De novo design, synthesis and evaluation of benzylpiperazine derivatives as highly selective binders of Mcl-1

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DOI: 10.1002/cmdc.201300316

De Novo Design, Synthesis and Evaluation of

Benzylpiperazine Derivatives as Highly Selective Binders

of Mcl-1

Xiao Ding,^[a]Yan Li,^[b]Li Lv,^[a]Mi Zhou,^[b]Li Han,^[b]Zhengxi Zhang,^[b]Qian Ba,^[c]Jingquan Li,^[c]

Hui Wang,^[c]Hong Liu,*^[a]and Renxiao Wang*^[b]

Considerable efforts have been made to the development of

small-molecule inhibitors of antiapoptotic B-cell lymphoma 2

(Bcl-2) family proteins (such as Bcl-2, Bcl-x_L, and Mcl-1) as

a new class of anticancer therapies. Unlike general inhibitors of

the entire family, selective inhibitors of each member protein

can hopefully reduce the adverse side effects in chemotherapy

treatments of cancers overexpressing different Bcl-2 family

proteins. In this study, we designed four series of benzylpipera-

zine derivatives as plausible Bcl-2 inhibitors based on the out-

comes of a computational algorithm. A total of 81 compounds

were synthesized, and their binding affinities to Bcl-2, Bcl-x_L,

and Mcl-1 measured. Encouragingly, 22 compounds exhibited

binding affinities in the micromolar range (K_i<20 mm) to at

least one target protein. Moreover, some compounds were ob-

served to be highly selective binders to Mcl-1 with no detecta-

ble binding to Bcl-2 or Bcl-x_L, among which the most potent

one has a K_ivalue of 0.18 mm for Mcl-1. Binding modes of four

selected compounds to Mcl-1 and Bcl-x_Lwere derived through

molecular docking and molecular dynamics simulations. It

seems that the binding affinity and selectivity of these com-

pounds can be reasonably interpreted with these models. Our

study demonstrated the possibility for obtaining selective Mcl-

1 inhibitors with relatively simple chemical scaffolds. The active

compounds identified by us could be used as lead compounds

for developing even more potent selective Mcl-1 inhibitors

with potential pharmaceutical applications.

Introduction

Apoptosis, or programmed cell death, is an essential physio-

logical process required by the development and maintenance

of tissue homeostasis. Disregulation of this process is known

to be associated with cancer.^[1–5]The B-cell lymphoma 2 (Bcl-2)

family of proteins are a major group of apoptosis regulators,

which includes both antiapoptotic members, such as Bcl-2, Bcl-

x_L, Mcl-1, and Bcl-w, as well as proapoptotic members, such as

Bak, Bax, and other BH3-only proteins.^[6]Overexpression of an-

tiapoptotic Bcl-2 family proteins contributes to the resistance

to chemotherapies and ratiotherapies in various cancer cells.

Therefore, targeting Bcl-2 family proteins has become an at-

tractive approach towards the invention of new cancer thera-

pies since the beginning of this century.^[7,8]

A number of small-molecule inhibitors of Bcl-2 family pro-

teins have been reported during the past decade.^[9–30]The

chemical structures and binding data of some potent Bcl-2 in-

hibitors are summarized in Figure 1. Interestingly, quite

a number of Bcl-2 inhibitors are more potent towards Bcl-2 or

Bcl-x_Lthan Mcl-1. For example, ABT-737 and its orally active an-

alogue ABT-263 (Navitoclax) were found to be highly potent

inhibitors of Bcl-2, Bcl-x_Land Bcl-w (K_i<0.001 mm), but only

modestly targeted Mcl-1 (K_i=0.55 mm).^[9–11]They thus lack effi-

cacy on some types of cancer cells with Mcl-1 overexpression.

Indeed, downregulation of Mcl-1 makes cancer cells sensitive

to ABT-737 or other cytotoxic agents.^[31–35]In contrast, identifi-

cation of inhibitors of other known antiapoptotic members,

such as Mcl-1, is relatively unaddressed. It is important to men-

tion that though all antiapoptotic Bcl-2 family proteins have

similar 3D structures, the sequence identities among them are

only modest, typically around 30–70%, implying that they may

target different types of tissues during apoptosis and could

also differ in responses to different stress stimuli.^[36]For exam-

ple, it is known that Bcl-2 and Bcl-x_Lare essential for regulating

B-lymphocytes and platelet survival.^[37,38]Hence, administration

of anticancer drugs interacting with them might cause either

lymphopaenia or thrombocytopaenia to patients in which the

two proteins are not overexpressed. In contrast, Mcl-1 and

Bcl-w are essential for the sustainable production of haemato-

[a] Dr. X. Ding, L. Lv, Prof. H. Liu

CAS Key Laboratory of Receptor Research

Shanghai Institute of Materia Medica

Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences

555 Zu Chong Zhi Road

Shanghai 201203 (P.R. China)

E-mail: hliu@mail.shcnc.ac.cn

[b] Dr. Y. Li, M. Zhou, Dr. L. Han, Z. Zhang, Prof. R. Wang

State Key Laboratory of Bioorganic Chemistry

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences

345 Lingling Road

Shanghai 200032 (P.R. China)

E-mail: wangrx@mail.sioc.ac.cn

[c] Dr. Q. Ba, Dr. J. Li, Prof. H. Wang

Institute of Nutritional Sciences

Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences

319 Yueyang Road

Shanghai 200031 (P.R. China)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201300316.

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Figure 1. Known small-molecule inhibitors of antiapoptotic Bcl-2 family proteins.

poetic stem cells and sperm cells, respectively.^[39,40]It is reason-

able to expect that specific inhibitors of each antiapoptotic

Bcl-2 family protein are able to reduce the adverse side effects

in chemotherapy treatments of cancers overexpressing differ-

ent Bcl-2 family proteins.

efficacy of conventional anticancer chemotherapies on cancer

cells with Mcl-1 overexpression. Such compounds could also

be used as molecular tools to further explore the biological

mechanisms of Mcl-1, which is actually not fully understood so

far.

To date, some efforts have been made on targeting Mcl-

1 for cancer treatment,^[41]such as, downregulation of Mcl-1 by

cyclin-dependent kinase inhibitors, debiquitinase inhibitors, or

treatment of antisense oligonucleotides. Some small-molecule

inhibitors of Bcl-2/Bcl-x_Lalso have decent binding affinities to

Mcl-1. However, only a few selective Mcl-1 inhibitors have

been reported. To the best of our knowledge, the only potent

selective Mcl-1 inhibitor was reported by Friberg et al.^[28](K_i<

100 nm) with roughly 100-fold selectivity over Bcl-2 or Bcl-x_L.

Here, we propose that besides developing general inhibitors of

Bcl-2 family proteins, developing selective Mcl-1 inhibitors rep-

resents an alternative strategy for enhancing the therapeutic

Mcl-1 as well as other Bcl-2 family proteins conduct their

biological functions through protein–protein interactions with

proapoptotic Bcl-2 family proteins.^[6]Designing small-molecule

inhibitors of protein–protein interactions is more challenging

in drug discovery. A major reason is that protein–protein bind-

ing interfaces are normally wider and flatter, lacking well-de-

fined “binding pockets”.^[42–45]In our previous study, we relied

on structure-based virtual screening to discover lead com-

pounds, which led to the development of more potent Bcl-2

inhibitors, for example, DCBL55.^[22]In this study, we decided to

adopt the de novo design strategy to generate the structural

scaffolds of our lead compounds instead, since this strategy

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can in principle explore a larger chemical space. It is also more

convenient to incorporate human expertise during this pro-

cess.

identify conserved residue clusters. We then screened over

25000 protein–ligand complexes in PDB for such residue clus-

ters as well as the suitable chemical fragments matching them.

Based on the outcomes of this analysis, four series of benzylpi-

perazine derivatives, in total 81 compounds, were designed

and synthesized. Binding affinities of these compounds to Mcl-

1, Bcl-2 and Bcl-x_Lwere measured in a fluorescence polariza-

tion (FP)-based binding assay. A number of obtained com-

pounds exhibited binding to Mcl-1 with inhibition constants

(K_i) ranging between 0.18–20.3 mm. Interestingly, some of them

are selective binders to Mcl-1 with no detectable binding to

Bcl-2 or Bcl-x_L. The structure–activity relationships (SARs) of

these compounds were analyzed and further interpreted by

the binding modes derived through molecular modeling. Our

study provides valuable lead compounds for the development

of even more potent selective Mcl-1 inhibitors. Such com-

pounds could help to explore the biological role of Mcl-1 in

normal physiology and tumor maintenance and might comple-

ment other Bcl-2 inhibitors in therapeutic applications.

A popular approach for deriving inhibitors of protein–pro-

tein interactions is through so-called fragment-based design.

Normally, the starting point of this approach is low-weight

ligand molecules (MW<300) identified by NMR screening or

other experimental means. For example, a successful applica-

tion of this approach has been demonstrated in the develop-

ment of ABT-737/ABT-263.^[9,10]In this study, we employed

a computational approach to apply fragment-based design.

The key idea of this approach is illustrated in Figure 2 whereas

Results and Discussion

Design of four compound series and their SARs

Bcl-2 family proteins are good targets for applying fragment-

based design since the BH3-domain binding site on them can

be divided into several subsites. As examples, the binding sites

on Bcl-x_Land Mcl-1 are shown in Figure 3. In general, we divid-

ed the binding site into three subsites, P-site, Q-site, and L-site.

The P-site is essentially hydrophobic, mainly consisting of

Leu108, Val126 and Phe97 residues in the case of Bcl-x_L, and

Met231, Leu235, Val249 residues in the case of Mcl-1. The Q-

site is a more hybridized site, which mainly consists of Arg100,

Asn136 and Tyr195 residues in the case of Bcl-x_L, and His224,

Asn260, Phe319 residues in the case of Mcl-1. The L-site refers

to the region between the P-site and the Q-site, where a linker

fragment is to be placed in order to connect the chemical frag-

ments fitting to the P-site and Q-site.

Figure 2. Illustration of a fragment-based computational approach for de-

signing small-molecule inhibitors of protein–protein interactions. (1) Con-

served residue clusters are identified on a given protein–protein binding in-

terface. (2) A library of protein–ligand complex structures is searched for

same or similar residue clusters, and chemical fragments interacting with

these residue clusters are retrieved. (3) Small-molecule binders are sketched

by assembling suitable chemical fragments.

By applying a fragment-based design approach described in

the Experimental Section, we obtained several types of chemi-

cal fragments from PDB that match the residue clusters at

each subsite (Figure 4). It should be mentioned that in order

to increase the chance for obtaining active compounds, we

considered Mcl-1 as well as Bcl-x_Lat this step. These fragments

were then docked into each corresponding subsite to examine

their fitness. A few of them are shown in Figure 3 as examples.

During the next step, we selected some hydrophobic moieties,

such as, phenyl, biphenyl, and piperazine groups, for the P-

site, a phenyl moiety with polar substituent groups for the Q-

site, and benzamide and tetrazole groups as linkers. Later, the

tetrazole group was replaced by a triazole group since the

latter was more convenient for synthesis through “click

chemistry”. These separated fragments were assembled to-

gether to form complete molecules of proper sizes for fitting

to the BH3-binding groove.

detailed descriptions are given elsewhere.^[46]Briefly, our as-

sumption is that critical residues at the binding interface of

protein–protein complexes exist in conserved clusters. Such

residue clusters form microenvironments on the binding inter-

face, which are able to host certain chemical fragments. Given

the abundant structural information available from public do-

mains such as the Protein Data Bank (PDB),^[47]one can find

suitable chemical fragments from known protein–ligand com-

plexes by mapping the same (or similar) residue clusters. If

there are multiple conserved residue clusters on a protein–pro-

tein binding interface, it is then possible to retrieve multiple

chemical fragments in this way and then assemble them into

complete molecules.

Following this approach, we analyzed the binding sites on

Mcl-1 and Bcl-x_Lwith a set of in-house computer programs to

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Synthesis

Four series (A–D), including a total of 81 compounds, were

synthesized in our study (Figure 4). The synthetic routes for

these compounds are illustrated in Scheme 1, 2, 3, and 4, re-

spectively. Compounds A1–A26 were synthesized through the

route outlined in Scheme 1. Compound 1 was obtained from

the reaction with tert-butoxycarbonyl (Boc)-piperazine and cor-

responding benzyl bromide derivatives. Benzylpiperazine 2a–t

were obtained from a Suzuki coupling reaction between 1 and

different phenylboronic acids, followed by deprotection of the

Boc group with trifluoroacetic acid (TFA). Compounds 3a–k

were obtained through amide coupling reaction of methyl 4-

(aminomethyl)benzoate and different acids or S_N2 reactions be-

tween methyl 4-(aminomethyl)benzoate and halide com-

pounds, which was followed by hydrolysis of the ester. Final

products A1–A26 were obtained by amide coupling of 2a–t

and 3a–k.

Compounds B1–B17 were synthesized through the route

outlined in Scheme 2. Compounds 4a–g were obtained by

amide coupling reaction between methyl 3-aminobenzoate

and different acids, which was followed by hydrolysis of the

ester. Final products B1–B17 were obtained by amide coupling

of 2a–t and 4a–g.

Compounds C1–C12 were synthesized through the route

outlined in Scheme 3. Ethyl 2-bromoacetate was prepared

from commercially available (3-aminophenyl)methanol or 3-

(hydroxymethyl)phenol. It was followed by the bromination

with N-bromosuccinimide (NBS) and nucleophilic displacement

with azide to yield 7a,b. Hydrolyzing 7a,b yielded the key in-

termediates 8a,b. Compounds 9a–h were obtained by treating

benzylpiperazine 2a–t with 3-

Figure 3. Illustration of the three subsites in the BH3-binding groove on

a) Bcl-x_L(PDBID: 1PQ1) and b) Mcl-1 (PDBID: 2NL9) and some chemical frag-

ments matching these subsites retrieved from PDB.

bromoprop-1-yne. Click reaction

of azide 8a,b and alkyne 9a–h

afforded final products C1–C12.

Scheme 4 outlines the route

for the synthesis of compounds

D1–D26. Amide coupling of

benzylpiperazine 2a–t afforded

from Scheme 1 with several

commercially available acids

yielded compounds D1–D12.

Treating benzylpiperazine 2a–t

with anhydride, followed by

amide coupling with commer-

cially available amines, afforded

compounds C13–C24. Intermedi-

ate 11 was afforded through the

reaction of phthalic anhydride

with 1-(biphenyl-3-ylmethyl)pi-

perazine. Finally, D25 and D26

were obtained from the amide

coupling reaction between 11

and 3,4,5-trimethoxyaniline or

2,4-dimethoxyaniline, respective-

Figure 4. Fragment-based design of the compounds described in our study. The fragments actually used in our

compounds are shown in red in the boxes on the left. The representative chemical structures in four series are

shown on the right. Exact chemical structures of all compounds are given in Tables 1–4.

ly.

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Scheme 1. Reagents and conditions: a) TEA, CH₂Cl₂; b) Boronic acid or ester, Pd(PPh₃)₄, Na₂CO₃, DME/EtOH/H₂O; c) TFA, CH₂Cl₂; d) EDCI, TEA, HOBT, CH₂Cl₂;

e) TEA, CH₂Cl₂; f) NaOH, CH₃OH, H₂O; g) HATU, DIPEA, DMF.

Chemical structures and bind-

ing affinity data of compounds

A1–A26 are summarized in

Table 1. Our binding assay re-

sults indicate that a number of

compounds in this series exhibit-

ed binding affinities to Mcl-1, of

which six compounds (A1, A7,

A8, A14, A15 and A17) have K_i

values below 15 mm. In particu-

Scheme 2. Reagents and conditions: a) EDCI, TEA, HOBT, CH₂Cl₂; b) NaOH, CH₃OH, H2O; c) 2a–t, HATU, DIPEA, DMF.

lar, A1 is the most potent com-

Biological evaluation

pound with K_i=0.18 mm. It seems that the biphenyl group is

the optimal fragment for the P-site. Moving the attachment of

the biphenyl group from ortho- (A1) to meta- (A2) or para-po-

sition (A3) abolishes binding affinity. This indicates that the

bulky biphenyl group probably locates in a narrow cavity and

thus needs to take a precise orientation. Binding affinities are

also lost when some simple halogen groups are added to the

ortho-biphenyl group (A4–A6), which further supports our

speculation. When the ortho-biphenyl group is changed to a b-

naphthyl (A7) or 3,4,5-trimethoxybenzyl group (A8), K_ivalues

All compounds were then tested in a FP-based binding assay

to measure their binding affinities to three antiapoptotic Bcl-2

family proteins, including Bcl-x_L, Bcl-2, and Mcl-1. Technically,

these compounds were tested first at three doses (1, 10, and

50 mm). If a compound exhibited obvious dose-dependent in-

hibition rates within this range and inhibited over 50% at

50 mm, it was then tested at a series of doses to derive accu-

rate K_ivalues.

Scheme 3. Reagents and conditions: a) Ethyl 2-bromoacetate, K₂CO₃, acetone; b) NBS, PPh₃, THF; c) NaN₃, acetone; d) LiOH, THF/H₂O; e) 3-bromoprop-1-yne,

K₂CO₃, CH₂Cl₂; f) CuSO₄, l-abscorbic acid, tert-butyl alcohol/H₂O.

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increase significantly to 14.5 and 4.9 mm, respectively.

As for the fragment occupying the Q-site, a wider

range of chemical groups were attempted since this

subsite is relatively open. However, as indicated in

Table 1, most of them were not successful. Binding

affinities to Mcl-1 are only observed when this frag-

ment is either

a 2-(3,4,5-trimethoxyphenyl)acetic

group (A1, A7, A8, A17) or 3-carboxylbenzyl group

(A14, A15). This observation suggests that a phenyl

group with appropriate hydrogen-bond-acceptor

substituent groups is desired for this subsite. Interest-

ingly, the selective Mcl-1 binders in this series (A1

and A7) have no detectable binding to Bcl-x_Lor Bcl-

2. There are also general binders of Bcl-2 family pro-

teins (A8, A14, and A15) that do not have obvious

selectivities among all three tested proteins.

Chemical structures and binding affinity data of

compounds B1–B17 and C1–C12 are summarized in

Table 2 and Table 3, respectively. No compound in

these two series exhibits an obvious binding affinity

to any of the three Bcl-2 family proteins. Note that in

these two series, the P-groups and Q-groups are simi-

lar to their counterparts in series A. Thus, the 3-ami-

nobenzyl group as well as the triazole group is not

a good linker to connect the chemical moieties that

are supposed to fit into the P- and Q-site. Compared

to the effective linker group in series A, we speculate

that the 3-aminobenzyl group lacks the desired con-

formational flexibility due

Scheme 4. Reagents and conditions: a) HATU, DIPEA, DMF; b) CH₂Cl₂, 408C; c) Amines,

HATU, DIPEA, DMF; d) CH₂Cl₂, 408C; e) Amines, HATU, DIPEA, DMF.

to the absence of one

Table 1. Chemical structures and binding data of compounds A1–A26.

critical methylene unit,

whereas

the

triazole

group is simply too short.

It is true that although

the computational ap-

proach employed in our

study is able to retrieve

plausible chemical frag-

ments for each well-de-

fined subsite (Figure 1), it

does not automatically

suggest how these frag-

ments can be connected

appropriately. Thus, linker

groups were still chosen

manually by chemists

in our study. In the case

of series B and C, our

choices of the linker were

unfortunately not suc-

cessful.

Mcl-1

Bcl-x_L

Bcl-2

Compd

P¹

Q¹

Inhibition K_i[mm]^[b]Inhibition K_i[mm]^[b]Inhibition K_i[mm]^[b]

[%]^[a]

A1

66

0.18ꢀ0.05

N.A.

ꢁ6

N.A.

40

N.A.

A2

A3

A4

A5

54

52

48

9

ꢁ15

1

47

32

31

15

N.A.

Chemical

structures

and binding affinity data

of compounds D1–D26

are

summarized

in

N.A.

ꢁ6

Table 4. Structures in this

series are actually similar

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to those in series A. The

major difference is that

the phenyl moiety for the

linker was replaced by

Table 1. (Continued)

a

short

hydrocarbon

chain, that is, ꢁ(CH₂)₂ꢁ or

ꢁ(CH₂)₃ꢁ, in series D. It is

thus not surprising to ob-

serve that 16 compounds

in this series also exhibit-

ed obvious binding affini-

ties to Mcl-1, with K_i

values lower than 20 mm.

The most potent com-

pounds are D14 (K_i=

0.32 mm) and D16 (K_i=

0.35 mm), suggesting that

it is a reasonable design

where the linker group is

connected to the sub-

Mcl-1

Bcl-x_L

Bcl-2

Compd

P¹

Q¹

Inhibition K_i[mm]^[b]Inhibition K_i[mm]^[b]Inhibition K_i[mm]^[b]

[%]^[a]

A6

43

N.A.

ꢁ12

N.A.

22

N.A.

A7

A8

A9

66

78

10

14.5ꢀ2.3

ꢁ2

78

39

59

48

4.9ꢀ1.2

2.1ꢀ0.4

4.0ꢀ1.1

stituent

phenyl

ring

through an additional

amide group. Generally

speaking, the compounds

in this series are selective

binders to Mcl-1, which

are basically inactive on

Bcl-x_Lor Bcl-2. The only

exception is D11, which

exhibited roughly 12-fold

selectivity towards Bcl-x_L

(K_i=0.26 mm) over Mcl-

1 (K_i=3.2 mm).

N.A.

ꢁ41

N.A.

A10

A11

A12

A13

A14

A15

A16

36

49

22

29

90

83

44

N.A.

ꢁ30

ꢁ8

N.A.

42

32

8

N.A.

ꢁ9

N.A.

Analysis of the binding

modes of the active

compounds with Mcl-

1 protein

N.A.

ꢁ35

67

N.A.

19

50

41

N.A.

3.3ꢀ1.3

2.9ꢀ0.5

N.A.

9.0ꢀ3.1

5.9ꢀ0.6

N.A.

3.8ꢀ0.3

3.1ꢀ0.8

N.A.

The compounds tested in

this study were designed

using a fragment-based

computational approach

plus intuitive modifica-

tions. It is encouraging to

observe that at least

some of them actually

have promising binding

affinities to Bcl-2 family

proteins, demonstrating

that our de novo design

was effective. In order to

derive the binding modes

for the active com-

pounds, we selected

compound D11 to moni-

tor the chemical shift

75

ꢁ19

A17

A18

60

16

3.2ꢀ2.0

ꢁ12

N.A.

44

2

N.A.

6

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observed between the

chemical shift patterns be-

tween D11 and ABT-737

Table 1. (Continued)

(Figure 5b),

which

is

a strong indication that

D11 binds to Bcl-x_Lat the

same site as ABT-737. To il-

lustrate this point further,

the nine residues associat-

Mcl-1

Bcl-x_L

Bcl-2

Compd

P¹

Q¹

Inhibition K_i[mm]^[b]Inhibition K_i[mm]^[b]Inhibition K_i[mm]^[b]

[%]^[a]

ed with the highest

W

A19

20

N.A.

