompound 8, from compound 7 as boronic acid precursor andcompound 5 (38 mg, 0.113 mmol). Column chromatography(cyclohexane/EtOAc, 98:2 to 90:10) provided 11 (55 mg,0.082 mmol, 73%) as a white solid. Mp ¼ 78e87 �C; IR (ATR): 1607,1436, 1261, 1238 cm�1; 1H NMR (400 MHz, acetone-d6): 1.06 (3H, t,J ¼ 7.5 Hz), 1.08 (3H, t, J ¼ 7.5 Hz), 1.42 (6H, d, J ¼ 7 Hz), 1.48 (6H, d,J ¼ 7 Hz), 1.81e1.93 (4H, m), 3.70e3.82 (9H, m), 3.93 (1H, sep,J ¼ 7 Hz), 4.53 (4H, t, J ¼ 6.5 Hz), 7.24 (1H, d, J ¼ 9.5 Hz), 7.47 (1H,ddd, J1 ¼8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.64 (1H, d, J ¼ 1.5 Hz), 7.69 (1H,ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.81 (1H, d, J ¼ 1.5 Hz), 7.82(1H, s), 7.86 (1H, d, J ¼ 8.5 Hz), 7.98 (1H, d, J ¼ 7.5 Hz), 8.01 (1H, s),8.39 (1H, d, J¼ 9.5 Hz), 8.39 (1H, s), 8.56 (1H, s); 13C NMR (100MHz,acetone-d6): 11.2, 11.3, 24.0 (2C), 24.1 (2C) (CH3), 23.1 (2C), 46.4(2C), 67.4 (2C), 68.4, 68.5 (CH2), 29.1, 29.3 (CH), 110.2, 121.3, 122.9,125.1, 126.3, 126.4, 127.6, 128.8, 130.4, 133.9, 134.8, 139.3 (CHarom),121.4, 123.6, 126.6, 127.21, 127.25, 138.9, 139.1, 144.9, 145.7, 147.0,147.5, 149.3, 158.3, 160.2, 160.3 (Carom); HRMS (ESþ) calcd forC43H49N4O3 (M þ H)þ 669.3805, found 669.3794.

3.1.9. 2-Chloro-20-ethoxy-50-isopropyl-3,70-biquinoline 15Amixture of compound 13 (706mg, 2.40mmol), boronic acid 12

(623 mg, 3.00 mmol), Pd(OAc)2 (54 mg, 0.24 mmol), P(tBu)3(120 mL, 0.49 mmol) and Na2CO3 (763 mg, 7.2 mmol) in dioxane(7.5 mL) and water (3 mL) was refluxed overnight. After cooling andconcentration under reduced pressure, water was added and theproduct was extracted with EtOAc. The assembled organic fractionswere dried over MgSO4 and evaporated under reduced pressure.The residue was purified by column chromatography (cyclohexane/EtOAc, 99.5:0.5 to 9:1) to give 15 (517 mg, 1.37 mmol, 57%) asa white solid. Mp ¼ 47e50 �C; IR (ATR): 1609, 1302, 1290, 1253,1044 cm�1; 1H NMR (400 MHz, CDCl3): 1.42 (6H, d, J ¼ 7 Hz), 1.46(3H, t, J ¼ 7 Hz), 3.69 (1H, sep, J ¼ 7 Hz), 4.55 (2H, q, J ¼ 7 Hz), 6.97(1H, d, J ¼ 9 Hz), 7.49 (1H, d, J ¼ 1.5 Hz), 7.61 (1H, ddd, J1 ¼ 8 Hz,J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.78 (1H, ddd, J1 ¼8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz),7.83 (1H, d, J ¼ 1.5 Hz), 7.87 (1H, d, J¼ 8 Hz), 8.10 (1H, d, J ¼ 8.5 Hz),8.24 (1H, s), 8.34 (1H, d, J ¼ 9 Hz); 13C NMR (100 MHz, CDCl3): 14.8,23.8 (2C) (CH3), 61.9 (CH2), 28.8 (CH),113.5,121.8, 126.4,127.5, 127.7,128.5, 130.6, 134.5, 139.2 (CHarom), 122.7, 127.4, 135.0, 138.6, 145.1,147.1, 147.2, 149.7, 162.2 (Carom); HRMS (ESþ) calcd forC23H22

35ClN2O (M þ H)þ 377.1421, found 377.1432.

3.1.10. 2-Chloro-50-isopropyl-20-propoxy-3,70-biquinoline 16Same procedure as described above in the preparation of

compound 15: compound 14 (405 mg, 1.31 mmol), 15 h. Columnchromatography (cyclohexane/EtOAc, 99.5:0.5 to 9:1) provided 16(288 mg, 0.74 mmol, 56%) as a white solid. Mp ¼ 41e43 �C; IR(ATR): 1609, 1419, 1303, 1289, 1257 cm�1; 1H NMR (400 MHz,CDCl3): 1.07 (3H, t, J ¼ 7.5 Hz), 1.42 (6H, d, J ¼ 6.5 Hz), 1.87 (2H, sex,J¼ 7 Hz), 3.69 (1H, sep, J¼ 6.5 Hz), 4.44 (2H, t, J¼ 6.5 Hz), 6.98 (1H,d, J ¼ 9 Hz), 7.49 (1H, s), 7.61 (1H, t, J ¼ 7.5 Hz), 7.78 (1H, t,


J¼ 7.5 Hz), 7.84 (1H, s), 7.87 (1H, d, J¼ 8 Hz), 8.10 (1H, d, J¼ 8.5 Hz),8.24 (1H, s), 8.34 (1H, d, J ¼ 9 Hz); 13C NMR (100 MHz, CDCl3): 10.8,23.8 (2C) (CH3), 22.5, 67.7 (CH2), 28.8 (CH), 113.5, 121.8, 126.4, 127.5,127.7, 128.5, 130.6, 134.4, 139.2 (CHarom), 122.7, 127.4, 135.0, 138.6,145.1, 147.1, 147.2, 149.7, 162.4 (Carom); HRMS (ESþ) calcd forC24H24

35ClN2O (M þ H)þ 391.1577, found 391.1583.

3.1.11. 50-Isopropyl-20-ethoxy-2-pyrrolidin-1-yl-3,70-biquinoline 17Amixture of compound 15 (215 mg, 0.57 mmol) and pyrrolidine

(1 mL) was refluxed for 2 h. After cooling and evaporation underreduced pressure, the residue was purified by column chromatog-raphy (CH2Cl2/EtOAc, 10:0 to 9:1) to give 17 (159 mg, 0.39 mmol,68%) as a white solid. Mp ¼ 66e70 �C; IR (ATR): 1608, 1429, 1410,1290 cm�1; 1H NMR (400 MHz, acetone-d6): 1.39 (6H, d, J ¼ 7 Hz),1.43 (3H, d, J ¼ 7 Hz), 1.75e1.80 (4H, m), 3.25e3.31 (4H, m), 3.78(1H, sep, J ¼ 7 Hz), 4.53 (2H, q, J ¼ 7 Hz), 6.98 (1H, d, J ¼ 9 Hz), 7.25(1H, t, J ¼ 7.5 Hz), 7.49 (1H, d, J ¼ 1.5 Hz), 7.56 (1H, ddd, J1 ¼ 8 Hz,J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.72 (1H, d, J ¼ 8.5 Hz), 7.75e7.79 (2H, m), 8.00(1H, s), 8.50 (1H, d, J ¼ 9 Hz); 13C NMR (100 MHz, acetone-d6): 14.9,23.9 (2C) (CH3), 26.2 (2C), 50.7 (2C), 62.1 (CH2), 29.1 (CH), 113.4,122.1, 122.9, 125.4, 127.16, 128.4, 130.0, 135.5, 139.6 (CHarom), 122.7,124.6, 127.18, 143.0, 145.9, 148.1, 148.3, 156.7, 162.9 (Carom); HRMS(ESþ) calcd for C27H30N3O (M þ H)þ 412.2389, found 412.2391.

