Scaffold repurposing of fendiline: Identification of potent KRAS plasma membrane localization inhibitors

European Journal of Medicinal Chemistry 217 (2021) 1 13381

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Scaffold repurposing of fendiline: Identiﬁcation of potent KRAS

plasma membrane localization inhibitors

,

Pingyuan Wang ^{a 1}, Dharini van der Hoeven ^{c 1}, Na Ye ^{a 1}, Haiying Chen ^a, Zhiqing Liu ^a,

Xiaoping Ma ^b, Dina Montufar-Solis ^b, Kristen M. Rehl ^d, Kwang-Jin Cho ^d, Sabita Thapa ^c,

Wei Chen ^b, Ransome van der Hoeven ^c, Jeffrey A. Frost ^b, John F. Hancock ^b^**, Jia Zhou ^a

,

, *

^aChemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, 77555, USA

^bDepartment of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX,

77030, USA

^cDepartment of Diagnostic and Biomedical Sciences, School of Dentistry, The University of Texas Health Science Center at Houston, Houston, TX, 77054, USA

^dDepartment of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH, 45435, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

KRAS plays an essential role in regulating cell proliferation, differentiation, migration and survival.

Mutated KRAS is a major driver of malignant transformation in multiple human cancers. We showed

previously that fendiline (6) is an effective inhibitor of KRAS plasma membrane (PM) localization and

function. In this study, we designed, synthesized and evaluated a series of new fendiline analogs to

optimize its drug properties. Systemic structure-activity relationship studies by scaffold repurposing led

to the discovery of several more active KRAS PM localization inhibitors such as compounds 12f (NY0244),

12h (NY0331) and 22 (NY0335) which exhibit nanomolar potencies. These compounds inhibited onco-

genic KRAS-driven cancer cell proliferation at single-digit micromolar concentrations in vitro. In vivo

studies in a xenograft model of pancreatic cancer revealed that 12h and 22 suppressed oncogenic KRAS-

expressing MiaPaCa-2 tumor growth at a low dose range of 1e5 mg/kg with no vasodilatory effects,

indicating their potential as chemical probes and anticancer therapeutics.

Received 8 December 2020

Received in revised form

4 March 2021

Accepted 8 March 2021

Available online 15 March 2021

Keywords:

KRAS

Plasma membrane localization

Scaffold repurposing

Fendiline

Pancreatic cancer

1. Introduction

mutated, accounting for ~85% of all RAS mutations [1,7e9]. Devel-

opment of therapeutic small molecule inhibitors of KRAS is there-

RAS is a small membrane-localized GTPase that operates as a

molecular switch by oscillating between active GTP-bound and

inactive GDP-bound states to regulate cell proliferation, differen-

tiation, migration and survival [1e4]. In human cells, three main

RAS isoforms are ubiquitously expressed: HRAS, NRAS and KRAS

[5,6]. RAS point mutations occur in about 20% of all human cancers

which constitutively activate RAS by locking the protein in the GTP-

bound state. Among the RAS isoforms, KRAS is the most frequently

fore critically important for the treatment of KRAS-driven cancers.

Signiﬁcant efforts have been made to pharmacologically target

KRAS albeit mainly through the development of inhibitors of ki-

nases downstream of KRAS [7,10e15]. Direct targeting of KRAS has

proven more challenging [4], although signiﬁcant progress has

been achieved with efﬁcacious small molecules that block the in-

teractions between KRAS and the RAS exchange factor son of sev-

enless (SOS) [16,1 7], and molecules that speciﬁcally target KRAS

G12C [18], allowing the development of KRAS G12C irreversible

allosteric inhibitors [10,19e22]. Several potent and highly selective

KRAS G12C inhibitors (e.g. AMG510 [23] and MRTX849 [24]) have

currently advanced into human clinical trials. Nevertheless, such

compounds are only useful for treatment of the 15% of KRAS tumors

that harbor a G12C mutation [25].

* Corresponding author. Chemical Biology Program, Department of Pharma-

cology and Toxicology, University of Texas Medical Branch, Galveston, TX, 77555,

USA.

** Corresponding author. Department of Integrative Biology and Pharmacology,

McGovern Medical School, The University of Texas Health Science Center at

Houston, Houston, TX, 77054, USA.

Newly synthesized KRAS undergoes post-translational modiﬁ-

cation of the C-terminal CAAX motif (C ¼ cysteine; A ¼ isoleucine;

X ¼ serine or methionine) that is required for localization to the

E-mail addresses: john.f.hancock@uth.tmc.edu (J.F. Hancock), jizhou@utmb.edu

(J. Zhou).

1

These authors contributed equally to the manuscript.

https://doi.org/10.1016/j.ejmech.2021.113381

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Abbreviations

Cer

WT

ceramide

wild-type

RAS

Rat sarcoma

CC3

cleaved caspase 3

HRAS

NRAS

KRAS

SOS

Harvey RAS

Neuroblastoma RAS

Kirsten RAS

MDCK

pERK

GFP

Madin-Darby Canine Kidney epithelial

phosphorylated protein kinase RNA-like ER kinase

green ﬂuorescent protein

son of sevenless

GFP-Lys lysenin tagged with GFP

PM

plasma membrane

farnesyltransferase

Ras-converting enzyme 1

isoprenylcysteine carboxylmethyltransferase 1

THF

DCE

DIPEA

EDCI

tetrahydrofuran

1,2-dichloroethane

N,N-diisopropylethylamine

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

FTase

RCE1

ICMT1

PDE

d

phosphodiesterase d

RE

recycling endosome

phosphatidylserine

acid sphingomyelinase

plasma membrane

pharmacokinetics

HOBt

TLC

UV

1-hydroxybenzotriazole

thin layer chromatography

ultraviolet

PtdSer

ASM

PM

PK

SAR

SM

TMS

tetramethylsilane

HRMS

HPLC

LactC2

high-resolution mass spectrometry

high-performance liquid chromatography

lactadherin-C2 domain

structure-activity relationship

sphingomyelin

inner leaﬂet of the plasma membrane (PM). After farnesylation by

farnesyltransferase (FTase), the AAX residues are cleaved off by

RAS-converting enzyme 1 (RCE1), and the now C-terminal farne-

sylated cysteine is methylated by isoprenylcysteine carbox-

ylmethyltransferase 1 (ICMT1) [7,26,27]. PM localization is essential

for KRAS function, and therefore, blocking oncogenic KRAS-driven

signaling by preventing PM localization represents an appealing

strategy [28]. Initial attempts to block KRAS processing and PM

localization using FTase inhibitors such as Tipifarnib [29] (1, Fig. 1),

Lonafarnib [30] (2, Fig. 1) and BMS214662 [31] (3, Fig. 1) were un-

successful because KRAS is alternatively prenylated by geranylger-

anyltransferase type I (GGTase I) in FTI treated cells [32,33]. Recent

ﬁndings that KRAS released from endosomes is captured by the

chaperone protein phosphodiesterase

the recycling endosome (RE) for forward transport back to the PM

led to the development of small molecule PDE inhibitors such as

d (PDEd) that delivers it to

d

Fig. 1. Structures of reported representative inhibitors 1e7 targeting KRAS PM localization. Three potential pharmacological motifs of lead PM localization inhibitor 7 were depicted

for the structural optimization presented in this work.

2

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Deltarasin (4, Fig. 1) and Deltazinone 1 (5, Fig. 1), as inhibitors of

KRAS PM localization [34,35]. However, there are toxicity concerns

obtained by further substitution of 11d with the corresponding

acyclic or cyclic amines.

because PDE

d

has a crucial role in regulating the function of many

Substitutions in the other diphenyl group in P2 were explored

subsequently. In the meantime, we kept the 1-ethyl-4-

methylpiperazine group which was proven to be the optimal

moiety of P3 in the next SAR studies. As outlined in Scheme 1,

further simpliﬁed diphenyl ring analogs 14a-b were produced

through an alternative approach, in which amino linker was

introduced, leading to compound 13, followed by installation of

allyl or cinnamyl groups.

other prenylated GTPases [10]. Inhibitors of RCE1 and ICMT are

under development, but these agents will also target CAAX pro-

cessing of geranylgeranylated RHO family GTPases [36e38]. Thus

the development of potent and selective KRAS PM localization in-

hibitors is still an urgent need.

Repurposing FDA-approved non-cancer drugs as KRAS in-

hibitors presents an efﬁcient strategy for anticancer agent devel-

opment because such compounds have well-investigated

pharmacokinetic and toxicological proﬁles [39,40]. In this context

we recently reported that fendiline (6, Fig. 1) has attractive activity

as an inhibitor of KRAS PM localization [41]. Compound 6, identi-

ﬁed through a high-content screening of the Prestwick chemical

library, is a calcium channel blocker that was previously used as a

coronary vasodilator for the treatment of angina. The R isomer of

compound 6 selectively mislocalized KRAS from the PM with an

Diverse substituents on different positions of the benzyl ring of

the P1 moiety were then extensively explored. The syntheses of

compounds 15a-h are depicted in Scheme 2. Compounds 15a-h

were prepared following a similar procedure of 12f starting from

compounds 9b-i. As shown in Scheme 3, novel compounds 18, 20,

22 and 25 were designed via introducing different 4-

methylpiperazine bearing linkers into 10a. Alkylation of 10a with

methyl 2-chloroacetate following the similar procedure to that of

11c afforded intermediate 16. Compound 18 was obtained by hy-

drolysis of 16 to compound 17, followed by coupling of intermediate

17 with 1-methylpiperazine. Reaction of 10a with 2-chloroacetyl

chloride in the presence of Et₃N afforded intermediate 19. Com-

pound 20 was produced via a similar procedure to that of com-

pound 12f. The intermediates 21 and 23 were obtained by

alkylation of 10a with 1-bromo-3-chloropropane and 4-chloro-1,1-

diethoxybutane, respectively. Starting from intermediate 21,

following a similar procedure to that of 12f produced the ﬁnal

compound 22. Deprotection of intermediate 23 using aqueous HCl

resulted in compound 24. Compound 25 was obtained by coupling

of aldehyde 24 with 1-methylpiperazine under a standard reduc-

tive amination condition.

IC₅₀of approximately 5.6

zation of HRAS or NRAS [41]. Mechanistic studies indicated that

compound decreased PM phosphatidylserine (PtdSer) and

mM, but had no effect on the PM locali-

6

cholesterol levels through inhibition of acid sphingomyelinase

(ASM) and further showed that KRAS mislocalization was a direct

consequence of reduced PM PtdSer content [42,43]. Compound 7

(Fig. 1) was identiﬁed after initial chemical structural modiﬁcations

of 6, which exhibited 10-fold increased potency. However, com-

pound 7 showed no capability to kill the mutant KRAS expressing

cancer cells even at a high concentration of 30 mM likely due to its

high cLogP value indicating high lipophilicity and poor druglike

properties for further preclinical development [44].

As part of our ongoing anticancer drug discovery efforts, we aim

to develop potent and selective KRAS PM localization inhibitors as

useful pharmacological tools and anticancer drug candidates for

potential clinical use. Inspired by our previous work, compound 7

was selected as a lead compound for further structure-activity

relationship (SAR) studies. In the present work, further structural

optimization of 7 by scaffold repurposing, and simpliﬁcation ap-

proaches were used to design a new class of diphenylprop-2-en-1-

amines as novel potent KRAS PM localization inhibitors. To this end,

the three pharmacophore moieties (P1 highlighted in blue, P2

highlighted in purple and P3 highlighted in red; Fig. 1) were sys-

tematically modiﬁed. The general synthetic routes of these newly

synthesized PM localization inhibitors are outlined in Schemes 1e3

to establish a meaningful SAR.

2.2. Biology

2.2.1. In vitro evaluation of potency and efﬁcacy in KRAS

mislocalization assays

All newly synthesized compounds were evaluated for their

ability to mislocalize GFP-tagged oncogenic mutant KRAS (mGFP-

KRAS G12V) from the PM of Madin-Darby Canine Kidney epithelial

(MDCK) cells to determine their potency (IC₅₀values), efﬁcacy

(E_max), which measures the fractional extent of mislocalization

from the PM and cytotoxicity at 30 mM concentration. We previ-

ously observed that a combination of potency in the KRAS mis-

localization assay together with high-dose cytotoxicity to the KRAS

expressing MDCK cells correlated best with selective toxicity

against mutant KRAS expressing cancer cells [41,44]. The likely

explanation for this observation relates to work showing that

lipophilic compounds with a free amino group are not infrequently

functional ASM inhibitors. Such compounds, which are concen-

trated in acidic lysosomes and then protonated, displace

membrane-bound ASM and acid ceramidase to the lumen where

the enzymes are degraded [45,46]. Oncogenic transformation also

remodels SM metabolism reducing cellular SM levels, triggering

increased expression of SM synthases and reduced expression of

ASM [47,48]. This remodeling is an adaptive mechanism to reduce

lysosomal fragility and lysosomal cell death, to which transformed

cells seem predisposed, and which are directly correlated with

lysosomal SM content [48]. Together these observations can

explain why transformed cells are generically more sensitive to

ASM inhibitors than cognate non-transformed cells [47,48]. Our

work further shows that KRAS transformed cells are globally more

sensitive to 6 than non-KRAS transformed cells [41,42,49]. Our

hypothesis is therefore that concomitant loss of KRAS function

further enhances sensitivity to lysosomal cell death in KRAS

addicted cells. Generic sensitivity to lysosomal cell death is marked

2. Results and discussion

2.1. Chemistry

The ﬁrst strategy we proposed was to remove the hydrophobic

diphenyl moiety and modify the linker of P3 (highlighted in red,

Fig. 1) to reduce the cLogP value. We attempted to introduce a more

polar nitrogen atom at the terminal of P3 moiety to improve the

aqueous solubility and form N,N-dialkylamino, or cyclic amino

groups to replace the highly lipophilic diphenyl moiety to generate

a series of compounds 11, 12 and 14, as shown in Scheme 1.

Commercially available (R)-1-(4-methoxyphenyl)ethylamine (9a)

was chosen as the starting synthetic material. The key intermediate

10a was prepared via stepwise reductive amination of 9a and b-

phenylcinnamylaldehyde as the sole product in a yield of 68%.

Alkylation of amine 10a with corresponding alkylating agents

yielded compounds 11a, 11c, 11e and 11f in the presence of K₂CO₃

and KI at reﬂux. Further reductive amination of 10a with acetal-

dehyde and 2-chloroacetaldehyde using NaBH(OAc)₃as a reducing

agent provided compounds 11b and 11d. Compounds 12a-i were

3

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Scheme 1. Synthetic route of (R)-N-substituted-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenylprop-2-en-1-amine derivatives 11a-c, 12a-i and 14a-b. Reagents and conditions: (a) i)

-phenylcinnamylaldehyde, 4 Å molecular sieves, anhydrous Na₂SO₄, THF, rt, 48 h; ii) NaBH₄, MeOH, 0 ^ꢀC, 2 h, 68% for two steps; (b) for 11a, MeI, K₂CO₃, MeCN, reﬂux, overnight,

b

34%; for 11b, CH₃CHO, NaBH(OAc)₃, HOAc, DCE, rt, 24 h, 80%; for 11c, allyl bromide, K₂CO₃, acetone, 50 ^ꢀC, 6 h, 77%; for 11d, 45 wt% 2-chloroacetaldehyde aq., NaBH(OAc)₃, HOAc,

DCE, rt, 2 h, 90%; for 11e, 2-bromoethan-1-ol, DIPEA, MeCN, 80 ^ꢀC, 24 h, 18%; and for 11f, 1-bromo-2-ﬂuoroethane, DIPEA, DMF, 80 ^ꢀC, 16 h, 70%; (c) for 12a-i, R²H, K₂CO₃, KI, MeCN,

reﬂux, overnight, 31e69%; (d) tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate, K₂CO₃, KI, MeCN, 50 ^ꢀC to reﬂux, quant.; (e) for 14a, cinnamylaldehyde, NaBH(OAc)₃, phos-

photungstic acid hydrate, DCE, rt, 48 h, 45% for two steps; for 14b, allyl bromide, K₂CO₃, KI, MeCN, 50 ^ꢀC, 3 h, 41% for two steps; (f) i) 6 N HCl, EtOH, 0 ^ꢀC to rt, overnight; ii) 37 wt%

formaldehyde aq., NaBH(OAc)₃, HOAc, DCE, rt, 24 h, 67% for two steps for 14a, 64% for two steps for 14b.