ꢁ9

N.A.

33

N.A.

values upon the addition

of D11 are shown on the

Bcl-x_Lstructure in Fig-

ure 5c. Most of them

indeed scatter around the

BH3-binding pocket, sug-

gesting that D11 binds to

Bcl-x_Lin the expected

manner. Because Mcl-

1 and Bcl-x_Lshares a cer-

tain level of structural sim-

ilarity, it is reasonable to

assume that the Mcl-

1 binders obtained in our

study also bind to Mcl-1 re-

sembling the binding

mode of D11 to Bcl-x_L.

A20

14

ꢁ19

28

A21

A22

24

34

N.A.

4

N.A.

31

38

N.A.

14

A23

ꢁ2

N.A.

ꢁ35

N.A.

ꢁ5

N.A.

We then employed mo-

lecular modeling to derive

the binding modes at the

atomic level for the active

compounds obtained in

our study. Two com-

pounds from series A (A1

and A15) and two from

series D (D11 and D12)

were considered for this

A24

A25

5

N.A.

0

6

N.A.

13

N.A.

11

ꢁ3

A26

8

N.A.

2

N.A.

2

N.A.

purpose.

These

com-

pounds include selective

Mcl-1 binders (A1 and

D12) as well as general

binders to all three tested

proteins (A15 and D11).

The predicted binding

[a] Inhibition at 50 mm test compound. [b] Data represent the mean ꢀSD and are derived from three parallel measure-

ments. N.A.: No activity, that is, obvious binding was not observed in the initial screening or not confirmed in the sub-

sequent measurements using multiple doses.

changes upon its complexation with Bcl-x_Lthrough ¹⁵N-

HSQC NMR experiment. D11 was chosen for this experiment

because it was the most potent compound (K_i=0.26 mm) on

Bcl-x_L. Besides, good solubility of this compound in dichloro-

methane made it suitable for NMR measurements. The same

¹⁵N-HSQC NMR experiment was not performed using Mcl-1 in

our study because ¹⁵N-labeled Mcl-1 protein was not available

to us at that time.

The ¹⁵N-HSQC spectrum of D11 is shown in Figure 5a. ABT-

737 was used as a positive control, and its ¹⁵N-HSQC spectrum

is given in the Supporting Information. One can see that many

residues on Bcl-x_Lexhibited apparent chemical shifts upon the

addition of D11. More importantly, an overall similarity can be

modes of these four compounds in complex with Mcl-1 and

Bcl-x_Lare shown in Figure 6 and Figure 7. It is naive to expect

that every binding data obtained in our study can be ex-

plained perfectly by these predicted binding modes. Instead,

we hope to interpret the overall trends in the SARs as well as

the selectivities of our compounds.

In order to fit the P-site, which is essentially hydrophobic,

we chose a biphenyl group for most of our compounds. Such

a moiety was among the general suggestion given by our

computational approach (Figure 4). The choice of this particu-

lar group was inspired by the molecular structure of ABT-737

(Figure 1). One can see in Figure 6 and 7 that this group has

the right size and shape to fit the P-site. The Q-site, however,

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Table 2. Chemical structures and binding data of compounds B1–B17.

Table 2. (Continued)

Inhibition [%]^[a]

Mcl-1 Bcl-x_LBcl-2

Inhibition [%]^[a]

Mcl-1 Bcl-x_LBcl-2

Compd

P²

Q²

Compd

P²

Q²

B14

22

ꢁ2

ꢁ6

7

B1

15

0

ꢁ8

ꢁ4

5

B15

B16

ꢁ1 ꢁ23

B2

B3

ꢁ2

8

ꢁ17

ꢁ7

32 ꢁ10

10

13

B17

11 ꢁ31 ꢁ10

[a] Inhibition at 50 mm test compound.

B4

5

16

is hybridized in nature, consisting of nonpolar as well as polar

residues. We mainly chose a phenyl group decorated with hy-

drogen-bond donor or acceptor substituent groups for fitting

this site. The most notable difference among series A–D is the

linker moiety connecting these two parts in the molecular

structure. Note that all of the active compounds observed in

our study belong to series A or D, whereas no compound has

obvious binding affinities to any of the three proteins in ser-

ies B or C. This indicates that the linker in series B or C is not

suitable (Figure 4). In series C, the linker is obviously too short

to allow the chemical fragments at both sides to reach the P-

and the Q-site on the target protein simultaneously. In series B,

the linker is a phenyl ring with two amide groups attached at

the meta-position, which is about the same length as the link-

ers in series A or D. Nevertheless, due to the conjugation be-

tween the two amide groups and the phenyl ring, the linker in

series B is essentially flat, lacking the conformational flexibility

to allow the chemical fragments at both sides to fit comforta-

bly into the P- and the Q-site.

B5

B6

14

ꢁ8

ꢁ3

ꢁ1 ꢁ27

B7

B8

B9

30 ꢁ13

ꢁ5 ꢁ20

4

ꢁ4

7

1

ꢁ15

Our predicted binding modes suggest that the linker moiety

does not merely fill up the space between the P- and the Q-

site. They could also be involved in critical interactions with

the target protein. In the complex of Mcl-1 with A1 for exam-

ple, one amide group on the linker moiety forms a C=O···HꢁN

hydrogen bond with Arg263 above the binding groove on

Mcl-1; whereas the other amide group on the linker moiety

forms a NꢁH···O hydrogen bond with Thr266 at the bottom of

the binding groove. The occupancy of both hydrogen bonds

was above 50% during molecular dynamics (MD) simulation.

This probably explains why A1 is the most potent compound

for Mcl-1 among all compounds. A15 has the same hydrogen-

bond donor/acceptor groups on its linker moiety as A1. How-

ever, due to the different connection between the biphenyl

B10

B11

B12

B13

ꢁ8 ꢁ31

ꢁ4 ꢁ32

ꢁ3

ꢁ4

ꢁ1

7

5

2

ꢁ19

ꢁ14

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Arg263 and Thr266. In fact, the occupancy of both hydrogen

bonds was below 20% during MD simulation. But when the

target protein is Bcl-x_L, the carboxyl group on the substituent

phenyl ring on A15 can form a charged hydrogen bond (i.e.,

salt bridge) with Arg100 at the Q-site in the binding groove.

This critical interaction is missing in the case of A1 because

there is no negatively charged, strong hydrogen acceptor

group at the equivalent part on A1. Besides, our predicted

binding mode of A1 with Bcl-x_Lindicates that the relatively

bulky Q-moiety on A1, which is a phenyl ring plus three me-

thoxy groups, is actually not well accommodated at this part

of the binding groove. The binding modes shown in both

Figure 6 and 7 are the outcomes after 3 ns-long MD simula-

tions. We assume that one would observe the dissociation of

A1 from Bcl-x_Lif a much longer MD simulation was performed.

Compound D11 is a general binder to all three proteins,

while D12 is a selective binder to Mcl-1. Note that the only dif-

ference between the chemical structures of these two com-

pounds lies in the substituent groups on the phenyl ring fit-

ting the Q-site: In the case of D11, the substituent groups are

two hydroxy groups, which can act as hydrogen-bond donors

or acceptors; while in the case of D12, the substituent groups

are two methoxy groups, which can act only as weak hydro-

gen-bond acceptors. Our predicted binding mode of D11 with

Mcl-1 suggests that it forms an OꢁH···O=C hydrogen bond

with the terminal amide group on the side chain of residue

Asn260 (Figure 7). D12 also forms an O···HꢁN hydrogen bond

with the terminal amide group on the same residue. But this

hydrogen bond was observed to be unstable during MD simu-

lation and is not seen in the final binding mode shown in

Figure 7. This observation is consistent with the fact that D11

has a stronger affinity to Mcl-1 (K_i=3.2 mm) than D12 (K_i=

9.4 mm).

When the target protein is Bcl-x_L, our predicted binding

mode of D11 reveals certain similarities to that of ABT-737.

This is not surprising because these two compounds both

have a benzyl substituted piperazine moiety at the center of

their chemical structures (Figure 1 and 7). Superimposed bind-

ing modes of D11 and ABT-737 to Bcl-x_Lare given in Figure S5

in the Supporting Information. One can see there that (1) the

terminal biphenyl moiety on both compounds occupies the

hydrophobic P-site, and (2) the substituted piperazine moiety

on both compounds bridges the P-site and the L-site. How-

ever, the Q-moieties on D11 and ABT-737 are totally different.

ABT-737 has a more complicated and branched structure fit-

ting this subsite, which extends further to the C terminus of

Bcl-x_L. In contrast, D11 has a simple substituted phenol moiety

fitting to this subsite, which forms an OꢁH···O=C hydrogen

bond with the terminal carboxylic group on the side chain of

residue Glu96. The Q-moiety on D12 cannot form any hydro-

gen bond with the residues at the Q-site since the hydroxy

groups are blocked on this molecule. In theory, the Q-moiety

on D12 has only dispersed hydrophobic contacts with the sur-

rounding residues. But the Q-site on Bcl-x_Lis much less hydro-

phobic as compared with Mcl-1. Thus, the hydrophobic Q-

moiety on D12 loses its advantage here, which explains why

Table 3. Chemical structures and binding data of compounds C1–C12.

Inhibition [%]^[a]

Mcl-1 Bcl-x_LBcl-2

Compd

P³

Q³

C1

1

ꢁ6

7

6

C2

13

5

ꢁ5

C3

C4

C5

ꢁ19

12

16

26 ꢁ10

3

ꢁ27 ꢁ12

C6

C7

C8

ꢁ27 ꢁ29 ꢁ15

ꢁ4 ꢁ29

8

7

6

ꢁ14

C9

ꢁ4

ꢁ8

27

5

C10

C11

C12

32 ꢁ43

11 ꢁ28

ꢁ5

10

14

[a] Inhibition at 50 mm test compound.

group and the piperazine ring in A15, the binding pose of A15

to Mcl-1 is very different from that of A1 (Figure 6). Conse-

quently, A15 is not able to maintain the hydrogen bonds with

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D12 is essentially in-

active on Bcl-x_L. This

is actually similar to

the case of A1.

Table 4. Chemical Structures and binding data of compounds D1–D26.

It is known that the

BH3-binding groove

Mcl-1

Bcl-x_L

Bcl-2

Compd

P⁴

Q⁴

n Inhibition K_i[mm]^[b]Inhibition

K_i

[mm]^[b]

Inhibition K_i[mm]^[b]

on Bcl-2 family pro-

teins undergoes nota-

[%]^[a]

ble

conformational

changes upon ligand

binding. Such differ-

ence can be observed

on the predicted

complex structures of

several selected com-

pounds even though

our MD simulations

were not extensive.

Our predicted binding

modes suggest that

the selective binding

of A1 and D12 to

Mcl-1 can be ex-

plained by their spe-

cific interactions with

certain residues on

Mcl-1. They have the

right structural scaf-

folds, which favor

D1

2

13

N.A.

ꢁ7

N.A.

25

34

N.A.

D2

D3

D4

D5

D6

D7

69

0

2.5ꢀ0.7

20

6

N.A.

1

2

N.A.

11

43

35

36

32

N.A.

63

52

65

35

18.2ꢀ6.0

20.0ꢀ4.9

12.6ꢀ3.7

N.A.

ꢁ14

ꢁ7

ꢁ14

1

such

interactions

upon binding to Mcl-

1 but not Bcl-x_L.

As mentioned in

above, Friberg and

coworkers recently re-

ported

a

class of

D8

2

49

N.A.

ꢁ12

N.A.

46

N.A.

potent selective Mcl-

1 inhibitors,^[28]which

is to our knowledge

the only work of this

kind so far. Several

most potent com-

pounds reported in

their study are given

in Figure 8. One can

see that their com-

pounds are structural-

ly quite different from

ours, implying that

Mcl-1 is a molecular

target that allows li-

gands in versatile

D9

2

65

59

1.2ꢀ0.9

11

16

N.A.

42

37

N.A.

D10

11.6ꢀ1.8

D11

D12

2

49

55

3.2ꢀ1.4

9.4ꢀ4.0

52

0.26ꢀ0.15

74

37

3.6ꢀ1.7

chemotypes.

The

ꢁ15

N.A.

most potent com-

pounds obtained in

our study (e.g., A1,

D14, and D16) are es-

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sentially on the same

level as theirs in

terms of binding af-

finities to Mcl-1. Be-

sides, these com-

Table 4. (Continued)

pounds seem to be

Mcl-1

Bcl-x_L

Bcl-2

Compd

P⁴

Q⁴

n Inhibition K_i[mm]^[b]Inhibition

K_i

Inhibition K_i[mm]^[b]

selective Mcl-1 binders

since they do not

[%]^[a]

[mm]^[b]

[%]^[a]

show

detectable

binding to Bcl-2 or

Bcl-x_L. In terms of

ligand efficiency (LE),

which is computed as

ꢁlogK_idivided by the

number of nonhydro-

gen atoms in the

ligand molecule, our

most potent com-

pounds on Mcl-1 (A1,

D14, and D16) have

LE values of 0.15,

0.17, and 0.17, respec-

tively; whereas Fri-

D13

2

56

7.4ꢀ1.3

ꢁ4

N.A.

40

48

N.A.

D14

61

0.32ꢀ0.14

9

N.A.

D15

D16

D17

2

3

59

51

64

0.95ꢀ0.25

0.35ꢀ0.18

1.8ꢀ0.3

20

N.A.

42

38

42

N.A.

1

berg’s

compounds

53, 57, and 60 have

LE values of 0.28,

0.24, and 0.26, respec-

tively. Therefore, our

compounds are less

“efficient” compared

to Friberg’s com-

pounds. There is still

plenty of room for

improving the poten-

cy of our compounds

through structural op-

timization.

9

D18

D19

D20

D21

D22

3

2

49

31

40

49

50

N.A.

15

ꢁ23

ꢁ10

ꢁ18

ꢁ32

N.A.

30

33

41

39

40

N.A.

In order to under-

stand the ligand effi-

ciency gap between

our compounds and

Friberg’s compounds,

we compared their

binding modes to

Mcl-1. The crystal

structure of Friberg’s

compound 53 in

complex with Mcl-1 is

shown in Figure 9. A

remarkable feature of

this complex structure

is the conformational

change upon ligand

binding at the P-site,

especially in the heli-

cal region between

Val243 to Phe254.

N.A.

10.6ꢀ5.2

15.9ꢀ8.8

D23

3

43

N.A.

4

N.A.

33

N.A.

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study can antagonize

the biological func-

Table 4. (Continued)

tions

of

overex-

pressed Mcl-1 in cells

or even in vivo. These

compounds

could

Mcl-1

Bcl-x_L

Bcl-2

Inhibition K_i[mm]^[b]

[%]^[a]

Compd

P⁴

Q⁴

n Inhibition K_i[mm]^[b]Inhibition

K_i

serve as lead com-

pounds for the devel-

opment of potent

Mcl-1 inhibitors with

pharmaceutical appli-

cations.

[%]^[a]

[mm]^[b]

D24

3

ꢁ34

N.A.

ꢁ30

N.A.

ꢁ2

N.A.

Conclusions

In this study, we at-

tempted to obtain

small-molecule inhibi-

tors of Bcl-2 family

proteins by de novo

design. We divided

D25

9

N.A.

ꢁ26

N.A.

18

N.A.

the

BH3-binding

groove on Bcl-x_Land

Mcl-1 into three sub-

sites,

and

then

D26

53

2.0ꢀ1.3

ꢁ51

N.A.

39

N.A.

sketched molecules

that consist of suita-

ble fragments fitting

these subsites. Our

design was aided by

the use of a computa-

[a] Inhibition at 50 mm test compound. [b] Data represent the mean ꢀSD and are derived from three parallel measurements.

N.A.: No activity, that is, obvious binding was not observed in the initial screening or not confirmed in the subsequent

measurements using multiple doses.

tional

algorithm,

which was developed

to analyze the critical

This phenomenon is quite unique among other known Mcl-

1 complex structures. An associated consequence is that there

is not a typical Q-site inside the binding groove on this Mcl-

1 structure. In fact, 53 does not stretch into that part of the

binding groove at all. The substituted phenoxyl moiety on 53

and part of the connecting chain fill up the hydrophobic P-

site, which is equivalent to the terminal biphenyl moiety on

our compound A1. The indole ring on 53 resides at the L-site.

Notably, the carboxylic group on this indole ring forms a salt

bridge with the side chain of Arg263, a conserved residue

among all antiapoptotic Bcl-2 family proteins. This important

interaction with Arg263 is missing in all of our compounds,

which accounts for the higher ligand efficiencies of Friberg’s

compounds. Comparison of the binding modes of our com-

pounds and Friberg’s compounds suggests that the L-moiety

on our compounds needs further optimization, and the Q-

moiety can be much simplified.

residue clusters on protein–protein binding interface and re-

trieve suitable chemical fragments matching them. A total of

81 compounds were synthesized, and their binding affinities to

Bcl-2, Bcl-x_L, and Mcl-1 were measured. Twenty-two com-

pounds exhibited binding affinities at the micromolar level

(K_i<20 mm) for at least one target protein, indicating that our

design strategy was successful. Interestingly, though it was not

our original aim, some of our compounds were observed to be

selective binders to Mcl-1. For example, the most potent one

(A1) has an inhibition constant (K_i) of 0.18 mm for Mcl-1 and vir-

tually no binding to Bcl-x_Lor Bcl-2. Molecular modeling was

employed to derive the binding modes of several selected

compounds, including selective binders of Mcl-1 and general

binders of all three tested proteins. It seems that the selectivity

to Mcl-1 can be explained by their different interaction pat-

terns with Mcl-1 and Bcl-x_L. Nevertheless, our study provides

a proof-of-concept demonstration that it is possible to obtain

highly selective inhibitors of Mcl-1 with relatively simple chem-

ical structures.

Our work and Friberg’s work collectively demonstrate that

development of small-molecule selective inhibitors of Mcl-1 is

possible. Such molecules do not need to be as sophisticated

as ABT-737 or ABT-263. Relatively simple molecules may fulfill

this goal. Of course, a panel of mechanism-based studies is still

needed to determine if the active compounds obtained in our

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tiplet (m), and broad (br). Low- and high-resolution mass

spectra (LRMS and HRMS) are given with electric, electro-

spray, and matrix-assisted laser desorption ionization (EI,

ESI, and MALDI) produced by a Finnigan MAT-95, LCQ-

DECA spectrometer and IonSpec 4.7 T. The purity of final

compounds was assessed with analytical HPLC and ob-

served to be>95%. An Agilent 1100 series HPLC with an

Agilent Zorbax Eclipse SB-C18 (4.6ꢁ150 mm, 5 mm parti-

cle sizes) reversed-phase column was used for analytical

HPLC analyses. The eluent was an A/B gradient, where

A=H₂O and B=CH₃OH. Retention time and relative

purity of all compounds are given in the Supporting In-

formation.

General procedure I for the synthesis of 2a–t: 1-(Bi-

phenyl-2-ylmethyl)piperazine (2a): 1-tert-butoxycarbon-

yl (Boc)-piperazine (4.0 mmol) and Et₃N (6.0 mmol) were

added to

a

solution of 2-bromobenzyl bromide

(4.0 mmol) in CH₂Cl₂(60 mL). After heating at reflux for

6 h, the reaction mixture was diluted with H₂O (100 mL)

and extracted with CH₂Cl₂. The organic phase was

washed with H₂O and brine, dried over Na₂SO₄, filtered

and concentrated in vacuo. The residue was purified by

chromatography with n-pentane/EtOAc (8:1 v/v) to

obtain the intermediate tert-butyl 4-(2-bromobenzyl) pi-

perazine-1-carboxylate (LC-MS: 355 [M+H]⁺), which was

then dissolved in dimethoxyethane (DME)/EtOH/H₂O

(1:1:1 v/v/v, 3 mL), followed by addition of phenylboronic

acid (2.1 mmol), Pd(PPh₃)₄(0.028 mmol) and anhyd

Na₂CO₃(5.4 mmol). The reaction mixture was stirred at

1008C for 30 min under microwave irradiation. The sol-

vent was evaporated in vacuo and the residue was puri-

fied by chromatography with n-pentane/EtOAc (8:1, v/v)

to get tert-butyl 4-([1,1’-biphenyl]-2-ylmethyl)piperazine-

1-carboxylate (LC-MS: 353 [M+H]⁺). After dissolution in

CH₂Cl₂, trifluoroacetic acid (TFA; 15.5 mmol) was added

and the mixture was heated at reflux for 2 h. The reac-

tion mixture was diluted with saturated NaHCO₃

(100 mL) and extracted with CH₂Cl₂. The combined or-

ganic phases were washed with H₂O, brine, dried over

Na₂SO₄. The solvent was evaporated in vacuo to obtain

2a as a yellow oil (440 mg, 62%): ¹H NMR (300 Hz,

CDCl₃): d=7.88 (d, J=7.2 Hz, 1H), 7.70–7.62 (m, 2H),

7.48 (m, 1H), 7.40–7.29 (m, 4H), 7.19 (d, J=7.4 Hz, 1H),

3.62 (s, 2H), 2.75 (brs, 4H), 2.45 ppm (brs, 4H); MS (ESI):

m/z 253 [M+H]⁺.

Figure 5. a) Superimposed ¹H-¹⁵N-HSQC spectra of free Bcl-x_L(in green) and Bcl-x_Lin

complex with D11 (in red). The nine residues with the highest W values are labeled ex-

plicitly. b) Refined chemical shift perturbation maps for all backbone amide nitrogen

atoms on Bcl-x_Lin complex with D11. c) A binding mode of D11 derived through molec-

ular modeling. D11 is shown in the stick model. The backbone of Bcl-x_Lis shown as gray

ribbons, where locations of the nine residues with the highest W values are indicated by

purple spheres.

1-(Biphenyl-3-ylmethyl)piperazine (2b): Prepared ac-

cording to general procedure I from 3-bromobenzyl bro-

1

mide (455 mg, 63%): H NMR (300 Hz, CDCl₃): d=7.58 (s,

1H), 7.45 (d, J=7.4 Hz, 2H), 7.40–7.31 (m, 4H), 7.20 (d,

J=6.8 Hz, 2H), 3.56 (s, 2H), 2.55 (brs, 4H), 2.48 (brs, 2H),

2.38 ppm (brs, 2H); MS (ESI): m/z 253 [M+H]⁺.

Experimental Section

1-(Biphenyl-4-ylmethyl)piperazine (2c): Prepared according to

general procedure I from 4-bromobenzyl bromide (426 mg, 60%):

¹H NMR (300 Hz, CDCl₃): d=7.82 (d, J=7.8 Hz, 1H), 7.67–7.58 (m,

2H), 7.40–7.29 (m, 5H), 7.19 (d, J=6.8 Hz, 1H), 3.68 (s, 2H), 2.78

(brs, 4H), 2.45 (brs, 2H), 2.26 ppm (brs, 2H); MS (ESI): m/z 253

[M+H]⁺.

