Synthesis and in Vitro Anticancer Activity of the First Class of Dual Inhibitors of REV-ERBβ and Autophagy

Article abstract of DOI:10.1021/acs.jmedchem.5b00511

Autophagy inhibition is emerging as a promising anticancer strategy. We recently reported that the circadian nuclear receptor REV-ERBβ plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol, 1 (ARN5187), as a novel dual inhibitor of REV-ERBβ and autophagy. 1 had improved cytotoxicity against BT-474 breast cancer cells compared to chloroquine, a clinically relevant autophagy inhibitor. Here, we present the results of structure-activity studies, based around 1, that disclose the first class of dual inhibitors of REV-ERBβ and autophagy. This study led to identification of 18 and 28, which were more effective REV-ERBβ antagonists than 1 and were more cytotoxic to BT-474. The combination of optimal chemical and structural moieties of these analogs generated 30, which elicited 15-fold greater REV-ERBβ inhibitory and cytotoxic activities compared to 1. Furthermore, 30 induced death in a panel of tumor cell lines at doses 5-50 times lower than an equitoxic amount of chloroquine but did not affect the viability of normal mammary epithelial cells.

Full text of DOI:10.1021/acs.jmedchem.5b00511

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Article
Synthesis and in Vitro Anticancer Activity of the First
Class of Dual Inhibitors of REV-ERB# and Autophagy
Esther Torrente, Chiara Parodi, Luisa Ercolani, Claudia De
Mei, Alessio Ferrari, Rita Scarpelli, and Benedetto Grimaldi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00511 • Publication Date (Web): 02 Jul 2015
Downloaded from http://pubs.acs.org on July 6, 2015
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Synthesis and in Vitro Anticancer Activity of the
First Class of Dual Inhibitors of REVꢀERBβ and
Autophagy
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,‡
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Esther Torrente, Chiara Parodi, Luisa Ercolani, Claudia De Mei, Alessio Ferrari, Rita
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Scarpelli, and Benedetto Grimaldi
§
Department of Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia,
Via Morego 30, 16163, Genoa, Italy
KEYWORDS: anticancer activity, anticancer agents, ARN5187, autophagy, BMAL1, breast
cancer, circadian, combined anticancer strategy, dual inhibitor, liver cancer, lysosomotropic
agents, nuclear receptors, prostate cancer, REVꢀERB, REVꢀERB antagonist.
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ABSTRACT: Autophagy inhibition is emerging as a promising anticancer strategy. We recently
reported that the circadian nuclear receptor REVꢀERBβ plays an unexpected role in sustaining
cancer cell survival when the autophagy flux is compromised. We also identified 4ꢀ[[[1ꢀ(2ꢀ
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fluorophenyl)cyclopentyl]amino]methyl]ꢀ2ꢀ[(4ꢀmethylpiperazinꢀ1ꢀyl)methyl]phenol,
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(ARN5187) as a novel dual inhibitor of REVꢀERBβ and autophagy. 1 had improved cytotoxicity
against BTꢀ474 breast cancer cells compared to chloroquine, a clinically relevant autophagy
inhibitor. Here, we present the results of structureꢀactivity studies, based around 1, that disclose
the first class of dual inhibitors of REVꢀERBβ and autophagy. This study led to identification of
18 and 28, which were more effective REVꢀERBβ antagonists than 1, and were more cytotoxic
to BTꢀ474. The combination of optimal chemical and structural moieties of these analogs
generated 30, which elicited 15ꢀfold greater REVꢀERBβꢀinhibitory and cytotoxic activities
compared to 1. Furthermore, 30 induced death in a panel of tumor cell lines at doses 5–50 times
lower than an equitoxic amount of chloroquine, but did not affect the viability of normal
mammary epithelial cells.