3.1.12. 20-Ethoxy-50-isopropyl-2-morpholin-4-yl-3,70-biquinoline18Same procedure as described above in the preparation of

compound 17, from compound 15 (71 mg, 0.19 mmol) and mor-pholine (1 mL); 24 h. Column chromatography (cyclohexane/EtOAc,99.5:0.5 to 95:5) provided 18 (52 mg, 0.12 mmol, 65%) as a paleyellow solid. Mp¼ 56e58 �C; IR (ATR): 1608,1407,1250,1234,1120,1044 cm�1; 1H NMR (400 MHz, acetone-d6): 1.41e1.46 (9H, m),3.19e3.24 (4H, m), 3.59e3.64 (4H, m), 3.79 (1H, sep, J ¼ 7 Hz), 4.55(2H, q, J ¼ 7 Hz), 6.99 (1H, d, J ¼ 9 Hz), 7.41 (1H, t, J ¼ 7.5 Hz), 7.66(1H, t, J ¼ 7.5 Hz), 7.81 (1H, s), 7.84 (1H, d, J ¼ 8.5 Hz), 7.89 (1H, d,J ¼ 8 Hz), 7.92 (1H, s), 8.17 (1H, s), 8.50 (1H, d, J ¼ 9 Hz); 13C NMR(100 MHz, acetone-d6): 14.9, 24.1 (2C) (CH3), 50.5 (2C), 62.1, 67.2(2C) (CH2), 29.3 (CH), 113.5, 121.0, 124.96, 125.01, 128.1, 128.6, 130.3,135.6, 140.3 (CHarom), 123.1, 126.4, 129.2, 142.2, 146.2, 147.5, 148.8,159.5, 162.8 (Carom); HRMS (ESþ) calcd for C27H30N3O2 (M þ H)þ

428.2338, found 428.2348.

3.1.13. 50-Isopropyl-20-propoxy-2-pyrrolidin-1-yl-3,70-biquinoline19Same procedure as described above in the preparation of

compound 17, from compound 16 (10 mg, 0.026 mmol) and pyr-rolidine (1 mL); 2 h. Column chromatography (cyclohexane/EtOAc,9:1) provided 19 (10 mg, 0.023 mmol, 92%) as a white solid.Mp ¼ 62e64 �C; IR (ATR): 1608, 1430, 1409, 1289, 1259 cm�1; 1HNMR (400 MHz, CDCl3): 1.08 (3H, t, J ¼ 7.5 Hz), 1.36 (6H, d,J ¼ 6.5 Hz), 1.71e1.80 (4H, br s), 1.87 (2H, sex, J ¼ 7 Hz), 3.25e3.34(4H, br s), 3.65 (1H, sep, J¼ 6.5 Hz), 4.45 (2H, t, J¼ 6.5 Hz), 6.94 (1H,d, J ¼ 9 Hz), 7.22 (1H, t, J ¼ 7.5 Hz), 7.39 (1H, s), 7.55 (1H, t,J¼ 7.5 Hz), 7.63 (1H, d, J¼ 8 Hz), 7.78 (1H, d, J¼ 8.5 Hz), 7.82 (1H, s),7.89 (1H, s), 8.30 (1H, d, J ¼ 9 Hz); 13C NMR (100 MHz, CDCl3): 10.8,23.8 (2C) (CH3), 22.5, 25.8 (2C), 50.2 (2C), 67.6 (CH2), 28.7 (CH),112.7, 121.4, 122.3, 124.6, 126.3, 127.5, 129.5, 134.4, 139.3 (CHarom),121.9, 123.7, 126.4, 142.5, 144.8, 147.3, 147.5, 156.2, 162.4 (Carom);HRMS (ESþ) calcd for C28H32N3O (M þ H)þ 426.2545, found426.2561.

3.1.14. 50-Isopropyl-2-morpholin-4-yl-20-propoxy-3,70-biquinoline20Same procedure as described above in the preparation of

compound 17, from compound 16 (32 mg, 0.082 mmol) and mor-pholine (1 mL); 6 h. Column chromatography (cyclohexane/EtOAc,95:5) provided 20 (27 mg, 0.061 mmol, 75%) as a pale yellow solid.Mp ¼ 61e65 �C; IR (ATR): 1608, 1426, 1407, 1289, 1255, 1250, 1234,

1120 cm�1; 1H NMR (400 MHz, acetone-d6): 1.07 (3H, t, J ¼ 7.5 Hz),1.44 (6H, d, J ¼ 7 Hz), 1.87 (2H, sex, J ¼ 7 Hz), 3.19e3.24 (4H, m),3.59e3.64 (4H, m), 3.79 (1H, sep, J ¼ 7 Hz), 4.46 (2H, t, J ¼ 6.5 Hz),7.01 (1H, d, J ¼ 9 Hz), 7.41 (1H, ddd, J1 ¼ 8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz),7.66 (1H, ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.80 (1H, d,J¼ 1.5 Hz), 7.84 (1H, d, J¼ 8.5 Hz), 7.89 (1H, d, J¼ 8 Hz), 7.92 (1H, s),8.17 (1H, s), 8.50 (1H, d, J ¼ 9 Hz); 13C NMR (100 MHz, acetone-d6):10.9, 24.1 (2C) (CH3), 23.0, 50.5 (2C), 67.2 (2C), 68.0 (CH2), 29.3 (CH),113.5, 121.0, 124.98, 125.01, 128.1, 128.6, 130.3, 135.6, 140.3 (CHarom),123.2, 126.4, 129.2, 142.2, 146.2, 147.5, 148.8, 159.5, 163.0 (Carom);HRMS (ESþ) calcd for C28H32N3O2 (M þ H)þ 442.2495, found442.2503.

3.1.15. 20,200-Diethoxy-50,500-diisopropyl-2-pyrrolidin-1-yl-3,70:3,700-terquinoline 21

Same procedure as described above in the preparation ofcompound 8, from compound 17 as boronic acid precursor andcompound 13 (21 mg, 0.071 mmol). Compound 21 (31 mg,0.050 mmol, 70%) was isolated as a pale yellow solid. Mp ¼ 72e75 �C; IR (ATR): 1609, 1427, 1250, 1047 cm�1; 1H NMR (400 MHz,acetone-d6): 1.41e1.47 (18H, m), 1.77e1.84 (4H, m), 3.29e3.35 (4H,m), 3.80 (1H, sep, J ¼ 7 Hz), 3.94 (1H, sep, J ¼ 7 Hz), 4.55 (2H, q,J ¼ 7 Hz), 4.63 (2H, q, J ¼ 7 Hz), 7.01 (1H, d, J ¼ 9 Hz), 7.26 (1H, t,J¼ 7.5 Hz), 7.54e7.60 (2H, m), 7.73 (1H, d, J¼ 8.5 Hz), 7.77e7.81 (2H,m), 7.84 (1H, s), 7.94 (1H, s), 8.05 (1H, s), 8.52 (1H, d, J ¼ 9 Hz), 8.60(1H, s); 13C NMR (100MHz, acetone-d6): 14.9,15.0, 24.04 (2C), 24.05(2C) (CH3), 26.2 (2C), 50.8 (2C), 62.1, 62.6 (CH2), 29.1, 29.2 (CH),113.6, 122.4, 122.9 (2C), 125.0, 126.59, 127.18, 128.4, 130.1, 135.0,135.5, 139.6 (CHarom), 123.12, 123.15, 124.7, 126.60, 127.21, 139.3,143.1, 145.3, 146.3, 147.7, 148.1, 148.3, 156.8, 160.1, 162.7 (Carom);HRMS (ESþ) calcd for C41H45N4O2 (M þ H)þ 625.3543, found625.3539.

3.1.16. 50,500-Diisopropyl-20,200-dipropoxy-2-pyrrolidin-1-yl-3,70:3,700-terquinoline 22

Same procedure as described above in the preparation ofcompound 8, from compound 19 as boronic acid precursor andcompound 14 (30 mg, 0.097 mmol). Column chromatography(cyclohexane/EtOAc, 99.5:0.5 to 98:2) provided compound 22(62 mg, 0.095 mmol, 98%) as a pale yellow solid. Mp¼ 66e71 �C; IR(ATR): 1609, 1424, 1253, 1241 cm�1; 1H NMR (400 MHz, acetone-d6): 1.07 (3H, t, J ¼ 7.5 Hz), 1.08 (3H, t, J ¼ 7.5 Hz), 1.40e1.47 (12H,m), 1.76e1.92 (8H, m), 3.29e3.36 (4H, m), 3.80 (1H, sep, J ¼ 6.5 Hz),3.93 (1H, sep, J ¼ 7 Hz), 4.46 (2H, t, J ¼ 6.5 Hz), 4.54 (2H, t,J¼ 6.5 Hz), 7.02 (1H, d, J ¼ 9 Hz), 7.26 (1H, t, J ¼ 7.5 Hz), 7.56 (1H, s),7.54e7.60 (1H, m), 7.74 (1H, d, J ¼ 8.5 Hz), 7.76e7.81 (2H, m), 7.84(1H, s), 7.96 (1H, s), 8.04 (1H, s), 8.51 (1H, d, J ¼ 9 Hz), 8.59 (1H, s);13C NMR (100 MHz, acetone-d6): 10.9, 11.3, 24.0 (2C), 24.1 (2C)(CH3), 23.0, 23.1, 26.2 (2C), 50.8 (2C), 68.0, 68.5 (CH2), 29.1, 29.3(CH), 113.6, 122.4, 122.8, 123.0, 125.0, 126.7, 127.21, 128.4, 130.1,135.1, 135.5, 139.6 (CHarom), 123.1, 123.2, 124.7, 126.7, 127.24, 139.3,143.2, 145.3, 146.3, 147.7, 148.1, 148.3, 156.8, 160.3, 162.9 (Carom);HRMS (ESþ) calcd for C43H49N4O2 (M þ H)þ 653.3856, found653.3839.