Synthesis

All reagents were purchased from Lancaster, Acros, and Shanghai

Chemical Reagent, and were used without further purification. An-

alytical thin-layer chromatography (TLC) was performed with

HSGF 254 (150–200 mm thickness; Yantai Huiyou, China). NMR

spectroscopy was performed on a Bruker AMX-400 and AMX-300

(TMS as internal standard). Chemical shifts are reported in parts

per million (ppm, d) downfield from TMS. Proton coupling patterns

are described as singlet (s), doublet (d), triplet (t), quartet (q), mul-

1-((2’-Chlorobiphenyl-2-yl)methyl)piperazine (2d): Prepared ac-

cording to general procedure I from 2-bromobenzyl bromide and

2-chlorophenylboronic acid (517 mg, 64%):¹H NMR (300 Hz,

[D₆]DMSO): d=7.89 (d, J=7.5 Hz, 1H), 7.47–7.35 (m, 6H), 7.30–7.29

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(100 mL) and extracted with

CH₂Cl₂. The organic phase was

washed with H₂O, brine, dried over

Na₂SO₄, filtered and concentrated

in vacuo. The intermediate (LC-MS:

327 [M+H]⁺; 3.1 mmol), purified

by chromatography with n-pen-

tane/EtOAc (8:1 v/v), was dissolved

in CH₂Cl₂. TFA (15.5 mmol) was

added, and the solution was

heated at reflux for 2 h. The reac-

tion mixture was diluted with satu-

rated NaHCO₃(100 mL) and ex-

tracted with CH₂Cl₂. The combined

organic phases were washed with

H₂O, brine and dried over Na₂SO₄

and concentrated in vacuo. Com-

pound 2g was afforded as a white

solid (769 mg, 85%): ¹H NMR

(300 Hz, [D₆]DMSO): d=8.27 (d, J=

7.5 Hz, 1H), 7.86–7.83 (m, 2H),

8.94–7.83 (m, 4H), 3.84 (s, 2H),

2.52 (brs, 4H), 2.38 ppm (brs, 4H);

MS (ESI): m/z 227 [M+H]⁺.

1-((4’-Chlorobiphenyl-2-yl)me-

thyl)piperazine (2h): Prepared ac-

cording to general procedure I

from 4-chlorophenylboronic acid

(532 mg, 66%): ¹H NMR (300 Hz,

CDCl₃): d=7.73 (d, J=7.8 Hz, 2H),

7.54 (m, 4H), 7.30 (m, 2H), 3.66 (s,

2H), 2.85 (brs, 4H), 2.45 ppm (brs,

4H); MS (ESI): m/z 287 [M+H]⁺.

1-((4’-Chlorobiphenyl-3-yl)me-

thyl)piperazine (2i): Prepared ac-

cording to general procedure I

from 3-bromobenzyl bromide and

4-chlorophenylboronic

acid

Figure 6. Chemical structures of A1 and A15 and their binding modes to Mcl-1 and Bcl-x_Lderived through molec-

ular modeling.

(524 mg, 65%): ¹H NMR (300 Hz,

CDCl₃): d=8.01 (d, J=7.8 Hz, 1H),

7.95 (d, J=7.6 Hz, 1H), 7.80 (s, 1H),

7.45 (t, J=7.4 Hz, 2H), 7.25–7.39

(m, 3H), 3.63 (s, 2H), 2.51 (brs,

4H), 2.43 ppm (brs, 4H); MS (ESI): m/z 287 [M+H]⁺.

(m, 1H), 3.26 (s, 2H), 2.67 (brs, 4H), 2.24 ppm (brs, 4H); MS (ESI):

m/z 287 [M+H]⁺.

1-((3-Fluorobiphenyl-4-yl)methyl)piperazine (2j): Prepared ac-

cording to general procedure I from 2-fluoro-4-bromobenzyl bro-

mide (58%): ¹H NMR (300 Hz, CDCl₃): d=7.65 (d, J=7.4 Hz,1H),

7.60 (s, 1H),7.67–7.60 (m, 2H), 7.55 (d, J=7.2 Hz,1H), 7.40–7.32 (m,

3H), 3.65 (s, 2H), 2.98 (brs, 4H), 2.65 (brs, 2H), 2.50 ppm (brs, 2H);

MS (ESI): m/z 271 [M+H]⁺.

1-((3’-Chloro-4’-fluorobiphenyl-2-yl)methyl)piperazine (2e): Pre-

pared according to general procedure I from 2-bromobenzyl bro-

mide and 3-chloro-4-fluorophenylboronic acid (566 mg, 66%):

¹H NMR (300 Hz, [D₆]DMSO): d=7.54–7.51 (m, 1H), 7.41–7.36 (m,

3H), 7.34–7.28 (m, 2H), 7.13–7.11 (m, 1H), 3.13 (s, 2H), 2.54 (brs,

4H), 2.01 ppm (brs, 4H); MS (ESI): m/z 305 [M+H]⁺.

1-((3-Methoxybiphenyl-4-yl)methyl)piperazine (2k): Prepared ac-

1-((3-Fluorobiphenyl-2-yl)methyl)piperazine (2 f): Prepared ac-

cording to general procedure I from 2-fluoro-6-bromobenzyl bro-

mide and phenylboronic acid (471 mg, 62%): ¹H NMR (300 Hz,

[D₆]DMSO): d=7.72–7.70 (m, 2H), 7.53–7.46 (m, 5H), 7.42–7.40 (m,

1H), 3.58 (s, 2H), 2.51 (brs, 4H), 2.48 ppm (brs, 4H); MS (ESI): m/z

271 [M+H]⁺.

cording to general procedure I from 2-methoxy-4-bromobenzyl

1

bromide (66%): H NMR (300 Hz, CDCl₃): d=7.78 (d, J=7.6 Hz,1H),

7.70 (s, 1H), 7.68–7.60 (m, 2H), 7.49 (d, J=7.2 Hz, 2H), 7.45–7.38

(m, 2H), 3.98 (s, 3H), 3.65 (s, 2H), 2.78 (brs, 4H), 2.45 (brs, 2H),

2.28 ppm (brs, 2H); MS (ESI): m/z 283 [M+H]⁺.

1-(Naphthalen-2-ylmethyl)piperazine

(2g):

1-Boc-piperazine

1-((3-Methylbiphenyl-4-yl)methyl)piperazine (2l): Prepared ac-

(4.0 mmol) and Et₃N (6.0 mmol) was added to a solution of 2-(bro-

momethyl)naphthalene (4.0 mmol) in CH₂Cl₂(60 mL). After heating

at reflux for 6 h, the reaction mixture was diluted with H₂O

cording to general procedure I from 2-methyl-4-bromobenzyl bro-

mide (62%): H NMR (300 Hz, CDCl₃): d=7.85 (s, 1H), 7.82 (d, J=

7.6 Hz,1H), 7.67–7.60 (m, 2H), 7.40–7.32 (m, 3H), 7.19 (d, J=

1

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4H), 3.99 (s, 3H), 3.68 (s, 2H), 2.55

(brs, 4H), 2.24(brs, 2H), 2.18 ppm

(brs, 2H); MS (ESI): m/z 283 [M+

H]⁺.

1-((4’-Nitrobiphenyl-2-yl)methyl)-

piperazine (2n): Prepared accord-

ing to general procedure I from 2-

bromobenzyl bromide and 4-nitro-

1

phenylboronic acid (55%): H NMR

(300 Hz, CDCl₃): d=8.29 (d, J=

8.0 Hz, 2H), 7.82 (d, J=7.8 Hz, 2H),

7.57–7.46 (m, 4H), 3.66 (s, 2H),

2.79 (brs, 4H), 2.55 ppm (brs, 4H);

MS (ESI): m/z 298 [M+H]⁺.

1-((4’-Chlorobiphenyl-3-yl)me-

thyl)piperazine (2o): Prepared ac-

cording to general procedure I

from 3-bromobenzyl bromide and

4-chlorophenylboronic acid (54%):

¹H NMR (300 Hz, CDCl₃): d=8.01

(d, J=7.8 Hz, 1H), 7.95 (d, J=

7.6 Hz, 1H), 7.80 (s, 1H), 7.45 (t, J=

7.4 Hz, 2H), 7.25–7.39 (m, 3H), 3.63

(s, 2H), 2.51 (brs, 4H), 2.43 ppm

(brs, 4H); MS (ESI): m/z 287 [M+

H]⁺.

1-((4’-Fluorobiphenyl-3-yl)me-

thyl)piperazine (2p): Prepared ac-

cording to general procedure I

from 3-bromobenzyl bromide and

4-fluorophenylboronic acid (56%):

¹H NMR (300 Hz, CDCl₃): d=7.81 (s,

1H), 7.68–7.58 (m, 4H), 7.29 (t, J=

7.8 Hz, 1H), 7.18 (d, J=7.4 Hz, 2H),

3.59 (s, 2H), 2.47 (brs, 2H), 2.38

(brs, 2H), 2.29 ppm (brs, 4H); MS

(ESI): m/z 271 [M+H]⁺.

Figure 7. Chemical structures of D11 and D12 and their binding modes to Mcl-1 and Bcl-x_Lderived through mo-

lecular modeling.

1-((4’-Methoxybiphenyl-3-yl)me-

thyl)piperazine (2q): Prepared according to general pro-

cedure I from 3-bromobenzyl bromide and 4-methoxy-

phenylboronic acid (57%): ¹H NMR (300 Hz, CDCl₃): d=

7.70 (s, 1H), 7.40–7.36 (m, 2H), 7.30 (d, J=7.6 Hz, 1H),

7.20–7.12 (m, 4H), 3.89 (s, 3H), 3.68 (s, 2H), 2.82 (brs,

2H), 2.75 (brs, 2H), 2.38 (brs, 2H), 2.29 ppm (brs, 2H);

MS (ESI): m/z 283 [M+H]⁺.

1-((4’-Nitrobiphenyl-3-yl)methyl)piperazine (2r): Pre-

pared according to general procedure I from 3-bromo-

benzyl bromide and 4-nitrophenylboronic acid (53%):

¹H NMR (300 Hz, CDCl₃): d=8.22 (d, J=7.8 Hz, 2H), 7.85

(s, 1H), 7.76 (d, J=7.9 Hz, 2H), 7.26–7.35 (m, 3H), 3.69 (s,

2H), 2.71 (brs, 4H), 2.45 (brs, 2H), 2.38 ppm (brs, 2H);

MS (ESI): m/z 298 [M+H]⁺.

Figure 8. Chemical structures and binding data of several most potent Mcl-1 inhibitors

recently reported by Friberg et al.^[28]

1-((4’-Fluorobiphenyl-2-yl)methyl)piperazine (2s): Pre-

pared according to general procedure I from 2-bromo-

benzyl bromide and 4-fluorophenylboronic acid (59%): ¹H NMR

(300 Hz, CDCl₃): d=7.70 (d, J=7.9 Hz, 1H), 7.30–7.37 (m, 4H), 7.29–

7.33 (m, 2H), 7.21 (d, J=7.6 Hz, 1H), 3.58 (s, 2H), 2.65 (brs, 2H),

2.57 (brs, 2H), 2.49 ppm (brs, 4H); MS (ESI): m/z 271 [M+H]⁺.

7.2 Hz,1H), 3.65 (s, 2H), 2.80 (brs, 4H), 2.50 (brs, 2H), 2.38 (s, 3H),

2.26 ppm (brs, 2H); MS (ESI): m/z 267 [M+H]⁺.

1-((4’-Methoxybiphenyl-2-yl)methyl)piperazine (2m): Prepared

according to general procedure I from 2-bromobenzyl bromide

1

and 4-methoxyphenylboronic acid (60%): H NMR (300 Hz, CDCl₃):

1-((3’-Fluorobiphenyl-2-yl)methyl)piperazine (2t): Prepared ac-

d=7.68 (d, J=8.1 Hz, 2H), 7.50 (t, J=7.8 Hz, 2H), 7.05–7.15 (m,

cording to general procedure I from 2-bromobenzyl bromide and

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4-((3-(Methoxycarbonyl)benzylamino)methyl)benzoic acid (3d):

Prepared according to general procedure II from methyl 3-(bromo-

methyl)benzoate (315 mg, 62%): ¹H NMR (300 Hz, [D₆]DMSO): d=

8.10 (d, J=7.8 Hz, 1H),8.01 (s, 1H), 7.92 (d, J=8.1 Hz, 1H), 7.78 (s,

1H), 7.45–7.36 (m, 4H), 3.99 (s, 3H), 3.75 (s, 2H), 3.52 ppm (s, 2H);

MS (ESI): m/z 299 [MꢁH]^ꢁ.

4-((2-(4-Methoxyphenyl)acetamido)methyl)benzoic acid (3e):

Prepared according to general procedure II from 2-(4-methoxyphe-

1

nyl)acetic acid (740 mg, 78%): H NMR (300 Hz, [D₆]DMSO): d=8.44

(s, 1H), 7.75 (d, J=7.5 Hz, 2H), 7.20 (d, J=8.1 Hz, 2H), 7.08 (d, J=

8.4 Hz, 2H), 6.87 (d, J=8.7 Hz,2H), 4.24 (s, 2H), 3.74 (s, 3H),

1.40 ppm (s, 2H); MS (ESI): m/z 298 [MꢁH]^ꢁ.

4-((2-(4-Fluorophenyl)acetamido)methyl)benzoic acid (3 f): Pre-

pared according to general procedure II from 2-(4-fluorophenyl)-

1

acetic acid (366 mg, 75%): H NMR (300 Hz, [D₆]DMSO): d=8.48 (s,

1H), 7.78 (d, J=7.2 Hz, 2H), 7.31 (d, J=7.2 Hz, 2H), 7.14 (m, J=

7.2 Hz, 4H), 4.28 (s, 2H), 3.48 ppm (s, 2H); MS (ESI): m/z 286

[MꢁH]^ꢁ.

4-((2-(Thiophen-2-yl)acetamido)methyl)benzoic acid (3g): Pre-

pared according to general procedure II from 2-(thiophen-3-yl)ace-

tic acid (322 mg, 69%): ¹H NMR (300 Hz, [D₆]DMSO): d=8.69 (s,

1H), 7.89 (d, J=7.8 Hz, 2H), 7.36 (m, 3H), 6.96 (m, 2H), 4.35 (s, 2H),

3.82 ppm (s, 2H); MS (ESI): m/z 274 [MꢁH]^ꢁ.

Figure 9. Crystal structure of Mcl-1 in complex with Friberg’s compound 53

(PDBID 4HW2) where the backbone of Mcl-1 is represented by green rib-

bons and 53 is shown in the stick model.

4-((3-Methylbutanamido)methyl)benzoic acid (3h): Prepared ac-

cording to general procedure II from 3-methylbutanoic acid

(300 mg, 75%): ¹H NMR (300 Hz, [D₆]DMSO): d=8.39 (s, 1H), 7.88

(d, J=8.1 Hz, 2H), 7.34 (d, J=7.8 Hz, 2H), 4.32 (s, 2H), 2.03 (brs,

3H), 1.06 ppm (brs, 6H); MS (ESI): m/z 234 [MꢁH]^ꢁ.

3-fluorophenylboronic acid (76%): ¹H NMR (400 Hz, CDCl₃): d=

7.41–7.44 (m, 1H), 7.36–7.38 (m, 1H), 7.34–7.35 (m, 1H), 7.31–7.34

(m, 1H), 7.25–7.27 (m, 1H), 7.14–7.18 (m, 1H), 7.10–7.12 (m, 1H),

7.02–7.08 (m, 1H), 6.26 (s, 1H), 3.43 (s, 2H), 3.03 (t, J=4.8 Hz, 4H),

2.55 ppm (brs, 4H); MS (ESI): m/z 271 [M+H]⁺.

4-((3-(2-Ethoxy-2-oxoethoxy)benzamido)methyl)benzoic

acid

General procedure II for the synthesis of 3a–l: 4-((2-(3,4,5-Trime-

(3i): Prepared according to general procedure II from 3-(2-ethoxy-

2-oxoethoxy)benzoic acid (314 mg, 70%): ¹H NMR (300 Hz,

[D₆]DMSO): d=8.12 (d, J=7.8 Hz, 1H), 8.01 (s, 1H), 7.82–7.71 (m,

3H), 7.18 (d, J=8.0 Hz, 2H),6.91 (s, 1H), 4.85 (s, 2H), 4.11 (q, J=

6.9 Hz, 2H), 3.58 (s, 2H), 1.26 ppm (t, J=6.9 Hz, 3H); MS (ESI): m/z

356 [MꢁH]^ꢁ.

thoxyphenyl)acetamido)methyl)benzoic

acid

(3a):

Et₃N

(2.6 mmol) was added to a solution of 2-(3,4,5-trimethoxyphenyl)-

acetic acid (1.7 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodii-

mide (EDCI; 2.6 mmol) and hydroxybenzotriazole (HOBt; 1.7 mmol)

in CH₂Cl₂(60 mL), followed by methyl 4-(aminomethyl)benzoate

(1.4 mmol). After heating at reflux for 4 h, the reaction mixture was

diluted with H₂O (100 mL) and extracted with CH₂Cl₂. The organic

phase was washed with saturated NaHCO₃and brine, dried over

Na₂SO₄, filtered and concentrated in vacuo. The crude compound

(LC-MS: 373 [M+H]⁺) was then dissolved in CH₃OH (20 mL), and

10% NaOH solution (20 mL) was added. The mixture was stirred at

608C for 2 h. CH₃OH was evaporated in vacuo, the reaction mixture

was diluted with H₂O (100 mL) and extracted with CH₂Cl₂(3ꢁ). The

water layer was acidified with 6n HCl. The formed precipitate was

collected by filtration, washed with H₂O, and dried on the sintered

4-((3-(2-Bromo-4,5-dimethoxyphenyl)propanamido)methyl) ben-

zoic acid (3j): Prepared according to general procedure II from 3-

(2-bromo-4,5-dimethoxyphenyl)propanoic acid (536 mg, 72%):

¹H NMR (300 Hz, [D₆]DMSO): d=8.10 (d, J=8.1 Hz, 1H), 7.91 (s,

1H), 7.57–7.48 (m, 2H), 7.05 (d, J=7.8 Hz, 1H), 6.85 (s, 1H), 4.25 (s,

2H), 3.98 (s, 6H), 3.01 (t, J=7.2 Hz, 2H), 2.58 ppm (t, J=7.2 Hz,

2H); MS (ESI): m/z 420 [MꢁH]^ꢁ.

4-((3-(4’-Chloro-4,5-dimethoxybiphenyl-2-yl)propanamido)meth-

yl) benzoic acid (3k): Prepared according to general procedure II

1

glass to yield 3a as a white solid (728 mg, 74%): H NMR (300 Hz,

from

3-(4’-chloro-4,5-dimethoxybiphenyl-2-yl)propanoic

acid

[D₆]DMSO): d=8.01 (d, J=7.8 Hz,1H), 7.89 (d, J=7.8 Hz, 1H), 7.80–

7.72 (m, 2H), 6.55 (s, 2H), 4.12 (s, 2H), 3.98 (s, 9H), 3.88 ppm (s,

2H); MS (ESI): m/z 358 [MꢁH]^ꢁ.

4-((3-(2,3-Dimethoxyphenyl)propanamido)methyl)benzoic acid

(3b): Prepared according to general procedure II from 3-(3,4-dime-

thoxyphenyl)propanoic acid (420 mg, 72%): ¹H NMR (300 Hz,

[D₆]DMSO): d=8.09 (d, J=7.8 Hz,1H), 7.89 (s, 1H), 7.62–7.55 (m,

3H), 6.85 (m, 2H), 4.32 (s, 2H), 3.97 (s, 6H), 3.12 (t, J=7.6 Hz, 2H),

2.49 ppm (t, J=7.6 Hz, 2H); MS (ESI): m/z 342 [MꢁH]^ꢁ.

4-((3-Hydroxybenzamido)methyl)benzoic acid (3c): Prepared ac-

cording to general procedure II from 3-hydroxybenzoic acid

(142 mg, 62%): ¹H NMR (300 Hz, [D₆]DMSO): d=8.10 (d, J=

7.8 Hz,1H), 7.92 (s, 1H), 7.75–7.63 (m, 4H), 7.23 (d, J=7.6 Hz, 2H),

5.52 (s, 1H), 4.35 ppm (s, 2H); MS (ESI): m/z 270 [MꢁH]^ꢁ.

1

(591 mg, 74%): H NMR (300 Hz, CDCl₃): d=8.11 (d, J=7.8 Hz, 1H),

7.90 (s, 1H), 7.62–7.51 (m, 4H), 7.12 (s, 1H), 7.03–6.85 (m, 3H), 4.32

(s, 2H), 3.97 (s, 6H), 3.12 (t, J=7.6 Hz, 2H), 2.49 ppm (t, J=7.6 Hz,

2H); MS (ESI): m/z 452 [MꢁH]^ꢁ.

General procedure III for the synthesis of 4a–g: 3-(2-(6-Bromo-

2,3,4-trimethoxyphenyl)acetamido)benzoic acid (4a): Et₃N

(2.6 mmol) was added to a solution of 2-(2-bromo-3,4,5-trimethoxy-

phenyl)acetic acid (1.7 mmol), EDCI (2.6 mmol) and HOBt

(1.7 mmol), followed by methyl 3-aminobenzoate (1.4 mmol). After

heating at reflux for 4 h, the reaction mixture was diluted with H₂O

(100 mL) and extracted with CH₂Cl₂. The combined organic phases

were washed with saturated NaHCO₃and brine, dried over Na₂SO₄,

filtered and concentrated in vacuo. The crude compound (LC-MS:

438 [M+H]⁺) was dissolved in CH₃OH (20 mL), and 10% NaOH so-

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lution (20 mL) was added. The mixture was stirred at 608C for 2 h.

After CH₃OH was evaporated in vacuo, the reaction mixture was di-

luted with H₂O (100 mL) and extracted with CH₂Cl₂(3ꢁ). The water

layer was acidified with 6n HCl. The formed precipitate was col-

lected by filtration, washed with H₂O, and dried on the sintered

tion mixture was stirred at 608C for 2 h. After filtration, the solvent

was evaporated in vacuo. The crude compound (LC-MS: 222 [M+

H]⁺) was dissolved in THF (20 mL), followed by the addition of H₂O

(20 mL) and LiOH (18.5 mmol). The reaction mixture was stirred at

RT for 12 h. 1n HCl solution was added, and the formed precipi-

tate was collected by filtration, washed with H₂O, and dried on the

sintered glass to yield 8a as white solid (390 mg, 73%): ¹H NMR

(300 Hz, CDCl₃): d=7.35 (m, 1H), 7.21 (s, 1H), 7.10 (d, J=7.4 Hz,

2H), 4.56 (s, 2H), 2.98 ppm (s, 2H); MS (ESI): m/z 206 [MꢁH]^ꢁ.

1

glass to yield 4a as a white solid (504 mg, 70%): H NMR (300 Hz,

CDCl₃): d=8.10 (s, 1H), 7.98–7.81 (m, 3H), 6.55 (s, 1H), 4.01 (s, 9H),

3.78 ppm (s, 2H); MS (ESI): m/z 422 [MꢁH]^ꢁ.