11e displayed a similar efﬁcacy to that of 7 (E_max¼ 0.72) but a better

cLogP (5.18 vs 9.32). Like compound 7, compound 11e did not show

any cytotoxicity at 30

rine to yield compound 11f also led to a 20-fold loss of potency

M). Comparing 12a to 11e, replacing the hydroxyl

mM. Changing the hydroxyl group to a ﬂuo-

(IC₅₀¼ 10.1

m

group with a dimethylamino group resulted in a dramatic decrease

of both potency and efﬁcacy. Conversely, when a diethylamino

group (12b), or nitrogen containing 6-member ring (12c-e) was

introduced, potency equivalent to 11e was retained, while efﬁcacy

was slightly improved. Compound 12e exhibited decent potency

and excellent efﬁcacy (IC₅₀¼ 1.3

m

M, E_max¼ 0.90) as well as cyto-

Scheme 2. Syntheses of N-(1-(substituted phenyl)ethyl)-N-(2-(4-methylpiperazin-1-

yl)ethyl)-3,3-diphenylprop-2-en-1-amine derivatives 15a-h. Reagents and condi-

toxicity at 30 M. Inspired by the result from 12e, we then started to

m

modify the piperazine ring by introducing a methyl group (12f) or

an oxygen atom (12g). The results indicated that 12f was the best

compound of this series with an IC₅₀of 0.1 mM (5-fold more potent

than 7), and slightly enhanced efﬁcacy (E_max¼ 0.75) in the KRAS

mislocalization assay. In addition, 12f also has a better cLogP than 7,

suggesting that it may have a better druglike proﬁle. Introducing an

tions: (a) i) b-phenylcinnamylaldehyde, 4 Å molecular sieves, anhydrous Na₂SO₄, THF,

rt, 48 h; ii) NaBH₄, MeOH, 0 ^ꢀC, 2 h, used directly for next step; (b) 45 wt% 2-

chloroacetaldehyde aq., NaBH(OAc)₃, HOAc, DCE, rt, 2 h, used directly for next step;

(c)1-methylpiperazine, K₂CO₃, KI, MeCN, 50 ^ꢀC to reﬂux, overnight, 15e53% for three

steps.

oxygen atom on the piperazine (12g, IC₅₀¼ 7.7

mM) was not

by 30

mM toxicity, whereas the additional KRAS speciﬁcity is

tolerated, leading to a 6-fold loss of potency compared to 12e.

Decreasing the ring size (six-member piperazine ring) of 12f into a

pyrrolidine ring (12h) or an azetidine ring (12i) resulted in 3-fold

marked by potency in the KRAS mislocalization assay, which

equates to loss of KRAS function, but both activities of a compound

are required to demonstrate efﬁcacy against KRAS addicted tumors.

(IC₅₀¼ 0.3

mM) and 5-fold (IC₅₀¼ 0.5

mM) reductions in potency,

For this reason, we preserved cytotoxicity at 30 mM in our evalua-

respectively. Compounds 14a and 14b with simpliﬁed side chain A2

and A3 exhibited a complete loss of in vitro activity, indicating that

at least one diphenyl ring system is required to retain KRAS PM

mislocalization activity. Taken together, these results suggest that

only one diphenyl ring system is necessary and the methylpiper-

azine motif is more favorable for KRAS mislocalization activity.

Next, we kept methylpiperazine intact as the P3 moiety and

investigated the SAR of the P1 moiety. We ﬁrst studied the effect of

the chiral methyl group on in vitro activity. As shown in Table 2,

compound 15a with an (S)-enantiomer was less potent than its (R)-

enantiomer (12f), exhibiting a 12-fold decreased potency with an

tions, together with potency and efﬁcacy in the KRAS mislocaliza-

tion assay, as indicated in the tables.

Compound 7, which has an IC₅₀value of 0.5 mM for KRAS mis-

localization was used as reference compound for comparison. We

initially focused on reducing the cLogP value of 7 (Table 1) with the

aim to increase its druglike properties. Therefore, we removed the

diphenyl moiety of P3 and alkylated with a methyl group leading to

compound 11a, which resulted in a 7-fold loss of potency in the

KRAS mislocalization assay compared to 7 (IC₅₀¼ 3.3

mM).

Extending the length of the alkyl linker (e.g., compounds 11b and

11c) resulted in a further substantial loss of potency. However, the

compound regained enhanced activity when a hydroxyl group

(11e) was introduced, which resulted in a 2.5-fold increase in KRAS

IC₅₀of 1.2

mM, but with a slight improvement on efﬁcacy

(E_max¼ 0.83). With the favorable R-conﬁguration of methyl group,

compounds 15b and 15c were synthesized to probe the impact of

the position of methoxy group in the phenyl ring on potency. As

mislocalization potency compared to 7 (IC₅₀¼ 0.2

mM). Compound

4

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Scheme 3. Syntheses of (R)-N-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine derivatives 18, 20, 22 and 25. Reagents and

conditions: (a) methyl 2-chloroacetate, K₂CO₃, KI, MeCN, reﬂux, overnight, 92%; (b) i) LiOH, THF, rt, overnight; ii) 2 N HCl, 93%; (c) 1-methylpiperazine, EDCI, HOBt, DIPEA, CH₂Cl₂, rt,

overnight, 83%; (d) 2-chloroacetyl chloride, Et₃N, rt, 2 h, 72%; (e) 1-methylpiperazine, K₂CO₃, KI, MeCN, 50 ^ꢀC to reﬂux, overnight, 44% for 20, 60% for 22; (f) 1-bromo-3-

chloropropane, DIPEA, DMF, 80 ^ꢀC, 16 h, 24%; (g) 4-chloro-1,1-diethoxybutane, K₂CO₃, KI, MeCN, reﬂux,

2 d, 43%; (h) 6 N 2 h, 92%; (i) 1-

HCl, 1,4-dioxane, 50 ^ꢀC,

methylpiperazine, NaBH(OAc)₃, DCE, rt, overnight, 52%.

shown in Table 2, a methoxy group at the para-position is better

than at the ortho- and meta-positions. Diverse substituents on the

phenyl ring of the P1 moiety were also investigated and are sum-

marized in Table 2. Replacement of the methoxy group with

hydrogen, ﬂuorine, chlorine or a nitro group (e.g., 15d~1h) led to 6-

to 112-fold decrease in potency. Overall, we can conclude that the

(R)-enantiomer is more favorable than the (S)-enantiomer and the

methoxy group is essential for KRAS mislocalization potency. The

substituents and the positions are also important for potency, with

derivatives, MDCK cells stably co-expressing mCherry-CAAX and

mGFP-LactC2, a probe for PtdSer, were treated with the compounds

for 48 h and analyzed by quantitative confocal microscopy. The

results show that the new analogs disrupt the PM localization of

mGFP-LactC2, and by inference PtdSer, with potencies (measured

as IC₅₀) very similar to their respective potencies for mislocalizing

KRASG12V from the PM (Fig. 2A).

Compound 6 depletes PtdSer from the PM by indirectly inhib-

iting the activity of ASM, an enzyme that hydrolyzes sphingomyelin

(SM) to ceramide (Cer) [49]. ASM-inhibition causes SM accumula-

tion and aberrant endo-lysosomal function that in turn depletes the

PM of PtdSer. To determine if the new derivatives of compound 6

also functioned through the same mechanism, we measured inhi-

bition of ASM using a sphingomyelinase activity assay. Our results

show that all new analogs inhibit ASM with higher potency than

compound 6 (Fig. 2B). Compound 12h was the most potent com-

pound in inhibiting ASM. Western blot analysis to determine the

effect of the compounds on the cellular levels of ASM revealed that

compounds 12f and 12h reduced the levels of ASM while the others

had no detectable effect (Fig. 2C). To conﬁrm that KRAS mis-

localization occurred as a consequence of ASM inhibition, we

supplemented new analog-treated cells with exogenous ASM. As

shown in Fig. 2D, acute ASM supplementation partially corrected

the mislocalization of KRASG12V from the PM induced by the

compounds. These results recapitulate earlier observations with

compound 6.

electron-donating

substituents

preferred

over

electron-

withdrawing groups, and substitution at the para-position being

superior to that at the ortho- or meta-positions.

Finally, we investigated the signiﬁcance of linker changes on

potency. As shown in Table 3, we replaced each methylene group

with a carbonyl group to generate compounds 18 and 20. Neither of

these compounds were more potent than 12f. However, extending

the length of the linker by adding a methylene group (compound

22) resulted in a substantially increased KRAS mislocalization po-

tency from 0.1

mM to 20 nM whilst retaining good efﬁcacy

(E_max¼ 0.81). However, when we further extended the linker with

two methylene groups, the resulting compound 25 displayed

signiﬁcantly decreased potency. Collectively, the type, shape, and

length of the linker are all critical for potency, with the propane

linker appearing to be the most favorable.

2.2.2. In vitro mechanism of action studies of selected new analogs

For further analysis we selected analogs that exhibited an IC₅₀of

less than 0.5

MDCK cells expressing KRASG12V cells at 30

m

M, an E_maxgreater than 0.75, and had cytotoxicity to

2.2.3. In vitro biological activities against KRAS-driven cell lines

The growth inhibitory effects of these newly synthesized PM

localization inhibitors were evaluated against mouse embryonic

ﬁbroblast cells expressing oncogenic mutant BRAF V600E (wild

type KRAS), or KRAS G12V or KRAS G12D, using cell proliferation

assays. All compounds tested selectively inhibited proliferation of

oncogenic mutant KRAS-expressing MEF cells, but importantly had

no effect on proliferation of the mutant BRAF-expressing cells

(Fig. 3). Compounds 12f, 12h and 22 were also more potent than

mM, which as dis-

cussed above are a set of parameters that correlate well with the

ability of the compound to inhibit proliferation of oncogenic KRAS

expressing cancer cell lines. This set of new analogs included

compounds 12f, 12h and 22. Fendiline (6) was included as the

positive control. Compound 6 causes KRAS mislocalization by

depleting PtdSer from the inner leaﬂet of the PM [41,42,49]. To

determine if the same mechanism operates with the new

5

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Table 1

The IC₅₀and E_maxvalues of (R)-N-substituted-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenylprop-2-en-1-amine KRAS PM localization inhibitors.

.

R¹

R³

IC₅₀

(m

M)^a

E_max

cLogP^c

Cytotoxic at 30 mM

b

Compound

7

A1

-Me

-Et

A1

0.5

3.3

10.6

13.0

0.74 0.03

0.75 0.01

0.84 0.02

0.47 0.01

9.32

5.82

6.21

6.38

No

11a

11b

11c

11e

11f

12a

-CH₂CH₂OH

-CH₂CH₂F

A1

0.2

10.1

11.2

0.72 0.03

0.52 0.04

0.66 0.01

5.18

6.16

5.75

No

Yes

12b

12c

12d

A1

1.2

0.6

4.4

0.89 0.01

0.83 0.02

0.86 0.02

6.53

6.68

5.52

Yes

No

12e

12f

12g

A1

1.3

0.1

7.7

0.86 0.02

0.75 0.01

0.81 0.02

5.10

5.44

4.62

Yes

No

12h

A1

0.3

0.85 0.04

6.29

Yes

12i

A1

A2

0.5

0.69 0.02

0.84 0.01

5.90

4.09

Yes

No

14a

14.8

14b

A3

59.3

0.76 0.02

2.49

No

a

IC₅₀: 50% inhibitory concentration for KRASG12V mislocalization.

E_max: Maximal effects elicited by the compounds. IC₅₀and E_maxvalues were calculated from three independent experiments.

cLogP was calculated based on a web calculator, http://biosig.unimelb.edu.au/pkcsm/prediction.

b

c

6 at inhibiting proliferation of the mutant KRAS-expressing MEFs.

Similar results were also obtained with pancreatic, endometrial,

colon and lung cancer cell lines. Overall, the newly synthesized

compounds exhibited better anti-proliferation activity than com-

pound 6 in all KRAS-driven cancer cell lines tested (Table 4).

Concordant with results in the KRAS mislocalization assay, the

selected PM localization inhibitors were more potent at inhibiting

the proliferation of pancreatic cancer cell lines with an oncogenic

KRAS mutation than a pancreatic cancer cell line expressing wild-

type (WT) KRAS. A similar selective inhibition of proliferation of

oncogenic mutant KRAS expressing cells was also observed in

endometrial, lung and colon cancer cell lines. Compounds 6 and 7

displayed weak activities against mutant KRAS pancreatic cancer

cells (MiaPaCa-2, MOH and MPanc96) whereas compounds 12f,12h

and 22 signiﬁcantly inhibited proliferation of these three pancreatic

cancer cell lines at single-digit micromolar concentrations

(Table 4). Whilst increased activity compared to 6 was observed

towards the WT KRAS expressing BxPC-3 cell line, the new com-

pounds remained more potent inhibitors of mutant KRAS

expressing cells. Similarly, while compounds 12f, 12h and 22 had

no effect on proliferation of WT KRAS-expressing endometrial

cancer cell lines KLE and ESS-1 up to 30 mM (data not shown), and

6

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Table 2

The IC₅₀and E_maxvalues of N-(1-(Substituted phenyl)ethyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine KRAS PM localization inhibitors.

.

R⁴, *

IC₅₀

(m

M)^a

E_max

cLogP^c

Cytotoxic at 30 mM

b

Compound

7

0.5

0.1

1.2

3.8

2.8

0.6

1.5

3.8

11.2

5.2

0.74 0.03

0.75 0.01

0.83 0.02

0.86 0.01

0.75 0.04

0.79 0.01

0.83 0.02

0.70 0.02

0.77 0.07

0.62 0.02

9.32

5.44

5.43

5.57

6.08

5.34

No

12f

15a

15b

15c

15d

15e

15f

15g

15h

-4-OMe, (R)

-4-OMe, (S)

-2-OMe, (R)

-3-OMe, (R)

-H, (R)

-4-F, (R)

-4-Cl, (R)

-3-Cl, (R)

-4-NO_2,(R)

Yes

a

IC₅₀: 50% inhibitory concentration for KRASG12V mislocalization.

E_max: Maximal effects elicited by the compounds. IC₅₀and E_maxvalues were calculated from three independent experiments.

cLogP was calculated based on a web calculator, http://biosig.unimelb.edu.au/pkcsm/prediction.

b

c

moderately inhibited proliferation of Ishikawa cell line, they dis-

played more potent anti-proliferative activity against KRAS mutant

endometrial cancer cells (Hec1A and Hec1B) (Table 4). All three

new compounds were also signiﬁcantly more potent than 6 and 7

in blocking proliferation of mutant KRAS endometrial cancer cells

(Table 4). Similar results were evident in assays with colon and lung

cancer cells. Compound 7 was inactive against all cell lines tested. 6,

12f, 12h and 22 were all more potent inhibitors of KRAS mutant

cancer cell lines than WT KRAS cancer cell lines, and in all cases 12f,

12h and 22 were more potent inhibitors than 6 (Table 4). Taken

together, these data show that compared to lead compounds 6 and

7, the new analogs: 12f, 12h and 22 exhibited signiﬁcantly

improved anti-proliferative activities against all KRAS-driven can-

cer cells tested.

2.2.4. Assessment of in vivo biological activity

To test the capacity of the new compounds to block KRAS

signaling in vivo we ﬁrst used the well-validated invertebrate

model system C.elegans, which has a single RAS gene, let-60, that is

a KRAS ortholog [50e53]. Activating mutations in let-60 (e.g. LET-

60 G13D (n1046)) induce

a readily quantiﬁable multi-vulva

phenotype [51]. We therefore determined if treatment of these

worms with the new analogs would suppress the multi-vulva

phenotype. L1 larvae were cultured in M9 buffer containing the

E. coli strain OP50 in presence of DMSO or compounds. After 4e5

days, worms reached the adult stage and were scored for the

presence of the multi-vulva phenotype. Compound 6, which we

previously showed to potently inhibit the multi-vulva phenotype,

was used as the positive control. All of the compounds tested dose-

Table 3

The IC₅₀and E_maxvalues of ((R)-N-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine KRAS PM localization inhibitors.

.