INTRODUCTION
Cancer is the second leading cause of mortality in industrialized countries, highlighting an
urgent need to discover novel therapies and approaches to cure this disease. Inhibition of
autophagy has recently emerged as a promising anticancer strategy. Autophagy is a selfꢀ
degradation process by which cells consume their own macromolecules or organelles to survive
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starvation and stress. In cancer, the role of autophagy is complex: it may either inhibit or
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ꢀ4
promote tumor growth, depending on the cellular context. For example, autophagy may
suppress tumor growth in the tumor initiation stage, whereas in established tumors autophagy
likely fulfills the metabolic needs of cancer cells. This is since, during oncogene activation or
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nutrient limitation, autophagy provides a mechanism to recycle intracellular carbon and
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nitrogen. In addition, recent studies reveal a direct role of autophagyꢀrelated proteins in
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regulating the stability and function of oncogenic factors, such as MCL1 and CMYC.
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The efficacy of autophagy inhibition in cancer therapy is well documented both in human
cancer cell lines and xenograft models, and lysosomotropic autophagy inhibitors, such as (±)ꢀN4ꢀ
(
7ꢀchloroꢀ4ꢀquinolyl)ꢀN1,N1ꢀdiethylꢀpentaneꢀ1,4ꢀdiamine (chloroquine, CQ, Figure 1), are
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currently being evaluated in numerous cancer clinical trials. Lysosomotropic agents block
autophagy by affecting lysosomal function and preventing the complete maturation of
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autophagosomes.
We recently reported that REVꢀERBβ, a nuclear receptor involved in circadian signaling, plays
an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised.
Accordingly, genetic or pharmacological inhibition of REVꢀERBβ sensitizes cancer cells to
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cytotoxicity induced by CQ. We thus hypothesized that combined pharmacological inhibition of
autophagy and REVꢀERBβ may offer a novel anticancer approach.
To pursue this idea, we screened an internal compound collection, and identified 4ꢀ[[[1ꢀ(2ꢀ
fluorophenyl)cyclopentyl]amino]methyl]ꢀ2ꢀ[(4ꢀmethylpiperazinꢀ1ꢀyl)methyl]phenol,
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(
ARN5187) (Figure 1) . Subsequent cellꢀbased assays revealed that 1 has both lysosomotropic
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and REVꢀERBβꢀinhibitory activities.
1 is a novel autophagy inhibitor that disrupts lysosomal function, blocks the autophagy process
at the late stage, and reduces cancer cell viability. In addition, 1 inhibits REVꢀERBβꢀmediated
transcriptional repression.
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and CQ have similar lysosomotropic activity and are equally effective with regard to
autophagy inhibition. However, 1 was significantly more cytotoxic than CQ toward BTꢀ474
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breast cancer cells, while it had negligible effects on nonꢀcancer human mammary epithelial cell
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(HMEC) viability.
In the present work, we describe structure–activity relationship (SAR) studies of 1 elucidating
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the main chemical and structural features within this class of diarylalkylamines that are crucial
for in vitro anticancer activity (Figure 2). Compounds with improved activity compared to 1
were then investigated as to whether their cytotoxicity was dependent on the ability to inhibit
REVꢀERBβ, the ability to block autophagy, or both.
The planned strategy of our SAR exploration is summarized in Figure 2 and involved the
evaluation of: a) the role of the secondary amine; b) the role of different substituents on the A
and B phenyl rings; c) the influence of the cycloalkyl group and d) the role of the heterocyclic
moiety.
These studies identified compounds 18 and 28, which showed improved REVꢀERBβ
antagonism and increased anticancer activity against BTꢀ474 cells compared to 1. Combining the
optimal chemical and structural moieties of 18 and 28 generated compound 30, which reduced
both REVꢀERBβ activity and BTꢀ474 viability about 15ꢀfold better than the hit 1. Remarkably,
30 was cytotoxic to a panel of tumor cell lines at concentrations from 5 to 50 times lower than an
equitoxic dose of CQ, yet did not affect the viability of normal HMEC cells. Finally, genetic
inhibition of REVꢀERBβ confirmed that this nuclear receptor is required for tumor cell survival
in the context of a CQꢀmediated autophagy blockade.
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RESULTS AND DISCUSSION
CHEMISTRY
In order to rapidly have access to a diversified structural modification of our class of
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diarylalkylamines by standard reductive amination reaction (Scheme 1), we exploited
previously reported synthetic procedures for the preparation of differently substituted amines
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1aꢀk (Scheme 2) and aldehydes 32aꢀj (Scheme 3), 33aꢀe (Scheme 4), and 34aꢀb (Scheme 5).