3-(2-(4’-Chloro-3,4,5-trimethoxy-[1,1’-biphenyl]-2yl)acetamido)-

benzoic acid (4b): Prepared according to general procedure III

from 2-(4’-chloro-4,5,6-trimethoxy-3-methylbiphenyl-2-yl)acetic acid

2-(3-(Azidomethyl)phenylamino)acetic acid (8b): Prepared ac-

cording to general procedure IV from 3-aminobenzyl alcohol

1

(504 mg, 62%): H NMR (300 Hz, CDCl₃): d=8.36 (s, 1H), 7.98 (d, J=

(61%): H NMR (300 Hz, CDCl₃): d=7.25 (m, 1H), 7.12 (d, J=7.8 Hz,

8.0 Hz, 2H), 7.75 (d, J=7.8 Hz, 2H), 7.68–7.56 (m, 3H), 6.58 (s, 1H),

3.98 (s, 9H), 3.78 ppm (s, 2H); MS (ESI): m/z 454 [MꢁH]^ꢁ.

2H), 7.01 (s, 1H), 4.21 (s, 2H), 2.87 ppm (s, 2H); MS (ESI): m/z 205

[MꢁH]^ꢁ.

3-(3,5-Dimethoxybenzamido)benzoic acid (4c): Prepared accord-

General procedure V for the synthesis of 9a–h: 1-((4’-Methoxy-

ing to general procedure III from 3,5-dimethoxybenzoic acid

(394 mg, 77%): H NMR (300 Hz, CDCl₃): d=8.35 (s, 1H), 7.95–7.84

(m, 3H), 6.87 (s, 1H), 6.59 (s, 2H), 3.98 ppm (s, 6H); MS (ESI): m/z

300 [MꢁH]^ꢁ.

biphenyl-2-yl)methyl)-4-(prop-2-ynyl)piperazine

(9a):

K₂CO₃

1

(3.50 mmol) and 3-bromo-1-propyne (1.75 mmol) was added to

a mixture 2m (1.75 mmol) in CH₂Cl₂(40 mL). After heating at reflux

for 12 h, the reaction mixture was diluted with H₂O (100 mL) and

extracted with CH₂Cl₂. The combined organic phases were washed

with H₂O and brine, dried over Na₂SO₄, filtered and concentrated

in vacuo. The crude compound was purified by chromatography

with n-pentane/EtOAc (8:1 v/v) to afford 9a as a white solid

3-(2-(3,5-Dimethoxyphenyl)acetamido)benzoic acid (4d): Pre-

pared according to general procedure III from 2-(3,5-dimethoxy-

1

phenyl)acetic acid (397 mg, 74%): H NMR (300 Hz, CDCl₃): d=8.35

(s, 1H), 7.85–7.76 (m, 3H), 6.65 (s, 1H), 6.52 (s, 2H), 4.01 (s, 2H),

3.89 ppm (s, 6H); MS (ESI): m/z 314 [MꢁH]^ꢁ.

1

(510 mg, 90%): H NMR (300 Hz, CDCl₃): d=7.67 (d, J=7.8 Hz, 2H),

7.51 (t, J=8.0 Hz, 2H), 7.05–7.15 (m, 4H), 3.99 (s, 3H), 3.68 (s, 2H),

3.52 (s, 2H), 2.55 (brs, 4H), 2.24 (brs, 2H), 2.18 (brs, 2H), 2.02 ppm

(m, 1H); MS (ESI): m/z 321 [M+H]⁺.

3-(2-Cyclohexylacetamido)benzoic acid (4e): Prepared according

to general procedure III from 2-cyclohexylacetic acid (320 mg,

1

72%): H NMR (300 Hz, CDCl₃): d=8.45 (s, 1H), 7.95 (d, J=7.8 Hz,

1-((4’-Nitrobiphenyl-2-yl)methyl)-4-(prop-2-ynyl)piperazine (9b):

Prepared according to general procedure V from 2n (91%):

¹H NMR (300 Hz, CDCl₃): d=8.31 (d, J=8.0 Hz, 2H), 7.92 (d, J=

7.8 Hz, 2H), 7.60–7.46 (m, 4H), 3.66 (s, 2H), 3.58 (s, 2H), 2.79 (brs,

4H), 2.68 (m, 1H), 2.50 (brs, 2H), 2.39 ppm (brs, 2H); MS (ESI): m/z

336 [M+H]⁺.

2H), 7.84–7.76 (m, 1H), 2.35 (d, J=6.5 Hz, 2H), 2.09–1.94 (m, 1H),

1.19–1.14 (m, 4H), 1.07–0.98 ppm (m, 6H); MS (ESI): m/z 260

[MꢁH]^ꢁ.

3-(2-Naphthamido)benzoic acid (4 f): Prepared according to gen-

eral procedure III from 2-naphthoic acid (366 mg, 74%): ¹H NMR

(300 Hz, CDCl₃): d=8.56 (s, 1H), 8.23 (s, 2H), 8.01 (d, J=7.8 Hz,

2H), 7.64–7.59 (m, 3H), 7.48–7.39 ppm (m, 3H); MS (ESI): m/z 290

[MꢁH]^ꢁ.

3-(2-(Naphthalen-2-yl)acetamido)benzoic acid (4g): Prepared ac-

cording to general procedure III from 2-(naphthalen-2-yl)acetic acid

(389 mg, 75%): ¹H NMR (300 Hz, CDCl₃): d=8.45 (s, 1H), 8.18 (s,

2H), 8.00 (d, J=8.0 Hz, 2H), 7.70–7.61 (m, 3H), 7.45–7.36 (m, 3H),

4.10 ppm (s, 2H); MS (ESI): m/z 304 [MꢁH]^ꢁ.

General procedure IV for the synthesis of 8a,b: 2-(3-(Azidome-

thyl)phenoxy)acetic acid (8a): K₂CO₃(16.2 mmol) and ethyl bro-

moacetate (8.1 mmol) was added to a solution of 3-hydroxybenzyl

alcohol (8.1 mmol) in acetone (40 mL). After heating at reflux for

12 h, the reaction mixture was diluted with H₂O (100 mL) and ex-

tracted with EtOAc. The combined organic phases were washed

with H₂O and brine, dried over Na₂SO₄, filtered and concentrated

in vacuo. The crude compound was purified by chromatography

with n-pentane/EtOAc (4:1 v/v) to afford 5a as a white solid (LC-

MS: 197 [M+H]⁺). Ph₃P (4.2 mmol) was added to a solution of 5a

(3.8 mmol) in dry tetrahydrofuran (THF; 50 mL) under a nitrogen

atmosphere. N-bromosuccinimide (NBS; 4.2 mmol) was added to

the reaction mixture 5 min later. After stirring at RT for 2 h, the sol-

vent was evaporated in vacuo. H₂O (50 mL) was added, and the

aqueous layer was back-extracted with CH₂Cl₂. The combined or-

ganic phases were washed with H₂O and brine, dried over Na₂SO₄,

filtered and concentrated in vacuo. The crude compound was puri-

fied by chromatography with n-pentane/EtOAc (8:1 v/v) to afford

6a as a white solid (LC-MS: 259 [M+H]⁺). NaN₃(5.5 mmol) was

added to a solution of 6a (3.7 mmol) in acetone (40 mL). The reac-

1-((4’-Chlorobiphenyl-3-yl)methyl)-4-(prop-2-ynyl)piperazine

(9c): Prepared according to general procedure V from 2o (89%):

¹H NMR (300 Hz, CDCl₃): d=8.10 (d, J=7.8 Hz, 1H), 7.85 (d, J=

7.6 Hz, 1H), 7.78 (s, 1H), 7.45 (t, J=7.4 Hz, 1H), 7.25–7.39 (m, 4H),

3.90 (s, 2H), 3.63 (s, 2H), 2.51 (brs, 4H), 2.43 (m, 2H), 2.56 (m, 1H),

2.32 ppm (m, 2H); MS (ESI): m/z 325 [M+H]⁺.

1-((4’-Fluorobiphenyl-3-yl)methyl)-4-(prop-2-ynyl)piperazine

(9d): Prepared according to general procedure V from 2p (92%):

¹H NMR (300 Hz, CDCl₃): d=7.90 (s, 1H), 7.68–7.58 (m, 4H), 7.31 (t,

J=8.1 Hz, 1H), 7.20 (d, J=7.8 Hz, 2H), 4.00 (s, 2H), 3.59 (s, 2H),

2.61 (m, 1H), 2.47 (brs, 4H), 2.38 (brs, 2H), 2.29 ppm (brs, 2H); MS

(ESI): m/z 309 [M+H]⁺.

1-((4’-Methoxybiphenyl-3-yl)methyl)-4-(prop-2-ynyl)piperazine

(9e): Prepared according to general procedure V from 2q (90%):

¹H NMR (300 Hz, CDCl₃): d=7.65 (s, 1H), 7.42–7.38 (m, 4H), 7.28 (d,

J=7.6 Hz, 1H), 7.20–7.12 (m, 2H), 3.89 (s, 3H), 3.68 (s, 2H), 3.59 (s,

2H), 2.82 (brs, 2H), 2.75 (brs, 2H), 2.38 (brs, 2H), 2.29 (brs, 2H),

2.34 ppm (m, 1H); MS (ESI): m/z 321 [M+H]⁺.

1-((4’-Nitrobiphenyl-3-yl)methyl)-4-(prop-2-ynyl)piperazine (9 f):

1

Prepared according to general procedure V from 2r (87%): H NMR

(300 Hz, CDCl₃): d=8.29 (d, J=7.8 Hz, 2H), 7.98 (s, 1H), 7.86 (d, J=

7.9 Hz, 2H), 7.58–7.43 (m, 3H), 3.78 (s, 2H), 3.69 (s, 2H), 2.71 (brs,

4H), 2.45 (brs, 2H), 2.38 (brs, 2H), 2.28 ppm (m, 1H); MS (ESI): m/z

336 [M+H]⁺.

1-((4’-Chlorobiphenyl-2-yl)methyl)-4-(prop-2-ynyl)piperazine

(9g): Prepared according to general procedure V from 2h (90%):

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¹H NMR (300 Hz, CDCl₃): d=7.73 (d, J=7.8 Hz, 2H), 7.54 (m, 4H),

7.30 (m, 2H), 3.89 (s, 2H), 3.66 (s, 2H), 2.85 (brs, 4H), 2.65 (m, 1H),

2. 35 (brs, 2H), 2.24 ppm (brs, 2H); MS (ESI): m/z 325 [M+H]⁺.

added, and the reaction mixture was stirred at ꢁ108C for 2 h and

then warmed to RT and stirred overnight. The solvent was evapo-

rated in vacuo, and the mixture was diluted with H₂O (100 mL) and

extracted with CH₂Cl₂. The organic phases were washed with satu-

rated NaHCO₃and brine, dried over Na₂SO₄, filtered and concen-

trated in vacuo. The crude compound was purified by chromatog-

raphy with CH₂Cl₂/CH₃OH (20:1 v/v) to afford A1 as a white solid

1-((4’-Fluorobiphenyl-2-yl)methyl)-4-(prop-2-ynyl)piperazine

(9h): Prepared according to general procedure V from 2s (93%):

¹H NMR (300 Hz, CDCl₃): d=7.67 (d, J=7.9 Hz, 1H), 7.37–7.29 (m,

4H), 7.33–7.19 (m, 2H), 7.02 (d, J=7.6 Hz, 1H), 3.76 (s, 2H), 3.58 (s,

2H), 2.65 (brs, 4H), 2.58 (m, 1H), 2.49 (brs, 2H), 2.38 ppm (brs,

2H); MS (ESI): m/z 309 [M+H]⁺.

1

(114 mg, 80%): HPLC: 96.16%, t_R=1.462 min; H NMR (CDCl₃): d=

7.51–7.49 (m, 1H), 7.39–7.32 (m, 6H), 7.31–7.27 (m, 2H), 7.17 (d,

J=8.4 Hz,2H), 6.48 (d, J=7.5 Hz,4H), 4.00 (s, 2H), 3.84 (s, 3H), 3.82

(s, 6H), 3.73 (t, J=6.3 Hz, 2H), 3.55 (s, 2H), 3.44 (s, 2H), 3.26 (t, J=

6.3 Hz, 2H), 2.39–2.24 ppm (br, 4H); MS (ESI): m/z 594 [M+H]⁺;

HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₃₉N₃O₅: 594.2890, observed:

594.2979.

General procedure VI for the synthesis of 10a–e: 4-(4-{[3-(4-Flu-

orophenyl)phenyl]methyl}piperazin-1-yl)-4-oxobutanoic

acid

(10a): Dihydrofuran-2,5-dione (0.47 mmol) was added to a mixture

of 2p (0.39 mmol) in CH₂Cl₂(2 mL). After heating to 408C for

about 1.5 h, the starting material disappeared as monitored by

TLC. The reaction mixture was diluted with H₂O (20 mL) and ex-

tracted with CH₂Cl₂. The combined organic phases were washed

with brine, dried over Na₂SO₄, filtered and concentrated in vacuo.

The crude compound was purified by chromatography with

CH₂Cl₂/MeOH (20:1 v/v) to afford 10a as a yellow oil (76 mg, 52%):

¹H NMR (400 Hz, CDCl₃): d=7.55–7.57 (m, 1H), 7.54 (m, 1H), 7.51 (s,

1H), 7.47 (d, J=7.6 Hz, 1H), 7.38–7.42 (t, J=7.2 Hz, 1H), 7.29–7.31

(d, J=7.6 Hz, 1H), 7.11–7.15 (t, J=8.8 Hz, 2H), 3.82 (s, 2H), 3.66 (m,

4H), 3.53–3.55 (t, J=4.2 Hz, 2H), 2.62–2.67 (m, 4H), 2.53–2.56 ppm

(m, 4H); MS (ESI): m/z 371 [M+H]⁺.

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-2-

(3,4,5-trimethoxyphenyl)acetamide (A2): Prepared according to

general procedure VII from 2b and 3a (115 mg, 81%): HPLC:

97.60%, t_R=1.331 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.65 (m,

1H), 7.71–7.68 (m, 3H), 7.53–7.48 (m, 3H), 7.43-.30 (m, 6H), 6.59 (s,

2H), 4.32 (d, J=5.4 Hz, 2H), 3.74 (s, 6H), 3.63 (s, 3H), 3.42 (brs,

4H), 3.34 (brs, 4H), 2.51 (brs, 2H), 2.02 ppm (brs, 2H); MS (ESI):

m/z 594 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₄₀N₃O₅:

594.2890, observed: 594.2995.

N-(4-(4-(Biphenyl-4-ylmethyl)piperazine-1-carbonyl)benzyl)-2-

(3,4,5-trimethoxyphenyl)acetamide (A3): Prepared according to

general procedure VII from 2c and 3a (120 mg, 84%): HPLC:

95.06%, t_R=1.323 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.57–8.54

(m, 1H), 7.70–7.67 (m, 3H), 7.51–7.30 (m, 9H), 6.59 (s, 2H), 4.32 (d,

J=5.4 Hz, 2H), 3.74 (s, 6H), 3.63 (s, 3H), 3.52–3.47 (m, 4H), 3.43

(brs, 2H), 2.52 (brs, 2H), 2.01 ppm (brs, 2H); MS (ESI): m/z 594

[M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₄₀N₃O₅: 594.2890,

observed: 594.2999.

4-(4-{[3-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-4-oxobu-

tanoic acid (10b): Prepared according to general procedure VI

1

from 2o (80%): H NMR (CDCl₃): d=7.56 (s, 1H), 7.54 (m, 1H), 7.53

(m, 1H), 7.49–7.51 (d, J=7.6 Hz, 1H), 7.40–7.44 (m, 3H), 7.33–7.35

(d, J=7.6 Hz, 1H), 3.70 (s, 4H), 3.59 (s, 2H), 2.59–2.69 ppm (m, 8H);

MS (ESI): m/z 387 [M+H]⁺.

4-Oxo-4-{4-[(3-phenylphenyl)methyl]piperazin-1-yl}butanoic acid

(10c): Prepared according to general procedure VI from 2b (91%):

¹H NMR (CDCl₃): d=8.45 (s, 1H), 7.58 (s, 1H), 7.57 (s, 1H), 7.54 (s,

1H), 7.50 (d, J=8.0 Hz, 1H), 7.42 (t, J=8.0 Hz, 2H), 7.37 (d, J=

7.6 Hz, 1H), 7.32 (t, J=7.6 Hz, 1H), 7.28 (d, J=7.6 Hz, 1H), 3.63 (s,

2H), 3.61 (brs, 2H), 3.48 (brs, 2H), 2.50–2.56 ppm (m, 8H); MS

(ESI): m/z 353 [M+H]⁺.

N-(4-(4-((2’-Chlorobiphenyl-2-yl)methyl)piperazine-1-carbonyl)-

benzyl)-2-(3,4,5-trimethoxyphenyl)acetamide (A4): Prepared ac-

cording to general procedure VII from 2d and 3a (121 mg, 80%):

HPLC: 96.62%, t_R=1.747 min; ¹H NMR (300 Hz, [D₆]DMSO): d=

8.56–8.52 (m, 1H), 7.82 (d, J=6.9 Hz, 1H), 7.51–7.45 (m, 5H), 7.40–

7.31 (m, 5H), 6.59 (s, 2H), 4.31 (d, J=6.0 Hz, 2H), 3.74 (s, 6H), 3.64

(s, 3H), 3.50 (s, 2H), 3.42 (s, 2H), 3.36 (brs, 4H), 2.52 ppm (brs, 4H);

MS (ESI): m/z 628 [M+H]⁺; HRMS (ESI): m/z [M]⁺calcd for

C₃₆H₃₉ClN₃O₅: 627.2500, observed: 627.2511.

5-Oxo-5-{4-[(3-phenylphenyl)methyl]piperazin-1-yl}pentanoic

acid (10d): Prepared according to general procedure VI from 2b

and dihydro-2H-pyran-2,6(3H)-dione (89%): ¹H NMR (CDCl₃): d=

9.90 (s, 1H), 7.59–7.60 (m, 1H), 7.58 (s, 1H), 7.55 (m, 2H), 7.43–7.46

(m, 2H), 7.40–7.42 (t, J=6.8 Hz, 1H), 7.33–7.37 (m, 1H), 7.29–7.31

(d, J=7.7 Hz, 1H), 3.70–3.74 (m, 4H), 3.56 (s, 2H), 2.62 (s, 4H),

2.37–2.42 (m, 4H), 1.88–1.95 ppm (m, 2H); MS (ESI): m/z 367 [M+

H]⁺.

N-(4-(4-((3’-Chloro-4’-fluorobiphenyl-2-yl)methyl)piperazine-1-

carbonyl)benzyl)-2-(3,4,5-trimethoxyphenyl)acetamide (A5): Pre-

pared according to general procedure VII from 2e and 3a (110 mg,

1

71%): HPLC: 100%, t_R=2.246 min; H NMR (300 Hz, [D₆]DMSO): d=

5-(4-{[3-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-5-oxo-

pentanoic acid (10e): Prepared according to general procedure VI

from 2o and dihydro-2H-pyran-2,6(3H)-dione (75%): ¹H NMR

(CDCl₃): d=7.52 (m, 1H), 7.50–7.51 (m, 2H), 7.47–7.49 (m, 1H), 7.42

(d, J=3.2 Hz, 1H), 7.40–7.41 (m, 1H), 7.38–7.39 (d, J=3.2 Hz, 1H),

7.29–7.31 (d, J=7.6 Hz, 1H), 3.65 (m, 4H), 3.50–3.53 (t, J=4.8 Hz,

2H), 2.51–2.56 (m, 4H), 2.37–2.41 (m, 4H), 1.89–1.96 ppm (m, 2H);

MS (ESI): m/z 401 [M+H]⁺.

8.61–8.59 (m, 1H), 7.55–7.52 (m, 1H), 7.42–7.26 (m, 9H), 6.59 (s,

2H), 4.27 (d, J=4.5 Hz, 2H), 3.73 (s, 6H), 3.63 (s, 3H), 3.42–3.19

(brs, 6H), 3.16 (brs, 2H), 2.13 ppm (brs, 4H); MS (ESI): m/z 646

[M+H]⁺; HRMS (ESI): m/z [M]⁺calcd for C₃₆H₃₈ClFN₃O₅: 645.2406,

observed: 645.2417.

N-(4-(4-((5-Fluorobiphenyl-2-yl)methyl)piperazine-1-carbonyl)-

benzyl)-2-(3,4,5-trimethoxyphenyl)acetamide (A6): Prepared ac-

cording to general procedure VII from 2 f and 3a (106 mg, 72%):

HPLC: 98.55%, t_R=1.351 min; ¹H NMR (300 Hz, [D₆]DMSO): d=

8.55–8.53 (m, 1H), 7.73–7.70 (m, 2H), 7.51–7.40 (m, 5H), 7.34–7.29

(m, 5H), 6.59 (s, 2H), 4.31 (d, J=5.7 Hz, 2H), 3.74 (s, 6H), 3.69 (s,

3H), 3.63 (s, 2H), 3.53 (s, 2H), 3.49 (brs, 4H), 2.46 ppm (brs, 4H);

MS (ESI): m/z 612 [M+H]⁺; HRMS (ESI): m/z [M]⁺calcd for

C₃₆H₃₉FN₃O₅: 611.2795, observed: 611.2799.

General procedure VII for the synthesis of A1–A27, B1–B3, B5–

B17: N-(4-(4-(biphenyl-2-ylmethyl)piperazine-1-carbonyl)benzyl)-

2-(3,4,5-trimethoxyphenyl)acetamide (A1): 1-[Bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro-

phosphate (HATU; 1.2 mmol) and N,N-diisopropylethylamine

(DIPEA; 1.5 mmol) was added to a solution of 3a (1.0 mmol) in dry

DMF (10 mL). After stirring at ꢁ108C for 30 min, 2a (0.8 mmol) was

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N-(4-(4-(Naphthalen-2-ylmethyl)piperazine-1-carbonyl)benzyl)-2-

(3,4,5-trimethoxyphenyl)acetamide (A7): Prepared according to

general procedure VII from 2g and 3a (110 mg, 84%): HPLC:

100%, t_R=1.158 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.58 (brs,

1H), 8.28 (d, J=6.9 Hz, 1H), 7.94–7.84 (m, 2H), 7.54–7.45 (m, 4H),

7.35–7.29 (m, 4H), 6.59 (s, 2H), 4.31 (d, J=5.4 Hz, 2H), 3.92 (s, 2H),

3.74 (s, 6H), 3.69 (s, 3H), 3.46 (s, 2H), 3.42 (brs, 4H), 2.48 ppm (brs,

4H); MS (ESI): m/z 568 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₃₄H₃₈N₃O₅: 568.2733, observed: 568.2741.

4H); MS (ESI): m/z 506 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₃₂H₃₁N₃O₃: 506.2365, observed: 506.2330.