M)^a

E_max

cLogP^c

Cytotoxic at 30 mM

b

Compound

X

Y

IC₅₀

(m

7

0.5

0.1

1.1

7.1

0.74 0.03

0.75 0.01

0.73 0.02

0.51 0.02

0.81 0.01

0.81 0.03

9.32

5.44

4.96

5.83

6.22

No

Yes

No

Yes

12f

18

20

22

25

CH₂

CO

CH₂

CO

CH₂

(CH₂)₂

(CH₂)₃

0.02

ND^d

a

IC₅₀: 50% inhibitory concentration for KRASG12V mislocalization.

E_max: Maximal effects elicited by the compounds. IC₅₀and E_maxvalues were calculated from three independent experiments. cLogP was calculated based on a web

b

calculator, http://biosig.unimelb.edu.au/pkcsm/prediction.

d

ND means not determined within the concentration range tested.

7

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Fig. 2. (A) Dose response curves quantifying the extent of KRASG12V and LactC2 mislocalization after 48 h treatment with compounds. Mislocalization was quantiﬁed using

Manders coefﬁcients as the fraction of mCherry-CAAX co-localizing with mGFPeKRASG12V or GFP-LactC2. (B) ASM activity in cell stably expressing GFP-tagged SMPD1 treated with

DMSO or 5

m

M compound for 48 h. (C) Representative western blots and quantitation of SMPD1-GFP levels in cells treated with 5

mM compounds for 48 h. (D) Representative

confocal images of MDCK cells stably co-expressing mGFP-KRASG12V and mCherry-CAAX treated with 10

m

M compound 6 or 1 M of compounds 12f, 12h or 22 for 48 h (-ASM),

m

then incubated with recombinant ASM in the continued presence of drugs for a further 60 min (þASM). Mislocalization was quantiﬁed using Manders coefﬁcients as the fraction of

mCherry-CAAX co-localizing with mGFPeKRASG12V.

dependently reverted the multi-vulva phenotype to a single-vulva

WT phenotype (Fig. 4).

We next determined the effect of the selected PM localization

inhibitors on the growth of oncogenic KRAS expressing MiaPaCa-

2 cells implanted subcutaneously into the ﬂanks of nu/nu immu-

nosuppressed mice. Given that compounds 12h and 22 had good

potency in inhibiting in vitro proliferation and suppressing the multi-

vulva phenotype in C.elegans, those two were chosen to be tested in

the in vivo tumor growth assays. The animals were randomized into

control and PM localization inhibitor treated groups (10 mice per

group). Treatment was initiated when the tumors reached a mean

volume of 100 mm³. PM localization inhibitors were tested at 5, 2.5

8

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Fig. 3. Dose response curves quantifying cell number of isogenic MEF cells expressing oncogenic mutant BRAF or KRAS following 72 h treatment with compounds. Shown in table

are the corresponding IC₅₀values in mM.

Table 4

Selected potent PM localization inhibitors anti-proliferative activities of pancreatic, endometrial, colon and lung cancer cells.

IC₅₀

(

m

M)^acancer Cell lines

6

7

12f

12 h

22

Pancreatic

BxPC-3 (WT)

MiaPaCa-2 (Mut)

MOH (Mut)

MPanc96 (Mut)

Ishikawa (WT)

Hec1A (Mut)

28.6

9.2

>30

5.7

3.0

3.7

4.2

>30

2.6

3.3

5.1

3.3

>30

28.2

8.2

3.8

5.5

3.1

3.6

3.8

17.33

2.3

3.0

4.2

3.0

5.3

2.8

3.8

4.2

11.0

2.3

3.9

4.4

3.5

>30

23.5

8.2

4.1

11.1

10.8

>30

9.48

9.8

14.5

7.8

>30

29.6

24.3

11.4

Endometrial

Hec1B (Mut)

Caco-2 (WT)

Colon

Lung

SK-CO-1 (Mut)

NCI H1975 (WT)

NCI H522 (WT)

NCI H1299 (WT)

NCI H23 (Mut)

>30

27.3

8.0

3.73

a

IC₅₀values were calculated from three independent experiments. >30 indicates that the IC₅₀is greater than the highest dose (30

mM) tested.

and 1 mg/kg doses, administered once daily intraperitoneally for 5

days, with 2 days of no treatment in between for the duration of the

experiment. Administration of compounds 12h and 22 at 5 and

2.5 mg/kg decreased the rate of growth of MiaPaCa-2 cells xenografts

in nude mice (Fig. 5A), to the same degree of inhibition previously

observed with compound 6 at the dose of 12.5 mg/kg [42]. Com-

pounds 12h also inhibited tumor growth at lower doses tested, at

1 mg/kg (Fig. 5A). However, at the same dose, of 1 mg/kg 12h had no

effect on the growth of the wild type KRAS-expressing BxPC3 tumors

(Fig. 5B). There was no observed toxicity in any of the compound-

treated groups during the experiment and, accordingly, there was

no signiﬁcant difference in the body weight of the animals in the

different groups. These results demonstrate that compounds 12h

and 22 can selectively reduce the growth of oncogenic KRAS-

transformed tumors in vivo.

lysenin tagged with GFP (GFP-Lys). In DMSO-treated tumor sec-

tions, weak GFP- Lys staining was observed. The GFP-Lys staining

was increased in tumors from compounds 6 and 12h-treated mice

(12.5 mg/kg) (Fig. 5C), suggesting accumulation of SM with com-

pound treatment. The SM staining was signiﬁcantly greater with

compound 12h than with compound 6, which corresponds with

increased potency of compound 12h. Compound 22 did not

enhance GFP-Lys staining. Compounds 12h and 22 reduced phos-

phorylated ERK (pERK) levels and increased cleaved caspase 3

(CC3) in tumors whereas compound 6 had no signiﬁcant effect

suggesting that the new analogs are more potent in inhibiting KRAS

signaling and inducing apoptosis of tumor cells (Fig. 5D and E). We

also observed that the blood vessels were dilated in compound 6

treated tumors, whereas the blood vessel diameters were not

changed in compound 12h and 22 treated tumors (Fig. 5F). These

results suggest that while compounds 12h and 22 retained the

KRAS inhibitory function, they lost the Ca^2þchannel blocking

function of compound 6.

To determine if the reduction in tumor growth elicited by the

compounds was mediated by inhibition of ASM, we stained the

tissue sections for SM using a non-toxic recombinant fragment of

9

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

concentrations. Moreover, compounds 12f, 12h, and 22 showed

higher anti-proliferation potency in KRAS mutant cancer cells than

wild KRAS wide type cancer cells. In nude mice bearing pancreatic

cancer xenografts, compound 12 h at a very low dose of 1.0 mg/kg

suppressed MiaPaCa-2 xenograft tumor growth and was more

potent than compound 6 [42]. Compound 12h also had no effect on

the growth of KRAS WT BxPC3 xenograft tumor growth, indicating

selective toxicity towards KRAS mutant tumors, suggesting selec-

tivity towards KRAS-dependent cancer cells. Histological analysis of

tumors also suggested that 12h and 22 likely have no effects on

vasomotor tone, unlike the parent compound fendiline (6), sug-

gesting that while maintaining the KRAS inhibitor function, these

compounds have lost the calcium channel blocking property of

fendiline. Our mechanistic studies demonstrated that these new

molecules mislocalize K-Ras at least in part through inhibition of

ASM with higher potency than compound 6. These compounds,

which are concentrated in acidic lysosomes and then protonated,

displace membrane-bound ASM and acid ceramidase to the lumen

where the enzymes are degraded. To conﬁrm that KRAS mis-

localization occurred as a consequence of ASM inhibition, we

supplemented compound-treated cells with exogenous ASM and

showed that ASM supplementation partially corrected the mis-

localization of KRASG12V from the PM induced by the compounds.

Therefore, collectively, our data show that this novel series of KRAS

PM localization inhibitors may prove useful as chemical probes and

pharmacological tools for elucidating KRAS-mediated signaling

pathways and KRAS-associated functions. Further studies of

selected drug candidates in various xenograft tumor models and via

different administrations as well as further systematic optimization

based upon these identiﬁed advanced chemical leads to enhance

the overall druglike properties such as pharmacokinetic (PK) and

toxicity characteristics will be pursued for the preclinical drug

development towards KRAS-dependent cancer therapeutics.

Fig. 4. Strain let-60 (n1046) L1 larvae were cultured in M9 buffer containing the E. coli

strain OP50 in presence of vehicle (DMSO) or compounds (0.1, 0.3, 1, 3, 10 or 30 mM).

After 4e5 days, when worms reached the adult stage, they were scored for the pres-

ence of the multi-vulva phenotype using DIC/Nomarski microscopy.

4. Experimental section

3. Conclusions

4.1. Chemistry

In summary, we have designed, synthesized and biologically

evaluated

a series of N-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-

All commercially available starting materials and solvents were

reagent grade and used without further puriﬁcation. Reactions

were performed under a nitrogen atmosphere in dry glassware

with magnetic stirring. Preparative column chromatography was

performed using silica gel 60, particle size 0.063e0.200 mm

(70e230 mesh, ﬂash). Analytical TLC was carried out employing

silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the

developed chromatograms was performed with detection by UV

(254 nm). NMR spectra were recorded on a Bruker-600 (¹H,

300 MHz; ¹³C, 75 MHz) spectrometer. ¹H and ¹³C NMR spectra were

recorded with TMS as an internal reference. Chemical shifts

downﬁeld from TMS were expressed in ppm, and J values were

given in Hz. High-resolution mass spectra (HRMS) were obtained

from Thermo Fisher LTQ Orbitrap Elite mass spectrometer. Param-

eters include the following: nano ESI spray voltage was 1.8 kV,

capillary temperature was 275 ^ꢀC, and the resolution was 60000;

ionization was achieved by positive mode. Purity of ﬁnal com-

pounds was determined by analytical HPLC, which was carried out

on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-20A

methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine de-

rivatives by scaffold repurposing of 6 as a novel class of potent KRAS

PM localization inhibitors. This is the ﬁrst report of systematic SAR

study of repurposed 6 and its analog 7, and the P1, P2 and P3

pharmacological moieties of 7 are extensively explored to identify

promising KRAS PM localization inhibitors. Our study shows that

only one diphenyl ring system is essential and the methylpiper-

azine motif is more favorable for the ligand to mislocalize KRAS.

Meanwhile, the type, shape, and length of the linker are all critical

for potency. Additionally, we found that the (R)-enantiomer is

preferred over (S)-enantiomer for the KRAS mislocalization. Finally,

our SAR study revealed that the substituents and the substitution

position are also critical for anti-KRAS potency: electron-donating

substitutes are superior to electron-withdrawing groups, and the

para-position is more favorable than the ortho- and meta-positions.

Several novel molecules such as compounds 12f (NY0244), 12h

(NY0331) and 22 (NY0335) were identiﬁed as potent KRAS PM

localization inhibitors with nanomolar level IC₅₀values while the

parent compound 6 mislocalizes KRAS at the low micromolar po-

tency. These compounds also have a more favorable cLogP in

comparison with that of compound 7. The growth inhibitory effects

of 12f, 12h, and 22 against pancreatic, endometrial, colon and lung

cancer cells revealed that these molecules signiﬁcantly inhibited

KRAS-driven cancer cell proliferation at single-digit micromolar

UV/vis). HPLC analysis conditions: Waters

mBondapak C18

(300 mm ꢁ 3.9 mm), ﬂow rate 0.5 mL/min, UV detection at 270 and

254 nm, linear gradient from 10% acetonitrile in water (0.1% TFA) to

100% acetonitrile (0.1% TFA) in 20 min, followed by 30 min of the

last-named solvent. All biologically evaluated compounds are >95%

pure.

10

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

Fig. 5. Nu/nu mice implanted with MiaPaCa-2 tumors (A) or BxPC3 (B) were treated with vehicle (DMSO) or compounds intraperitoneally and tumor sizes were measured with an

externalcaliper every 3e4 days. (C) Tumor sections were stained with GFP-Lys. (D) Tumor sections were stained with antibodies against pERK. (E) Tumor sections were stained with

cleaved caspase 3. (F) Blood vessel lumen area was also measured in tumor sections.

11

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

4.1.1. (R)-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenylprop-2-en-1-

amine (10a)

procedure to that of compound 11a from 3-bromo-propene. The

title compound was obtained as a colorless oil. ¹H NMR (300 MHz,

A RB ﬂask with activated 4 Å molecular sieves and dried anhy-

drous Na₂SO₄was charged with THF (8 mL), (R)-1-(4-methoxy-

CDCl₃)

d

7.42e7.23 (m, 10H), 7.17 (dd, J ¼ 7.7, 1.8 Hz, 2H), 6.89e6.81

(m, 2H), 6.24 (t, J ¼ 6.6 Hz,1H), 5.83 (m,1H), 5.18e5.04 (m, 2H), 3.91

(q, J ¼ 6.7 Hz, 1H), 3.83 (s, 3H), 3.22 (t, J ¼ 6.5 Hz, 2H), 3.09 (dd,

J ¼ 12.6, 6.3 Hz, 2H), 1.29 (d, J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz,

phenyl)-ethylamine 9a (360 mg, 2.4 mmol) and

b-phenyl-

cinnamylaldehyde (500 mg, 2.4 mmol) under a nitrogen atmo-

sphere. The reaction mixture was stirred at rt for 48 h, and then

removed the solvent and reﬁlled with MeOH (10 mL). To the so-

lution was added NaBH₄(140 mg, 3.7 mmol) at 0 ^ꢀC and reacted for

2 h. After removing the solvent, the residue was diluted with water

(10 mL) and extracted with EtOAc (20 mL ꢁ 3). The combined

organic phase was acidized with 4 N aq. HCl to pH < 3 and extracted

with Et₂O (15 mL ꢁ 3). The water phase was separated and basiﬁed

with 10 N aq. NaOH to pH > 9, and then extracted with EA

(15 mL ꢁ 3). The combined EtOAc extracts were washed with brine,

dried with NaSO₄, ﬁltered, and condensed by rotary evaporation to

yield 10a as a colorless oil used directly for next step (560 mg, 68%).

CDCl₃)

d 158.3, 142.8, 142.5, 139.8, 136.7, 136.1, 129.9, 128.7, 128.5,

128.1, 128.0, 127.4, 127.1 (2C), 116.7, 113.4, 58.1, 55.2, 53.0, 48.6, 16.9.

HRMS (ESI) calcd for C₂₇H₃₀NO 384.2327 (M þ H)^þ, found

384.2328.

4.1.5. (R)-N-(2-chloroethyl)-N-(1-(4-methoxyphenyl)ethyl)-3,3-

diphenylprop-2-en-1-amine (11d)

Compound 11d (365 mg, 90%) was prepared by a similar pro-

cedure to that of compound 11b from 45 wt% 2-chloroacetaldehyde

aqueous solution. The title compound was obtained as a colorless

oil. ¹H NMR (300 MHz, CDCl₃)

d 7.43e7.22 (m, 10H), 7.16 (d,

¹H NMR (300 MHz, CDCl₃)

d

7.33e7.25 (m, 3H), 7.24e7.18 (m, 5H),

J ¼ 6.7 Hz, 2H), 6.86 (d, J ¼ 7.6 Hz, 2H), 6.21 (t, J ¼ 6.2 Hz, 1H),

3.96e3.86 (q, J ¼ 9.0 Hz, 1H), 3.83 (s, 3H), 3.46e3.33 (m, 2H),

3.31e3.15 (m, 2H), 2.79 (m, 2H), 1.30 (d, J ¼ 6.3 Hz, 3H)$¹³C NMR

7.17e7.11 (m, 2H), 7.11e7.04 (m, 2H), 6.83e6.76 (m, 2H), 6.17 (t,

J ¼ 6.9 Hz,1H), 3.75 (s, 3H), 3.70 (q, J ¼ 6.5 Hz,1H), 3.17 (d, J ¼ 7.0 Hz,

2H), 2.06 (s, 1H), 1.29 (d, J ¼ 6.5 Hz, 3H)$¹³C NMR (75 MHz, CDCl₃)

(75 MHz, CDCl₃)

d 158.5, 143.50, 142.2, 139.5, 135.9, 129.9, 128.6,

d

158.6, 143.4, 142.3, 139.6, 137.2, 129.8, 128.1, 127.7, 127.6, 127.5,

128.2, 127.6, 127.4, 127.3, 113.5, 59.1, 55.3, 52.2, 49.8, 42.5, 16.9.