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The cycloalkyl amines, 31a-h, were prepared through a threeꢀstep synthetic sequence,
consisting of a nucleophilic addition of the appropriate organolithium species or Grignard
reagent to the commercially available cycloalkyl ketone (Scheme 2). This yielded the
corresponding phenylcycloalkanols 35a-h, which, after treatment with sodium azide (NaN ) in
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the presence of trifluoracetic acid (TFA), provided tertiary azide intermediates. Lithium
aluminum hydride (LiAlH ) was used as a reducing agent to directly convert these intermediates
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into the corresponding amines 31a-h.
Amines 31i-k are commercially available, and 31k can be also readily synthesized in a oneꢀpot
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procedure by slight modification of a previously reported method.
Specifically,
methylmagnesium bromide is added to commercially available 2ꢀfluorobenzenitrile in the
presence of titanium tetraisoproxide (Ti(OiꢀPr) ), under microwave irradiation conditions
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(Scheme 2).
The synthesis of 3ꢀsubstitutedꢀ4ꢀhydroxyꢀbenzaldehydes 32a-j was performed by using a
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Mannich reaction between 4ꢀhydroxyꢀbenzaldehyde and the appropriate commercially
available amines, in the presence of formaldehyde (HCHO), as shown in Scheme 3.
Alternatively, benzaldehydes 33a-e were synthesized following a threeꢀstep sequence, which
consists of a reductive amination reaction between the appropriate commercially available cyclic
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amines and methyl formylbenzoates in the presence of NaBH(OAc) to give esters 37a-f. A
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reductionꢀoxidation procedure, which employed LiAlH followed by manganese dioxide
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MnO ), was used to convert 37a-d and 37f into aldehydes 33aꢀe (Scheme 4).
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Benzaldehydes 34a-b were prepared following a fiveꢀstep synthetic procedure, consisting of a
WittigꢀHorner reaction between 38 and the appropriate commercially available cycloalkanone
9a-b. This was performed using sodium hydride (NaH) as a base, and yielded alkenes 40a-b.
The alkenes were converted into aldehydes 34a-b by a catalytic hydrogenation reaction, in the
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presence of triethylsilane (Et SiH) and palladium on carbon (Pd/C), followed by a reductionꢀ
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oxidation procedure, by the use of LiAlH and MnO (Scheme 5).
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Compound 2 (Entry 2, Table 1) was prepared in a oneꢀpot procedure consisting of a reductive
amination reaction between amine 31b and aldehyde 32a (Schemes 2 and 3), in the presence of
NaBH(OAc) , followed by the addition of HCHO.
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Compound 3 (Entry 3, Table 1) was obtained by performing a standard coupling reaction
between amine 31b and acid 41 (Schemes 2 and 4), in the presence of 1ꢀ
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bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate
(HATU) as a coupling reagent. The carboxylic acid intermediate, 41, was prepared by basic
hydrolysis of the corresponding methyl ester 37e (Scheme 4).
BIOLOGICAL EVALUATION
We first investigated the chemical and structural properties of the diarylalkylamine class
exemplified by hit 1 (Figure 2), that were required for the selective induction of cytoxicity in
cancer versus normal cells. Analogs were thus tested for in vitro activity against the human
breast cancer cell line, BTꢀ474, and normal human mammary epithelial cells (HMEC). BTꢀ474
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were adopted as we previously used them to characterize the REVꢀERBβꢀ and autophagyꢀ
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inhibitory activity of 1.
We started our SAR analysis by comparing the relative importance of the secondary amine in 1
to that of the tertiary amine 2, and of the amide 3 (Table 1). As previously reported, 1 was
selectively cytotoxic to BTꢀ474, whereas both 2 and 3 had negligible effects on both BTꢀ474 and
HMEC viability at all doses tested. These data demonstrate that secondary amine functionality is
essential for cytotoxicity. We then assessed the contribution of the cycloalkyl ring by preparing
compounds 4ꢀ6 (Table 1). The removal of a cyclopentyl ring (compound 4) or the insertion of
monoꢀ or diꢀmethyl substituents at the benzylic position (compounds 5 and 6) led to loss of
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anticancer activity (compounds with IC > 100 µM were considered inactive).