3.1.17. 20-Ethoxy-50,500-diisopropyl-200-propoxy-2-pyrrolidin-1-yl-3,70:3,700-terquinoline 23

Same procedure as described above in the preparation ofcompound 8, from compound 17 as boronic acid precursor andcompound 14 (22.6 mg, 0.073 mmol). Column chromatography(cyclohexane/EtOAc, 99.5:0.5 to 98:2) provided compound 23(20 mg, 0.031 mmol, 43%) as a pale yellow solid. Mp¼ 84e89 �C; IR(ATR): 1610,1425,1252,1047 cm�1; 1HNMR (400MHz, acetone-d6):1.07 (3H, t, J ¼ 7.5 Hz), 1.440 (6H, d, J ¼ 7 Hz), 1.444 (3H, t, J ¼ 7 Hz),1.45 (6H, d, J¼7Hz),1.78e1.83 (4H,m),1.87 (2H, sex, J¼ 7Hz), 3.30e


3.35 (4H,m), 3.80 (1H, sep, J¼7Hz), 3.94 (1H, sep, J¼7Hz), 4.46 (2H,t, J¼6.5Hz), 4.63 (2H, q, J¼7Hz), 7.02 (1H, d, J¼9Hz), 7.26 (1H, ddd,J1 ¼ 8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.56 (1H, d, J ¼ 2 Hz), 7.57 (1H, ddd,J1¼8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.73 (1H, d, J¼ 8.5 Hz), 7.80 (1H, d,J ¼ 1.5 Hz), 7.77e7.81 (1H, m), 7.84 (1H, d, J ¼ 1 Hz), 7.94 (1H, d,J ¼ 1 Hz), 8.05 (1H, s), 8.52 (1H, d, J ¼ 9 Hz), 8.60 (1H, s); 13C NMR(100 MHz, acetone-d6): 10.9, 15.0, 24.0 (2C), 24.1 (2C) (CH3), 23.0,26.2 (2C), 50.8 (2C), 62.6, 68.0 (CH2), 29.1, 29.2 (CH), 113.5, 122.4,122.9 (2C), 125.0, 126.58, 127.18, 128.4, 130.1, 135.0, 135.5, 139.6(CHarom), 123.1, 123.2, 124.7, 126.61, 127.21, 139.3, 143.1, 145.3, 146.3,147.7, 148.1, 148.3, 156.8, 160.1, 162.9 (Carom); HRMS (ESþ) calcd forC42H47N4O2 (M þ H)þ 639.3699, found 639.3719.

3.1.18. 200-Ethoxy-50,500-diisopropyl-20-propoxy-2-(pyrrolidin-1-yl)-3,70:3,700-terquinoline 24

Same procedure as described above in the preparation ofcompound 8, from compound 19 as boronic acid precursor andcompound 13 (38 mg, 0.129 mmol). Column chromatography(cyclohexane/EtOAc, 99.5:0.5 to 95:5) provided compound 24(68 mg, 0.106 mmol, 82%) as a pale yellow solid. Mp ¼ 67e69 �C; IR(ATR): 1609, 1427, 1252 cm�1; 1H NMR (400 MHz, acetone-d6): 1.06(3H, t, J ¼ 7.5 Hz), 1.41e1.47 (15H, m), 1.76e1.84 (4H, m), 1.86 (2H,sex, J ¼ 7 Hz), 3.29e3.36 (4H, m), 3.80 (1H, sep, J ¼ 7 Hz), 3.93 (1H,sep, J ¼ 7 Hz), 4.51e4.58 (4H, m), 7.00 (1H, d, J ¼ 9 Hz), 7.26 (1H, t,J¼ 7.5 Hz), 7.54e7.60 (2H, m), 7.73 (1H, d, J¼ 8.5 Hz), 7.77e7.81 (2H,m), 7.84 (1H, s), 7.95 (1H, s), 8.04 (1H, s), 8.51 (1H, d, J ¼ 9 Hz), 8.59(1H, s); 13C NMR (100 MHz, acetone-d6): 11.3, 14.9, 24.0 (2C), 24.1(2C) (CH3), 23.1, 26.2 (2C), 50.7 (2C), 62.1, 68.5 (CH2), 29.0, 29.3 (CH),113.6, 122.4, 122.8, 122.9, 125.0, 126.7, 127.20, 128.4, 130.0, 135.0,135.4, 139.6 (CHarom), 123.1, 123.2, 124.6, 126.7, 127.21, 139.3, 143.1,145.3, 146.2, 147.7, 148.1, 148.3, 156.7, 160.3, 162.7 (Carom); HRMS(ESþ) calcd for C42H47N4O2 (M þ H)þ 639.3699, found 639.3707.

3.1.19. 50,500-Diisopropyl-2-morpholin-4-yl-20,200-dipropoxy-3,70:3,700-terquinoline 25

Same procedure as described above in the preparation ofcompound 8, from compound 20 as boronic acid precursor andcompound 14 (14.8 mg, 0.048 mmol). Column chromatography(cyclohexane/EtOAc, 99.5:0.5 to 98:2) provided compound 25(23 mg, 0.034 mmol, 72%) as a pale yellow solid. Mp ¼ 89e91 �C; IR(ATR): 1608, 1429, 1407, 1257, 1235 cm�1; 1H NMR (400 MHz,acetone-d6): 1.07 (6H, 2t, J ¼ 7.5 Hz), 1.45 (6H, d, J ¼ 7 Hz), 1.49 (6H,d, J ¼ 7 Hz), 1.81e1.93 (4H, m), 3.23e3.27 (4H, m), 3.63e3.67 (4H,m), 3.81 (1H, sep, J ¼ 7 Hz), 3.95 (1H, sep, J ¼ 7 Hz), 4.46 (2H, t,J ¼ 6.5 Hz), 4.56 (2H, t, J ¼ 6.5 Hz), 7.02 (1H, d, J ¼ 9 Hz), 7.43 (1H, t,J ¼ 7.5 Hz), 7.67 (1H, ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.79 (1H,d, J ¼ 1.5 Hz), 7.84e7.88 (2H, m), 7.92 (1H, d, J ¼ 8 Hz), 7.95 (1H, s),8.00 (1H, s), 8.21 (1H, s), 8.52 (1H, d, J¼ 9 Hz), 8.60 (1H, s); 13C NMR(100 MHz, acetone-d6): 10.9, 11.3, 24.0 (2C), 24.2 (2C) (CH3), 23.0,23.1, 50.5 (2C), 67.2 (2C), 68.0, 68.5 (CH2), 29.2, 29.3 (CH), 113.5,121.3, 122.7, 124.5,125.0, 126.7, 128.1, 128.6, 130.3, 135.1, 135.5,140.3(CHarom), 123.2, 123.6, 126.4, 126.8, 129.2, 139.2, 142.3, 145.3, 146.5,147.5, 148.20, 148.24, 159.5, 160.2, 162.9 (Carom); HRMS (ESþ) calcdfor C43H49N4O3 (M þ H)þ 669.3805, found 669.3785.