HRMS (ESI) calcd for C₂₆H₂₉ClNO 406.1938 (M þ H)^þ, found

406.1932.

127.3, 127.2, 113.8, 57.2, 55.3, 46.4, 24.2.

4.1.2. (R)-N-(1-(4-methoxyphenyl)ethyl)-N-methyl-3,3-

diphenylprop-2-en-1-amine (11a)

4.1.6. (R)-2-((3,3-Diphenylallyl) (1-(4-methoxyphenyl)ethyl)

amino)ethanol (11e)

Compound 11e (14 mg, 18%) was prepared by a similar pro-

cedure to that of compound 11a from 2-bromoethanol with DIPEA

as the base. The title compound was obtained as a colorless oil. ¹H

To a solution of 10a (34.3 mg, 0.1 mmol) in MeCN (2 mL) was

added K₂CO₃(27.6 mg, 0.2 mmol) and iodomethane (21.3 mg,

0.15 mmol). Then the mixture was stirred at reﬂux overnight. After

removing the solvent, the residue was diluted with water (8 mL)

and extracted with EtOAc (15 mL ꢁ 3). The combined EtOAc extracts

were washed with brine, dried over NaSO₄, ﬁltered, and condensed

by rotary evaporation to yield an oil. This material was further

puriﬁed by preparative TLC plates using EtOAc/hexane as the eluant

to yield a colorless oil 11a (12 mg, 34%). ¹H NMR (300 MHz, CDCl₃)

NMR (300 MHz, CDCl₃) d 7.42e7.34 (m, 3H), 7.32e7.24 (m, 5H), 7.17

(t, J ¼ 7.6 Hz, 4H), 6.83 (d, J ¼ 8.6 Hz, 2H), 6.18 (t, J ¼ 6.7 Hz, 1H), 3.99

(q, J ¼ 6.9 Hz,1H), 3.81 (s, 3H), 3.46 (q, J ¼ 5.4 Hz, 2H), 3.35e3.14 (m,

2H), 2.70e2.59 (m, 1H), 2.56e2.47 (m, 1H), 2.39 (s, 1H), 1.30 (d,

J ¼ 6.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃)

d 158.5, 143.8, 142.2, 139.5,

d

7.40e7.19 (m, 10H), 7.15 (dd, J ¼ 7.7, 1.8 Hz, 2H), 6.88e6.79 (m, 2H),

134.7, 129.8, 128.8, 128.2, 127.4, 127.3, 113.6, 58.4, 58.1, 55.2, 50.7,

48.6, 15.8. HRMS (ESI) calcd for C₂₆H₃₀NO₂388.2277 (M þ H)^þ,

found 388.2273.

6.25 (t, J ¼ 6.7 Hz, 1H), 3.82 (s, 3H), 3.55 (q, J ¼ 6.7 Hz, 1H), 3.18 (dd,

J ¼ 14.5, 6.4 Hz, 1H), 3.02 (dd, J ¼ 14.4, 7.1 Hz, 1H), 2.21 (s, 3H), 1.31

(d, J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 158.4, 143.2, 142.4,

139.7, 136.0, 129.9, 128.6, 128.1, 128.0, 127.6, 127.3, 127.1 (2C), 113.5,

62.2, 55.2, 53.4, 38.8, 18.8. HRMS (ESI) calcd for C₂₅H₂₈NO 358.2171

(M þ H)^þ, found 358.2169.

4.1.7. (R)-N-(2-Fluoroethyl)-N-(1-(4-methoxyphenyl)ethyl)-3,3-

diphenylprop-2-en-1-amine (11f)

Compound 11f (27 mg, 70%) was prepared by a similar pro-

cedure to that of compound 11a from 1-bromo-2-ﬂuoroethane

with DIPEA as the base. The title compound was obtained as a

4.1.3. (R)-N-ethyl-N-(1-(4-methoxyphenyl)ethyl)-3,3-

diphenylprop-2-en-1-amine (11b)

colorless oil. ¹H NMR (300 MHz, CDCl₃)

d 7.43e7.26 (m,10H), 7.17 (d,

To a solution of 10a (20 mg, 0.04 mmol) and acetaldehyde

(0.01 mL, 0.20 mmol) in 1,2-dichloroethane (4 mL) was added

acetic acid (cat.). 10 min later, NaBH(OAc)₃(20 mg, 0.09 mmol) was

added to the stirred reaction solution. The ﬂask was stirred at rt for

24 h. The reaction was worked up by the addition of sat. aq. NaHCO₃

and EtOAc. The combined EtOAc extracts were washed with brine,

dried over Na₂SO₄, ﬁltered, and condensed under pressure. The

residue was further puriﬁed by preparative TLC plates using DCM/

MeOH (25/1) as the eluant to yield 11b (17 mg, 80%) as a yellow oil.

J ¼ 7.1 Hz, 2H), 6.86 (d, J ¼ 7.7 Hz, 2H), 6.25 (t, J ¼ 6.6 Hz,1H), 4.45 (t,

J ¼ 5.5 Hz,1H), 4.29 (t, J ¼ 5.5 Hz,1H), 3.95 (q, J ¼ 6.3 Hz,1H), 3.83 (s,

3H), 3.30 (t, J ¼ 6.5 Hz, 2H), 2.90e2.62 (m, 2H), 1.30 (d, J ¼ 6.7 Hz,

3H). ¹³C NMR (75 MHz, CDCl₃)

d 158.4, 143.3, 142.3, 139.6, 135.9,

129.9, 128.7, 128.2 (2C), 128.1, 127.9, 127.4, 127.2 (2C), 113.5, 83.1

(J ¼ 166.5 Hz), 59.0, 55.2, 49.9, 49.7 (J ¼ 21 Hz), 16.7. HRMS (ESI)

calcd for C₂₆H₂₉NFO 390.2233 (M þ H)^þ, found 390.2235.

4.1.8. (R)-N¹-(3,3-diphenylallyl)-N¹-(1-(4-methoxyphenyl)ethyl)-

N²,N²-dimethylethane-1,2-diamine (12a)

¹H NMR (300 MHz, CDCl₃)

d 7.43e7.33 (m, 3H), 7.31e7.23 (m, 7H),

7.19e7.12 (m, 2H), 6.88e6.81 (m, 2H), 6.24 (t, J ¼ 6.6 Hz, 1H),

3.89e3.78 (m, 1H), 3.82 (s, 3H), 3.28e3.13 (m, 2H), 2.66e2.47 (m,

2H), 1.28 (d, J ¼ 6.7 Hz, 3H), 0.94 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR

A mixture of K₂CO₃(23 mg, 0.17 mmol), KI (cat.) and 11d (34 mg,

0.08 mmol) in MeCN (5 mL) was heated at reﬂux for 1 h. Then

dimethylamine (0.01 mL, 0.17 mmol) was added to the mixture and

stirred at reﬂux overnight. After completion of the reaction, the

contents were cooled to rt, poured into ice water and then

extracted with EtOAc. The combined organic phase was washed

with brine, dried over Na₂SO₄, and then concentrated under

reduced pressure. The residue was puriﬁed by preparative TLC

plates (DCM/MeOH ¼ 15/1) to give 12a (12 mg, 37%) as a colorless

(75 MHz, CDCl₃) d 158.3, 142.5, 139.8, 129.9, 128.6, 128.1, 128.1 (2C),

127.3, 127.1, 127.0, 113.4, 58.6, 55.2, 48.3, 43.6, 17.8, 12.1. HRMS (ESI)

calcd for C₂₆H₃₀NO 372.2322 (M þ H)^þ, found 372.2315.

4.1.4. (R)-N-allyl-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenylprop-

2-en-1-amine (11c)

Compound 11c (29 mg, 77%) was prepared by a similar

oil. ¹H NMR (300 MHz, CDCl₃)

d 7.41e7.32 (m, 3H), 7.31e7.22 (m,

12

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

7H), 7.15 (dd, J ¼ 7.7, 1.7 Hz, 2H), 6.87e6.79 (m, 2H), 6.23 (t,

J ¼ 6.6 Hz,1H), 3.87 (q, J ¼ 6.7 Hz,1H), 3.81 (s, 3H), 3.26 (qd, J ¼ 15.2,

6.7 Hz, 2H), 2.65e2.48 (m, 2H), 2.40e2.21 (m, 2H), 2.15 (s, 6H), 1.29

similar to that used to prepare compound 12a from 1-

methylpiperazine. The title compound was obtained as a colorless

oil. ¹H NMR (300 MHz, CDCl₃)

d

7.40e7.22 (m, 10H), 7.15 (dd, J ¼ 7.6,

(d, J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d

158.3, 143.0, 142.5,

1.8 Hz, 2H), 6.86e6.79 (m, 2H), 6.22 (t, J ¼ 6.6 Hz, 1H), 3.89 (q,

J ¼ 6.7 Hz, 1H), 3.80 (s, 3H), 3.33e3.16 (m, 2H), 2.71e2.29 (m, 12H),

2.28 (s, 3H), 1.27 (d, J ¼ 6.8 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

139.7, 136.4, 130.0, 128.7, 128.1 (2C), 128.0, 127.4, 127.2, 127.1, 113.4,

59.2, 58.1, 55.2, 49.28, 48.0, 45.8, 17.3. HRMS (ESI) calcd for

C

₂₈H₃₅N₂O 415.2749 (M þ H)^þ, found 415.2738.

d 158.3, 142.9, 142.5, 139.7, 136.3, 129.9, 128.7, 128.3, 128.1, 128.0,

127.4, 127.1 (2C), 113.4, 59.0, 57.2, 55.2, 55.0, 53.5, 49.5, 47.3, 46.0,

17.0. HRMS (ESI) calcd for C₃₁H₄₀N₃O 470.3171 (M þ H)^þ, found

470.3172.

4.1.9. (R)-N¹-(3,3-diphenylallyl)-N²,N²-diethyl-N¹-(1-(4-

methoxyphenyl)ethyl)ethane-1,2-diamine (12b)

Compound 12b (30 mg, 69%) was prepared by a procedure

similar to that used to prepare compound 12a from diethylamine.

The title compound was obtained as a colorless oil. ¹H NMR

4.1.14. (R)-4-(2-((3,3-Diphenylallyl) (1-(4-methoxyphenyl)ethyl)

amino)ethyl)piperazin-2-one (12g)

(300 MHz, CDCl₃)

d

7.40e7.21 (m, 10H), 7.15 (d, J ¼ 6.1 Hz, 2H), 6.83

Compound 12g (18 mg, 38%) was prepared by a procedure

similar to that used to prepare compound 12a from piperazin-2-

one. The title compound was obtained as a colorless oil. ¹H NMR

(d, J ¼ 7.6 Hz, 2H), 6.23 (t, J ¼ 6.1 Hz,1H), 3.88 (q, J ¼ 6.5 Hz,1H), 3.81

(s, 3H), 3.34e3.14 (m, 2H), 2.58e2.38 (m, 8H), 1.28 (d, J ¼ 6.3 Hz,

3H), 0.95 (t, J ¼ 7.0 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃)

d

158.3, 142.8,

(300 MHz, CDCl₃)

d

7.41e7.21 (m, 10H), 7.15 (d, J ¼ 7.0 Hz, 2H), 6.83

142.5, 139.7, 136.4, 130.0, 128.7, 128.4, 128.1, 128.0, 127.4, 127.1 (2C),

113.4, 59.1, 55.3, 51.9, 49.8, 48.1, 47.4, 16.9, 11.7. HRMS (ESI) calcd for

(d, J ¼ 8.0 Hz, 2H), 6.47 (s, 1H), 6.21 (t, J ¼ 6.5 Hz, 1H), 3.89 (q,

J ¼ 6.3 Hz, 1H), 3.81 (s, 3H), 3.37e3.16 (m, 4H), 3.04 (s, 2H),

2.64e2.47 (m, 4H), 2.37 (d, J ¼ 6.5 Hz, 2H), 1.27 (d, J ¼ 6.5 Hz, 3H).

C

₃₀H₃₉N₂O 443.3062 (M þ H)^þ, found 443.3059.

¹³C NMR (75 MHz, CDCl₃)

d 169.6, 158.4, 143.3, 142.4, 139.6, 136.1,

4.1.10. (R)-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenyl-N-(2-

(piperidin-1-yl)ethyl)prop-2-en-1-amine (12c)

Compound 12c (14 mg, 32%) was prepared by a procedure

similar to that used to prepare compound 12a from piperidine. The

title compound was obtained as a colorless oil. ¹H NMR (300 MHz,

129.9, 128.7, 128.2, 128.1, 127.6, 127.4, 127.3, 127.2, 113.4, 59.0, 57.3,

56.0, 55.2, 49.3, 49.2, 47.2, 41.3, 17.0. HRMS (ESI) calcd for

C

₃₀H₃₆N₃O₂470.2808 (M þ H)^þ, found 470.2806.

4.1.15. (R)-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenyl-N-(2-

(pyrrolidin-1-yl)ethyl)prop-2-en-1-amine (12h)

Compound 12h (26 mg, 59%) was prepared by a procedure

similar to that used to prepare compound 12a from pyrrolidine. The

title compound was obtained as a colorless oil. ¹H NMR (300 MHz,

CDCl₃)

d

7.42e7.21 (m, 10H), 7.16 (d, J ¼ 7.1 Hz, 2H), 6.83 (d,

J ¼ 7.6 Hz, 2H), 6.23 (t, J ¼ 6.4 Hz,1H), 3.89 (q, J ¼ 6.2 Hz,1H), 3.81 (s,

3H), 3.24 (t, J ¼ 6.5 Hz, 2H), 2.69e2.52 (m, 2H), 2.45e2.23 (m, 6H),

1.63e1.35 (m, 4H), 1.46e1.34 (m, 2H), 1.28 (d, J ¼ 6.7 Hz, 3H). ¹³

C

NMR (75 MHz, CDCl₃)

d

158.3, 142.7, 142.5, 139.7, 136.3, 130.0, 128.7,

CDCl₃)

d

7.43e7.20 (m, 10H), 7.15 (d, J ¼ 6.7 Hz, 2H), 6.82 (d,

128.5, 128.1 (2C), 127.4, 127.1 (2C), 113.4, 58.9, 58.1, 55.2, 55.0, 49.6,

47.4, 25.9, 24.38, 16.8. HRMS (ESI) calcd for C₃₁H₃₉N₂O 455.3062

(M þ H)^þ, found 455.3062.

J ¼ 7.8 Hz, 2H), 6.22 (t, J ¼ 6.2 Hz, 1H), 3.88 (dd, J ¼ 13.2, 6.5 Hz, 1H),

3.81 (s, 3H), 3.35e3.14 (m, 2H), 2.73e2.48 (m, 4H), 2.47e2.35 (m,

4H), 1.84e1.64 (m, 4H), 1.28 (d, J ¼ 6.1 Hz, 3H). ¹³C NMR (75 MHz,

CDCl₃)

d 158.3, 142.8, 142.5, 139.7, 136.3, 130.0, 128.7, 128.3, 128.1,

4.1.11. (R)-N-(1-(4-methoxyphenyl)ethyl)-N-(2-morpholinoethyl)-

3,3-diphenylprop-2-en-1-amine (12d)

Compound 12d (14 mg, 31%) was prepared by a procedure

similar to that used to prepare compound 12a from morpholine.

The title compound was obtained as a colorless oil. ¹H NMR

128.0, 127.4, 127.1 (2C), 113.4, 59.0, 55.2 (2C), 54.5, 49.6, 49.0, 23.4,

17.1. HRMS (ESI) calcd for C₃₀H₃₇N₂O 441.2906 (M þ H)^þ, found

441.2906.