3-((4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzylami-

no)methyl)benzoic acid (A14): According to the general procedur-

e VII, we obtained the ester intermediate from 3d and 2c as crude

material. The crude was dissolved in MeOH (10 mL) and 2n aq

NaOH (10 mL) was added. The reaction was stirred for a further

3 h, and then 1n HCl was added until a white solid formed. The

mixture was then filtered, and the solid was dried to give A14 as

1

a white solid (76 mg, 61%): HPLC: 97.34%, t_R=1.105 min; H NMR

N-(4-(4-(3,4,5-Trimethoxybenzyl)piperazine-1-carbonyl)benzyl)-2-

(3,4,5-trimethoxyphenyl)acetamide (A8): Prepared according to

general procedure VII from 1-(3,4,5-trimethoxybenzyl)piperazine

and 3a (123 mg, 81%): HPLC: 98.94%, t_R=0.738 min; ¹H NMR

(300 Hz, [D₆]DMSO): d=7.34–7.31 (m, 4H), 6.99 (d, J=8.4 Hz, 1H),

6.77 (d, J=9.0 Hz, 1H), 6.59 (s, 2H), 4.31 (d, J=5.7 Hz, 2H), 3.78 (s,

6H), 3.74 (s, 9H), 3.69 (s, 3H), 3.55 (brs, 4H), 3.55–3.42 (m, 4H),

2.38 ppm (brs, 4H); MS (ESI): m/z 608 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₃H₄₁N₃O₈: 608.2794, observed: 608.2799.

(300 Hz, CDCl₃): d=8.03 (s, 2H), 7.91 (d, J=7.8 Hz, 2H), 7.62–7.58

(m, 4H), 7.56 (s, 1H), 7.44–7.39 (m, 7H), 7.30 (d, J=7.2 Hz, 1H),

3.81 (s, 2H), 3.60 (s, 4H), 3.57 (s, 2H), 3.44 (s, 2H), 2.58–2.43 ppm

(br, 4H); MS (ESI): m/z 518 [MꢁH]^ꢁ; HRMS (ESI): m/z [M+H]⁺calcd

for C₃₃H₃₄N₃O₃: 520.2522, observed: 520.2531.

3-((4-(4-(Biphenyl-4-ylmethyl)piperazine-1-carbonyl)benzylami-

no)methyl)benzoic acid (A15): Prepared according to general pro-

cedure VII from 3d and 2b as described above for A14, to A14 as

1

a white solid (79 mg, 63%): HPLC: 98.53%, t_R=1.119 min; H NMR

N-(4-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)benzyl)-3-

(3,4-dimethoxyphenyl)propanamide (A9): Prepared according to

general procedure VII from 2a and 3b (104 mg, 75%): HPLC:

98.39%, t_R=1.597 min; ¹H NMR (300 Hz, CDCl₃): d=7.52–7.49(m,

1H), 7.39–7.31 (m, 6H), 7.21 (d, J=8.1 Hz, 2H), 7.06 (d, J=7.5 Hz,

2H), 7.80–6.71 (m, 5H), 4.34 (d, J=6.0 Hz, 2H), 3.86 (s, 3H), 3.82 (s,

3H), 3.69 (t, J=6.9 Hz, 2H), 3.44 (s, 2H), 3.29 (t, J=6.9 Hz, 2H), 2.92

(t, J=6.3 Hz, 2H), 2.51 (t, J=7.2 Hz, 2H), 2.39–2.31 ppm (br, 4H);

MS (ESI): m/z 578 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₃₈H₃₉N₃O₄: 578.2941, observed: 578.3046.

(300 Hz, CDCl₃): d=7.78 (d, J=9.0 Hz, 2H), 7.56 (t, J=8.7 Hz, 4H),

7.45–7.31 (m, 8H), 7.26 (s, 1H), 6.89 (d, J=9.0 Hz, 2H), 4.64 (s, 2H),

4.59 (d, J=6.0 Hz, 2H), 3.79 (brs, 2H), 3.57 (s, 2H), 3.43 (brs, 2H),

2.55–2.41 ppm (br, 4H); MS (ESI): m/z 518 [MꢁH]^ꢁ; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₃H₃₃N₃O₃: 520.2522, observed: 520.2582.

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-2-(4-

methoxyphenyl)acetamide (A16): Prepared according to general

procedure VII from 2b and 3e (113 mg, 88%): HPLC: 96.31%, t_R=

1.573 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.53–8.50 (m, 1H),

7.55–7.28 (m, 8H), 7.26–7.17 (m, 7H), 6.88–6.84 (m, 2H), 4.27 (d,

J=4.5 Hz, 2H), 3.72 (s, 3H), 3.56–3.35 (m, 4H), 3.48–3.40 (m, 4H),

3.23 ppm (brs, 4H); MS (ESI): m/z 534 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₄H₃₆N₃O₃: 534.2678, observed: 534.2697.

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-3-

(3,4-dimethoxyphenyl)propanamide (A10): Prepared according to

general procedure VII from 2b and 3b (96 mg, 69%): HPLC:

95.88%, t_R=1.469 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.39 (m,

1H), 7.64 (m, 4H), 7.45–7.18 (m, 8H), 6.81–6.71 (m, 3H), 4.27 (s,

2H), 3.69 (s, 6H), 3.55 (brs, 4H), 3.17–3.10 (m, 2H), 2.96–2.70 (m,

2H), 2.43 ppm (br, 4H); MS (ESI): m/z 578 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₈H₄₀N₃O₄: 578.2941, observed: 578.2956.

N-(4-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)benzyl)-2-(4-

methoxyphenyl)acetamide (A17): Prepared according to general

procedure VII from 2a and 3e (104 mg, 81%): HPLC: 96.55%, t_R=

1

1.749 min; H NMR (300 Hz, [D₆]DMSO): d=8.51 (m, 1H), 7.55–7.53

(m,1H), 7.47–7.38 (m, 7H), 7.35–7.81 (m, 7H), 6.88–6.85 (m, 2H),

4.28 (d, J=5.4 Hz, 2H), 3.73 (s, 3H), 3.52 (brs, 6H), 3.41–3.30 (m,

2H), 2.82 ppm (m, 4H); MS (ESI): m/z 534 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₄H₃₆N₃O₃: 534.2678, observed: 534.2684.

N-(4-(4-(Biphenyl-4-ylmethyl)piperazine-1-carbonyl)benzyl)-3-

(3,4-dimethoxyphenyl)propanamide (A11): Prepared according to

general procedure VII from 2c and 3b (100 mg, 72%): HPLC:

97.05%, t_R=1.472 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.38–8.35

(m, 1H), 7.68–7.57 (m, 4H), 7.51–7.28 (m, 7H), 7.18–7.16 (m, 2H),

6.85–6.70 (m, 3H), 4.29 (d, J=5.4 Hz, 2H), 3.70 (s, 6H), 3.77–3.64

(m, 4H), 3.52 (brs, 2H), 3.18–3.11 (m, 2H), 2.82–2.51 (m, 2H), 2.47–

2.42 ppm (br, 4H); MS (ESI): m/z 578 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₈H₄₀N₃O₄: 578.2941, observed: 578.2963.

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-2-(4-

fluorophenyl)acetamide (A18): Prepared according to general pro-

cedure VII from 2b and 3 f (100 mg, 81%): HPLC: 99.72%, t_R=

1

1.693 min; H NMR (300 Hz, [D₆]DMSO): d=8.64 (m, 1H), 7.68–7.62

(m,4H), 7.50–7.40 (m, 3H), 7.36–7.27 (m, 8H), 7.16–7.10 (m, 2H),

4.30 (d, J=5.7 Hz, 2H), 3.56 (s, 2H), 3.49 (s, 2H), 3.60–3.49 (m, 4H),

2.51 ppm (brs, 4H); MS (ESI): m/z 522 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₃H₃₃FN₃O₃: 522.2749, observed: 522.2757.

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-3-hy-

droxybenzamide (A12): Prepared according to general procedure

VII from 2b and 3c (83 mg, 68%): HPLC: 98.20%, t_R=1.126 min;

¹H NMR (300 Hz, CDCl₃): d=8.84 (s, 1H), 7.78 (d, J=8.7 Hz, 2H),

7.65 (d, J=7.5 Hz, 2H), 7.58 (s, 2H), 7.54–7.31 (m, 8H), 6.82 (d, J=

8.4 Hz, 2H), 4.47 (d, J=5.1 Hz, 2H), 3.75–3.58 (br, 4H), 2.51 (s, 2H),

2.43 ppm (br, 4H); MS (ESI): m/z 506 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₂H₃₁N₃O₃: 506.2365, observed: 506.2425.

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-2-

(thiophen-3-yl)acetamide (A19): Prepared according to general

procedure VII from 2b and 3g (104 mg, 85%): HPLC: 98.60%, t_R=

1

1.492 min; H NMR (300 Hz, [D₆]DMSO): d=8.67 (m, 1H), 7.69–7.62

(m, 4H), 7.50–7.30 (m, 10H), 6.98–6.94 (m, 2H), 4.31 (d, J=5.7 Hz,

2H), 3.73 (s, 2H), 3.52 (s, 2H), 3.60–3.30 (m, 4H), 2.421 ppm (brs,

4H); MS (ESI): m/z 510 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₃₁H₃₂N₃O₂S: 510.2137, observed: 510.2144.

N-(4-(4-(Biphenyl-4-ylmethyl)piperazine-1-carbonyl)benzyl)-3-hy-

droxybenzamide (A13): Prepared according to general procedure

VII from 2c and 3c (75 mg, 62%): HPLC: 97.61%, t_R=1.154 min;

¹H NMR (300 Hz, CDCl₃): d=8.84 (s, 1H), 7.78 (d, J=8.4 Hz,2H), 7.65

(t, J=8.7 Hz, 5H), 7.49–7.36 (m, 7H), 6.81 (d, J=9.0 Hz, 2H), 4.48

(d, J=6.0 Hz, 2H), 3.66–3.55 (br, 4H), 2.51 (s, 2H), 2.41 ppm (br,

N-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzyl)-3-

methylbutanamide (A20): Prepared according to general proce-

dure VII from 2b and 3h (79 mg, 70%): HPLC: 95.13%, t_R=

1.831 min; ¹H NMR (300 Hz, [D₆]DMSO): d=8.33 (m, 1H), 7.52

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(m,1H), 7.45–7.38 (m, 7H), 7.35–7.23 (m, 5H), 4.28 (d, J=6.0 Hz,

2H), 3.41 (s, 2H), 3.28 (s, 2H), 2.28 (brs, 4H), 2.03–1.98 (brs,

4H),2.02 (m, 3H), 0.88 ppm (d, J=6 Hz, 6H); MS (ESI): m/z 470 [M+

H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₀H₃₆N₃O₂: 470.2729, ob-

served: 470.2739.

(brs, 2H), 7.39–7.30 (m, 9H), 7.25 (d, J=8.1 Hz, 3H), 7.20 (s, 1H),

7.06 (d, J=7.8 Hz, 2H), 6.82 (s, 1H), 6.67 (s, 1H), 4.31 (d, J=6.0 Hz,

2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.68 (brs, 2H), 3.40 (s, 2H), 3.40–3.32

(br, 2H), 2.91 (t, J=7.2 Hz, 2H), 2.38–2.33 (brs, 4H), 2.30 ppm (t,

J=7.5 Hz, 2H); MS (ESI): m/z 688 [M+H]⁺; HRMS (ESI): m/z [M+

H]⁺calcd for C₄₂H₄₂ClN₃O₄: 688.2864, observed: 688.2908.

Ethyl 2-(3-(4-(4-(biphenyl-3-ylmethyl)piperazine-1-carbonyl)ben-

zylcarbamoyl)phenoxy)acetate (A21): Prepared according to gen-

eral procedure VII from 2a and 3i (92 mg, 65%): HPLC: 98.22%,

2-(2-Bromo-3,4,5-trimethoxyphenyl)-N-(3-(4-((4’-chlorobiphenyl-

2-yl)methyl)piperazine-1-carbonyl)phenyl)acetamide (B1): Pre-

pared according to general procedure VII from 2h and 4a

(136 mg, 82%): HPLC: 100%, t_R=0.503 min; ¹H NMR (300 Hz,

CDCl₃): d=7.87 (s, 1H), 7.62 (d, J=8.1 Hz, 1H), 7.54 (d, J=9.0 Hz,

3H), 7.45 (s, 2H), 7.39 (d, J=8.1 Hz, 3H), 7.30 (t, J=7.5 Hz, 2H),

7.08 (d, J=9.0 Hz, 1H), 4.19 (s, 1H), 4.12 (s, 1H), 3.95 (s, 3H), 3.91

(s, 6H), 3.79 (brs, 2H), 3.60 (s, 2H), 3.48 (brs, 2H), 2.56–2.44 ppm

(br, 4H); MS (ESI): m/z 692 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺

calcd for C₃₅H₃₆ClN₃O₅Br: 692.1527, observed: 692.1537.

1

t_R=2.577 min; H NMR (300 Hz, CDCl₃): d=7.59–7.53 (m, 5H), 7.48

(t, J=7.8 Hz, 2H), 7.39 (t, J=7.8 Hz, 3H), 7.29 (t, J=7.2 Hz, 3H),

7.08 (d, J=7.2 Hz, 2H), 6.97 (d, J=8.4 Hz, 2H), 4.49 (d, J=5.7 Hz,

2H), 4.10 (q, J=6.9 Hz, 2H), 3.73 (brs, 2H), 3.36 (s, 2H), 3.37 (brs,

2H), 3.49–3.36 (br, 4H), 2.01 (s, 2H), 1.23 (t, J=6.9 Hz, 3H); MS

(ESI): m/z 592 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₃₆H₃₇N₃O₅: 592.2733, observed: 592.2768.

Ethyl 2-(3-(4-(4-(biphenyl-4-ylmethyl)piperazine-1-carbonyl)ben-

zylcarbamoyl)phenoxy)acetate (A22): Prepared according to gen-

eral procedure VII from 2b and 3i (97 mg, 68%): HPLC: 97.85%,

2-(2-Bromo-3,4,5-trimethoxyphenyl)-N-(3-(4-((4’-chlorobiphenyl-

3-yl)methyl)piperazine-1-carbonyl)phenyl)acetamide (B2): Pre-

pared according to general procedure VII from 2i and 4a (138 mg,

82%): HPLC: 99.93%, t_R=0.526 min; ¹H NMR (300 Hz, CDCl₃): d=

7.60 (d, J=7.5 Hz, 4H), 7.50 (d, J=7.5 Hz, 1H), 7.44 (s, 2H), 7.40 (d,

J=7.2 Hz, 1H), 7.37–7.29 (m, 4H), 7.10 (t, J=7.5 Hz, 1H), 4.21 (s,

2H), 3.98 (s, 3H), 3.93 (s, 6H), 3.86 (s, 2H), 3.40 (s, 4H), 2.40 (s, 2H),

2.29 ppm (s, 2H); MS (ESI): m/z 692 [M+H]⁺; HRMS (ESI): m/z [M+

H]⁺calcd for C₃₅H₃₆ClN₃O₅Br: 692.1527, observed: 692.1544.

1

t_R=1.628 min; H NMR (300 Hz, CDCl₃): d=7.77 (d, J=9.3 Hz, 2H),

7.57 (t, J=8.1 Hz, 3H), 7.50–7.39 (m, 4H), 7.37–7.28 (m, 6H), 6.91

(d, J=8.7 Hz, 2H), 4.65 (s, 2H), 4.26 (q, J=6.9 Hz, 2H), 3.78 (brs,

2H), 3.60 (s, 2H), 3.42 (brs, 2H), 2.56–2.41 (br, 4H), 1.75 (s, 2H),

1.29 ppm (t, J=6.9 Hz, 3H); MS (ESI): m/z 592 [M+H]⁺; HRMS (ESI):

m/z [M+H]⁺calcd for C₃₆H₃₇N₃O₅: 592.2733, observed: 592.2792.

2-(3-(4-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)benzylcar-

bamoyl)phenoxy)acetic acid (A23): 2n NaOH (2 mL) was added to

a solution of A21 in CH₂CH₃OH (2 mL). The reaction mixture was

heated at reflux for 2 h, and CH₂CH₃OH was evaporated in vacuo.

1n HCl was added, and the precipitate was filtrated and washed

with H₂O to get A23 as a white solid (44 mg, 92%): HPLC: 99.12%,

N-(3-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)phenyl)-2-(4’-

chloro-4,5,6-trimethoxybiphenyl-2-yl)acetamide (B3): Prepared

according to general procedure VII from 2a and 4b (136 mg,

1

82%): HPLC: 100%, t_R=4.226 min; H NMR (300 Hz, CDCl₃): d=7.67

(d, J=7.5 Hz, 1H), 7.63–7.61 (m, 3H), 7.53 (d, J=7.5 Hz, 2H), 7.48–

7.41 (m, 5H), 7.32 (d, J=8.4 Hz, 3H), 7.23 (d, J=8.4 Hz, 3H), 7.03

(d, J=7.8 Hz, 1H), 4.44 (s, 1H), 4.10 (s, 1H), 3.923 (s, 3H), 3.89 (s,

3H), 3.87 (s, 3H), 3.66 (br, 2H), 3.43 (s, 2H), 3.31–3.27 (br, 2H),

2.38–2.25 ppm (br, 4H); MS (ESI): m/z 690 [M+H]⁺; HRMS (ESI): m/

z [M+H]⁺calcd for C₄₁H₄₁ClN₃O₅: 690.2735, observed: 690.2749.

1

t_R=0.429 min; H NMR (300 Hz, CDCl₃): d=7.78 (d, J=8.7 Hz, 2H),

7.56 (t, J=8.4 Hz, 5H), 7.25 (s, 1H), 7.45–7.33 (m, 7H), 6.91 (d, J=

9.0 Hz, 2H), 4.64 (s, 2H), 4.59 (d, J=6.0 Hz, 2H), 3.79 (brs, 2H), 3.57

(s, 2H), 3.43 (brs, 2H), 3.55–2.41 ppm (br, 4H); MS (ESI): m/z 562

[MꢁH]^ꢁ; HRMS (ESI): m/z [M+H]⁺calcd for C₃₄H₃₃N₃O₅: 564.2420,

observed: 564.2483.

N-(3-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)phenyl)-2-(4’-

2-(3-(4-(4-(Biphenyl-4-ylmethyl)piperazine-1-carbonyl)benzylcar-

bamoyl)phenoxy)acetic acid (A24): Prepared according to the pro-

cedure described for A23 from A22 (46 mg, 96%): HPLC: 97.98%,

chloro-4,5,6-trihydroxybiphenyl-2-yl)acetamide

(B4):

BBr₃

(2.25 mmol) was added to a solution of B3 (0.15 mmol) in dry

CH₂Cl₂at ꢁ788C. After stirring at ꢁ788C for 12 h, the reaction mix-

ture was diluted, and acidified with 1n HCl. The mixture was ex-

tracted with CH₂Cl₂. The combined organic phases were washed

with saturated NaHCO₃and brine, dried over Na₂SO₄, filtered and

concentrated in vacuo. The crude compound was purified by chro-

matography with CH₂Cl₂/CH₃OH (10:1 v/v) to afford B4 as a white

1

t_R=0.391 min; H NMR (300 Hz, CDCl₃): d=7.61–7.55 (m, 5H), 7.50–

7.40 (m, 3H), 7.38–7.26 (m, 5H), 7.11 (d, J=8.4 Hz, 2H), 7.00 (d, J=

8.7 Hz, 2H), 4.51 (d, J=5.7 Hz, 2H), 3.76 (s, 2H), 3.58 (s, 2H), 3.39

(s, 2H), 2.52–2.39 (br, 4H), 2.04 ppm (s, 2H); MS (ESI): m/z 562

[MꢁH]^ꢁ; HRMS (ESI): m/z [M+H]⁺calcd for C₃₄H₃₃N₃O₅: 564.2420,

observed: 564.2456.

1

solid (68 mg, 70%): HPLC: 95.33%, t_R=1.628 min; H NMR (300 Hz,

CDCl₃): d=8.31 (s, 1H), 7.43 (d, J=8.4 Hz, 4H), 7.36 (d, J=7.5 Hz,

4H), 7.33–7.26 (m, 5H), 7.18 (d, J=7.5 Hz, 2H), 6.71 (s, 1H), 6.54 (s,

1H), 4.34 (s, 2H), 4.24 (d, J=5.1 Hz, 2H), 4.06–4.00 (br, 2H), 2.60 (s,

2H), 2.26 ppm (t, J=7.2 Hz, 4H); MS (ESI): m/z 562 [MꢁH]^ꢁ.

N-(4-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)benzyl)-3-(2-

bromo-4,5-dimethoxyphenyl)propanamide (A25): Prepared ac-

cording to general procedure VII from 2a and 3j (111 mg, 75%):

1

HPLC: 100%, t_R=0.417 min; H NMR (300 Hz, CDCl₃): d=7.52–7.49

(m, 1H), 7.41–7.29 (m, 8H), 7.25 (s, 1H), 7.12 (d, J=8.1 Hz, 2H),

6.98 (s, 1H), 6.78 (s, 1H), 5.92 (t, J=6.3 Hz,1H), 4.39 (d, J=6.0 Hz,

2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.71 (t, J=6.6 Hz, 2H), 3.44 (s, 2H),

3.31 (t, J=6.6 Hz, 2H), 3.04 (t, J=7.5 Hz, 2H), 2.52 (t, J=7.5 Hz,

2H), 2.39–2.27 ppm (br, 4H); MS (ESI): m/z 656 [M+H]⁺; HRMS

(ESI): m/z [M+H]⁺calcd for C₃₆H₃₈BrN₃O₄: 656.2046, observed:

656.2088.

N-(3-(4-((3-Fluorobiphenyl-4-yl)methyl)piperazine-1-carbonyl)-

phenyl)-3,5-dimethoxybenzamide (B5): Prepared according to

general procedure VII from 2j and 4c (113 mg, 85%): HPLC:

96.42%, t_R=5.870 min; ¹H NMR (300 Hz, CDCl₃): d=9.10 (s, 1H),

7.83 (d, J=7.8 Hz, 1H), 7.57 (t, J=8.4 Hz, 3H), 7.45–7.32 (m, 6H),

7.29 (s, 1H), 7.26–7.23 (m, 1H), 7.02 (s, 1H), 6.56 (s, 1H), 3.79 (s,

6H), 3.68 (brs, 2H), 3.62 (s, 2H), 3.45 (brs, 2H), 2.50–2.45 ppm (br,

4H); MS (ESI): m/z 554 [M+H]⁺.