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Based on these results, we investigated whether the size of the cycloalkyl group altered
cytotoxicity by preparing compounds 7 and 8 (Table 1). While the cyclobutyl analog 7 was
inactive, the cyclohexyl derivative 8 was as cytotoxic to BTꢀ474 cells (IC = 30.65 ± 2.06 ꢀM)
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as the original compound, 1. Furthermore, 8 was not toxic to HMEC (Table 1).
This preliminary analysis suggested that secondary cyclopentyl or cyclohexyl amine
derivatives, such as 1 and 8, provide promising scaffolds for further SAR studies. With this in
mind, we focused on chemical and structural variations in additional regions. We thus
determined the contribution of different R substituents on the piperazine ring by introducing
representative alkyl, aryl or acyl groups at the distal nitrogen atom (Table 2). Removal of the
methyl group (9) produced a cytotoxic activity comparable to 1. Moreover, either the
introduction of differently stericallyꢀhindered alkyls (11-13) or a phenyl group (14) slightly
reduced the cytotoxicity. In contrast, replacement of the methyl group with an acetyl group (10)
completely abolished activity.
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We then evaluated the nature and position of different substituents on the A and B phenyl rings
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Figure 2). To gain insights into the function of the orthoꢀF (R ) atom at the A phenyl ring, we
assessed the activity of the desꢀfluoro analog, 15, and a methoxy derivative, 16 (Table 3).
Surprisingly, variation of the electronic density on the A phenyl ring did not affect cytotoxicity
of either compound in BTꢀ474 cells. However, 16 had adverse toxic effects in HMEC (IC =
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7.37± 2.31 µM). In marked contrast, the movement of the fluorine atom to the metaꢀ and paraꢀ
positions (17 and 18) significantly increased cytotoxicity in BTꢀ474 (IC = 14.15 ± 2.81 µM and
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9.41 ± 0.62 µM, respectively), without affecting the viability of HMEC (Table 2). Notably, as
observed with compound 16, replacement of the fluorine atom at the metaꢀposition of 17 with a
methoxy group (19) did not alter cytotoxicity in cancer cells, but elicited undesired cytotoxic
effects in HMEC (Table 2).
The mechanistic explanation for the cytotoxicity we observed in HMEC remains to be
determined. However, the above results allowed us to exclude the methoxy group as a site for
further chemical optimization. In contrast, introduction of a fluorine atom at the metaꢀ or paraꢀ
position of the A phenyl ring appeared beneficial for in vitro anticancer activity.
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desꢀhydroxy analog 20 and the methoxy derivative 21 (Table 3). While 20 was equipotent to 1 in
BTꢀ474 cells (IC = 34.05 ± 3.40 µM), the methoxy analog 21 was inactive at the doses tested.
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Because the OH group at the B phenyl ring appeared dispensable for the biological activity,
and considering the easier synthetic accessibility of desꢀhydroxy derivatives, we further assessed
the contribution of the relative position of the two remaining substituents by synthesizing
compounds 22 and 23 (Table 4). Indicating an essential function of the relative metaꢀposition of
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the substituents on the B phenyl ring, these analogs had no effect on the viability of BTꢀ474 cells
at the doses tested.
To gain further insight into the function of the piperazine ring of our class of molecules, we
tested the analog series reported in Table 5. While replacement of the piperazine moiety with a
1ꢀpiperidine ring (26) did not affect the activity (IC = 35.62 ± 6.42 µM), the introduction of
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diethylaminoꢀ or morpholineꢀ groups (24 and 25) generated inactive compounds. Confirming
that the OH group at the B phenyl ring was dispensable for anticancer activity, its removal from
26 did not reduce the potency of the resulting derivative compound, 27, in BTꢀ474 cells (IC =
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9.96 ± 4.30 µM).