4.1.16. (R)-N-(2-(azetidin-1-yl)ethyl)-N-(1-(4-methoxyphenyl)

ethyl)-3,3-diphenylprop-2-en-1-amine (12i)

Compound 12i (25 mg, 59%) was prepared by a procedure

similar to that used to prepare compound 12a from azetidine hy-

drochloride. The title compound was obtained as a colorless oil. ¹H

(300 MHz, CDCl₃)

d

7.43e7.12 (m, 12H), 6.87 (d, J ¼ 8.0 Hz, 2H), 6.22

(t, J ¼ 6.5 Hz, 1H), 3.89 (q, J ¼ 6.4 Hz, 1H), 3.81 (s, 3H), 3.77e3.61 (m,

4H), 3.26 (t, J ¼ 6.2 Hz, 2H), 2.67e2.53 (m, 2H), 2.43e2.29 (m, 6H),

1.28 (d, J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 158.3, 143.0,

142.5, 139.7, 136.3, 129.9, 128.7, 128.1 (2C), 127.4, 127.2, 127.1, 113.4,

66.9, 59.0, 57.7, 55.2, 54.1, 49.4, 47.0, 16.9. HRMS (ESI) calcd for

NMR (300 MHz, CDCl₃)

d

7.41e7.30 (m, 4H), 7.24 (dd, J ¼ 10.7,

6.0 Hz, 6H), 7.15 (dd, J ¼ 7.7, 1.7 Hz, 2H), 6.85e6.79 (m, 2H), 6.21 (t,

J ¼ 6.7 Hz, 1H), 3.87 (q, J ¼ 6.9 Hz, 1H), 3.80 (s, 3H), 3.35e3.17 (m,

6H), 2.56e2.42 (m, 4H), 2.18e2.09 (m, 2H), 1.26 (d, J ¼ 6.9 Hz, 3H).

C

₃₀H₃₇N₂O₂457.2855 (M þ H)^þ, found 457.2853.

4.1.12. (R)-N-(1-(4-methoxyphenyl)ethyl)-3,3-diphenyl-N-(2-

(piperazin-1-yl)ethyl)prop-2-en-1-amine (12e)

Compound 12e (21 mg, 45%) was prepared by a procedure

similar to that used to prepare compound 12a from piperazine. The

title compound was obtained as a colorless oil. ¹H NMR (300 MHz,

¹³C NMR (75 MHz, CDCl₃)

d 158.4, 143.3, 142.3, 139.6, 129.9, 128.8,

128.1 (2C), 127.4, 127.2 (C), 113.4, 59.1, 55.2, 55.0, 49.6, 47.0, 29.7,

17.5, 16.9. HRMS (ESI) calcd for C₂₉H₃₅N₂O 427.2749 (M þ H)^þ,

found 427.2740.

CDCl₃)

d

7.39e7.21 (m, 10H), 7.15 (d, J ¼ 6.6 Hz, 2H), 6.82 (d,

4.1.17. tert-Butyl (R)-4-(2-((1-(4-methoxyphenyl)ethyl)amino)

ethyl)piperazine-1-carboxylate (13)

J ¼ 7.0 Hz, 2H), 6.21 (t, J ¼ 6.0 Hz,1H), 3.88 (q, J ¼ 5.8 Hz,1H), 3.80 (s,

3H), 3.34e3.16 (m, 2H), 2.92e2.79 (m, 4H), 2.66e2.45 (m, 2H),

2.43e2.18 (m, 7H), 1.26 (d, J ¼ 6.0 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

A mixture of K₂CO₃(690 mg, 5 mmol), KI (cat.) and tert-butyl 4-

(2-chloroethyl)piperazine-1-carboxylate (450 mg, 2.4 mmol) in

MeCN (10 mL) was heated at 50 ^ꢀC for 1 h. Then (R)-1-(4-

methoxyphenyl)ethanamine 9a (310 mg, 2 mmol) was added to

the mixture and stirred at 80 ^ꢀC overnight. After completion of the

reaction, the contents were cooled to rt, removed the solvent,

diluted with water and then extracted with EtOAc (30 mL). The

organic phase was washed with brine, dried over Na₂SO₄, and

concentrated to yield 450 mg of amine 13 used directly for next

step.

d

158.3, 142.9, 142.5, 139.7, 136.3, 129.9, 128.7, 128.3, 128.1 (2C),

127.4, 127.2, 127.1, 113.4, 58.9, 57.9, 55.2, 54.8, 49.5, 47.1, 45.9, 16.9.

HRMS (ESI) calcd for C₃₀H₃₈N₃O 456.3015 (M þ H)^þ, found

456.3069.

4.1.13. (R)-N-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-

methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (12f)

Compound 12f (20 mg, 43%) was prepared by a procedure

13

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

4.1.18. (R,E)-N-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-

136.9,136.0, 128.7, 116.6, 113.4, 79.5, 58.5, 57.4, 55.2, 53.9, 53.4, 46.8,

28.4, 16.6.

methylpiperazin-1-yl)ethyl)-3-phenylprop-2-en-1-amine (14a)

A RB ﬂask with magnetic stir bar was charged with 1,2-

dichloroethane (10 mL), phosphotungstic acid hydrate (cat.),

crude 13 (100 mg, 0.27 mmol) and cinnamylaldehyde (28 mg,

0.22 mmol). To the stirred reaction solution under a nitrogen at-

mosphere was added NaBH(OAc)₃(87 mg, 0.41 mmol). The ﬂask

was sealed under nitrogen and stirred at rt for 48 h. The reaction

was worked up by the addition of sat. aq. NaHCO₃and EtOAc. The

combined EtOAc extracts were washed with brine, dried over

NaSO₄, ﬁltered, and condensed by rotary evaporation to yield a

crude oil. This material was further puriﬁed by silica gel column

chromatography using EtOAc/Hex (2/1) as the eluant to yield tert-

butyl (R)-4-(2-(cinnamyl (1-(4-methoxyphenyl)ethyl)amino)ethyl)

-piperazine-1-carboxylate (45 mg, 45%) as a colorless oil. ¹H NMR

To a solution of tert-butyl (R)-4-(2-(allyl (1-(4-methoxyphenyl)

ethyl)amino)ethyl) piperazine-1-carboxylate (28 mg, 0.07 mmol) in

EtOH (2 mL) was added 6 N HCl (1 mL) at 0 ^ꢀC. The mixture was

stirred at rt overnight. Part of the solvent was removed, and then

the residue was adjusted to pH 7e8 with aq. NH₄OH and extracted

with EtOAc. The combined EtOAc extracts were washed with brine,

dried Na₂SO₄, ﬁltered, and condensed by rotary evaporation to yield

a colorless oil used directly in the next step. To a solution of pre-

vious colorless oil and 37 wt% formaldehyde aqueous solution

(0.03 mL, 0.35 mmol) in 1,2-dichloroethane (4 mL) was added

acetic acid (cat.). 10 min later, NaBH(OAc)₃(30 mg, 0.14 mmol) was

then added to the stirred reaction solution. The ﬂask was stirred at

rt for 24 h. The reaction was worked up by the addition of sat. aq.

NaHCO₃and EtOAc. The combined EtOAc extracts were washed

with brine, dried Na₂SO₄, ﬁltered, and condensed under reduced

pressure. The residue was further puriﬁed by preparative TLC plates

using DCM/MeOH (10/1) as the eluant to yield 14b (14 mg, 64% for

two steps) as a colorless oil. ¹H NMR (300 MHz, Chloroform-d)

(300 MHz, CDCl₃)

d

7.41e7.19 (m, 7H), 6.88 (d, J ¼ 8.3 Hz, 2H), 6.52

(d, J ¼ 16.0 Hz, 1H), 6.32e6.19 (m, 1H), 3.93 (q, J ¼ 6.6 Hz, 1H), 3.82

(s, 3H), 3.46e3.36 (m, 4H), 3.35e3.19 (m, 2H), 2.76e2.58 (m 2H),

2.52e2.42 (m, 2H), 2.40e2.26 (m, 4H), 1.47 (s, 9H), 1.39 (d,

J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 158.4, 154.7, 137.3, 136.0,

131.7,128.7 (2C),128.6,127.3,126.2,113.4, 79.5, 58.8, 57.5, 55.2, 53.4,

53.3, 47.0, 28.4, 16.7.

d

7.27 (d, J ¼ 7.2 Hz, 2H), 6.85 (d, J ¼ 7.9 Hz, 2H), 5.94e5.76 (m, 1H),

5.23e5.03 (m, 2H), 3.86 (q, J ¼ 9.0 Hz, 1H), 3.81 (s, 3H), 3.22e2.99

To

a

solution of tert-butyl (R)-4-(2-(cinnamyl (1-(4-

piperazine-1-carboxylate

(m, 2H), 2.71e2.33 (m, 12H), 2.27 (s, 3H), 1.33 (d, J ¼ 6.5 Hz, 3H). ¹³

C

methoxyphenyl)ethyl)amino)ethyl)

NMR (75 MHz, CDCl₃)

d 158.3, 137.0, 136.1, 128.7, 116.5, 113.3, 58.5,

(45 mg, 0.09 mmol) in EtOH (2 mL) was added 6 N HCl (1 mL) at

0 ^ꢀC. The mixture was stirred at rt overnight. Part of the solvent was

removed, and then the pH of the residue was adjusted to 7e8 with

aq NH₄OH and extracted with EtOAc. The combined EtOAc extracts

were washed with brine, dried NaSO₄, ﬁltered, and condensed by

rotary evaporation to yield a colorless oil used directly in the next

step. To a solution of previous colorless oil and 37 wt% formalde-

hyde aqueous solution (0.04 mL, 0.47 mmol) in 1,2-dichloroethane

(5 mL) was added acetic acid (cat.). 10 min later, NaBH(OAc)₃

(40 mg, 0.19 mmol) was then added to the stirred reaction solution.

The ﬂask was stirred at rt for 24 h. The reaction was worked up by

the addition of sat. aq. NaHCO₃and EtOAc. The combined EtOAc

extracts were washed with brine, dried over NaSO₄, ﬁltered, and

condensed under reduced pressure. The residue was further puri-

ﬁed by preparative TLC plates using DCM/MeOH (10/1) as the

eluant to yield 14a (24 mg, 67% for two step) as a yellow oil. ¹H NMR

57.3, 55.2, 55.0, 53.9, 53.6, 46.8, 46.0, 16.7. HRMS (ESI) calcd for

C

₁₉H₃₂N₃O 318.2545 (M þ H)^þ, found 318.2547.

4.1.20. (S)eN-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-

methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (15a)

A RB ﬂask with activated 4 Å molecular sieves and dried anhy-

drous Na₂SO₄was charged with THF (2 mL), (S)-1-(4-

methoxyphenyl)ethanamine 9b (15 mg, 0.1 mmol) and b-phenyl-

cinnamylaldehyde (21 mg, 0.1 mmol) under a nitrogen atmosphere.

The reaction mixture was stirred at rt for 48 h, and then removed

the solvent and reﬁlled with MeOH (2 mL). To the solution was

added NaBH₄(4 mg, 0.1 mmol) at 0 ^ꢀC and reacted for 2 h. After

removing the solvent, the residue was diluted with water (10 mL)

and extracted with EtOAc (10 mL ꢁ 3). The combined organic phase

was acidized with 4 N aq. HCl to pH < 3 and extracted with Et₂O

(15 mL ꢁ 3). The aqueous phase was separated and basiﬁed with

10 N aq. NaOH to pH > 9, and then extracted with EA (15 mL ꢁ 3).

The combined EtOAc extracts were washed with brine, dried with

NaSO₄, ﬁltered, and condensed by rotary evaporation to as a

colorless oil used directly in the next step. To a solution of the crude

(300 MHz, CDCl₃)

d

7.40e7.15 (m, 7H), 6.87 (d, J ¼ 7.9 Hz, 2H), 6.51

(d, J ¼ 15.8 Hz, 2H), 6.32e6.17 (m, 1H), 3.92 (q, J ¼ 6.8 Hz, 1H), 3.81

(s, 3H), 3.37e3.18 (m, 2H), 2.78e2.61 (m, 2H), 2.60e2.33 (m, 10H),

2.27 (s, 3H), 1.38 (d, J ¼ 6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d

158.4, 137.3, 136.0, 131.6, 128.8, 128.7, 128.5, 127.2, 126.2, 113.4,

colorless oil and acetaldehyde (50 mL, 0.10 mmol) in 1,2-

58.8, 57.4, 55.2, 55.0, 53.6, 53.4, 47.0, 46.0, 16.9. HRMS (ESI) calcd for

C

dichloroethane (2 mL) was added acetic acid (cat.). 10 min later,

NaBH(OAc)₃(21 mg, 0.1 mmol) was added to the stirred reaction

solution. The ﬂask was stirred at rt for 2 h. The reaction was worked

up by the addition of sat. aq. NaHCO₃and EtOAc. The combined

EtOAc extracts were washed with brine, dried over Na₂SO₄, ﬁltered,

and condensed under reduced pressure. The residue was added to a

solution of K₂CO₃(23 mg, 0.17 mmol), KI (cat.) in MeCN (5 mL). The

₂₅H₃₆N₃O 394.2858 (M þ H)^þ, found 394.2860.

4.1.19. (R)-N-(1-(4-methoxyphenyl)ethyl)-N-(2-(4-

methylpiperazin-1-yl)ethyl)prop-2-en-1-amine (14b)

To a solution of crude 13 (61 mg, 0.17 mmol) in MeCN (4 mL) was

added K₂CO₃(60 mg, 0.44 mmol), KI (cat.) and propargyl bromide

(0.03 mL, 0.33 mmol). Then the mixture was stirred at 50 ^ꢀC for 3 h.

After removing the solvent, the residue was diluted with water

(8 mL) and extracted with EtOAc (15 mL ꢁ 2). The combined EtOAc

extracts were washed with brine, dried Na₂SO₄, ﬁltered, and

condensed by rotary evaporation to yield a crude oil. This material

was further puriﬁed by preparative TLC plates using EtOAc/Hex as

the eluant to yield tert-butyl (R)-4-(2-(allyl (1-(4-methoxyphenyl)

ethyl)amino)ethyl)piperazine-1-carboxylate (28 mg, 41%). ¹H NMR

mixture solution was heated at reﬂux for

1 h. Then 1-

methylpiperazine (22 L, 0.2 mmol) was added to the mixture

m

and stirred at reﬂux overnight. After completion of the reaction, the

contents were cooled to rt, poured into ice water and then

extracted with EtOAc. The combined organic phase was washed

with brine, dried over Na₂SO₄, and then concentrated under

reduced pressure. The residue was puriﬁed by preparative TLC

plates (DCM/MeOH ¼ 15/1) to give 15a (25 mg, 53%) as a yellow oil.

(300 MHz, CDCl₃)

d

7.27 (d, J ¼ 7.5 Hz, 2H), 6.85 (d, J ¼ 7.6 Hz, 2H),

¹H NMR (300 MHz, CDCl₃)

d 7.40e7.32 (m, 3H), 7.29e7.23 (m, 7H),

5.85 (td, J ¼ 16.0, 6.2 Hz, 1H), 5.13 (dd, J ¼ 23.2, 13.6 Hz, 2H),

3.91e3.83 (q, J ¼ 6.0 Hz, 1H), 3.81 (s, 3H), 3.48e3.32 (m, 4H),

3.22e2.99 (m, 2H), 2.68e2.48 (m, 2H), 2.48e2.25 (m, 6H), 1.46 (s,

7.18e7.11 (m, 2H), 6.82 (d, J ¼ 8.7 Hz, 2H), 6.21 (t, J ¼ 6.6 Hz, 1H),

3.88 (q, J ¼ 6.8 Hz, 1H), 3.81 (s, 3H), 3.32e3.13 (m, 2H), 2.64e2.55

(m, 2H), 2.55e2.29 (m, 10H), 2.28 (s, 3H), 1.27 (d, J ¼ 6.8 Hz, 3H). ¹³

C

9H), 1.33 (d, J ¼ 6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d

158.3, 154.7,

NMR (75 MHz, CDCl₃) d 158.3, 142.9, 142.5, 139.7, 136.3, 129.9, 128.7,

14

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

128.3, 128.1 (4C), 127.4, 127.2, 127.1, 113.4, 59.0, 57.2, 55.2, 55.0, 53.5,

49.5, 47.3, 46.0, 17.0. HRMS (ESI) calcd for C₃₁H₄₀N₃O 470.3171

(M þ H)^þ, found 470.3163.