3.1.20. 200-Ethoxy-50,500-diisopropyl-2-morpholin-4-yl-20-propoxy-3,70:3,700-terquinoline 26

Same procedure as described above in the preparation ofcompound 8, from compound 20 as boronic acid precursor andcompound 13 (16.5 mg, 0.056 mmol). Column chromatography(cyclohexane/EtOAc, 99.5:0.5 to 95:5) provided compound 26(25 mg, 0.038 mmol, 68%) as a pale yellow solid. Mp ¼ 96e97 �C; IR(ATR): 1609, 1407, 1235, 1120 cm�1; 1H NMR (400 MHz, acetone-d6): 1.06 (3H, t, J ¼ 7.5 Hz), 1.41e1.47 (9H, m), 1.49 (6H, d, J ¼ 7 Hz),1.88 (2H, sex, J ¼ 7 Hz), 3.23e3.27 (4H, m), 3.63e3.67 (4H, m), 3.81

(1H, sep, J ¼ 7 Hz), 3.95 (1H, sep, J ¼ 7 Hz), 4.51e4.59 (4H, m) 7.01(1H, d, J ¼ 9 Hz), 7.43 (1H, t, J ¼ 7.5 Hz), 7.67 (1H, ddd, J1 ¼ 8.5 Hz,J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.79 (1H, d, J ¼ 1.5 Hz), 7.84e7.88 (2H, m), 7.92(1H, d, J ¼ 8 Hz), 7.95 (1H, s), 8.00 (1H, s), 8.22 (1H, s), 8.52 (1H, d,J ¼ 9 Hz), 8.60 (1H, s); 13C NMR (100 MHz, acetone-d6): 11.3, 14.9,24.0 (2C), 24.2 (2C) (CH3), 23.1, 50.5 (2C), 62.1, 67.2 (2C), 68.5 (CH2),29.3 (2C) (CH), 113.6, 121.3, 122.8, 124.6, 125.0, 126.76, 128.1, 128.6,130.3, 135.1, 135.5, 140.3 (CHarom), 123.2, 123.6, 126.4, 126.79, 129.2,139.3, 142.3, 145.3, 146.5, 147.5, 148.2, 148.3, 159.5, 160.2, 162.7(Carom); HRMS (ESþ) calcd for C42H47N4O3 (M þ H)þ 655.3648,found 655.3646.

3.1.21. 20-Ethoxy-50,500-diisopropyl-2,200-dipyrrolidin-1-yl-3,70:3,700-terquinoline 27

Same procedure as described above in the preparation ofcompound 8, from compound 17 as boronic acid precursor andcompound 4 (20 mg, 0.063 mmol). Column chromatography(cyclohexane/EtOAc, 98:2 to 90:10) provided compound 27 (25 mg,0.038 mmol, 61%) as a pale yellow solid. Mp ¼ 118e120 �C; IR(ATR): 1609, 1448, 1434 cm�1; 1H NMR (400 MHz, acetone-d6): 1.42(6H, d, J ¼ 7 Hz), 1.437 (6H, d, J ¼ 7 Hz), 1.441 (3H, t, J ¼ 7 Hz), 1.77e1.83 (4H, m), 2.01e2.10 (4H under the solvent signal), 3.29e3.35(4H, m), 3.61e3.67 (4H, m), 3.72 (1H, sep, J ¼ 7 Hz), 3.93 (1H, sep,J ¼ 7 Hz), 4.62 (2H, q, J ¼ 7 Hz), 6.92 (1H, d, J ¼ 9.5 Hz), 7.26 (1H, t,J ¼ 7.5 Hz), 7.53e7.60 (3H, m), 7.73 (1H, d, J ¼ 8.5 Hz), 7.77 (1H, s),7.79 (1H, d, J ¼ 8 Hz), 7.83 (1H, s), 8.04 (1H, s), 8.31 (1H, d,J ¼ 9.5 Hz), 8.55 (1H, s); 13C NMR (100 MHz, acetone-d6): 15.0, 24.0(2C) 24.1 (2C) (CH3), 26.1 (2C), 26.2 (2C), 47.4 (2C), 50.7 (2C), 62.5(CH2), 29.0, 29.1 (CH), 110.8, 120.1, 122.3, 122.9, 125.0, 125.9, 127.2,128.4, 130.0, 133.1, 134.7, 139.6 (CHarom), 120.6, 123.1, 124.6, 127.3(2C), 138.7, 142.9, 144.9, 146.1, 147.6, 148.1, 150.2, 156.3, 156.8, 160.3(Carom); HRMS (ESþ) calcd for C43H48N5O (MþH)þ 650.3859, found650.3834.

3.1.22. 50,500-Diisopropyl-20-propoxy-2,200-dipyrrolidin-1-yl-3,70:3,700-terquinoline 28

Same procedure as described above in the preparation ofcompound 8, from compound 19 as boronic acid precursor andcompound 4 (21.7 mg, 0.068 mmol). Column chromatography(cyclohexane/EtOAc, 99.6:0.4 to 95:5) provided compound 28(23 mg, 0.035 mmol, 51%) as a pale yellow solid. Mp ¼ 118e122 �C;IR (ATR): 1609, 1449, 1434 cm�1; 1H NMR (400 MHz, acetone-d6):1.06 (3H, t, J ¼ 7.5 Hz), 1.41 (6H, d, J ¼ 7 Hz), 1.43 (6H, d, J ¼ 7 Hz),1.77e1.82 (4H, m), 1.86 (2H, sex, J ¼ 7 Hz), 2.02e2.07 (4H under thesolvent signal), 3.29e3.35 (4H,m), 3.60e3.66 (4H,m), 3.72 (1H, sep,J¼ 7 Hz), 3.92 (1H, sep, J¼ 7 Hz), 4.52 (2H, t, J¼ 6.5 Hz), 6.90 (1H, d,J ¼ 9.5 Hz), 7.26 (1H, ddd, J1 ¼ 8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.54 (2H,2s), 7.57 (1H, ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.73 (1H, d,J ¼ 8.5 Hz), 7.78 (1H, dd, J1 ¼ 8 Hz, J2 ¼ 1 Hz), 7.78 (1H, d, J ¼ 1 Hz),7.83 (1H, d, J ¼ 1 Hz), 8.03 (1H, s), 8.30 (1H, d, J ¼ 9.5 Hz), 8.54 (1H,s); 13C NMR (100 MHz, acetone-d6): 11.3, 24.1 (4C) (CH3), 23.1, 26.16(2C), 26.23 (2C), 47.4 (2C), 50.7 (2C), 68.4 (CH2), 29.07, 29.11 (CH),110.8, 120.0, 122.3, 122.9, 125.0, 126.0, 127.2, 128.4, 130.0, 133.1,134.7, 139.6 (CHarom), 120.6, 123.2, 124.7, 127.28, 127.35,138.8, 142.9,144.9, 146.2, 147.6, 148.1, 150.2, 156.3, 156.8, 160.4 (Carom); HRMS(ESþ) calcd for C44H50N5O (M þ H)þ 664.4015, found 664.4029.

3.1.23. 20-Ethyl-50,500-diisopropyl-2,200-dimorpholin-4-yl-3,70:3,700-terquinoline 29

Same procedure as described above in the preparation ofcompound 8, from compound 18 as boronic acid precursor andcompound 5 (19 mg, 0.057 mmol). Column chromatography(cyclohexane/EtOAc, 98:2 to 90:10) provided compound 29 (37 mg,0.054 mmol, 96%) as a pale yellow solid. Mp ¼ 104e108 �C; IR(ATR): 1607, 1236, 1120 cm�1; 1H NMR (400 MHz, acetone-d6):


1.40e1.51 (15H, m), 3.22e3.27 (4H, m), 3.62e3.67 (4H, m), 3.70e3.82 (9H, m), 3.94 (1H, sep, J ¼ 7 Hz), 4.64 (2H, q, J ¼ 7 Hz), 7.24(1H, d, J ¼ 9.5 Hz), 7.42 (1H, t, J ¼ 7.5 Hz), 7.63e7.69 (2H, m), 7.81(1H, s), 7.83e7.87 (2H, m), 7.90 (1H, d, J ¼ 8 Hz), 7.99 (1H, s), 8.20(1H, s), 8.39 (1H, d, J ¼ 9.5 Hz), 8.56 (1H, s); 13C NMR (100 MHz,acetone-d6): 15.0, 24.0 (2C), 24.2 (2C) (CH3), 46.4 (2C), 50.6 (2C),62.6, 67.2 (2C), 67.4 (2C) (CH2), 29.1, 29.3 (CH), 110.3, 121.3, 121.4,124.6, 125.0, 126.3, 128.1, 128.6, 130.3, 133.9, 134.9, 140.3 (CHarom),121.4, 123.6, 126.5, 127.2, 129.2, 139.0, 142.2, 145.0, 146.5, 147.5,148.2, 149.4, 158.3, 159.5, 160.2 (Carom); HRMS (ESþ) calcd forC43H48N5O3 (M þ H)þ 682.3757, found 682.3782.