N-(4-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)benzyl)-3-(4’-

chloro-4,5-dimethoxybiphenyl-2-yl)propanamide (A26): Prepared

according to general procedure VII from 2a and 3k (117 mg, 72%):

3,5-Dimethoxy-N-(3-(4-((3-methoxybiphenyl-4-yl)methyl)pipera-

zine-1-carbonyl)phenyl)benzamide (B6): Prepared according to

general procedure VII from 2k and 4d (111 mg, 82%): HPLC:

1

HPLC: 100%, t_R=5.011 min; H NMR (300 Hz, CDCl₃): d=7.52–7.50

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96.27%, t_R=0.515 min; ¹H NMR (300 Hz, CDCl₃): d=7.74 (d, J=

8.1 Hz, 1H), 7.59 (s, 1H), 7.40–7.26 (m, 5H), 7.24–7.17 (m, 2H), 6.88

(s, 2H), 6.84–6.80 (m, 1H), 6.60 (s, 1H), 6.50–6.46 (m, 2H), 3.85 (s,

3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.69–3.60 (m, 2H), 3.51 (s, 2H), 3.42

(s, 2H), 2.50–2.38 ppm (br, 4H); MS (ESI): m/z 566 [M+H]⁺; HRMS

(ESI): m/z [M+H]⁺calcd for C₃₄H₃₆N₃O₅: 566.2655, observed:

566.2679.

7.36 (d, J=7.5 Hz, 3H), 7.30 (d, J=7.8 Hz, 2H), 7.11–7.06 (m, 2H),

6.89–6.84 (m, 1H), 3.82 (s,3H), 3.81 (s, 1H), 3.63 (s, 2H), 3.52 (s,

1H), 3.40 (s, 2H), 2.31 ppm (s, 4H); MS (ESI): m/z 556 [M+H]⁺;

HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₃₄N₃O₃: 556.2600, observed:

556.2609.

N-(3-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)phenyl)-2-

naphthamide (B13): Prepared according to general procedure VII

from 2b and 4 f (93 mg, 75%): HPLC: 96.68%, t_R=3.510 min;

¹H NMR (300 Hz, CDCl₃): d=8.77 (s, 1H), 8.39 (s, 1H), 7.92–7.82 (m,

5H), 7.63 (s, 1H), 7.56 (t, J=7.5 Hz, 2H), 7.49–7.47 (m, 1H), 7.39–

7.26 (m, 8H), 7.03 (d, J=7.5 Hz, 1H), 3.59 (s, 2H), 3.40 (s, 2H), 3.40–

3.38 (br, 2H), 2.28 ppm (s, 4H); MS (ESI): m/z 526 [M+H]⁺; HRMS

(ESI): m/z [M+H]⁺calcd for C₃₅H₃₂N₃O₂: 526.2495, observed:

526.2508.

2-(3,5-Dimethoxyphenyl)-N-(3-(4-((3-methylbiphenyl-4-yl)me-

thyl)piperazine-1-carbonyl)phenyl)acetamide (B7): Prepared ac-

cording to general procedure VII from 2l and 4d (112 mg, 83%):

1

HPLC: 95.27%, t_R=3.418 min; H NMR (300 Hz, CDCl₃): d=7.77 (s,

1H), 7.57 (d, J=8.1 Hz, 2H), 7.45–7.38 (m, 4H), 7.32–7.28 (m, 4H),

7.07 (t, J=7.5 Hz, 1H), 6.46 (s, 2H), 6.42 (s, 1H), 3.79 (s, 6H), 3.74 (s,

1H), 3.64 (s, 2H), 3.52 (s, 1H), 3.42 (s, 2H), 3.01 (s, 1H), 2.94 (s, 1H),

2.55–2.49 (br, 4H), 2.43 ppm (s, 3H); MS (ESI): m/z 564 [M+H]⁺;

HRMS (ESI): m/z [M+H]⁺calcd for C₃₅H₃₈N₃O₄: 564.2862, ob-

served:564.2869.

N-(3-(4-((3-Methoxybiphenyl-4-yl)methyl)piperazine-1-carbonyl)-

phenyl)-2-(naphthalen-2-yl)acetamide (B14): Prepared according

to general procedure VII from 2k and 4g (98 mg, 72%): HPLC:

97.34%, t_R=3.124 min; ¹H NMR (300 Hz, CDCl₃): d=7.89–7.82 (m,

3H), 7.78 (s, 1H), 7.54–7.50 (m, 3H), 7.43–7.35 (m, 6H), 7.29 (d, J=

7.8 Hz, 2H), 7.19 (s, 1H), 7.16 (s, 1H), 7.01 (d, J=7.5 Hz, 1H), 6.87–

6.84 (dd, J=5.7, 2.4 Hz, 1H), 3.89 (s, 2H), 3.85 (s, 3H), 3.67 (brs,

2H), 3.41 (s, 2H), 3.33 (brs, 2H), 2.39–2.27 ppm (br, 4H); MS (ESI):

m/z 570 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₇H₃₆N₃O₃:

570.2757, observed: 570.2769.

2-(3,5-Dimethoxyphenyl)-N-(3-(4-((3-methoxybiphenyl-4-yl)me-

thyl)piperazine-1-carbonyl)phenyl)acetamide (B8): Prepared ac-

cording to general procedure VII from 2k and 4d (107 mg, 77%):

1

HPLC: 96.93%, t_R=4.402 min; H NMR (300 Hz, CDCl₃): d=7.53 (d,

J=8.1 Hz, 1H), 7.40–7.33 (m, 3H), 7.29 (d, J=8.1 Hz, 4H), 7.17 (d,

J=8.4 Hz, 1H), 7.11 (s, 1H), 7.04 (d, J=7.2 Hz, 1H), 6.87–6.83 (dd,

J=5.7, 2.4 Hz, 1H), 6.44 (s, 2H), 6.43 (s, 1H), 3.85 (s, 3H), 3.80 (s,

6H), 3.72–3.66 (br, 2H), 3.66 (s, 2H), 3.42 (s, 2H), 3.38–3.32 (br, 2H),

2.40–2.94 ppm (br, 4H); MS (ESI): m/z 580 [M+H]⁺; HRMS (ESI):

m/z [M+H]⁺calcd for C₃₅H₃₈N₃O₅: 580.2811, observed: 580.2823.

N-(3-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)phenyl)-2-

(naphthalen-2-yl)acetamide (B15): Prepared according to general

procedure VII from 2a and 4g (91 mg, 72%): HPLC: 98.67%, t_R=

3.991 min; ¹H NMR (300 Hz, CDCl₃): d=8.09 (s, 1H), 7.82 (d, J=

8.4 Hz, 2H), 7.75 (s, 1H), 7.54 (d, J=8.1 Hz, 1H), 7.51–7.48 (m, 3H),

7.24 (s, 1H), 7.42–7.37 (m, 5H), 7.33–7.26 (m, 4H), 7.20 (d, J=

7.8 Hz, 1H), 6.93 (d, J=8.4 Hz, 1H), 3.83 (s, 2H), 3.67 (s, 2H), 3.42

(s, 2H), 3.31 (s, 2H), 2.37–2.25 ppm (br, 4H); MS (ESI): m/z 540 [M+

H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₃₄N₃O₂: 540.2651, ob-

served: 540.2667.

N-(3-(4-(Biphenyl-2-ylmethyl)piperazine-1-carbonyl)phenyl)-2-cy-

clohexylacetamide (B9): Prepared according to general procedure

VII from 2a and 4e (102 mg, 86%): HPLC: 98.56%, t_R=4.464 min;

¹H NMR (300 Hz, CDCl₃): d=7.68 (s, 1H),7.61 (d, J=8.4 Hz, 1H), 7.51

(s, 1H), 7.46 (s, 1H), 7.42–7.32 (m, 8H), 7.03 (d, J=8.1 Hz, 1H), 3.44

(s, 2H), 3.38 (s, 2H), 3.69 (s, 2H), 2.41–2.29 (br, 4H), 2.18 (d, J=

6.9 Hz, 2H), 1.88–1.64 (m, 6H), 1.34–1.12 (m, 3H), 1.03–0.92 ppm

(m, 2H); MS (ESI): m/z 496 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺

calcd for C₃₂H₃₈N₃O₂: 496.2964, observed: 496.2977.

N-(3-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)phenyl)-2-

(naphthalen-2-yl)acetamide (B16): Prepared according to general

procedure VII from 2b and 4g (92 mg, 70%): HPLC: 95.78%, t_R=

N-(3-(4-(Biphenyl-3-ylmethyl)piperazine-1-carbonyl)phenyl)-2-cy-

clohexylacetamide (B10): Prepared according to general procedur-

e VII from 2b and 4e (103 mg, 87%): HPLC: 99.17%, t_R=

1

3.412 min; H NMR (300 Hz, CDCl₃): d=7.86 (d, J=8.1 Hz, 2H), 7.78

(s, 1H), 7.69 (s, 1H), 7.59 (d, J=7.5 Hz, 2H), 7.54–7.47 (m, 6H), 7.44

(s, 1H), 7.42–7.35 (m, 4H), 7.31 (d, J=8.1 Hz, 1H), 7.23 (d, J=

7.8 Hz, 1H), 7.02 (d, J=7.5 Hz, 1H), 3.88 (s, 2H), 3.77 (s, 2H), 3.60

(s, 2H), 3.42 (s, 2H), 2.55–2.41 ppm (br, 2H); MS (ESI): m/z 540 [M+

H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₃₄N₃O₂: 540.2651, ob-

served: 540.2647.

1

2.339 min; H NMR (300 Hz, CDCl₃): d=7.62–7.59 (m, 3H), 7.55–7.47

(m, 3H), 7.45–7.34 (m, 4H), 7.31 (s, 1H), 7.28 (s, 1H), 7.08 (d, J=

7.5 Hz, 1H), 3.79 (s, 2H), 3.60 (s, 2H), 3.47 (s, 2H), 3.56–3.43 (br,

4H), 2.19 (d, J=6.9 Hz, 2H), 1.80–1.68 (m, 4H), 1.30–1.20 (m, 4H),

1.00–0.92 ppm (m, 3H); MS (ESI): m/z 496 [M+H]⁺; HRMS (ESI):

m/z [M+H]⁺calcd for C₃₂H₃₈N₃O₂: 496.2964, observed: 496.2989.

N-(3-(4-((3-Fluorobiphenyl-4-yl)methyl)piperazine-1-carbonyl)-

phenyl)-2-(naphthalen-2-yl)acetamide (B17): Prepared according

to general procedure VII from 2j and 4g (95 mg, 71%): HPLC:

96.46%, t_R=3.510 min; ¹H NMR (300 Hz, CDCl₃): d=8.25 (s, 1H),

7.83–7.75 (m, 5H), 7.62–7.57 (m, 3H), 7.50–7.45 (m, 6H), 7.26–7.19

(m, 3H), 6.96 (d, J=7.5 Hz, 1H), 3.83 (s, 2H), 3.78 (s, 2H), 3.64 (s,

2H), 3.42 (s, 2H), 2.57–2.43 ppm (br, 4H); MS (ESI): m/z 558 [M+

H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₆H₃₃FN₃O₂: 558.2557, ob-

served: 558.2569.

2-Cyclohexyl-N-(3-(4-((3-fluorobiphenyl-4-yl)methyl)piperazine-1-

carbonyl)phenyl)acetamide (B11): Prepared according to general

procedure VII from 2j and 4e (104 mg, 84%): HPLC: 95.63%, t_R=

4.409 min; ¹H NMR (300 Hz, CDCl₃): d=8.59 (s, 1H), 7.69 (d, J=

8.4 Hz, 1H), 7.55 (d, J=7.5 Hz, 2H), 7.47–7.41 (m, 4H), 7.38–7.33

(m, 2H), 7.29 (s, 1H), 7.02 (d, J=7.5 Hz, 1H), 3.80 (s, 2H), 3.64 (s,

2H), 3.46 (s, 2H), 2.59–2.45 (br, 4H), 2.16 (d, J=6.6 Hz, 2H), 1.78–

1.66 (m, 5H), 1.27–1.14 (m, 4H), 0.99–0.91 ppm (m, 2H); MS (ESI):

m/z 514 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₂H₃₇FN₃O₂:

514.2870, observed: 514.2881.

General procedure VIII for the synthesis of C1–C12: 2-(3-((4-((4-

((4’-Methoxybiphenyl-2-yl)methyl)piperazin-1-yl)methyl)-1H-

1,2,3-triazol-1-yl)methyl)phenoxy)acetic acid (C1): (+)-Sodium l-

ascorbate (0.03 mmol) and CuSO₄·6H₂O (0.03 mmol) was added to

a solution of 8a (0.3 mmol) and 9a (0.3 mmol) in (CH₃)₃COH/

H₂O.The reaction mixture was stirred at RT for 12 h. The solvent

was evaporated in vacuo and the crude product was purified by

N-(3-(4-((3-Methoxybiphenyl-4-yl)methyl)piperazine-1-carbonyl)-

phenyl)-2-naphthamide (B12): Prepared according to general pro-

cedure VII from 2k and 4 f (100 mg, 75%): HPLC: 95.13%, t_R=

2.810 min; ¹H NMR (300 Hz, CDCl₃): d=8.64 (s, 1H), 8.39 (s, 1H),

7.92 (s, 2H), 7.87 (t, J=7.8 Hz, 3H), 7.66 (s, 1H), 7.60–7.51 (m, 3H),

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chromatography with n-pentane/EtOAc (4:1 v/v) to afford C1 as

a white solid (132 mg, 80%): HPLC: 99.14%, t_R=0.506 min; H NMR

(300 Hz, CDCl₃): d=7.83 (s, 1H), 7.56 (s, 1H), 7.36–7.33 (m, 2H),

7.25–7.23 (m, 2H), 7.17 (d, J=8.7 Hz, 2H), 6.94 (d, J=8.4 Hz, 4H),

6.36 (s, 1H), 5.50 (s, 2H), 4.47 (s, 2H), 4.14 (s, 2H), 3.85 (s, 5H), 3.06

(s, 4H), 2.84 ppm (s, 4H); MS (ESI): m/z 526 [MꢁH]^ꢁ.

2H), 6.18 (s, 1H), 5.58 (s, 2H), 4.48 (s, 2H), 4.25 (s, 2H), 3.84 (s, 2H),

1

3.15 ppm (br, 8H); MS (ESI): m/z 513 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Methoxybiphenyl-2-yl)methyl)piperazin-1-yl)-

methyl)-1H-1,2,3-triazol-1-yl)methyl)phenylamino)acetic

acid

(C9): Prepared according to general procedure VIII from 8b and 9a

1

(62%): HPLC: 96.60%, t_R=2.530 min; H NMR (300 Hz, CDCl₃): d=

7.81 (s, 1H), 7.56 (s, 1H), 7.36–7.34 (m, 2H), 7.17 (d, J=8.1 Hz, 3H),

6.93 (d, J=8.1 Hz, 5H), 6.33 (s, 1H), 5.51 (s, 2H), 4.47 (s, 2H), 4.15

(s, 2H), 3.86 (s, 5H), 3.06–2.84 ppm (br, 8H); MS (ESI): m/z 525

[MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Nitrobiphenyl-2-yl)methyl)piperazin-1-yl)methyl)-

1H-1,2,3-triazol-1-yl)methyl)phenoxy)acetic acid (C2): Prepared

according to general procedure VIII from 8a and 9b (82%):

1

HPLC:100%, t_R=0.462 min; H NMR (300 Hz, CDCl₃): d=8.34 (d, J=

8.4 Hz, 2H), 7.74 (d, J=8.7 Hz, 2H), 7.62 (s, 2H), 7.48 (t, J=7.8 Hz,

2H), 7.02–6.83 (m, 5H), 5.56 (s, 2H), 4.62 (s, 1H), 4.48 (s, 2H), 4.30

(s, 1H), 3.87 (s, 2H), 3.18 (brs, 4H), 2.81 ppm (s, 4H); MS (ESI): m/z

541 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Chlorobiphenyl-3-yl)methyl)piperazin-1-yl)meth-

yl)-1H-1,2,3-triazol-1-yl)methyl)phenylamino)acetic acid (C10):

Prepared according to general procedure VIII from 8b and 9c

1

(65%): HPLC: 96.00%, t_R=0.693 min; H NMR (300 Hz, CDCl₃): d=

7.87 (s, 1H), 7.62 (d, J=8.4 Hz, 1H), 7.52 (d, J=9.0 Hz, 3H), 7.47–

7.37 (m, 5H), 7.31 (d, J=7.5 Hz, 2H), 6.33 (s, 1H), 5.51 (s, 2H), 4.49

(s, 2H), 4.22 (s, 2H), 4.01 (s, 1H), 3.69 (s, 2H), 3.13 (brs, 4H),

2.81 ppm (s, 4H); MS (ESI): m/z 529 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Chlorobiphenyl-3-yl)methyl)piperazin-1-yl)meth-

yl)-1H-1,2,3-triazol-1-yl)methyl)phenoxy)acetic acid (C3): Pre-

pared according to general procedure VIII from 8a and 9c (78%):

1

HPLC: 96.45%, t_R=3.512 min; H NMR (300 Hz, CDCl₃): d=7.93 (s,

1H), 7.52 (s, 2H), 7.38 (d, J=8.1 Hz, 4H), 7.22 (d, J=8.1 Hz, 3H),

6.94 (d, J=8.1 Hz, 2H), 6.34 (s, 1H), 5.52 (s, 2H), 4.48 (s, 2H), 4.23

(s, 2H), 3.70 (s, 2H), 3.22–2.99 (br, 4H), 2.94–2.82 ppm (br, 4H); MS

(ESI): m/z 530 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Methoxybiphenyl-3-yl)methyl)piperazin-1-yl)-

methyl)-1H-1,2,3-triazol-1-yl)methyl)phenylamino)acetic

acid

(C11): Prepared according to general procedure VIII from 8b and

9e (68%): HPLC: 96.58%, t_R=0.477 min; ¹H NMR (300 Hz, CDCl₃):

d=7.56–7.49 (m, 5H), 7.39 (t, J=7.8 Hz, 1H), 7.35 (s, 1H), 7.19 (d,

J=8.7 Hz, 1H), 6.98 (d, J=8.7 Hz, 3H), 6.92 (d, J=7.5 Hz, 1H), 6.22

(s, 1H), 5.53 (s, 2H), 4.81 (s, 1H), 4.44 (s, 2H), 4.00 (s, 2H), 3.91 (s,

2H), 3.85 (s, 3H), 2.83 (s, 4H), 2.60 ppm (s, 4H); MS (ESI): m/z 525

[MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Fluorobiphenyl-3-yl)methyl)piperazin-1-yl)meth-

yl)-1H-1,2,3-triazol-1-yl)methyl)phenoxy)acetic acid (C4): Pre-

pared according to general procedure VIII from 8a and 9d (72%):

HPLC: 96.83%, t_R=4.502 min; ¹H NMR (300 Hz, CDCl₃): d=7.55–

7.51 (m, 4H), 7.43–7.38 (m, 2H), 7.25–7.23 (m, 1H), 7.14 (t, J=

8.7 Hz, 3H), 7.00–6.91 (dd, J=11.1, 7.8 Hz, 2H), 6.24 (s, 1H), 5.53 (s,

2H), 4.43 (s, 2H), 4.02 (s, 2H), 3.90 (s, 2H), 2.87–2.82 (br, 4H), 2.82–

2.71 ppm (br, 4H); MS (ESI): m/z 514 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Nitrobiphenyl-3-yl)methyl)piperazin-1-yl)methyl)-

1H-1,2,3-triazol-1-yl)methyl)phenylamino)acetic acid (C12): Pre-

pared according to general procedure VIII from 8b and 9 f (68%):

1

HPLC: 97.13%, t_R=0.450 min; H NMR (300 Hz, CDCl₃): d=8.33 (d,

2-(3-((4-((4-((4’-Methoxybiphenyl-3-yl)methyl)piperazin-1-yl)-

methyl)-1H-1,2,3-triazol-1-yl)methyl)phenoxy)acetic acid (C5):

Prepared according to general procedure VIII from 8a and 9e

J=8.4 Hz, 2H), 7.74 (d, J=8.7 Hz, 2H), 7.63 (s, 3H), 7.48 (t, J=

8.1 Hz, 1H), 7.33 (t, J=7.8 Hz, 3H), 6.97 (d, J=8.4 Hz, 2H), 5.56 (s,

2H), 4.47 (s, 2H), 4.30 (s, 1H), 4.21 (s, 2H), 3.87 (s, 2H), 3.06 (s, 4H),

2.84 ppm (s, 4H); MS (ESI): m/z 540 [MꢁH]^ꢁ.

1

(74%): HPLC: 97.33%, t_R=2.533 min; H NMR (300 Hz, CDCl₃): d=

7.56–7.49 (m, 5H), 7.40 (d, J=7.8 Hz, 1H), 7.35 (s, 1H), 7.19 (d, J=

7.5 Hz, 1H), 6.98 (d, J=8.7 Hz, 3H), 6.92 (d, J=7.5 Hz, 1H), 6.22 (s,

1H), 5.53 (s, 2H), 4.75 (brs, 4H), 4.44 (s, 2H), 4.00 (s, 2H), 3.91 (s,

2H), 3.85 (s, 3H), 2.66 ppm (brs, 4H); MS (ESI): m/z 526 [MꢁH]^ꢁ.

General procedure IX for the synthesis of D1–D26: 1-(4-{[2-(4-

Chlorophenyl)phenyl]methyl}piperazin-1-yl)-3-(4-methoxyphe-

nyl)propan-1-one (D1): HATU (1.5 mmol) and DIPEA (1.5 mmol)

was added to a solution of 3-(4-methoxyphenyl)propanoic acid

(1.0 mmol) in dry DMF (10 mL). After stirring at ꢁ108C for 30 min,

2h (0.8 mmol) was added, and the reaction mixture was stirred at

ꢁ108C for 2 h and then warmed to RT and stirred overnight. The

solvent was evaporated in vacuo and the mixture was diluted with

H₂O (100 mL) and extracted with CH₂Cl₂. The organic phases were

washed with saturated NaHCO₃and brine, dried over Na₂SO₄, fil-

tered and concentrated in vacuo. The crude compound was puri-

fied by chromatography with CH₂Cl₂/CH₃OH (30:1 v/v) to afford D1

as a yellow oil (64%): HPLC: 97.89%, t_R=3.793 min; ¹H NMR

(400 MHz, CDCl₃): d=7.46 (d, J=6.0 Hz, 1H), 7.37 (m, 1H), 7.34–

7.36 (m, 2H), 7.33 (t, J=3.2 Hz, 2H), 7.31 (t, J=4.0 Hz,1H), 7.22–

7.24 (m, 1H), 7.11 (d, J=8.8 Hz, 2H), 6.81 (d, J=8.8 Hz, 2H), 3.77 (s,

3H), 3.55 (s, 2H), 3.36 (s, 2H), 3.31 (t, J=4.4 Hz, 2H), 2.88 (t, J=

7.2 Hz, 2H), 2.55 (t, J=8.0 Hz, 2H), 2.29 (t, J=4.8 Hz, 2H), 2.22 ppm

(s, 2H); MS (ESI): m/z 449 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd

for C₂₇H₂₉ClN₂O₂: 449.1918, observed: 449.2013.