1-(4-chlorophenyl)ethanamine 9g. The title compound was ob-

tained as a colorless oil. ¹H NMR (300 MHz, CDCl₃)

d 7.41e7.32 (m,

3H), 7.32e7.19 (m, 9H), 7.13 (dd, J ¼ 7.5, 1.8 Hz, 2H), 6.19 (t,

J ¼ 6.6 Hz, 1H), 3.89 (d, J ¼ 6.7 Hz, 1H), 3.23 (dd, J ¼ 6.5, 4.8 Hz, 2H),

2.63e2.34 (m, 12H), 2.29 (s, 3H), 1.27 (t, J ¼ 5.9 Hz, 3H). ¹³C NMR

4.1.21. (R)-N-(1-(2-methoxyphenyl)ethyl)-N-(2-(4-

methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (15b)

Compound 15b (11 mg, 23%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

1-(2-methoxyphenyl)ethanamine 9c. The title compound was ob-

(75 MHz, CDCl₃) d 143.2, 143.0, 142.3, 139.6, 132.2, 129.9, 129.0,

128.2,128.1, 127.8, 127.4, 127.3, 127.2, 59.0, 57.1, 55.0, 53.5, 49.5, 47.3,

46.0, 16.7. HRMS (ESI) calcd for C₃₀H₃₇N₃Cl 474.2676 (M þ H)^þ,

found 474.2675.

tained as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

d 7.44e7.13 (m,

12H), 6.96e6.82 (m, 2H), 6.29 (t, J ¼ 6.3 Hz, 1H), 4.40 (q, J ¼ 6.6 Hz,

1H), 3.81 (s, 3H), 3.41e3.23 (m, 2H), 2.76e2.61 (m, 2H), 2.56e2.29

(m, 10H), 2.27 (s, 3H), 1.26 (d, J ¼ 6.8 Hz, 3H)$¹³C NMR (75 MHz,

4.1.26. (R)-N-(1-(3-chlorophenyl)ethyl)-N-(2-(4-methylpiperazin-

1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (15g)

Compound 15g (17 mg, 36%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

1-(3-chlorophenyl)ethanamine 9h. The title compound was ob-

CDCl₃)

d 157.0, 142.6, 142.6, 139.7, 133.1, 129.9, 128.3, 128.1, 128.0,

127.7, 127.4 (2C), 127.1, 127.0, 120.4, 110.5, 56.6, 55.4, 55.1, 53.5, 52.7,

49.8, 47.6, 46.0, 18.7. HRMS (ESI) calcd for C₃₁H₄₀N₃O 470.3171

(M þ H)^þ, found 470.3167.

tained as a colorless oil. ¹H NMR (300 MHz, CDCl₃)

d 7.39e7.17 (m,

12H), 7.14 (d, J ¼ 7.0 Hz, 2H), 6.19 (t, J ¼ 6.3 Hz, 1H), 3.89 (q,

J ¼ 6.5 Hz, 1H), 3.34e3.13 (m, 2H), 2.58 (t, J ¼ 7.2 Hz, 2H), 2.54e2.30

(m, 10H), 2.28 (s, 3H), 1.26 (d, J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz,

4.1.22. (R)-N-(1-(3-methoxyphenyl)ethyl)-N-(2-(4-

methylpiperazin-1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (15c)

Compound 15c (15 mg, 32%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

1-(3-methoxyphenyl)ethanamine 9d. The title compound was ob-

CDCl₃) d 146.9, 143.2, 142.3, 139.5, 134.0, 129.9, 129.3, 128.1 (2C),

127.8, 127.7, 127.4, 127.2 (2C), 126.8, 125.8, 59.2, 57.1, 55.0, 53.6, 49.5,

47.4, 46.0, 16.6. HRMS (ESI) calcd for C₃₀H₃₇N₃Cl 474.2676 (M þ H)^þ,

found 474.2680.

tained as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

d 7.38e7.32 (m,

3H), 7.30e7.22 (m, 6H), 7.17e7.11 (m, 2H), 6.94 (d, J ¼ 8.8 Hz, 2H),

6.80e6.74 (m, 1H), 6.22 (t, J ¼ 6.6 Hz, 1H), 3.89 (q, J ¼ 6.6 Hz, 1H),

3.81 (s, 3H), 3.27 (dd, J ¼ 6.4, 3.8 Hz, 2H), 2.61 (t, J ¼ 7.5 Hz, 2H),

2.53e2.31 (m, 10H), 2.28 (s, 3H), 1.27 (d, J ¼ 6.5 Hz, 3H). ¹³C NMR

4.1.27. (R)-N-(2-(4-methylpiperazin-1-yl)ethyl)-N-(1-(4-

nitrophenyl)ethyl)-3,3-diphenylprop-2-en-1-amine (15h)

Compound 15h (7 mg, 15%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

1-(4-nitrophenyl)ethanamine 9i. The title compound was obtained

(75 MHz, CDCl₃) d 159.5,146.4,143.0,142.4,139.6,129.9,129.0,128.1

(3C), 128.0, 127.4, 127.2, 127.1 (2C), 113.4, 111.9, 59.8, 57.0, 55.2, 55.0,

53.4, 49.5, 47.4, 46.0, 17.2. HRMS (ESI) calcd for C₃₁H₄₀N₃O 470.3171

(M þ H)^þ, found 470.3171.

as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

d

8.13 (d, J ¼ 8.8 Hz, 2H),

7.52 (d, J ¼ 8.6 Hz, 2H), 7.37e7.22 (m, 8H), 7.15e7.08 (m, 2H), 6.18 (t,

J ¼ 6.7 Hz, 1H), 4.01 (q, J ¼ 6.7 Hz, 1H), 3.24 (dd, J ¼ 6.7, 3.6 Hz, 2H),

2.64e2.56 (m, 2H), 2.56e2.31 (m, 10H), 2.30 (s, 3H), 1.29 (d,

4.1.23. (R)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3,3-diphenyl-N-

(1-phenylethyl)prop-2-en-1-amine (15d)

J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 152.6, 146.8, 143.7, 142.2,

139.4, 129.8, 128.3, 128.2 (3C), 127.4, 127.3, 127.1, 123.4, 59.2, 57.0,

54.9, 53.4, 49.4, 47.4, 45.9, 16.5. HRMS (ESI) calcd for C₃₀H₃₇N₄O₂

485.2917 (M þ H)^þ, found 485.2917.

Compound 15d (18 mg, 41%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

1-phenylethanamine 9e. The title compound was obtained as a

colorless oil. ¹H NMR (300 MHz, CDCl₃)

d

7.39e7.22 (m, 13H),

4.1.28. (R)-Methyl 2-((3,3-diphenylallyl) (1-(4-methoxyphenyl)

ethyl)amino)acetate (16)

Compound 16 (764 mg, 92%) was prepared by a similar pro-

cedure to that of compound 11a from methyl chloroacetate. The

title compound was obtained as a colorless oil. ¹H NMR (300 MHz,

7.18e7.13 (m, 2H), 6.23 (t, J ¼ 6.6 Hz, 1H), 3.93 (d, J ¼ 6.7 Hz, 1H),

3.27 (t, J ¼ 6.3 Hz, 2H), 2.65e2.57 (m, 2H), 2.56e2.30 (m, 10H), 2.28

(s, 3H), 1.30 (d, J ¼ 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 144.4,

143.0, 142.5, 139.7, 129.9, 128.1 (4C), 127.7, 127.4, 127.2, 127.1, 126.7,

59.7, 57.1, 55.0, 53.5, 49.6, 47.4, 46.0, 17.0. HRMS (ESI) calcd for

CDCl₃)

d 7.43e7.27 (m, 10H), 7.21e7.13 (m, 2H), 6.93e6.85 (m, 2H),

C

₃₀H₃₈N₃440.3066 (M þ H)^þ, found 440.3066.

6.28 (t, J ¼ 6.9 Hz, 1H), 4.07 (q, J ¼ 6.7 Hz, 1H), 3.83 (s, 3H), 3.63 (s,

3H), 3.52e3.28 (m, 4H), 1.34 (d, J ¼ 6.7 Hz, 3H)$¹³C NMR (75 MHz,

4.1.24. (R)-N-(1-(4-Fluorophenyl)ethyl)-N-(2-(4-methylpiperazin-

1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (15e)

CDCl₃) d 172.6, 158.6, 143.8, 142.2, 139.5, 136.3, 129.9, 128.6, 128.2,

128.1, 127.4, 127.2 (2C), 113.6, 59.6, 55.2, 51.4, 51.3, 50.1, 19.3. HRMS

Compound 15e (14 mg, 31%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

1-(4-ﬂuorophenyl)ethanamine 9f. The title compound was ob-

(ESI) calcd for C₂₇H₃₀NO₃416.2226 (M þ H)^þ, found 416.2227.

4.1.29. (R)-2-((3,3-Diphenylallyl) (1-(4-methoxyphenyl)ethyl)

amino)acetic acid (17)

tained as a colorless oil. ¹H NMR (300 MHz, CDCl₃)

d 7.38e7.24 (m,

10H), 7.16e7.10 (m, 2H), 7.00e6.92 (m, 2H), 6.20 (t, J ¼ 6.6 Hz, 1H),

3.91 (q, J ¼ 6.7 Hz, 1H), 3.24 (t, J ¼ 6.3 Hz, 2H), 2.62e2.54 (m, 2H),

2.53e2.31 (m, 10H), 2.29 (s, 3H), 1.26 (d, J ¼ 6.7 Hz, 3H). ¹³C NMR

A solution of lithium hydroxide (84 mg, 2.0 mmol) in water

(1 mL) was added to the solution of ester 16 (415 mg, 1.0 mmol) in

THF (3 mL). The mixture was stirred overnight. After removal of the

solvent, the residue was diluted with water (2 mL), acidiﬁed with

6 N HCl to pH ¼ 5, and then extracted with EtOAc (15 mL ꢁ 2). The

combined organic layer was washed with brine (10 mL), dried,

ﬁltered, and then evaporated to yield 17 (374 mg, 93%) as a white

(75 MHz, CDCl₃)

d

161.7 (d, J ¼ 242.9 Hz), 143.2, 142.4, 140.0 (2C),

139.6, 129.9 (3C), 129.1, 129.0, 128.2 (2C), 128.1 (4C), 127.8, 127.4

(2C), 127.3, 127.2 (2C), 114.9, 114.6, 58.9, 57.1, 54.9, 53.4, 49.5, 47.2,

45.9, 16.8. HRMS (ESI) calcd for C₃₀H₃₇N₃F 458.2972 (M þ H)^þ,

found 458.2962.

solid. ¹H NMR (300 MHz, CDCl₃þ CD₃OD)

d 7.27e7.11 (m, 8H),

7.09e6.94 (m, 4H), 6.66 (d, J ¼ 8.7 Hz, 2H), 6.24 (t, J ¼ 6.5 Hz, 1H),

4.07 (q, J ¼ 6.9 Hz, 1H), 3.68 (s, 3H), 3.38 (dd, J ¼ 14.8, 5.9 Hz, 1H),

3.31e3.08 (m, 2H), 2.87 (d, J ¼ 16.1 Hz, 1H), 1.30 (d, J ¼ 6.9 Hz,

4.1.25. (R)-N-(1-(4-chlorophenyl)ethyl)-N-(2-(4-methylpiperazin-

1-yl)ethyl)-3,3-diphenylprop-2-en-1-amine (15f)

Compound 15f (13 mg, 28%) was prepared for three steps by a

procedure similar to that used to prepare compound 15a from (R)-

3H)$¹³C NMR (75 MHz, CDCl₃þ CD₃OD)

d 176.4, 158.8, 144.8, 141.8,

139.0, 131.2, 129.6, 129.5, 128.1, 127.4, 124.5, 113.5, 59.6, 55.1, 54.4,

15

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

50.4, 17.2. HRMS (ESI) calcd for C₂₆H₂₈NO₃402.2069 (M þ H)^þ,

d

158.4, 143.1, 142.4, 139.7, 136.0, 130.8, 129.9, 128.7 (2C), 128.4,

found 402.2082.

128.2, 128.1, 127.4, 127.2 (2C), 113.4, 58.4, 55.2, 48.8, 46.8, 43.3, 30.8,

16.4.

4.1.30. (R)-2-((3,3-Diphenylallyl) (1-(4-methoxyphenyl)ethyl)

amino)-1-(4-methylpiperazin-1-yl)ethanone (18)

4.1.34. (R)-N-(1-(4-methoxyphenyl)ethyl)-N-(3-(4-

To a solution of 17 (200 mg, 0.5 mmol) in DCM (4 mL), 1-

methylpiperazine (100 mg, 2 mmol), EDCI (192 mg, 2 mmol),

HOBt (68 mg, 0.5 mmol) and DIPEA (194 mg, 1.54 mmol) were

successively added. The mixture was stirred for overnight, diluted

with ice water (8 mL) and then extracted with EtOAc

(15 mL ꢁ 2).The combined organic phase was washed with brine,

dried over Na₂SO₄, and then concentrated under reduced pressure.

The residue was puriﬁed by preparative TLC plates (Hexane/EtOAc)

to give the 18 (200 mg, 83%) as a yellow oil. ¹H NMR (600 MHz,

methylpiperazin-1-yl)propyl)-3,3-diphenylprop-2-en-1-amine (22)

Compound 22 (29 mg, 60%) was prepared by a similar procedure

to that of compound 11a from 21. The title compound was obtained

as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

d 7.40e7.19 (m, 10H), 7.14

(d, J ¼ 6.8 Hz, 2H), 6.82 (d, J ¼ 8.5 Hz, 2H), 6.23e6.16 (m,1H), 3.85 (q,

J ¼ 6.2 Hz,1H), 3.80 (s, 3H), 3.19 (m, 2H), 2.65e2.21 (m,12H), 2.28 (s,

3H), 1.61e1.45 (m, 2H), 1.24 (d, J ¼ 6.6 Hz, 3H)$¹³C NMR (75 MHz,

CDCl₃)

d 158.2, 142.7, 142.5, 139.7, 136.5, 129.9, 128.7, 128.5, 128.1,

128.0, 127.3, 127.1 (2C), 113.3, 58.4, 56.6, 55.2, 55.1, 53.2, 48.7, 47.8,

46.1, 24.7, 16.9. HRMS (ESI) calcd for C₃₂H₄₂N₃O 484.3328 (M þ H)^þ,

found 484.3325.

CDCl₃)

d

7.38e7.27 (m, 6H), 7.26e7.24 (m, 2H), 7.22 (d, J ¼ 8.7 Hz,

2H), 7.14 (dd, J ¼ 8.1, 1.5 Hz, 2H), 6.84 (d, J ¼ 8.6 Hz, 2H), 6.23e6.18

(m, 1H), 4.03 (q, J ¼ 6.8 Hz, 1H), 3.81 (s, 3H), 3.57 (m, 2H), 3.48e3.44

(m, 2H), 3.27 (td, J ¼ 14.7, 6.8 Hz, 2H), 3.22 (d, J ¼ 3.0 Hz, 2H), 2.34

(m, 4H), 2.30 (s, 3H), 1.23 (d, J ¼ 6.9 Hz, 3H). ¹³C NMR (150 MHz,

4.1.35. (R)-N-(3,3-diphenylallyl)-4,4-diethoxy-N-(1-(4-

methoxyphenyl)ethyl)butan-1-amine (23)

Compound 23 (420 mg, 43%) was prepared by a similar pro-

cedure to that of compound 11a from 4-chlorobutyraldehyde

diethyl acetal. The title compound was obtained as a yellow oil. ¹H

CDCl₃)

d 169.7, 158.6, 143.9, 142.3, 139.4, 134.6, 129.9, 129.1, 128.2,

128.1, 127.4, 127.3, 113.4, 58.3, 55.3, 55.2, 54.7, 53.3, 49.5, 46.0, 45.1,

41.6, 14.6. HRMS (ESI) calcd for C₃₁H₃₈N₃O₂484.2964 (M þ H)^þ,

found 484.2979.