3.1.24. 50,500-Diisopropyl-2,200-dimorpholin-4-yl-20-propoxy-3,70:3,700-terquinoline 30

Same procedure as described above in the preparation ofcompound 8, from compound 20 as boronic acid precursor andcompound 5 (25 mg, 0.075 mmol). Column chromatography(cyclohexane/EtOAc, 98:2 to 90:10) provided compound 30 (21 mg,0.030 mmol, 40%) as a yellow solid. Mp ¼ 72e74 �C; IR (ATR): 1607,1236, 1120 cm�1; 1H NMR (400 MHz, acetone-d6): 1.07 (3H, t,J ¼ 7.5 Hz), 1.43 (6H, d, J ¼ 6.5 Hz), 1.49 (6H, d, J ¼ 7 Hz), 1.87 (2H,sex, J¼ 7 Hz), 3.23e3.27 (4H, m), 3.63e3.67 (4H, m), 3.71e3.82 (9H,m), 3.94 (1H, sep, J ¼ 7 Hz), 4.55 (2H, t, J ¼ 6.5 Hz), 7.26 (1H, d,J¼ 9.5 Hz), 7.43 (1H, ddd, J1 ¼8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.64 (1H, d,J¼ 1.5 Hz), 7.67 (1H, ddd, J1¼8.5 Hz, J2 ¼ 7 Hz, J3¼ 1.5 Hz), 7.82 (1H,d, J ¼ 1.5 Hz), 7.86 (1H, d, J ¼ 8 Hz), 7.86 (1H, d, J ¼ 1.5 Hz), 7.92 (1H,d, J ¼ 7.5 Hz), 7.99 (1H, d, J ¼ 1.5 Hz), 8.21 (1H, s), 8.40 (1H, d,J ¼ 9.5 Hz), 8.56 (1H, s); 13C NMR (100 MHz, acetone-d6): 11.3, 24.0(2C), 24.2 (2C) (CH3), 23.1, 46.4 (2C), 50.5 (2C), 67.2 (2C), 67.4 (2C),68.5 (CH2), 29.1, 29.3 (CH), 110.3, 121.2, 121.3, 124.6, 125.0, 126.4,128.1, 128.6, 130.3, 133.9, 134.9, 140.3 (CHarom), 121.4, 123.6, 126.5,127.2, 129.2, 139.0, 142.2, 144.9, 146.4, 147.5, 148.1, 149.3, 158.3,159.5, 160.3 (Carom); HRMS (ESþ) calcd for C44H50N5O3(M þ H)þ

696.3914, found 696.3934.

3.1.25. 20-Ethyl-50,500-diisopropyl-200-morpholin-4-yl-2-pyrrolidin-1-yl-3,70:3,700-terquinoline 31

Same procedure as described above in the preparation ofcompound 8, from compound 17 as boronic acid precursor andcompound 5 (19.5 mg, 0.058 mmol). Column chromatography(cyclohexane/EtOAc, 99:1 to 90:10) provided compound 31 (28 mg,0.042 mmol, 72%) as a pale yellow solid. Mp ¼ 88e92 �C; IR (ATR):1608, 1426, 1407, 1250, 1235, 1120, 1045 cm�1; 1H NMR (400 MHz,acetone-d6): 1.42 (6H, d, J ¼ 6.5 Hz), 1.436 (6H, d, J ¼ 7 Hz), 1.440(3H, t, J ¼ 7 Hz), 1.77e1.83 (4H, m), 3.29e3.35 (4H, m), 3.70e3.83(9H, m), 3.93 (1H, sep, J ¼ 7 Hz), 4.62 (2H, q, J ¼ 7 Hz), 7.25 (1H,d, J ¼ 9.5 Hz), 7.23e7.29 (1H, m), 7.55 (1H, d, J ¼ 1.5 Hz), 7.57 (1H,ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.65 (1H, d, J ¼ 1.5 Hz), 7.73(1H, d, J ¼ 8.5 Hz), 7.79 (1H, d, J ¼ 8 Hz), 7.81 (1H, s), 7.83 (1H, s),8.04 (1H, s), 8.39 (1H, d, J¼ 9.5 Hz), 8.57 (1H, s); 13C NMR (100MHz,acetone-d6): 15.0, 24.0 (4C) (CH3), 26.2 (2C), 46.3 (2C), 50.8 (2C),62.5, 67.4 (2C) (CH2), 29.0, 29.1 (CH), 110.2, 121.37, 122.4, 122.9,125.0, 126.2, 127.17, 128.4, 130.0, 133.8, 134.8, 139.6 (CHarom), 121.38,123.1, 124.6, 127.0, 127.23, 139.1, 143.0, 144.9, 146.2, 147.6, 148.1,149.3, 156.8, 158.3, 160.2 (Carom); HRMS (ESþ) calcd for C43H48N5O2(M þ H)þ 666.3808, found 666.3831.

3.1.26. 50,500-Diisopropyl-200-morpholin-4-yl-20-propoxy-2-pyrrolidin-1-yl-3,70:3,700-terquinoline 32

Same procedure as described above in the preparation ofcompound 8, from compound 19 as boronic acid precursor andcompound 5 (30 mg, 0.089 mmol). Column chromatography(cyclohexane/EtOAc, 99:1 to 90:10) provided compound 32(50 mg, 0.074 mmol, 82%) as a pale yellow solid. Mp ¼ 73e75 �C;IR (ATR): 1608, 1427, 1237 cm�1; 1H NMR (400 MHz, acetone-d6):

1.06 (3H, t, J ¼ 7.5 Hz), 1.43 (6H, d, J ¼ 6.5 Hz), 1.44 (6H, d,J ¼ 7 Hz), 1.78e1.83 (4H, m), 1.86 (2H, sex, J ¼ 7 Hz), 3.29e3.35(4H, m), 3.70e3.82 (9H, m), 3.93 (1H, sep, J ¼ 7 Hz), 4.53 (2H, t,J ¼ 6.5 Hz), 7.25 (1H, d, J ¼ 9.5 Hz), 7.24e7.29 (1H, m), 7.55 (1H, d,J ¼ 1.5 Hz), 7.57 (1H, ddd, J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.64(1H, d, J ¼ 1.5 Hz), 7.73 (1H, d, J ¼ 8.5 Hz), 7.79 (1H, d, J ¼ 8 Hz),7.82 (1H, s), 7.83 (1H, s), 8.04 (1H, s), 8.39 (1H, d, J ¼ 9.5 Hz), 8.56(1H, s); 13C NMR (100 MHz, acetone-d6): 11.3, 24.02 (2C), 24.05(2C) (CH3), 23.1, 26.2 (2C), 46.4 (2C), 50.7 (2C), 67.4 (2C), 68.4(CH2), 29.10, 29.11 (CH), 110.2, 121.2, 122.4, 122.9, 125.0, 126.4,127.19, 128.4, 130.0, 133.9, 134.8, 139.6 (CHarom), 121.4, 123.1, 124.7,127.1, 127.24, 139.1, 143.0, 144.9, 146.2, 147.6, 148.1, 149.3, 156.8,158.3, 160.3 (Carom); HRMS (ESþ) calcd for C44H50N5O2 (M þ H)þ

680.3965, found 680.3972.