2-(3-((4-((4-((4’-Nitrobiphenyl-3-yl)methyl)piperazin-1-yl)methyl)-

1H-1,2,3-triazol-1-yl)methyl)phenoxy)acetic acid (C6): Prepared

according to general procedure VIII from 8a and 9 f (75%): HPLC:

99.08%, t_R=0.489 min; ¹H NMR (300 Hz, CDCl₃): d=8.32 (d, J=

8.4 Hz, 2H), 7.73 (d, J=8.7 Hz, 2H), 7.62 (s, 3H), 7.48 (t, J=8.1 Hz,

1H), 7.33 (t, J=8.7 Hz, 3H), 6.97 (d, J=8.7 Hz, 2H), 5.57 (s, 2H),

4.47 (s, 2H), 4.22 (s, 2H), 3.87 (s, 2H), 3.06 ppm (brs, 8H); MS (ESI):

m/z 541 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)meth-

yl)-1H-1,2,3-triazol-1-yl)methyl)phenylamino)acetic acid (C7): Pre-

pared according to general procedure VIII from 8b and 9g (69%):

1

HPLC: 97.88%, t_R=0.465 min; H NMR (300 Hz, CDCl₃): d=7.93 (s,

1H), 7.52 (s, 1H), 7.38 (d, J=8.1 Hz, 5H), 7.22 (d, J=8.4 Hz, 3H),

6.93 (d, J=7.5 Hz, 2H), 6.34 (s, 1H), 5.52 (s, 2H), 4.48 (s, 2H), 4.23

(s, 2H), 3.98 (s, 1H), 3.70 (s, 2H), 2.97 (s, 4H), 2.72 ppm (s, 4H); MS

(ESI): m/z 529 [MꢁH]^ꢁ.

2-(3-((4-((4-((4’-Fluorobiphenyl-2-yl)methyl)piperazin-1-yl)meth-

yl)-1H-1,2,3-triazol-1-yl)methyl)phenylamino)acetic acid (C8): Pre-

pared according to general procedure VIII from 8b and 9h (66%):

1-(4-{[2-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-3-(3,4,5-

trimethoxyphenyl)propan-1-one (D2): Prepared according to gen-

eral procedure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid

1

HPLC: 99.32%, t_R=3.131 min; H NMR (300 Hz, CDCl₃): d=7.70 (s,

(70%): HPLC:97.10%, t_R=2.307 min; H NMR (400 MHz, CDCl₃): d=

1H), 7.55 (d, J=7.5 Hz, 4H), 7.48–7.28 (m, 5H), 6.96 (t, J=7.2 Hz,

7.38–7.48 (m, 1H), 7.25–7.38 (m, 6H), 7.23–7.24 (m, 1H), 6.78 (d,

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J=8.8 Hz, 2H), 3.86 (s, 6H), 3.85 (s, 3H), 3.56 (t, J=4.8 Hz, 2H), 3.37

(s, 2H), 3.32 (t, J=5.4 Hz, 2H), 2.90 (t, J=7.5 Hz, 2H), 2.57 (t, J=

7.2 Hz, 2H), 2.30 (t, J=5.1 Hz, 2H), 2.24 ppm (t, J=4.8 Hz, 2H); MS

(ESI): m/z 509 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₂₉H₃₃ClN₂O₄: 509.2129, observed: 509.2226.

2H), 3.56 (s, 2H), 3.41 (t, J=4.8 Hz, 2H), 2.91 (t, J=7.2 Hz, 2H), 2.61

(t, J=8.4 Hz, 2H), 2.44 (t, J=4.8 Hz, 2H), 2.35 ppm (t, J=4.8 Hz,

2H); MS (ESI): m/z 475 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₂₉H₃₄N₂O₄: 475.2519, observed: 475.2595.

1-(4-{[3-(4-Fluorophenyl)phenyl]methyl}piperazin-1-yl)-3-(3,4,5-

trimethoxyphenyl)propan-1-one (D9): Prepared according to gen-

eral procedure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid and

1-(4-{[2-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-2-(3,4,5-

trimethoxyphenyl)ethan-1-one (D3): Prepared according to gener-

al procedure IX from 2-(3,4,5-trimethoxyphenyl)acetic acid (62%):

1

2p (72%): HPLC: 97.27%, t_R=1.482 min; H NMR (400 MHz, CDCl₃):

1

HPLC: 97.09%, t_R=1.879 min; H NMR (400 MHz, CDCl₃): d=7.46–

d=7.56 (t, J=2.8 Hz, 1H), 7.54 (t, J=2.8 Hz, 1H), 7.50 (s, 1H), 7.46

(dt, J=1.2, 7.6 Hz, 1H), 7.39 (t, J=8.4 Hz, 1H), 7.28 (t, J=7.6 Hz,

1H), 7.12 (t, J=8.8 Hz, 2H), 6.43 (s, 2H), 3.84 (s, 6H), 3.82 (s, 3H),

3.67 (s, 2H), 3.57 (s, 2H), 3.42 (t, J=4.8 Hz, 2H), 2.91 (t, J=7.2 Hz,

2H), 2.61 (t, J=8.4 Hz, 2H), 2.46 (t, J=4.8 Hz, 2H), 2.36 ppm (s,

2H); MS (ESI): m/z 493 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₂₉H₃₃FN₂O₄: 493.2424, observed: 493.2476.

7.43 (m, 1H), 7.38–7.34 (m, 3H), 7.33–7.28 (m, 3H), 7.23–7.20 (m,

1H), 6.42 (s, 2H), 3.83 (s, 3H), 3.82 (s, 6H), 3.64 (s, 2H), 3.58 (t, J=

5.6 Hz, 2H), 3.40–3.38 (br, 2H), 3.36 (s, 2H), 2.31 (t, J=5.2 Hz, 2H),

2.20 ppm (t, J=5.2 Hz, 2H); MS (ESI): m/z 495 [M+H]⁺; HRMS (ESI):

m/z [M+H]⁺calcd for C₂₈H₃₁ClN₂O₄: 495.1917, observed: 495.2023.

1-(4-{[2-(4-Fluorophenyl)phenyl]methyl}piperazin-1-yl)-3-(3,4,5-

trimethoxyphenyl)propan-1-one (D4): Prepared according to gen-

eral procedure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid and

1-(4-{[3-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-3-(3,4,5-

trimethoxyphenyl)propan-1-one (D10): Prepared according to

general procedure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid

and 2o (61%): HPLC: 98.48%, t_R=2.122 min; ¹H NMR (400 MHz,

CDCl₃): d=7.53 (m, 1H), 7.51 (m, 2H), 7.46 (d, J=7.6 Hz, 1H), 7.41

(s, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 7.30 (d, J=7.6 Hz, 1H), 6.42 (s,

2H), 3.84 (s, 6H), 3.82 (s, 3H), 3.66 (s, 2H), 3.55 (s, 2H), 3.41 (t, J=

4.8 Hz, 2H), 2.91 (t, J=7.2 Hz, 2H), 2.61 (t, J=8.0 Hz, 2H), 2.44 (t,

J=4.8 Hz, 2H), 2.35 ppm (t, J=4.8 Hz, 2H); MS (ESI): m/z 509 [M+

H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₂₉H₃₃ClN₂O₄: 509.2129, ob-

served: 509.2238.

1

2s (70%): HPLC: 98.71%, t_R=1.637 min; H NMR (400 MHz, CDCl₃):

d=7.46 (s, 1H), 7.31–7.35 (m,4H), 7.22–7.24 (m, 1H), 7.07 (t, J=

8.8 Hz, 2H), 6.41 (s, 2H), 3.83 (s, 6H), 3.80 (s, 3H), 3.56 (s, 2H), 3.35

(m, 4H), 2.88 (t, J=7.2 Hz, 2H), 2.57 (t, J=8.4 Hz, 2H), 2.29 (s, 2H),

2.24 ppm (s, 2H); MS (ESI): m/z 493 [M+H]⁺; HRMS (ESI): m/z [M+

H]⁺calcd for C₂₉H₃₃FN₂O₄: 493.2424, observed: 493.2494.

1-(4-{[2-(3-Fluorophenyl)phenyl]methyl}piperazin-1-yl)-3-(3,4,5-

trimethoxyphenyl)propan-1-one (D5): Prepared according to gen-

eral procedure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid and

1

2t (65%): HPLC: 97.18%, t_R=1.711 min; H NMR (400 MHz, CDCl₃):

3-(3,4-Dihydroxyphenyl)-1-{4-[(3-phenylphenyl)methyl]piperazin-

1-yl}propan-1-one (D11): Prepared according to general procedur-

e IX from 3-(3,4-dihydroxyphenyl)propanoic acid and 2b (63%):

HPLC: 98.38%, t_R=1.010 min; ¹H NMR (400 MHz, CDCl₃): d=7.60

(m, 1H), 7.57 (m, 1H), 7.53 (s, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.44 (t,

J=7.2 Hz, 2H), 7.39 (t, J=7.6 Hz, 1H), 7.34 (tt, J=1.2, 7.6 Hz, 1H),

7.28 (d, J=7.6 Hz, 1H), 6.76 (d, J=8.0 Hz, 1H), 6.73 (d, J=1.6 Hz,

1H), 6.55 (dd, J=1.6, 8.0 Hz, 1H), 3.63 (s, 2H), 3.55 (s, 2H), 3.37 (t,

J=4.4 Hz, 2H), 2.82 (t, J=7.6 Hz, 2H), 2.59 (t, J=7.2 Hz, 2H), 2.44

(s, 2H), 2.26 (s, 2H), 2.18 ppm (s, 1H); MS (ESI): m/z 417 [M+H]⁺;

HRMS (ESI): m/z [M+H]⁺calcd for C₂₆H₂₈N₂O₃: 417.2100, observed:

417.2173.

d=7.47 (s, 1H), 7.33–7.38 (m,3H), 7.25–7.27 (m, 1H), 7.19 (m, 1H),

7.13 (dt, J=1.2, 7.2 Hz, 1H), 7.03–7.07 (m,1H), 6.41 (s, 2H), 3.84 (s,

6H), 3.81 (s, 3H), 3.58 (s, 2H), 3.38 (d, J=3.6 Hz, 4H), 2.89 (t, J=

6.8 Hz, 2H), 2.58 (t, J=8.4 Hz, 2H), 2.32 (s, 2H), 2.27 ppm (s, 2H);

MS (ESI): m/z 493 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for

C₂₉H₃₃FN₂O₄: 493.2424, observed: 493.2470.

1-(4-{[2-(3-Chloro-4-fluorophenyl)phenyl]methyl}piperazin-1-yl)-

3-(3,4,5-trimethoxyphenyl)propan-1-one (D6): Prepared accord-

ing to general procedure IX from 3-(3,4,5-trimethoxyphyl)propanoic

acid and 2e (62%): HPLC: 96.99%, t_R=2.493 min; ¹H NMR

(400 MHz, CDCl₃): d=7.69 (dd, J=2.0, 7.2 Hz, 1H), 7.38 (s,1H),

7.33–7.35 (m, 2H), 7.22–7.25 (m, 2H), 7.17 (t, J=8.8 Hz, 1H), 6.43 (s,

2H), 3.85 (s, 6H), 3.82 (s, 3H), 3.60 (s, 2H), 3.38 (t, J=4.0 Hz, 2H),

3.32 (s, 2H), 2.90 (t, J=7.6 Hz, 2H), 2.60 (t, J=8.4 Hz, 2H), 2.35 (s,

2H), 2.29 ppm (s, 2H); MS (ESI): m/z 527 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₂₉H₃₂ClFN₂O₄: 527.2035, observed: 527.2108.

3-(3,4-Dimethoxyphenyl)-1-{4-[(3-phenylphenyl)methyl]pipera-

zin-1-yl}propan-1-one (D12): Prepared according to general proce-

dure IX from 3-(3,4-dimethoxyphenyl)propanoic acid and 2b

1

(72%): HPLC: 98.51%, t_R=1.548 min; H NMR (400 MHz, CDCl₃): d=

7.60 (m, 1H), 7.58–7.59 (m, 1H), 7.54 (m, 1H), 7.49–7.52 (dt, J=1.2,

8.8 Hz, 1H), 7.43–7.46 (m, 2H), 7.40 (m, 1H), 7.33–7.37 (m, 1H),

7.29 (d, J=7.6 Hz, 1H), 6.79 (d, J=8.8 Hz, 1H), 6.74–6.76 (m, 2H),

3.86 (s, 3H), 3.85 (s, 3H), 3.65 (t, J=4.4 Hz, 2H), 3.55 (s, 2H), 3.39 (t,

J=5.2 Hz, 2H), 2.92 (t, J=7.2 Hz, 2H), 2.60 (t, J=8.0 Hz, 2H), 2.44

(t, J=4.8 Hz, 2H), 2.34 ppm (t, J=5.2 Hz, 2H); MS (ESI): m/z 445

[M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₂₈H₃₂N₂O₃: 445.2413,

observed: 445.2470.

1-{4-[(2-Phenylphenyl)methyl]piperazin-1-yl}-3-(3,4,5-trimethoxy-

phenyl)propan-1-one (D7): Prepared according to general proce-

dure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid and 2a (68%):

HPLC: 98.83%, t_R=1.664 min; ¹H NMR (400 MHz, CDCl₃): d=7.52

(m, 1H), 7.39 (m,1H), 7.38 (m, 1H), 7.34–7.35 (m, 3H), 7.31–7.33 (m,

2H), 7.25–7.27 (m, 1H), 6.41 (s, 2H), 3.83 (s, 6H), 3.80 (s, 3H), 3.66

(s, 2H), 3.43 (s, 2H), 3.34 (t, J=4.4 Hz, 2H), 2.88 (t, J=7.2 Hz, 2H),

2.57 (t, J=8.0 Hz, 2H), 2.30 (t, J=4.8 Hz, 2H), 2.25 ppm (t, J=

4.8 Hz, 2H); MS (ESI): m/z 475 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺

calcd for C₂₉H₃₄N₂O₄: 475.2519, observed: 475.2579.

4-Oxo-4-{4-[(3-phenylphenyl)methyl]piperazin-1-yl}-N-(3,4,5-tri-

methoxyphenyl)butanamide (D13): Prepared according to general

procedure IX from 10c and 3,4,5-trimethoxyaniline (78%): HPLC:

1

1-{4-[(3-Phenylphenyl)methyl]piperazin-1-yl}-3-(3,4,5-trimethoxy-

phenyl)propan-1-one (D8): Prepared according to general proce-

dure IX from 3-(3,4,5-trimethoxyphyl)propanoic acid and 2b (78%):

HPLC: 98.37%, t_R=1.515 min; ¹H NMR (400 MHz, CDCl₃): d=7.61

(m, 1H), 7.59 (m, 1H), 7.54 (m, 1H), 7.50 (d, J=7.6 Hz, 1H), 7.44 (t,

J=7.2 Hz, 2H), 7.40 (t, J=7.6 Hz, 1H), 7.35 (m, 1H), 7.28 (d, J=

7.6 Hz, 1H),6.43 (s, 2H), 3.84 (s, 6H), 3.82 (s, 3H), 3.66 (t, J=4.8 Hz,

96.44%, t_R=1.268 min; H NMR (400 MHz, CDCl₃): d=8.58 (s, 1H),

7.61 (s, 1H), 7.58 (s, 1H), 7.54 (s, 1H), 7.50 (d, J=7.6 Hz, 1H), 7.45

(m, 2H), 7.40 (d, J=6.0 Hz, 1H), 7.37 (d, J=7.6 Hz, 1H), 7.27 (d, J=

6.0 Hz, 1H), 6.83 (s, 2H), 3.82 (s, 6H), 3.78 (s, 3H), 3.66 (s, 2H), 3.58

(s, 2H), 3.51 (t, J=6.4 Hz, 2H), 2.72 (d, J=4.6 Hz, 4H), 2.47 ppm

(brs, 4H); MS (ESI): m/z 518 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺

calcd for C₃₀H₃₅N₃O₅: 518.2577, observed: 518.2630.

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4-(4-{[3-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-4-oxo-N-

(3,4,5-trimethoxyphenyl)butanamide (D14): Prepared according

to general procedure IX from 10b and 3,4,5-trimethoxyaniline

2.45 (m, 8H), 1.97–2.04 ppm (m, 2H); MS (ESI): m/z 472 [M+H]⁺;

HRMS (ESI): m/z [M+H]⁺calcd for C₂₉H₃₃N₃O₃: 472.2522, observed:

472.2584.

1

(63%): HPLC: 100%, t_R=1.728 min; H NMR (400 MHz, CDCl₃): d=

N-(3-Hydroxyphenyl)-4-oxo-4-{4-[(3-phenylphenyl)methyl]pipera-

zin-1-yl}butanamide (D20): Prepared according to general proce-

dure IX from 10c and 3-hydroxyaniline (62%): HPLC: 97.38%, t_R=

9.06 (s, 1H), 7.52–7.53 (m, 1H), 7.50–7.51 (m, 2H), 7.45–7.47 (d, J=

7.6 Hz, 1H), 7.41 (m, 2H), 7.37–7.39 (m, 1H), 7.29–7.31 (d, J=

7.6 Hz, 1H), 6.83 (s, 2H), 3.77 (s, 6H), 3.76 (s, 3H), 3.64 (s, 2H), 3.56

(s, 2H), 3.53 (t, J=4.8 Hz, 2H), 2.74–2.76 (m, 4H), 2.47 (t, J=4.8 Hz,

2H), 2.42 ppm (t, J=4.8 Hz, 2H); MS (ESI): m/z 552 [M+H]⁺; HRMS

(ESI): m/z [M+H]⁺calcd for C₃₀H₃₄ClN₃O₅: 552.2187, observed:

552.2278.

1

1.030 min; H NMR (400 MHz, CDCl₃): d=9.83 (s, 1H), 9.35 (s, 1H),

7.66 (d, J=7.6 Hz, 2H), 7.57 (d, J=9.6 Hz, 2H), 7.47 (m, 2H), 7.42

(d, J=7.6 Hz, 1H), 7.38 (d, J=7.2 Hz, 1H), 7.32 (d, J=7.2 Hz, 1H),

7.19 (s, 1H), 7.04 (t, J=8.1 Hz, 1H), 6.95 (d, J=8.0 Hz, 1H), 6.42 (dd,

J=1.2, 7.6 Hz, 1H), 3.56 (s, 2H), 3.46 (d, J=3.6 Hz, 4H), 2.60 (d, J=

5.2 Hz, 2H), 2.52 (m, 2H), 2.40 (s, 2H), 2.34 ppm (s, 2H); MS (ESI):

m/z 444 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₂₇H₂₉ClN₃O₃:

444.2209, observed: 444.2294.

4-(4-{[3-(4-Fluorophenyl)phenyl]methyl}piperazin-1-yl)-4-oxo-N-

(3,4,5-trimethoxyphenyl)butanamide (D15): Prepared according

to general procedure IX from 10a and 3,4,5-trimethoxyaniline

1

(78%): HPLC: 100%, t_R=1.237 min; H NMR (400 MHz, CDCl₃): d=

N-[(3,5-Dimethoxyphenyl)methyl]-4-oxo-4-{4-[(3-phenylphenyl)-

methyl]piperazin-1-yl}butanamide (D21): Prepared according to

general procedure IX from 10c and (3,5-dimethoxyphehyl)methan-

amine (71%): HPLC: 99.01%, t_R=1.505 min; ¹H NMR (400 MHz,

CDCl₃): d=7.61 (s, 1H), 7.59 (s, 1H), 7.54 (s, 1H), 7.51 (d, J=8.0 Hz,

1H), 7.44 (m,2H), 7.41 (d, J=6.4 Hz, 1H), 7.36 (d, J=7.6 Hz, 1H),

7.29 (d, J=7.6 Hz, 1H), 6.81 (t, J=7.2 Hz, 1H), 6.41 (d, J=2.0 Hz,

2H), 6.32 (t, J=2.8 Hz, 1H), 4.35 (d, J=6.0 Hz, 2H), 3.74 (s, 6H),

3.52–3.56 (m, 4H), 3.47 (t, J=6.8 Hz, 2H), 2.66 (t, J=8.4 Hz, 2H),

2.56 (t, J=7.6 Hz, 2H), 2.44 (t, J=6.8 Hz, 2H), 2.38 ppm (t, J=

6.4 Hz, 2H); MS (ESI): m/z 502 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺

calcd for C₃₀H₃₅N₃O₄: 502.2628, observed: 502.2734.

8.83 (s, 1H), 7.52–7.55 (m, 2H), 7.48 (s, 1H), 7.45 (dt, J=1.2, 7.6 Hz,

1H), 7.38 (t, J=7.6 Hz, 1H), 7.27 (d, J=7.6 Hz, 1H), 7.11 (t, J=

8.8 Hz, 2H), 6.81 (s, 2H), 3.70 (s, 6H), 3.76 (s, 3H), 3.64(s, 2H), 3.56

(s, 2H), 3.52 (t, J=4.8 Hz, 2H), 2.73 (m, 4H), 2.48 (s, 2H), 2.43 ppm

(t, J=4.8 Hz, 2H); MS (ESI): m/z 536 [M+H]⁺; HRMS (ESI): m/z [M+

Na]⁺calcd for C₃₀H₃₄FN₃O₅: 558.2482, observed: 558.2343.

5-Oxo-5-{4-[(3-phenylphenyl)methyl]piperazin-1-yl}-N-(3,4,5-tri-

methoxyphenyl)pentanamide (D16): Prepared according to gen-

eral procedure IX from 10d and 3,4,5-trimethoxyaniline (76%):

1

HPLC: 99.49%, t_R=1.338 min; H NMR (400 MHz, CDCl₃): d=8.39 (s,

1H), 7.63 (d, J=1.6 Hz, 1H), 7.59 (m, 1H), 7.55 (m, 1H), 7.51–7.53

(m, 1H), 7.43–7.47 (m, 2H), 7.41 (t, J=7.6 Hz, 1H), 7.34–7.38 (m,

1H), 7.30 (d, J=7.6 Hz, 1H), 6.89 (s, 2H), 3.84 (s, 6H), 3.81 (s, 3H),

3.67 (s, 2H), 3.60 (s, 2H), 3.51 (t, J=5.2 Hz, 2H), 2.43–2.51 (m, 8H),

1.99–2.06 ppm (m, 2H); MS (ESI): m/z 532 [M+H]⁺; HRMS (ESI):

m/z [M+H]⁺calcd for C₃₁H₃₇N₃O₅: 532.2733, observed: 532.2834.

N-[(2-Methoxyphenyl)methyl]-4-oxo-4-{4-[(3-phenylphenyl)me-

thyl]piperazin-1-yl}butanamide (D22): Prepared according to gen-

eral procedure IX from 10c and (2-methoxyphehyl)methanamine

1

(64%): HPLC: 98.63%, t_R=1.616 min; H NMR (400 MHz, CDCl₃): d=

7.61 (m, 1H), 7.58 (m, 1H), 7.54 (s, 1H), 7.50 (dt, J=1.2, 7.6 Hz, 1H),

7.43 (td, J=1.6, 7.2 Hz, 2H), 7.39 (t, J=7.6 Hz, 1H), 7.34 (tt, J=1.2,

7.2 Hz, 1H), 7.29 (d, J=7.6 Hz, 1H), 7.23 (m, 2H), 6.89 (td, J=0.8,

7.6 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.56 (s, 1H), 4.41 (d, J=6.0 Hz,

2H), 3.82 (s, 3H), 3.58 (t, J=4.8 Hz, 2H), 3.56 (s, 2H), 3.46 (t, J=

4.8 Hz, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.52 (t, J=6.8 Hz, 2H), 2.44 (t,

J=4.8 Hz, 2H), 2.40 ppm (t, J=5.2 Hz, 2H); MS (ESI): m/z 472 [M+

H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₂₉H₃₃N₃O₃: 472.2522, ob-

served: 472.2568.