NMR (300 MHz, CDCl₃)

d

7.43e7.31 (m, 3H), 7.29e7.19 (t, J ¼ 8.8 Hz,

4.1.31. (R)-2-Chloro-N-(3,3-diphenylallyl)-N-(1-(4-methoxyphenyl)

ethyl)acetamide (19)

7H), 7.14 (d, J ¼ 6.3 Hz, 2H), 6.82 (d, J ¼ 7.3 Hz, 2H), 6.22 (d,

J ¼ 6.0 Hz,1H), 4.39 (t, J ¼ 6.0 Hz,1H), 3.86 (q, J ¼ 6.0 Hz,1H), 3.81 (s,

3H), 3.68e3.54 (m, 2H), 3.52e3.37 (m, 2H), 3.25e3.09 (m, 2H),

2.54e2.31 (m, 2H), 1.61e1.50 (m, 2H), 1.47e1.36 (m, 2H), 1.28e1.18

To a solution of 10a (343 mg, 1 mmol) in CH₂Cl₂(4 mL) was

added Et₃N (0.4 mL, 3 mmol), then 2-chloroacetyl chloride (134 mg,

1.2 mmol) was added to the stirred reaction solution. The mixture

was stirred at rt for 2 h. The reaction was worked up by the addition

of sat. aq. NaHCO₃and EtOAc. The combined EtOAc extracts were

washed with brine, dried over Na₂SO₄, ﬁltered, and condensed

under reduced pressure. The residue was further puriﬁed by pre-

parative TLC plates using Hexane/EtOAc (5/1) as the eluant to yield

19 (303 mg, 72%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

(m, 9H). ¹³C NMR (75 MHz, CDCl₃)

d 158.2, 142.7, 142.5, 139.8, 136.5,

129.9, 128.7, 128.1, 128.0, 127.3, 127.1, 113.3, 102.9, 60.9, 60.8, 58.4,

55.2, 49.4, 48.7, 31.3, 22.6, 16.7, 15.4. HRMS (ESI) calcd for

C

₃₂H₄₂NO₃488.3165 (M þ H)^þ, found 488.3165.

4.1.36. (R)-4-((3,3-Diphenylallyl) (1-(4-methoxyphenyl)ethyl)

amino)butanal (24)

d

7.46e7.34 (m, 3H), 7.30e7.21 (m, 4H), 7.12 (td, J ¼ 7.4, 7.0, 3.0 Hz,

5H), 6.84 (dd, J ¼ 18.4, 8.3 Hz, 2H), 6.04 (q, J ¼ 7.0 Hz, 1H), 3.99 (s,

To a solution of compound 23 (300 mg, 0.62 mmol) in 1,4-

dioxane (9 mL) was added 6 N HCl (9 mL) at 0 ^ꢀC. The mixture

was stirred at 50 ^ꢀC for 2 h. After cooling to rt, the mixture was

poured into cold NH₄OH aq. and extracted with EtOAc (20 mL ꢁ 2).

The combined EtOAc extracts were washed with brine, dried

Na₂SO₄, ﬁltered, and condensed by rotary evaporation. The residue

was further puriﬁed by preparative TLC plates to give 24 (236 mg,

2H), 3.92e3.68 (m, 5H), 1.55 (d, J ¼ 7.1 Hz, 3H). ¹³C NMR (75 MHz,

CDCl₃)

d 166.8, 159.1, 144.1, 141.2, 138.2, 132.0, 129.8, 129.7, 128.9,

128.6, 128.3, 128.1, 127.9, 127.5, 125.4, 114.0, 55.3, 51.4, 42.4, 41.9,

16.6.

4.1.32. (R)-N-(3,3-diphenylallyl)-N-(1-(4-methoxyphenyl)ethyl)-2-

(4-methylpiperazin-1-yl)acetamide (20)

92%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

d

9.71 (t, J ¼ 1.7 Hz,

Compound 20 (106 mg, 44%) was prepared by a similar pro-

cedure to that of compound 11a from 19. The title compound was

1H), 7.40e7.17 (m, 12H), 6.89e6.81 (m, 2H), 6.21 (t, J ¼ 6.7 Hz, 1H),

3.91 (q, J ¼ 6.8 Hz, 1H), 3.82 (s, 3H), 3.21 (t, J ¼ 6.6 Hz, 2H),

obtained as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

d 7.42e7.30 (m,

2.52e2.31 (m, 4H), 1.75e1.59 (m, 2H), 1.26 (d, J ¼ 6.8 Hz, 3H). ¹³

C

3H), 7.29e6.99 (m, 9H), 6.80 (dd, J ¼ 20.6, 8.7 Hz, 2H), 6.09e5.92

(m, 1H), 5.57 (dt, J ¼ 79.7, 6.6 Hz, 1H), 3.98 (ddd, J ¼ 13.9, 8.6, 5.9 Hz,

1H), 3.89e3.58 (m, 4H), 3.44e2.86 (m, 2H), 2.68e2.24 (m,11H),1.49

NMR (75 MHz, CDCl₃) d 202.6, 158.4,143.3,142.4,139.7, 135.9,129.9,

128.8, 128.2, 128.1, 127.9, 127.4, 127.2 (2C), 113.4, 58.2, 55.2, 48.6,

48.5, 41.7, 20.5, 16.2.

(dd, J ¼ 13.1, 7.0 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 169.5, 169.0,

158.8,143.1,142.1,141.7,138.5,132.9,129.9,128.8,128.3,128.2,128.1,

127.9, 127.8, 127.6, 127.5, 127.4, 127.2, 126.7, 113.8, 62.2, 60.6, 55.3,

55.0, 54.8, 53.2, 50.5, 45.8, 41.8, 29.7, 16.9. HRMS (ESI) calcd for

4.1.37. (R)-N-(3,3-diphenylallyl)-N-(1-(4-methoxyphenyl)ethyl)-4-

(4-methylpiperazin-1-yl)butan-1-amine (25)

C

₃₁H₃₈N₃O₂484.2964 (M þ H)^þ, found 484.2976.

Compound 25 (26 mg, 52%) was prepared by a similar procedure

to that of compound 11b from 24. The title compound was obtained

4.1.33. (R)-N-(3-chloropropyl)-N-(1-(4-methoxyphenyl)ethyl)-3,3-

diphenylprop-2-en-1-amine (21)

as a colorless oil. ¹H NMR (300 MHz, CDCl₃)

d 7.41e7.32 (m, 3H),

7.29e7.21 (m, 7H), 7.14 (dd, J ¼ 7.7, 1.7 Hz, 2H), 6.82 (d, J ¼ 8.7 Hz,

2H), 6.21 (t, J ¼ 6.6 Hz, 1H), 3.84 (t, J ¼ 6.6 Hz, 1H), 3.81 (s, 3H), 3.18

(dd, J ¼ 6.4, 5.4 Hz, 2H), 2.60e2.32 (m, 10H), 2.30 (s, 3H), 2.29e2.23

(m, 2H), 1.46e1.31 (m, 4H), 1.25 (d, J ¼ 6.7 Hz, 3H). ¹³C NMR

Compound 21 (101 mg, 24%) was prepared by a similar pro-

cedure to that of compound 11a from 1-bromo-3-chloropropane

with DIPEA as the base. The title compound was obtained as a

yellow oil. ¹H NMR (300 MHz, CDCl₃)

d

7.28 (m, 10H), 6.85 (d,

(75 MHz, CDCl₃) d 158.2, 142.7, 142.5, 139.8, 136.6, 129.9, 128.7,

J ¼ 7.9 Hz, 2H), 6.21 (t, J ¼ 6.4 Hz, 1H), 3.95e3.85 (m, 1H), 3.82 (s,

3H), 3.66e3.48 (m, 2H), 3.28e3.10 (m, 2H), 2.70e2.44 (m, 2H),

1.91e1.70 (m, 2H), 1.29 (d, J ¼ 6.8 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

128.6, 128.1, 128.0, 127.3, 127.1 (2C), 113.3, 58.6, 58.5, 55.2, 55.1, 53.2,

49.6, 48.7, 46.1, 25.3, 24.7, 17.0. HRMS (ESI) calcd for C₃₃H₄₄N₃O

498.3484 (M þ H)^þ, found 498.3475.

16

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

4.2. Cell lines and cell culture

plus protease inhibitors. SDS-PAGE and immunoblotting were

performed using lysates containing 20 mg of total protein. Signals

Madin-Darby Canine Kidney epithelial cells (MDCK) stably co-

expressing mGFP-tagged full length oncogenic mutant KRAS

(GFP-KRASG12V) or mGFP-LactC2 (peptide probe for phosphati-

dylserine) and mCherry-tagged to the amino acids 179e189 of

HRAS C181S, C184S (mCherry-CAAX, localizes primarily to endo-

membranes) were grown in DMEM-high glucose/sodium pyruvate/

10% FBS. The medium was supplemented with 1 ꢁ Penicillin/

Streptomycin for ﬂuorescence microscopy assays, to avoid any

microbial contamination from the test compounds added. KLE and

Hec50 cells were maintained in DMEMeF-12 medium supple-

mented with 10% FBS. Hec-1A and Hec-1B cells were grown in

McCoy’s 5a medium supplemented with 10% FBS. ESS-1 cells were

grown in RPMI 1640 medium supplemented with 20% FBS.

MPanc96 cells were grown in DMEM supplemented with 10% FBS,

MiaPaCa-2 cells in DMEM supplemented with 10% FBS and 2.5%

horse serum and all other cell lines were grown in RPMI 1640

supplemented with 10% FBS. All cancer cell media were supple-

mented with ꢁ Penicillin/Streptomycin. All cell lines were grown at

37 ^ꢀC with 5% CO₂.

were detected by enhanced chemiluminescence (Thermo Fisher

Scientiﬁc) and imaged using a FluorChemQ imager (Alpha Inotech).

Quantiﬁcation of intensities was performed using FluorChemQ

software.

4.6. ASM add back assay

1.75 ꢁ 10⁵MDCK cells stably co-expressing mGFPeKRASG12V

and mCherry-CAAX were seeded on a glass coverslip in a 12-well

plate and grown with or without compounds for 48 h.

Medium was replaced with fresh medium with or without

compounds containing 2 units/mL ASM and the incubation was

continued for 60 min. The coverslips were mounted in mowiol and

imaged by confocal microscopy (Nikon A1) using a 60ꢁ objective.

4.7. Proliferation assay

Cells were seeded in 96-well plates. After 24 h, fresh growth

medium supplemented with 0.1% vehicle (DMSO), or various con-

centrations of drugs was added and cells were grown for 72 h. Cell

numbers were quantiﬁed using the CyQUANT® Cell Proliferation

Assay Kit (Molecular Probes, Life Technologies), according to the

manufacturer’s protocol. Fluorescence measurement was used as a

measure of live cell number.

4.3. KRAS/LactC2 mislocalization assay

MDCK co-expressing GFP-KRASG12V or lactadherin-C2 domain

(LactC2) and mCherry-CAAX were grown on coverslips, treated

with 0.1% vehicle (DMSO) or various concentrations of drugs for

48 h, and ﬁxed with 4% paraformaldehyde. The coverslips were

mounted in mowiol and imaged by confocal microscopy (Nikon A1)

using a 60 ꢁ objective. Using ImageJ software v1.42q, images were

converted to 8-bit, and a threshold to a control pixel of each image

was set. As a measure of KRAS/LactC2 mislocalization, the fraction

of mCherry-CAAX co-localizing with mGFP-KRASG12V was calcu-

lated using a Manders coefﬁcient plugin downloaded from Wright

Cell Image Facility. The fraction of mCherry-CAAX co-localizing

with mGFP-RASG12V (Mander’s coefﬁcient) is proportional to

KRAS mislocalization.

4.8. C. elegans vulva quantiﬁcation assay

Strain let-60 or lin-1 L1 larvae were cultured in M9 buffer con-

taining Escherichia coli (E.coli) OP50 in presence of DMSO or com-

pounds. After 4e5 days, when worms reached the adult stage, they

were scored for the presence of the multi-vulva phenotype using a

DIC/Nomarski microscope.

4.9. In vivo tumor growth assay

All animal studies were performed under an Institutional Ani-

mal Care and Use Committee (IACUC) approved animal protocol

(AWC-15-0101), in accordance with the National Institutes of

Health Guide for the Care and Use of Laboratory Animals. Early

passage MiaPaCa-2 cells were harvested, and 2 ꢁ 10⁶cells were

implanted into the right ﬂanks of female nu/nu mice. The animals

were randomized into control and treated groups (10 mice per

group). Tumor volume (V) was measured with an external caliper

every 3e4 days and it was calculated as V ¼ 0.52 (length ꢁ width²).

Compound treatment was initiated when the tumor sizes reached

100 mm³. Compounds (1, 2.5 and 5 mg/kg) were injected daily

intraperitoneally for 5 days, with 2 days of no treatment in be-

tween. All treatments were continued until any of the subcutane-

ous tumors reached 1500 mm³in volume, when all the animals

were sacriﬁced and the tumors removed.

4.4. ASM activity assay

The ASM activity assay was performed with Amplex Red SMase

Assay Kit (A12220; Invitrogen; Carlsbad, CA) according to the

manufacturer’s instructions. Brieﬂy, 10

g/ L concentration) from MDCK cells stably expressing human

ASM (SMPD1)-GFP prepared in lysis buffer B without DTT was

mixed with 40 L of low-pH buffer (50 mM sodium acetate, pH 5.0),

and loaded on a well of black 96- well plate. 5 L of 5 mM sphin-

mg of whole-cell lysates (at

1

m m

m

gomyelin was added to each well, and the plate was incubated in

dark in 37 ^ꢀC for 1 h. After the incubation, the pH was raised to 7.4

by adding 50 mL of Amplex Red reaction mixture [100 mM Amplex

Red reagent, 2 U/mL horseradish peroxidase, 0.2 U/mL choline

oxidase, 8 U/mL alkaline phosphatase in high-pH buffer (100 mM

Tris-HCl, pH 8.0)]. The plate was further incubated in the dark in

37 ^ꢀC for 60 min, and the ﬂuorescence was measured using BioTek

4.10. Immunohistochemistr

Synergy H1 microplate reader (excitation

l

¼ 540 nm, emission

l

¼ 590 nm). 0.1 U/mL SMase and low-pH buffer were used as a

4e5 mm sections were deparafﬁnized, rehydrated, and treated

positive and negative control, respectively. Human SMPD1 cDNA

(Cat# OHu18710D) was purchased from GenScript.

with 10 mM sodium citrate for heat induced antigen retrieval. After

quenching endogenous peroxidase, and blocking with 2.5% normal

goat serum (Vector Laboratories S-1012), sections were incubated

overnight at 4 ^ꢀC with primary antibody diluted in blocking solu-

tion as recommended by manufacturer (Cell Signaling). Controls

were incubated with diluted normal rabbit IgG. After incubation

with biotinylated secondary antibody diluted 1:250 in blocking

serum for 45 min at rt, sections were incubated with VECTASTAIN

ABC reagent for 30 min, and for development of the DAB

4.5. Western blotting

Cells treated with vehicle or compounds for 48 h were washed

in cold phosphate-buffered saline (PBS) and lysed in buffer con-

taining 50 mM TrisCl (pH 7.5), 75 mM NaCl, 25 mM NaF, 5 mM

MgCl₂, 5 mM EGTA, 1 mM dithiothreitol, 100 mM NaVO₄, 1% NP40

17

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

[7] J. Spiegel, P.M. Cromm, G. Zimmermann, T.N. Grossmann, H. Waldmann,

Small-molecule modulation of Ras signaling, Nat. Chem. Biol. 10 (2014)

613e622.

[8] S.A. Forbes, N. Bindal, S. Bamford, C. Cole, C.Y. Kok, D. Beare, M. Jia,

R. Shepherd, K. Leung, A. Menzies, J.W. Teague, P.J. Campbell, M.R. Stratton,

P.A. Futreal, COSMIC: mining complete cancer genomes in the Catalogue of

Somatic Mutations in Cancer, Nucleic Acids Res. 39 (2011) D945eD950.

[9] I.A. Prior, P.D. Lewis, C. Mattos, A comprehensive survey of Ras mutations in

cancer, Canc. Res. 72 (2012) 2457e2467.

[10] B. Papke, C.J. Der, Drugging RAS: Know the enemy, Science 355 (2017)

1158e1163.

[11] J.M. Ostrem, K.M. Shokat, Direct small-molecule inhibitors of KRAS: from

structural insights to mechanism-based design, Nat. Rev. Drug Discov. 15

(2016) 771e785.

chromogen the Quanto substrate was used. Slides were counter

stained with Hematoxylin. Five ﬁelds per stained section per

xenograft were photographed at 10X. CC3þ and pERK þ area and

number of events per ﬁeld were quantitated via automated image

analysis (NIS Elements BR analysis ver. 4.13.04). Vessel number and

lumen area per ﬁeld used the same software with manual settings

to trace vessel wall contours. Antibodies used are as follows: CC3:

Cell Signaling #9661, use at 1:300. (Cleaved Caspase-3 (Asp175)

Antibody. Rabbit polyclonal. pERK: Cell Signaling #4370: Phospho-

p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit

mAB. CD31 (PECAM-1) (D8V9E) XP Rabbit mAB (Cell signal # (Cell

signal #77699). Normal rabbit IgG: Santa Cruz #sc-3888. Concen-

[12] Y. Wang, C.E. Kaiser, B. Frett, H.Y. Li, Targeting mutant KRAS for anticancer

therapeutics: a review of novel small molecule modulators, J. Med. Chem. 56

(2013) 5219e5230.

tration is 50

mg/mL or 50 ng/mL. Prepared with 5 mg in 200 mL

[13] N. Prieto-Dominguez, C. Parnell, Y. Teng, Drugging the small GTPase pathways

in cancer treatment: promises and challenges, Cells 8 (2019) E255.