3.1.27. 20-Ethoxy-50,500-diisopropyl-2-morpholin-4-yl-200-pyrrolidin-1-yl-3,70:3,700-terquinoline 33

Same procedure as described above in the preparation ofcompound 8, from compound 18 as boronic acid precursor andcompound 4 (16 mg, 0.050 mmol). Column chromatography(cyclohexane/EtOAc, 95:5 to 80:20) provided compound 33 (16 mg,0.024 mmol, 48%) as a yellow solid. Mp ¼ 107e112 �C; IR (ATR):1609, 1429, 1250, 1047 cm�1; 1H NMR (400 MHz, acetone-d6): 1.42(6H, d, J¼ 7Hz),1.45 (3H, t, J¼ 7Hz),1.49 (6H, d, J¼ 7Hz), 2.02e2.08(4Hunder the solvent signal), 3.22e3.28 (4H,m), 3.59e3.69 (8H,m),3.73 (1H, sep, J¼ 7 Hz), 3.95 (1H, sep, J¼ 7 Hz), 4.64 (2H, q, J¼ 7 Hz),6.92 (1H, d, J¼ 9.5 Hz), 7.43 (1H, ddd, J1 ¼8 Hz, J2 ¼ 7 Hz, J3 ¼ 1 Hz),7.56 (1H, d, J¼1.5Hz), 7.67 (1H, ddd, J1¼8.5Hz, J2¼7Hz, J3¼1.5Hz),7.77 (1H, s), 7.84e7.88 (2H,m), 7.92 (1H, d, J¼ 8Hz), 7.99 (1H, s), 8.21(1H, s), 8.31 (1H, d, J ¼ 9.5 Hz), 8.55 (1H, s); 13C NMR (100 MHz,acetone-d6): 15.0, 24.1 (2C), 24.2 (2C) (CH3), 26.2 (2C), 47.4 (2C), 50.5(2C), 62.5, 67.2 (2C) (CH2), 29.0, 29.3 (CH), 110.9, 120.1, 121.2, 124.5,125.0, 125.9, 128.1, 128.6, 130.3, 133.1, 134.8, 140.2 (CHarom), 120.6,123.6,126.5,127.4,129.3,138.7,142.0,144.9,146.4,147.5,148.1,150.2,156.4, 159.5, 160.2 (Carom); HRMS (ESþ) calcd for C43H48N5O2(M þ H)þ 666.3808, found 666.3813.

3.1.28. 50,500-Diisopropyl-2-morpholin-4-yl-20-propoxy-200-pyrrolidin-1-yl-3,70:3,700-terquinoline 34

Same procedure as described above in the preparation ofcompound 8, from compound 20 as boronic acid precursor andcompound 4 (16.6 mg, 0.052 mmol). Column chromatography(cyclohexane/EtOAc, 95:5 to 80:20) provided compound 34 (20 mg,0.029 mmol, 57%) as a pale yellow solid. Mp ¼ 74e76 �C; IR (ATR):1609, 1450, 1406, 1235, 1121 cm�1; 1H NMR (400 MHz, acetone-d6):1.07 (3H, t, J ¼ 7.5 Hz), 1.42 (6H, d, J ¼ 6.5 Hz), 1.49 (6H, d, J ¼ 7 Hz),2.03e2.07 (4H under the solvent signal), 3.22e3.27 (4H, m), 3.59e3.68 (8H, m), 3.72 (1H, sep, J ¼ 7 Hz), 3.94 (1H, sep, J ¼ 7 Hz), 4.54(2H, t, J ¼ 6.5 Hz), 6.91 (1H, d, J ¼ 9.5 Hz), 7.42 (1H, ddd, J1 ¼ 8 Hz,J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.54 (1H, d, J ¼ 1.5 Hz), 7.66 (1H, ddd,J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.77 (1H, d, J ¼ 1.5 Hz), 7.84e7.87(2H, m), 7.91 (1H, d, J ¼ 8 Hz), 7.98 (1H, d, J ¼ 1.5 Hz), 8.20 (1H, s),8.30 (1H, d, J ¼ 9.5 Hz), 8.54 (1H, s); 13C NMR (100 MHz, acetone-d6): 11.3, 24.0 (2C), 24.2 (2C) (CH3), 23.1, 26.2 (2C), 47.4 (2C), 50.5(2C), 67.2 (2C), 68.4 (CH2), 29.1, 29.3 (CH), 110.8, 119.9, 121.2, 124.5,125.0, 126.0, 128.1, 128.6, 130.3, 133.1, 134.8, 140.2 (CHarom), 120.6,123.6, 126.5, 127.5, 129.2, 138.7, 142.0, 144.9, 146.4, 147.5, 148.1,150.2, 156.3, 159.5, 160.3 (Carom); HRMS (ESþ) calcd for C44H50N5O2

(M þ H)þ 680.3965, found 680.3968.

3.1.29. 7-Bromo-2-chloro-5-isopropylquinoline 35Same procedure as described above in the preparation of

compound 4: compound 1 (300 mg, 1.06 mmol), PtO2 (36 mg,0.16 mmol), EtOAc (16 mL). Column chromatography (cyclohexane/EtOAc, 99:1) provided 35 (267mg, 0.94mmol, 88%) as awhite solid.


Mp ¼ 42e46 �C; IR (ATR): 1592, 1584, 1122 cm�1; 1H NMR(400 MHz, DMSO-d6): 1.32 (3H, d, J ¼ 6.5 Hz), 3.74 (1H, hept,J¼ 6.5 Hz), 7.62e7.67 (1H, m), 7.65 (1H, s), 8.05 (1H, m), 8.68 (1H, d,J ¼ 9 Hz). 13C NMR (100 MHz, DMSO-d6): 23.2 (2C) (CH3), 28.0 (CH),122.6, 126.4, 128.0, 136.5 (CHarom), 123.7, 124.5, 148.2, 148.4, 150.6(Carom). HRMS (ESþ) calcd for C12H12

79Br35ClN (M þ H)þ 283.9842,found 283.9842.

3.1.30. 20-Chloro-50-isopropyl-2-propoxy-3,70-biquinoline 38Step A: boronic acid was prepared from compound 35 as

described above in the preparation of compound 8 (Step A). Step B:isopropanol (0.5 mL) was added to a mixture of tBuOK (43 mg,0.38 mmol) and PEPPSI-iPr (17.4 mg, 0.026 mmol) under argon. Themixture was stirred at room temperature for 10 min and a solutionof boronic acid from step A (2 equiv) in THF (0.5 mL) and thencompound 37 (72 mg, 0.25 mmol) were added. The reactionmixture was stirred at room temperature overnight. Water wasadded and the product was extracted with EtOAc. The assembledorganic fractions were dried over MgSO4 and evaporated underreduced pressure. The residue was purified by column chroma-tography (cyclohexane/EtOAc, 100:0 to 99:1) to give 38 (57 mg,0.15 mmol, 58%) as a white gum. IR (ATR): 1616, 1441, 1273, 1263,1241 cm�1; 1H NMR (400 MHz, CDCl3): 1.03 (3H, t, J ¼ 7.5 Hz), 1.44(6H, d, J ¼ 6.5 Hz), 1.85 (2H, sex, J ¼ 7 Hz), 3.70 (1H, sep, J ¼ 6.5 Hz),4.52 (2H, t, J ¼ 6.5 Hz), 7.38e7.45 (2H, m), 7.65 (1H, t, J ¼ 7.5 Hz),7.79 (1H, d, J ¼ 7.5 Hz), 7.85e7.90 (2H, m), 8.10 (1H, s), 8.12 (1H, s),8.42 (1H, d, J ¼ 9 Hz); 13C NMR (100 MHz, CDCl3): 11.0, 23.8 (2C)(CH3), 22.5, 68.1 (CH2), 28.9 (CH), 122.0, 124.4, 124.9, 126.96, 127.01,127.8, 129.8, 134.7, 138.8 (CHarom), 124.5, 125.5, 125.9, 139.6, 144.5,146.4, 148.8, 150.5, 159.5 (Carom); HRMS (ESþ) calcd forC24H24

35ClN2O (M þ H)þ 391.1577, found 391.1565.

3.1.31. 20-Chloro-2-ethoxy-5,50-diisopropyl-3,70-biquinoline 39Same procedure as described above in the preparation of

compound 38: compound 37 (63 mg, 0.221 mmol), 4 h. Columnchromatography (cyclohexane/EtOAc, 100:0 to 99:1) provided 39(55mg, 0.131mmol, 59%) as awhite solid. Mp¼ 34e37 �C; IR (ATR):1611, 1451, 1439, 1386, 1265, 1244 cm�1; 1H NMR (400 MHz, CDCl3):1.37e1.47 (15H, m), 3.61e3.77 (2H, m), 4.62 (2H, q, J ¼ 6.5 Hz), 7.34(1H, d, J ¼ 7 Hz), 7.43 (1H, d, J ¼ 8.5 Hz), 7.61 (1H, t, J ¼ 7.5 Hz), 7.73(1H, d, J ¼ 8.5 Hz), 7.95 (1H, s), 8.11 (1H, s), 8.40e8.47 (2H, m); 13CNMR (100 MHz, CDCl3): 14.8, 23.8 (2C) 23.9 (2C) (CH3), 62.1 (CH2),28.76, 28.81 (CH),120.3,121.9,125.16,125.19,126.7,129.8,134.8 (2C)(CHarom), 123.4, 124.5, 124.9, 140.2, 144.4, 145.6, 147.0, 148.9, 150.5,158.7 (Carom); HRMS (ESþ) calcd for C26H28

35ClN2O (M þ H)þ

419.1890, found 419.1889.