5-(4-{[3-(4-Chlorophenyl)phenyl]methyl}piperazin-1-yl)-5-oxo-N-

(3,4,5-trimethoxyphenyl)pentanamide (D17): Prepared according

to general procedure IX from 10e and 3,4,5-trimethoxyaniline

1

(52%): HPLC: 99.10%, t_R=1.810 min; H NMR (400 MHz, CDCl₃): d=

8.52 (s, 1H), 7.53 (m, 1H), 7.51 (m, 2H), 7.46–7.48 (m, 1H), 7.41 (m,

2H), 7.38–7.40 (m, 1H), 7.30–7.32 (d, J=7.6 Hz, 1H), 6.90 (s, 2H),

3.83 (s, 6H), 3.80 (s, 3H), 3.66 (s, 2H), 3.58 (s, 2H), 3.51 (t, J=4.8 Hz,

2H), 2.43–2.50 (m, 8H), 1.99–2.06 ppm (m, 2H); MS (ESI): m/z 566

[M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₁H₃₆ClN₃O₅:

566.2343, observed: 566.2427.

N-[(2,5-Dimethoxyphenyl)methyl]-5-oxo-5-{4-[(3-phenylphenyl)-

methyl]piperazin-1-yl}pentanamide (D23): Prepared according to

general procedure IX from 10d and (2,5-dimethoxyphehyl)methan-

amine (62%): HPLC: 99.01%, t_R=1.574 min; ¹H NMR (400 MHz,

CDCl₃): d=7.61 (m, 1H), 7.58 (m, 1H), 7.54 (s, 1H), 7.50 (dt, J=1.2,

7.6 Hz, 1H), 7.43 (t, J=7.2 Hz, 2H), 7.39 (m, 1H), 7.32–7.36 (m, 1H),

7.29 (d, J=7.6 Hz, 1H), 6.83 (d, J=2.8 Hz, 1H), 6.71–6.76 m, 2H),

6.36 (s, 1H), 4.38 (d, J=5.6 Hz, 2H), 3.77 (s, 3H), 3.71 (s, 3H), 3.59

(t, J=4.8 Hz, 2H), 3.56 (s, 2H), 3.42 (t, J=4.8 Hz, 2H), 2.41 (t, J=

4.8 Hz, 4H), 2.35 (t, J=7.2 Hz, 2H), 2.26 (t, J=7.2 Hz, 2H), 1.90–

1.97 ppm (m, 2H); MS (ESI): m/z 516 [M+H]⁺; HRMS (ESI): m/z

[M+H]⁺calcd for C₃₁H₃₇N₃O₄: 516.2784, observed: 516.2839.

N-(2,4-Dimethoxyphenyl)-5-oxo-5-{4-[(3-phenylphenyl)methyl]pi-

perazin-1-yl}pentanamide (D18): Prepared according to general

procedure IX from 10d and 2,4-dimethoxyaniline (64%): HPLC:

99.13%, t_R=1.650 min; ¹H NMR (400 MHz, CDCl₃): d=8.11 (d, J=

3.2 Hz, 1H), 7.92 (s, 1H), 7.61 (m, 1H), 7.59 (s, 1H), 7.54 (s, 1H), 7.50

(d, J=7.6 Hz, 1H), 7.44 (t, J=7.2 Hz, 2H), 7.39 (t, J=7.6 Hz, 1H),

7.32–7.36 (m, 1H), 7.29 (d, J=7.6 Hz, 1H), 6.77 (d, J=8.8, 1 Hz),

6.54–6.57 (dd, J=2.8, 8.8 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.64 (t,

J=4.4 Hz, 2H), 3.56 (s, 2H), 3.47 (t, J=5.2 Hz, 2H), 2.50 (t, J=

6.8 Hz, 2H), 2.42–2.45 (m, 6H), 2.01–2.08 ppm (m, 2H); MS (ESI):

m/z 502 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₀H₃₅N₃O₄:

502.2628, observed: 502.2711.

N-[(2-Methoxyphenyl)methyl]-5-oxo-5-{4-[(3-phenylphenyl)me-

thyl]piperazin-1-yl}pentanamide (D24): Prepared according to

general procedure IX from 10d and (2-methoxyphehyl)methana-

mine (75%): HPLC: 98.80%, t_R=1.631 min; ¹H NMR (400 MHz,

CDCl₃): d=7.61 (m, 1H), 7.58–7.59 (m, 1H), 7.54 (m, 1H), 7.49–7.51

(dt, J=2.8, 7.6 Hz, 1H), 7.44 (t, J=8.0 Hz, 2H), 7.39 (t, J=7.6 Hz,

1H), 7.32–7.37 (m, 1H), 7.28 (d, J=7.6 Hz, 1H), 7.23–7.25 (m, 1H),

7.20–7.22 (m, 1H), 6.86–6.90 (m, 1H), 6.83 (d, J=8.0 Hz, 1H), 6.32

(t, J=8.0 Hz, 1H), 4.42 (d, J=6.0 Hz, 2H), 3.82 (s, 3H), 3.59 (t, J=

4.8 Hz, 2H), 3.56 (s, 2H), 3.41 (t, J=4.8 Hz, 2H), 2.41 (t, J=4.8 Hz,

N-(3-Methoxyphenyl)-5-oxo-5-{4-[(3-phenylphenyl)methyl]piper-

azin-1-yl}pentanamide (D19): Prepared according to general pro-

cedure IX from 10d and 3-methoxyaniline (72%): HPLC: 100%, t_R=

1

1.644 min; H NMR (400 MHz, CDCl₃): d=8.74 (s, 1H), 7.58–7.60 (m,

2H), 7.53 (s, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.43 (t, 2H), 7.37 (t, J=

7.6 Hz, 1H), 7.32–7.36 (m, 2H), 7.28 (d, J=7.6 Hz, 1H), 7.16 (m, 1H),

7.04 (d, J=8.0 Hz, 1H), 6.61 (dd, J=2.4, 8.0 Hz, 1H), 3.75 (s, 3H),

3.63 (t, J=4.4 Hz, 2H), 3.55 (s, 2H), 3.46 (t, J=4.8 Hz, 2H), 2.42–

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4H), 2.34 (t, J=7.2 Hz, 2H), 2.25 (t, J=6.8 Hz, 2H), 1.89–1.96 ppm

(m, 2H); MS (ESI): m/z 486 [M+H]⁺; HRMS (ESI): m/z [M+H]⁺

calcd for C₃₀H₃₅N₃O₃: 486.2678, observed: 486.2785.

lel measurements was used to compute the inhibition ratio using

Equation (1).

FP_maxꢁ FP_test

ð1Þ

Inhibitionð%Þ ¼

FP_maxꢁ FP_NC

General procedure X for the synthesis of D25–D26: 2-({4-[(3-Phe-

nylphenyl)methyl]piperazin-1-yl}carbonyl)-N-(3,4,5-trimethoxy-

phenyl)benzamide (D25): Isobenzofuran-1,3-dione (1.1 mmol) was

added to a mixture of 2b (1.0 mmol) in CH₂Cl₂(3 mL). After heat-

ing to 408C overnight, the starting material disappeared as moni-

tored by TLC. The reaction mixture was concentrated in vacuo to

give crude compound 6. HATU (1.5 mmol) and DIPEA (1.5 mmol)

was added to a solution of 6 (1 mmol) in dry DMF. After stirring at

ꢁ108C for 30 min, 3,4,5-trimethoxyaniline (1.2 mmol) was added,

and the reaction mixture was stirred at ꢁ108C for 2 h and then

warmed to RT and stirred overnight. The solvent was evaporated in

vacuo and the mixture was diluted with H₂O (100 mL) and extract-

ed with CH₂Cl₂. The organic phases were washed with saturated

NaHCO₃and brine, dried over Na₂SO₄, filtered and concentrated in

vacuo. The crude compound was purified by chromatography with

CH₂Cl₂/CH₃OH (30:1 v/v) to afford D25 as a yellow oil (414 mg,

72%): HPLC: 100%, t_R=1.615 min; ¹H NMR (400 MHz, CDCl₃): d=

8.97 (s, 1H), 7.88 (d, J=5.6 Hz, 1H), 7.58 (s, 1H), 7.55 (s, 1H), 7.49–

7.50 (m, 2H), 7.48 (m,2H), 7.43 (m, 2H), 7.32–7.39 (m, 2H), 7.21–

7.23 (m, 2H), 6.96 (s, 2H), 3.88 (s, 6H), 3.83 (s, 3H), 3.47 (s, 2H),

3.23 (s, 2H), 2.35 (brs, 4H), 1.59 ppm (s, 2H); MS (ESI): m/z 566

[M+H]⁺; HRMS (ESI): m/z [M+H]⁺calcd for C₃₄H₃₅N₃O₅: 566.2577,

observed: 566.2650.

Here, FP_maxrefers to the FP value detected when the protein was

incubated with the Bid-BH3 peptide, FP_NCrefers to the FP value de-

tected from the negative control, that is, the Bid-BH3 peptide

alone, and FP_testrefers to the FP value detected when the test com-

pound was added at a certain concentration. If the test compound

showed obvious dose-dependent competitive binding to the

target protein and achieved an inhibition ratio over 50% at 50 mm,

it was further tested at seven different concentrations (i.e., 1 nm,

10 nm, 100 nm, 1 mm, 10 mm, 50 mm and 100 mm) to obtain a com-

plete dose-dependent binding curve. The binding curve was de-

rived through nonlinear fitting using GraphPad Prism software (ver-

sion 5). The concentration of the given compound at which 50%

of the bound peptide was displaced (IC₅₀) was derived from the

binding curve. The competitive inhibition constant (K_i) of the test

compound was calculated with a mathematical equation devel-

oped by Wang et al.^[50]assuming that it formed a binary complex

with the target protein.

Heteronuclear single quantum coherence (HSQC) NMR

The Bcl-x_Lprotein used for this purpose was a special truncated

construction of the full-length protein, with deletion of residues

45–84 on a long-loop region and residues 210–233 at the C termi-

nus. An 8ꢁHis tag was added to the N terminus. The correspond-

ing sequence was cloned into the pSJ2 vector (a modified vector

based on pET28a) at the EcoRI and XhoI sites, using the following

N-(2,4-Dimethoxyphenyl)-2-({4-[(3-phenylphenyl)methyl]pipera-

zin-1-yl}carbonyl)benzamide (D26): Prepared according to general

procedure X from 2,4-dimethoxyaniline (60%): HPLC: 100%, t_R=

2.192 min; ¹H NMR (400 MHz, CDCl₃): d=8.71 (s, 1H), 8.23 (d, J=

3.2 Hz, 1H), 7.78–7.81 (dd, J=1.6, 7.2 Hz, 1H), 7.56–7.59 (m, 2H),

7.51–7.54 (m, 2H), 7.48 (d, J=1.2 Hz, 2H), 7.43–7.46 (d, J=7.2 Hz,

2H), 7.41 (t, J=8.4 Hz, 1H), 7.31–7.36 (m, 3H), 6.82 (d, J=8.8 Hz,

1H), 6.61–6.65 (dd, J=3.2, 9.2 Hz, 1H), 3.84 (s, 3H), 3.83 (s, 3H),

3.47 (s, 2H), 3.25 (t, J=6.0 Hz, 2H), 2.37 (t, J=6.8 Hz, 4H),

1.57 ppm (s, 2H); MS (ESI): m/z 536 [M+H]⁺; HRMS (ESI): m/z [M+

H]⁺calcd for C₃₃H₃₃N₃O₄: 536.2471, observed: 536.2511.

oligonucleotides:

5’-TCTCGAATTCATGTCTCAGAGCAACCGGGA-3’

and 5’-GGTCCTCGAGTCAGCGTTCCTGGCCCTTTCG-3’. The protein

was expressed in E. coli BL21(DE3) cells. Cells were grown at 378C

in M9 medium containing ¹⁵N-NH₄Cl and ampicillin (1 mm) to an

OD₆₀₀0.6. Protein expression was induced by IPTG (0.4 mm) at

208C for 16 h. Cells were lysed in Tris-HCl (25 mm, pH 7.0) contain-

ing NaCl (100 mm) and PMSF (0.1 mgmL^ꢁ1). ¹⁵N-labeled His-TEV-

Bcl-x_Lprotein was purified from the soluble fraction using Ni-NTA

resin (Qiagen). The ¹⁵N-labeled His-TEV-Bcl-x_Lprotein was further

cleaved by TEV protease (0.5 mgmL^ꢁ1, 48C, 12 h) to obtain ¹⁵N-la-

beled Bcl-x_Lprotein. Then, the ¹⁵N-labeled Bcl-x_Lprotein was puri-

fied on Superdex75 (GE Healthcare) in phosphate-buffered saline

(PBS).

¹H NMR spectra of all target compounds, that is, A1–A26, B1–B17,

C1–C12, and D1–D26, are given in the Supporting Information.

Binding assay

¹⁵N-Heteronuclear single quantum correlation (HSQC) NMR spectra

were recorded on a Bruker DMX 600 MHz NMR spectrometer at

258C with samples containing 200–500 mm of the ¹⁵N-labeled pro-

tein. The ratio of each tested compound and the Bcl-x_Lprotein in

the sample was 1:1. The resulting NMR spectra were processed

with the nmrPipe software and analyzed with the Spark software

(version 3). The HSQC spectrum of free Bcl-x_L, which is publicly

available from the Biological Magnetic Resonance Data Bank

(http://www.bmrb.wisc.edu/, BMRB ID: 18250), was adopted as the

reference for assignment of chemical shifts.

A fluorescence polarization (FP)-based assay was employed in this

study to measure the binding affinities of small-molecule com-

pounds to three antiapoptotic Bcl-2 family proteins, including Mcl-

1, Bcl-x_L, and Bcl-2. A 26-residue peptide truncated from the BH3

domain of the Bid protein (QEDIIRNIARHLAQVGDSMDRSIPPG) was

modified by adding 5-carboxyfluorescein (5-FAM) on the N termi-

nus, which was used as the fluorescence tracer in this assay. In

each measurement, the target protein was incubated with the Bid-

BH3 peptide first, and then the competitive binding of the given

compound was characterized quantitatively by monitoring the

changes in FP signals upon the addition of the compound at dif-

ferent doses. This assay has been applied successfully in our previ-

ous studies on Bcl-2 family protein inhibitors.^[48,49]Details on the

preparation of the protein samples and experimental settings used

in FP measurement can be found in our recent publication.^[49]

In this study, each compound was initially screened at three con-

centrations (i.e., 1 mm, 10 mm, and 50 mm), against all three target

proteins (Mcl-1, Bcl-x_L, and Bcl-2). At each concentration, the aver-

age fluorescence polarization value (FP in mP units) of three paral-

In this study, compound D11 was selected to be tested using the

protocols described above. ABT-737, which is known to bind to the

BH3 binding groove on Bcl-x_L, was also tested as a reference.

Fragment-based design and molecular modeling

We recently developed a computational algorithm for mining the

so-called characteristic interaction patterns (CIPs) on protein–pro-

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tein binding interfaces.^[46]By definition, such a pattern is a cluster

of four interacting residues, which is conserved across different

protein–protein complexes. Our survey revealed that such patterns

are related to the “hot spot” regions with a significant probability.

Based on such information, small-molecule binders to a protein–

protein binding interface can be designed through a fragment-

based strategy. It can be done by finding protein-ligand complexes

sharing the same CIPs with the given target protein–protein com-

plex. For a pair of matched CIPs, the binding partner in the former

case (i.e., a chemical fragment) resembles the binding partner in

the latter case, that is, an amino acid residue (the “anchor”). If mul-

tiple CIPs exist on the given protein–protein binding interface, as-

sembling matched chemical fragments will yield complete ligand

molecules (Figure 2).

ments were assigned to these subsites according to their partner

residues in related hot-spot regions. Finally, four series of mole-

cules were manually designed by adding some synthetically feasi-

ble “linkers” to connect the selected fragments for each subsite

(Figure 4).

Molecular docking and molecular dynamics (MD) simulation were

employed to derive the possible binding modes to Bcl-x_Land Mcl-

1 for the active compounds described in this study. Several com-

pounds, including A1, A15, D11, and D12, were selected for this

purpose because their chemical structures are representative, and

they exhibit good binding affinities and interesting selectivities to

Mcl-1. Note that Bcl-2 has a very similar binding site to that of Bcl-

x_L, and thus extra modeling targeting Bcl-2 was not performed in

our study.

To achieve this goal, an extensive library of protein–ligand and pro-

tein–protein complex structures is needed. The Protein Data Bank

(PDB)^[47]is a good resource for this purpose. We downloaded the

entire PDB (a total of 78235 structures as released in January 2012)

from the RCSB web site (http://www.rcsb.org/), which includes

over 25000 valid protein–ligand complex structures. To reduce re-

dundancy, these protein–ligand complexes were clustered by se-

quence alignment of the protein molecules in them. The sequence

similarity cutoff used in clustering was set to 90%. Then, the

ligand molecule in each complex was dissected into fragments

with an in-house computer program. The rules in fragmentation

were as follows: (1) a substructure containing a single ring or fused

rings was defined as a fragment; (2) four continuous heavy atoms

on a chain were defined as a fragment; (3) any isolated atom or

group was merged into the nearest fragment. All of the resulting

fragments were further filtered by the “rules of three”, that is, mo-

lecular weightꢂ300, number of hydrogen-bond donorsꢂ3,

number of hydrogen-bond acceptorsꢂ3 and computed logP

valueꢂ3, to keep the valid ones. At the next stage, the pattern

mining algorithm developed by us was applied to all remaining

fragments in the protein–ligand complex library to detect their in-

teracting residues on the protein side. If the interacting residues

found for a certain fragment match a certain CIP on the given pro-

tein–protein binding interface, that fragment was retrieved as

a candidate.

Molecular docking was employed first to derive a rough binding

mode of the selected compounds to Bcl-x_Land Mcl-1. The complex

structure formed by human Bcl-x_Land ABT-737 (PDBID: 2YXJ) and

the complex structure formed between human Mcl-1 and the Bim-

BH3 peptide (PDBID: 2PQK) was used in this task. The molecular

structures of compound A1, A15, D11, and D12 were sketched

with the SYBYL software (version 8.1), and optimized with the

MMFF94 force field. Automatic docking of each molecule to Bcl-x_L

or Mcl-1 was performed by using the GOLD software (version 5.1,

released by Cambridge Crystallographic Data Centre). In each case,

a rough binding pose given by manual docking was provided as

the input. The binding site considered in docking for Bcl-x_Lprotein

was defined as all amino acid residues within 10 ꢂ from the refer-

ence ligand ABT-737. For Mcl-1 protein, the binding site was de-

fined as the amino acid residues within 10 ꢂ from Thr266 in the

binding pocket. The GOLD software employs a genetic algorithm

(GA) to conduct the docking process. In our study, the key parame-

ters for docking were as follows: the whole population was placed

on five separate islands with 100 individuals on each island; total

number of GA operations=100000; probabilities for crossover,

mutation and migration operations=95, 95 and 10, respectively;

scoring function=ChemScore. A total of 50 final binding poses

were generated for input molecule. These binding poses were fur-

ther clustered with a RMSD cutoff of 2.0 ꢂ using the “rms_analysis”

tool included in the GOLD software package. Among the binding

poses with the highest binding score in each cluster, one reasona-

ble binding pose was selected after visual examination. This selec-

tion was made also by referring to the known binding mode of

ABT-737, since the ¹⁵N-HSQC NMR spectrum of compound D11

suggested that our compounds adopt a binding mode similar to

ABT-737.

Bcl-2 family proteins execute their biological functions in apoptosis

through binding to BH3-only proteins. In order to identify the CIPs

on their binding interfaces, a number of complex structures

formed between Bcl-x_Lor Mcl-1 protein and BH3-only peptides

were analyzed. As for the complexes formed between Bcl-x_Land

BH3-peptides, PDBIDs 1PQ1, 2BZW, 2P1L, 2XA0, 2YJ1, 2YQ6, 2YQ7,

3FDL, 3IO8, 3P17, and 3R85 were considered; whereas for the com-

plexes formed between Mcl-1 and BH3-peptides, PDBIDs 2NL9,

2PQK, 3D7V, 3IO9, 3KJ0, 3KJ1, 3KJ2, 3KZ0, 3MK8, 3PK1, and 4G35

were considered. All of the CIPs identified on the protein–protein

binding interfaces in these complexes were used as queries to

search our library of protein–ligand complexes. All fragment-resi-

due interaction patterns included in this library were compared to

the CIP queries with computer programs, by which they were su-

perimposed with the CIP queries in pairs, and the root-mean-

square deviation (RMSD) values between them were calculated.

The hits for each query were sorted by their RMSD values in an as-

cending order. The top 30 hits for each query were examined visu-

ally. If a chemical fragment matched in terms of both spatial and

chemical properties with the “anchor” residue in the given CIP

query, it was considered as an appropriate candidate. Because we

divided the binding groove on Bcl-x_Lor Mcl-1 into three subsites,

that is, P-site, Q-site, and L-site (Figure 3), those candidate frag-

Binding modes of the selected compounds to Bcl-x_Land Mcl-1 pro-

teins produced by molecular docking were further evaluated by

MD simulations using the AMBER program (version 9, released by

University of California San Francisco, USA). Each MD simulation

was performed in explicit water for 3 ns. To set up each job, the

force field parameters applied to the small-molecule ligand were

prepared by applying the Antechamber module in AMBER. Atomic

partial charges on the small-molecule ligand were derived with the

RESP method based on the HF/6-31G* computation results given

by the Gaussian software (version 09, released by the Gaussian

Inc.). Atoms on the protein were assigned the PARM99 template

charges implemented in AMBER, and all ionizable residues were

set at the default protonation states at a neutral pH. The complex

structure was soaked in a box of TIP3P water molecules with

a margin of 10 ꢂ along each dimension. An appropriate number of

counter ions were added to neutralize the whole system.

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After these preparations, the complex structure was first relaxed by

100 cycles of steepest descent minimization, followed by 4900

cycles of conjugated gradient minimization. After that, the systems

were gradually heated up with the Berendsen algorithm from 0 K

to 300 K in 100 ps. Then, 3 ns long MD simulation was performed

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Published online on October 9, 2013

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ChemMedChem 2013, 8, 1986 – 2014 2014

Article Doi

De novo design, synthesis and evaluation of benzylpiperazine derivatives as highly selective binders of Mcl-1

DOI: 10.1002/cmdc.201300316

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Article abstract of DOI:10.1002/cmdc.201300316

Full text of DOI:10.1002/cmdc.201300316

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