[14] S. Lu, H. Jang, S. Gu, J. Zhang, R. Nussinov, Drugging Ras GTPase: a compre-

hensive mechanistic and signaling structural view, Chem. Soc. Rev. 45 (2016)

4929e4952.

blocking solution. Goat Anti-Rabbit IgG Biotin Conjugate: Calbio-

chem #OS03B, Concentration ¼ 1.2 mg/mL. Recommended to be

used for IHC at 1 mg/mL.

[15] P.M. Cromm, J. Spiegel, T.N. Grossmann, H. Waldmann, Direct modulation of

small GTPase activity and function, Angew. Chem. Int. Ed. 54 (2015)

13516e13537.

[16] E.S. Leshchiner, A. Parkhitko, G.H. Bird, J. Luccarelli, J.A. Bellairs, S. Escudero,

K. Opoku-Nsiah, M. Godes, N. Perrimon, L.D. Walensky, Direct inhibition of

oncogenic KRAS by hydrocarbon-stapled SOS1 helices, Proc. Natl. Acad. Sci. U.

S. A 112 (2015) 1761e1766.

4.11. Lysenin staining of tumor sections

To label intracellular SM, 3 m thick cryosections, ﬁxed in 4%

m

paraformaldehyde and quenched in 0.1 M NH₄Cl, were per-

meabilized with 0.5% saponin before overnight incubation (4 ^ꢀC)

[17] J.J. Winter, M. Anderson, K. Blades, C. Brassington, A.L. Breeze, C. Chresta,

K. Embrey, G. Fairley, P. Faulder, M.R. Finlay, J.G. Kettle, T. Nowak, R. Overman,

S.J. Patel, P. Perkins, L. Spadola, J. Tart, J.A. Tucker, G. Wrigley, Small molecule

binding sites on the Ras:SOS complex can be exploited for inhibition of Ras

activation, J. Med. Chem. 58 (2015) 2265e2274.

[18] J.C. Hunter, D. Gurbani, S.B. Ficarro, M.A. Carrasco, S.M. Lim, H.G. Choi, T. Xie,

J.A. Marto, Z. Chen, N.S. Gray, K.D. Westover, In situ selectivity pro ﬁ ling and

crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras

G12C, Proc. Natl. Acad. Sci. U. S. A 111 (2014) 8895e8900.

[19] M. Sanclemente, S. Francoz, L. Esteban-Burgos, E. Bousquet-Mur, M. Djurec,

P.P. Lopez-Casas, M. Hidalgo, C. Guerra, M. Drosten, M. Musteanu, M. Barbacid,

c-RAF ablation induces regression of advanced Kras/Trp53 mutant lung ade-

nocarcinomas by a mechanism independent of MAPK signaling, Canc. Cell 33

(2018) 217e228, e214.

[20] M. Roman, I. Baraibar, I. Lopez, E. Nadal, C. Rolfo, S. Vicent, I. Gil-Bazo, KRAS

oncogene in non-small cell lung cancer: clinical perspectives on the treatment

of an old target, Mol. Canc. 17 (2018) 33.

with MBP-GFP_Lysenin (50 mg/mL) in PBS with 0.5% saponin con-

taining DAPI (5 ng/mL). Samples mounted in FluorSave were pho-

tographed in the Nikon A1R Confocal Laser Microscope. GFP-

Lysenin binding was quantitated using NIS Elements BR analysis

ver. 4.13.04. The average value obtained in the negative control

pictures (background) was subtracted from each of the GFP pictures

(n ¼ 20).

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

[21] P. Lito, M. Solomon, L.S. Li, R. Hansen, N. Rosen, Allele-speci ﬁ c inhibitors

inactivate mutant KRAS G12C by a trapping mechanism, Science 351 (2016)

604e608.

Acknowledgment

[22] M.P. Patricelli, M.R. Janes, L.S. Li, R. Hansen, U. Peters, L.V. Kessler, Y. Chen,

J.M. Kucharski, J. Feng, T. Ely, J.H. Chen, S.J. Firdaus, A. Babbar, P. Ren, Y. Liu,

Selective inhibition of oncogenic KRAS output with small molecules targeting

the inactive state, Canc. Discov. 6 (2016) 316e329.

[23] J. Canon, K. Rex, A.Y. Saiki, C. Mohr, K. Cooke, D. Bagal, K. Gaida, T. Holt,

C.G. Knutson, N. Koppada, B.A. Lanman, J. Werner, A.S. Rapaport, T. San

Miguel, R. Ortiz, T. Osgood, J.R. Sun, X. Zhu, J.D. McCarter, L.P. Volak, B.E. Houk,

M.G. Fakih, B.H. O ’Neil, T.J. Price, G.S. Falchook, J. Desai, J. Kuo, R. Govindan,

D.S. Hong, W. Ouyang, H. Henary, T. Arvedson, V.J. Cee, J.R. Lipford, The clinical

KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity, Nature 575

(2019) 217e223.

This work was supported by the grants from the Cancer Pre-

vention and Research Institute of Texas (CPRIT) (DP150065 to JFH

and JZ; and RP200047 to JFH) the National Cancer Institute [R00-

CA188593 to KJC and KMR] and the National Natural Science

Foundation of China (81703330 to NY), the Natural Science Foun-

dation of Jiangsu Province (BK20170347 to NY), and the Priority

Academic Program Development of the Jiangsu Higher Education

Institutes (PAPD to NY). JZ is partly supported by the John D. Stobo,

M. D. Distinguished Chair Endowment Fund at UTMB.

[24] J. Hallin, L.D. Engstrom, L. Hargis, A. Calinisan, R. Aranda, D.M. Briere,

N. Sudhakar, V. Bowcut, B.R. Baer, J.A. Ballard, M.R. Burkard, J.B. Fell,

J.P. Fischer, G.P. Vigers, Y. Xue, S. Gatto, J. Fernandez-Banet, A. Pavlicek,

K. Velastagui, R.C. Chao, J. Barton, M. Pierobon, E. Baldelli, E.F. Patricoin 3rd,

Appendix A. Supplementary data

D.P. Cassidy, M.A. Marx,

Rybkin II, M.L. Johnson, S.I. Ou, P. Lito,

€

K.P. Papadopoulos, P.A. Janne, P. Olson, J.G. Christensen, The KRAS(G12C) in-

hibitor MRTX849 provides insight toward therapeutic susceptibility of KRAS-

mutant cancers in mouse models and patients, Canc. Discov. 10 (2020) 54e71.

[25] I.A. Prior, P.D. Lewis, C. Mattos, A comprehensive survey of Ras mutations in

cancer, Canc. Res. 72 (2012) 2457e2467.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13381.

References

[26] J.F. Hancock, Ras proteins: different signals from different locations, Nat. Rev.

Mol. Cell Biol. 4 (2003) 373e384.

[27] A.D. Cox, C.J. Der, M.R. Philips, Targeting RAS membrane association: back to

the future for anti-RAS drug discovery? Clin. Canc. Res. 21 (2015) 1819e1827.

[28] N. Ye, Q. Xu, W. Li, P. Wang, J. Zhou, Recent advances in developing K-Ras

plasma membrane localization inhibitors, Curr. Top. Med. Chem. 19 (2019)

2114e2127.

[1] J. Downward, Targeting RAS signalling pathways in cancer therapy, Nat. Rev.

Canc. 3 (2003) 11e22.

[2] K. Wennerberg, K.L. Rossman, C.J. Der, The Ras superfamily at a glance, J. Cell

Sci. 118 (2005) 843e846.

[3] Y. Pylayeva-Gupta, E. Grabocka, D. Bar-Sagi, RAS oncogenes: weaving

a

[29] J.M. Kraus, H.B. Tatipaka, S.A. McGuf ﬁ n, N.K. Chennamaneni, M. Karimi, J. Arif,

C.L. Verlinde, F.S. Buckner, M.H. Gelb, Second generation analogues of the

cancer drug clinical candidate tipifarnib for anti-Chagas disease drug dis-

covery, J. Med. Chem. 53 (2010) 3887e3898.

tumorigenic web, Nat. Rev. Canc. 11 (2011) 761e774.

[4] A.D. Cox, S.W. Fesik, A.C. Kimmelman, J. Luo, C.J. Der, Drugging the undrug-

gable RAS: mission possible? Nat. Rev. Drug Discov. 13 (2014) 828e851.

[5] A.M. Rojas, G. Fuentes, A. Rausell, A. Valencia, The Ras protein superfamily:

evolutionary tree and role of conserved amino acids, J. Cell Biol. 196 (2012)

189e201.

[30] E.J. Wang, W.W. Johnson, The farnesyl protein transferase inhibitor lonafarnib

(SCH6 6336) is an inhibitor of multidrug resistance proteins

1 and 2,

Chemotherapy 49 (2003) 303e308.

[6] J. Colicelli, Human RAS superfamily proteins and related GTPases, Sci. STKE

(2004) Re13, 2004.

[31] W.C. Rose, F.Y. Lee, C.R. Fairchild, M. Lynch, T. Monticello, R.A. Kramer,

18

P. Wang, D. van der Hoeven, N. Ye et al.

European Journal of Medicinal Chemistry 217 (2021) 113381

V. Manne, Preclinical antitumor activity of BMS-214662, a highly apoptotic

and novel farnesyltransferase inhibitor, Canc. Res. 61 (2001) 7507e7517.

[32] A.T. Baines, D. Xu, C.J. Der, Inhibition of Ras for cancer treatment: the search

continues, Future Med. Chem. 3 (2011) 1787e1808.

[33] N. Ye, J. Zhou, Kras - an evolving cancer target, Austin J. Cancer Clin. Res. 1

(2014).

[34] G. Zimmermann, B. Papke, S. Ismail, N. Vartak, A. Chandra, M. Hoffmann,

S.A. Hahn, G. Triola, A. Wittinghofer, P.I. Bastiaens, H. Waldmann, Small

molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS

signalling, Nature 497 (2013) 638e642.

Sphingomyelin metabolism is a regulator of K-Ras function, Mol. Cell Biol. 38

(2018) e00373-00317.

[43] W.E. Kattan, W. Chen, X. Ma, T.H. Lan, D. van der Hoeven, R. van der Hoeven,

J.F. Hancock, Targeting plasma membrane phosphatidylserine content to

inhibit oncogenic KRAS function, Life Sci. Alliance 2 (2019), e201900431.

[44] J.F. Hancock, D. van der Hoeven, G.V. Holland, J. Disha, Fendiline derivatives

and methods of use thereof, WO 2014031755A1 27 (2014). February.

[45] M. Adada, C. Luberto, D. Canals, Inhibitors of the sphingomyelin cycle:

sphingomyelin synthases and sphingomyelinases, Chem. Phys. Lipids 197

(2016) 45e59.

[35] B. Papke, S. Murarka, H.A. Vogel, P. Martin-Gago, M. Kovacevic, D.C. Truxius,

E.K. Fansa, S. Ismail, G. Zimmermann, K. Heinelt, C. Schultz-Fademrecht, A. Al

Saabi, M. Baumann, P. Nussbaumer, A. Wittinghofer, H. Waldmann,

P.I. Bastiaens, Identi ﬁ cation of pyrazolopyridazinones as PDEdelta inhibitors,

Nat. Commun. 7 (2016) 11360.

[36] M.O. Bergo, P. Ambroziak, C. Gregory, A. George, J.C. Otto, E. Kim, H. Nagase,

P.J. Casey, A. Balmain, S.G. Young, Absence of the CAAX endoprotease Rce1:

effects on cell growth and transformation, Mol. Cell Biol. 22 (2002) 171e181.

[37] S.P. Manandhar, E.R. Hildebrandt, W.K. Schmidt, Small-molecule inhibitors of

the Rce1p CaaX protease, J. Biomol. Screen 12 (2007) 983e993.

[38] A.M. Winter-Vann, R.A. Baron, W. Wong, J. dela Cruz, J.D. York, D.M. Gooden,

M.O. Bergo, S.G. Young, E.J. Toone, P.J. Casey, A small-molecule inhibitor of

isoprenylcysteine carboxyl methyltransferase with antitumor activity in

cancer cells, Proc. Natl. Acad. Sci. U. S. A 102 (2005) 4336e4341.

[39] S.C. Gupta, B. Sung, S. Prasad, L.J. Webb, B.B. Aggarwal, Cancer drug discovery

by repurposing: teaching new tricks to old dogs, Trends Pharmacol. Sci. 34

(2013) 508e517.

[46] A. Delgado, J. Casas, A. Llebaria, J.L. Abad, G. Fabrias, Inhibitors of sphingolipid

metabolism enzymes, Biochim. Biophys. Acta 1758 (2006) 1957e1977.

[47] A.S. Don, X.Y. Lim, T.A. Couttas, Re-con ﬁ guration of sphingolipid metabolism

by oncogenic transformation, Biomolecules 4 (2014) 315e353.

[48] N.H. Petersen, O.D. Olsen, L. Groth-Pedersen, A.M. Ellegaard, M. Bilgin,

S. Redmer, M.S. Ostenfeld, D. Ulanet, T.H. Dovmark, A. Lønborg, S.D. Vindeløv,

D. Hanahan, C. Arenz, C.S. Ejsing, T. Kirkegaard, M. Rohde, J. Nylandsted,

€€

€

M. Jaattela, Transformation-associated changes in sphingolipid metabolism

sensitize cells to lysosomal cell death induced by inhibitors of acid sphingo-

myelinase, Canc. Cell 24 (2013) 379e393.

[49] K.J. Cho, D. van der Hoeven, Y. Zhou, M. Maekawa, X. Ma, W. Chen, G.D. Fairn,

J.F. Hancock, Inhibition of acid sphingomyelinase depletes cellular phospha-

tidylserine and mislocalizes K-Ras from the plasma membrane, Mol. Cell Biol.

36 (2016) 363e374.

[50] C.J. Ceol, F. Stegmeier, M.M. Harrison, H.R. Horvitz, Identi ﬁ cation and classi-

ﬁ cation of genes that act antagonistically to let-60 Ras signaling in Caeno-

rhabditis elegans vulval development, Genetics 173 (2006) 709e726.

[51] D.J. Reiner, V. Gonzalez-Perez, C.J. Der, A.D. Cox, Use of Caenorhabditis elegans

to evaluate inhibitors of Ras function in vivo, Methods Enzymol. 439 (2008)

425e449.

[40] H. Chen, J. Wu, Y. Gao, H. Chen, J. Zhou, Scaffold repurposing of old drugs

towards new cancer drug discovery, Curr. Top. Med. Chem. 16 (2016)

2107e2114.

[41] D. van der Hoeven, K.J. Cho, X. Ma, S. Chigurupati, R.G. Parton, J.F. Hancock,

Fendiline inhibits K-Ras plasma membrane localization and blocks K-Ras

signal transmission, Mol. Cell Biol. 33 (2013) 237e251.

[42] D. van der Hoeven, K.J. Cho, Y. Zhou, X. Ma, W. Chen, A. Naji, D. Montufar-Solis,

Y. Zuo, S.E. Kovar, K.R. Levental, J.A. Frost, R. van der Hoeven, J.F. Hancock,

[52] G.J. Beitel, S.G. Clark, H.R. Horvitz, Caenorhabditis elegans ras gene let-60 acts

as a switch in the pathway of vulval induction, Nature 348 (1990) 503e509.

[53] M. Han, P.W. Sternberg, let-60, a gene that speci ﬁ es cell fates during C. elegans

vulval induction, encodes a ras protein, Cell 63 (1990) 921e931.

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Scaffold repurposing of fendiline: Identification of potent KRAS plasma membrane localization inhibitors

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