3.1.32. 50-Isopropyl-2-propoxy-20-pyrrolidin-1-yl-3,70-biquinoline 40Same procedure as described above in the preparation of

compound 2: compound 38 (45 mg, 0.12 mmol), pyrrolidine(1 mL), 1 h. Column chromatography (cyclohexane/EtOAc, 9:1)provided 40 (46 mg, 0.11 mmol, 94%) as a pale yellow solid.Mp ¼ 57e62 �C; IR (ATR): 1609, 1522, 1451, 1435, 1407, 1263 cm�1;1H NMR (400 MHz, acetone-d6): 1.06 (3H, t, J ¼ 7.5 Hz), 1.40 (6H, d,J ¼ 7 Hz), 1.84 (2H, sex, J ¼ 7 Hz), 2.02e2.07 (4H under the solventsignal), 3.58e3.65 (4H, m), 3.71 (1H, sep, J ¼ 7 Hz), 4.50 (2H, t,J ¼ 6.5 Hz), 6.89 (1H, d, J ¼ 9.5 Hz), 7.44 (1H, ddd, J1 ¼ 8 Hz,J2 ¼ 7 Hz, J3 ¼ 1 Hz), 7.51 (1H, d, J ¼ 1.5 Hz), 7.66 (1H, ddd,J1 ¼ 8.5 Hz, J2 ¼ 7 Hz, J3 ¼ 1.5 Hz), 7.76 (1H, s), 7.83 (1H, d,J ¼ 8.5 Hz), 7.95 (1H, d, J ¼ 8 Hz), 8.27e8.31 (2H, m); 13C NMR(100 MHz, acetone-d6): 11.3, 24.0 (2C) (CH3), 23.0, 26.1 (2C), 47.3(2C), 68.4 (CH2), 29.1 (CH), 110.8, 119.8, 125.0, 126.0, 127.5, 128.7,130.1, 133.1, 139.0 (CHarom), 120.6, 126.6, 127.8, 138.3, 145.0, 146.8,150.1, 156.3, 160.4 (Carom); HRMS (ESþ) calcd for C28H32N3O(M þ H)þ 426.2545, found 426.2564.

3.1.33. 50-Isopropyl-20-morpholin-4-yl-2-propoxy-3,70-biquinoline 41Same procedure as described above in the preparation of

compound 2: compound 38 (35 mg, 0.090 mmol), morpholine(1 mL), 3 h. Column chromatography (cyclohexane/EtOAc, 95:5 to80:20) provided 41 (34 mg, 0.077 mmol, 86%) as a white solid.Mp ¼ 67e69 �C; IR (ATR): 1607, 1436, 1409, 1268, 1261, 1237,1122 cm�1; 1H NMR (400 MHz, CDCl3): 1.04 (3H, t, J ¼ 7 Hz), 1.40(6H, d, J ¼ 6.5 Hz), 1.84 (2H, sex, J ¼ 7 Hz), 3.64 (1H, sep, J ¼ 6.5 Hz),3.69e3.76 (4H, m), 3.84e3.91 (4H, m), 4.50 (2H, t, J ¼ 6.5 Hz), 7.01(1H, d, J ¼ 9.5 Hz), 7.39 (1H, t, J ¼ 7.5 Hz), 7.56 (1H, s), 7.62 (1H, t,J ¼ 7.5 Hz), 7.76 (1H, d, J ¼ 8 Hz), 7.81 (1H, s), 7.87 (1H, d, J ¼ 8 Hz),8.14 (1H, s), 8.25 (1H, d, J¼ 9.5 Hz); 13C NMR (100MHz, CDCl3): 11.1,23.8 (2C) (CH3), 22.5, 45.8 (2C), 67.1 (2C), 68.0 (CH2), 28.6 (CH),109.0, 120.8, 124.2, 125.5, 126.9, 127.7, 129.4, 133.3, 138.4 (CHarom),120.7, 125.6, 127.0, 138.4, 144.0, 146.2, 148.4, 157.4, 159.9 (Carom);HRMS (ESþ) calcd for C28H32N3O2 (M þ H)þ 442.2495, found442.2492.

3.1.34. 2-Ethoxy-5,50-diisopropyl-20-pyrrolidin-1-yl-3,70-biquinoline 42

Same procedure as described above in the preparation ofcompound 2: compound 39 (25 mg, 0.060 mmol), pyrrolidine(1 mL), 1 h. Column chromatography (cyclohexane/EtOAc, 9:1)provided 42 (23 mg, 0.051 mmol, 85%) as a white gum. IR (ATR):1610, 1451, 1437, 1386, 1265 cm�1; 1H NMR (400 MHz, acetone-d6):1.37e1.45 (15H, m), 2.01e2.09 (4H under the solvent signal), 3.59e3.66 (4H, m), 3.71 (1H, sep, J ¼ 7 Hz), 3.86 (1H, sep, J ¼ 7 Hz), 4.58(2H, q, J ¼ 7 Hz), 6.91 (1H, d, J ¼ 9.5 Hz), 7.39 (1H, d, J ¼ 7 Hz), 7.52(1H, s), 7.62 (1H, t, J ¼ 8 Hz), 7.69 (1H, d, J ¼ 8 Hz), 7.73 (1H, s), 8.30(1H, d, J ¼ 9 Hz), 8.51 (1H, s); 13C NMR (400 MHz, acetone-d6): 14.9,24.1 (4C) (CH3), 29.0, 29.1 (CH), 26.2 (2C), 47.4 (2C), 62.4 (CH2),110.8, 120.1, 120.9, 125.8, 125.9, 130.1, 133.1, 134.8 (CHarom), 120.6,124.2, 127.2, 138.8, 144.9, 146.3, 147.4, 150.2, 156.3, 159.7 (Carom);HRMS (ESþ) calcd for C30H36N3O (M þ H)þ 454.2858, found454.2868.

3.1.35. 2-Ethoxy-5,50-diisopropyl-20-morpholin-4-yl-3,70-biquinoline 43

Same procedure as described above in the preparation ofcompound 2: compound 39 (35 mg, 0.084 mmol), morpholine(1 mL), 2 h. Column chromatography (cyclohexane/EtOAc, 95:5)provided 43 (18 mg, 0.038 mmol, 46%) as a white solid. Mp ¼ 62e67 �C; IR (ATR): 1607, 1434, 1409, 1266, 1244, 1237, 1122 cm�1; 1HNMR (400 MHz, acetone-d6): 1.37e1.46 (15H, m), 3.68e3.92 (10H,m), 4.58 (2H, q, J¼ 7 Hz), 7.25 (1H, d, J¼ 9 Hz), 7.40 (1H, d, J¼ 7 Hz),7.60e7.66 (2H, m), 7.70 (1H, d, J ¼ 8 Hz), 7.78 (1H, s), 8.38 (1H, d,J ¼ 9 Hz), 8.53 (1H, s); 13C NMR (400 MHz, acetone-d6): 14.9, 24.0(2C), 24.1 (2C) (CH3), 46.4 (2C), 62.4, 67.4 (2C) (CH2), 29.0, 29.1 (CH),110.2, 120.9, 121.38, 125.8, 126.3, 130.2, 133.8,134.9 (CHarom), 121.36,124.2, 126.9, 139.1, 144.9, 146.4, 147.5, 149.3, 158.3, 159.7 (Carom);HRMS (ESþ) calcd for C30H36N3O2 (M þ H)þ 470.2808, found470.2805.

3.1.36. 2,20-Dichloro-50-isopropyl-3,70-biquinoline 44Isopropanol (0.3 mL) was added to a mixture of tBuOK (23 mg,

0.205mmol) and PEPPSI-iPr (9.5 mg, 0.014mmol) under argon. Themixture was stirred at room temperature for 10 min and boronicacid 12 (1.7 equiv.), compound 35 (40 mg, 0.141 mmol) and THF(0.5 mL) were added. The mixture was stirred at room temperaturefor 4 h. Water was added and the product was extracted withEtOAc. The assembled organic fractions were dried over MgSO4 andevaporated under reduced pressure. The residue was purified bycolumn chromatography (cyclohexane/EtOAc 99:1 to 95:5) to give44 (22 mg, 0.060 mmol, 43%) as a white solid. Mp ¼ 55e59 �C; IR(ATR): 1616,1587,1558,1486 cm�1; 1H NMR (400MHz, CDCl3): 1.45


(6H, d, J ¼ 6.5 Hz), 3.72 (1H, sep, J ¼ 6.5 Hz), 7.48 (1H, d, J ¼ 9 Hz),7.63 (1H, t, J¼ 7.5 Hz), 7.69 (1H, s), 7.80 (1H, t, J¼ 7.5 Hz), 7.89 (1H, d,J ¼ 8 Hz), 8.03 (1H, s), 8.11 (1H, d, J ¼ 8.5 Hz), 8.23 (1H, s), 8.46 (1H,d, J ¼ 9 Hz); 13C NMR (100 MHz, CDCl3): 23.7 (2CH3), 28.9 (CH),122.6, 124.8, 127.6, 127.7, 127.8, 128.6, 131.0, 134.9, 139.3 (CHarom),124.8, 127.3, 134.2, 139.9, 145.4, 147.3, 148.5, 149.3, 151.0 (Carom);HRMS (ESþ) calcd for C21H17N2

35Cl2 (M þ H)þ 367.0769, found367.0766.

3.1.37. 50-Isopropyl-2,20-dipyrrolidin-1-yl-3,70-biquinoline 45Same procedure as described above in the preparation of

compound 2: compound 44 (18 mg, 0.049 mmol), pyrrolidine(1 mL), 1 h. Column chromatography (cyclohexane/EtOAc, 8:2)provided 45 (20 mg, 0.046 mmol, 93%) as a white solid. Mp ¼ 92e95 �C; IR (ATR): 1609, 1435 cm�1; 1H NMR (400 MHz, CDCl3): 1.34(6H, d, J ¼ 6.5 Hz), 1.71e1.77 (4H, m), 2.02e2.09 (4H, m), 3.28e3.34(4H, m), 3.56e3.70 (5H, m), 6.77 (1H, d, J ¼ 9 Hz), 7.18 (1H, s), 7.20(1H, t, J ¼ 7.5 Hz), 7.53 (1H, t, J ¼ 7.5 Hz), 7.61 (1H, d, J ¼ 8 Hz), 7.70(1H, s), 7.77 (1H, d, J ¼ 8 Hz), 7.89 (1H, s), 8.17 (1H, d, J ¼ 9 Hz); 13CNMR (100MHz, acetone-d6): 24.0 (2C) (CH3), 26.16 (2C), 26.20 (2C),47.4 (2C), 50.6 (2C) (CH2), 28.9 (CH), 110.7, 119.3, 122.8, 124.8, 127.1,128.3, 129.8, 133.2, 139.2 (CHarom), 120.1, 124.7, 127.9, 142.5, 145.6,148.1, 150.2, 156.5, 156.8 (Carom); HRMS (ESþ) calcd for C29H33N4

(M þ H)þ 437.2705, found 437.2726.

3.1.38. 50-Isopropyl-2,20-dimorpholin-4-yl-3,70-biquinoline 46Same procedure as described above in the preparation of

compound 2: compound 44 (14.0 mg, 0.038 mmol), morpholine(1mL),15 h. Column chromatography (cyclohexane/EtOAc, 9:1 to 7:3)provided 46 (16 mg, 0.034 mmol, 90%) as a pale yellow solid.Mp ¼ 108e112 �C; IR (ATR): 1607, 1407, 1242, 1234, 1120 cm�1; 1HNMR (400 MHz, CDCl3): 1.40 (6H, d, J ¼ 6.5 Hz), 3.24e3.32 (4H, br s),3.59e3.68 (5H,m), 3.71e3.78 (4H, br s), 3.85e3.92 (4H, br s), 7.02 (1H,d, J¼ 9.5 Hz), 7.36 (1H, t, J¼ 7 Hz), 7.59 (1H, s), 7.61 (1H, t, J¼ 7.5 Hz),7.70 (1H, d, J¼ 8 Hz), 7.79 (1H, s), 7.87 (1H, d, J¼ 8.5 Hz), 8.00 (1H, s),8.25 (1H, d, J ¼ 9.5 Hz); 13C NMR (100 MHz, CDCl3): 23.9 (2C) (CH3),45.6 (2C), 49.6 (2C), 67.0 (2C), 67.1 (2C) (CH2), 28.7 (CH), 108.9, 119.1,123.7, 124.3, 127.4, 127.5, 129.6, 133.5, 139.7 (CHarom), 120.6, 125.5,128.7, 141.8,144.9,146.7, 149.1, 157.4,158.7 (Carom); HRMS (ESþ) calcdfor C29H33N4O2 (M þ H)þ 469.2604, found 469.2612.

3.2. Biological evaluations

3.2.1. Cell lines and productsAll media and cell culture reagents were purchased from Invi-

trogen. BP3, IM9 and RS4.11 cells were cultured in RPMI completemedium supplemented with 10% fetal bovine serum, 2 mM gluta-mine, 10 mM Hepes and 40 mg/mL gentamicin. WT and DKO MEFwere cultured in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal bovine serum, 2 mM glutamine, 10 mMHepes and 40 mg/mL gentamicin. Blood samples from healthydonors were obtained from the Etablissement Français du Sang andPBMC were purified on FicollPAQUE gradient and cultured in RPMIcomplete medium supplemented with 10% fetal bovine serum,2 mM glutamine, 10 mM Hepes and 40 mg/mL gentamicin. 3T3 cellsoverexpressing human Bcl-xL were obtained by 3T3 infection withpMig-hBcl-xL vector. These cells were cultured in Dulbecco’smodified Eagle’s medium supplemented with 10% fetal bovineserum, 2 mM glutamine, 10 mM Hepes and 40 mg/mL gentamicin.

ABT-737 and Obatoclax were purchased from Euromedex,Gambogic acid from TEBU and Staurosporine from SIGMA.

3.2.2. Cell death assaysCells were treated with molecules for 24 h. Cell death was

evaluated by propidium iodide staining in combination or not with

Annexin V labeling and analyzed by FACS with the CellQuest Soft-ware (Becton Dickinson).

3.2.3. ELISA assaysBcl-xL/Bim, Bcl-2/Bim, Bfl-1/Bim and Mcl-1/Bim interactions

inhibition were evaluated using ELISA assay. Briefly, 96 well plateswere coated overnight at 4 �C with anti-GST antibody (USBio-logical) diluted in phosphate buffer saline to a final concentration of1 mg/mL. Plates were washed 3 times in washing buffer (PBS, 0.05%Tween) and blocked 1 h in blocking buffer (PBS, 1% BSA). Plateswere washed 3 times in washing buffer and GST-tagged recombi-nant Bcl-xL, Bcl-2, Bfl-1 or Mcl-1 proteins were incubated 2 h atroom temperature to a final concentration of 10 ng/mL in blockingbuffer. Plates were washed three times in washing buffer andmolecules were incubated 1 h at room temperature diluted atindicated concentrations in blocking buffer. Plates were emptiedand biotinylated Bim peptide was incubated 1 h at room temper-ature without washing to a final concentration of 1 nM in blockingbuffer. Plates were washed 3 times in washing buffer and HRP-streptavidin (R&D systems) was incubated 1:200 in blockingbuffer for 20 min at room temperature. Plates were washed 3 timesin washing buffer and substrate (R&D systems) was incubated atroom temperature until coloration development. Reaction wasstopped using 2 N sulfuric acid and OD was monitored at 450 nmusing an Infinite M200 plate reader. EC50 determinations wereperformed using GraphPad Prism software (GraphPad, Inc., SanDiego, CA).

Acknowledgments

The authors thank Bertrand Légeret for mass spectrometryanalysis and the contribution of the staff from the Flow cytometryfacility of SFR Biosciences Gerland-Lyon Sud (UMS344/US8) (http://www.ifr128.prd.fr/anglais/page_IFR128.htm). The French Ministèrede l’Enseignement Supérieur et de la Recherche is greatlyacknowledged for financial support (ES). This work was also sup-ported by institutional grants from INSERM and UCBL as well as theAssociation pour la Recherche sur le Cancer (NB).

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