As a further step, we replaced the Nꢀmethyl piperazine moiety with an Nꢀmethyl piperidine
group to create analog 28; this compound exhibited increased cytotoxicity in BTꢀ474 tumor cells
IC = 15.49 ± 0.74 µM) but was nonꢀtoxic in normal HMEC at the doses tested. As a last step
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of our investigation, we synthesized the methyl cyclohexyl analog 29; this compound had no
effects on cancer cell viability (Table 5).
Based on the improved cytotoxicity of 18 and 28 in BTꢀ474 cells, we decided to combine their
optimal chemical and structural moieties in a single compound in an attempt to produce a more
potent derivative. The resulting compound, 30, was significantly more potent (IC = 2.10 ± 0.19
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µM) than the parent 18 and 28, had 15ꢀfold higher activity than 1 in BTꢀ474, and remained nonꢀ
toxic to normal HMEC (Table 5).
We then assessed whether the increased in vitro anticancer activity of compounds 18, 28, and
30 was due to their ability to inhibit autophagy, REVꢀERBβ, or both. Indicating that these
analogs had a lysosomotropic activity similar to 1, all compounds were equipotent in terms of
reducing lysosomal pH in BTꢀ474 cells (IC ≈10 µM, Figure 3A).
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In marked contrast, compounds 18, 28, and 30 were significantly more effective in relieving
REVꢀERBꢀmediated transcriptional repression compared to 1, as assessed by a REVꢀERBβꢀ
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dependent luciferaseꢀbased reporter assay (Figure 3A). The ability of these compounds to
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antagonize this nuclear receptor correlated with their relative anticancer activity in BTꢀ474 cells.
Indeed, the most active compound (30) was at least one order of magnitude more potent than 1 in
REVꢀERBβ inhibition (IC = 1.34 ± 1.18 µM versus IC = 17.50 ± 1.08 µM toward REVꢀ
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ERBβ, respectively). Together, these data indicate that the improved cytotoxicity of 18, 28 and
30 was related to their enhanced REVꢀERBβꢀinhibitory activity.
A 4ꢀh treatment of BTꢀ474 cells with equimolar concentrations (10 µM) of 1, 18, 28, or 30
induced increases in the levels of LC3ꢀII protein (a marker of autophagy) that correlated with the
observed lysosomotropic and REVꢀERBβꢀinhibitory of these compounds (Figure 3B). This
supports the notion that they were equally effective in inhibiting autophagy. Conversely, there
was more variation between compounds with respect to their ability to deꢀrepress the REVꢀERBꢀ
regulated BMAL1 gene (Figure 3C). Overall, these analyses indicate that the increased in vitro
anticancer activities of 18, 28, and 30 are mainly due to their improved REVꢀERBβꢀinhibitory
activity.
When used as a single agent, CQ is only an efficient inhibitor of autophagy in cancer cells at
7
high micromolar doses. Therefore we reasoned that the dual activity of our compounds (i.e., the
combined inhibition of both autophagy and REVꢀERBβ) may render them more effective than
CQ when used as single agents. Thus, we compared the cytotoxicity of 30 and CQ in a panel of
human tumor cell lines, including breast carcinoma lines with different ERBB2 and ER
expression levels compared to BTꢀ474 cells (Table 6).
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Remarkably, 30 was significantly more cytotoxic than CQ in all the cell lines tested (Table 6).
Although we observed cell lineꢀdependent differences in the potency of 30, there was no
apparent influence of ERBB2 expression on compound cytotoxicity. Indeed, 30 equally affected
the viability of ERBB2ꢀpositive BTꢀ474 and ERBB2ꢀnegative MCFꢀ7 cells (IC = 2.10 ± 0.19
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µM and 2.19 ± 0.46 µM, respectively). In addition, there was no correlation between the in vitro
anticancer activity of 30 and the ER status of cancer cells (Table 6).
To further extend our analysis to nonꢀbreast cancer cells, we compared the activity of 30 and
CQ in both hepatocellular carcinoma HEPꢀG2 and prostatic adenocarcinoma LNCaP cells. This
analysis showed that 30 was about 10ꢀ and 4ꢀ fold more potent than CQ against HEPꢀG2 (30 IC50
=
2.73 ± 1.23 µM versus CQ IC = 25 ± 3.51 µM) and LNCaP (30 IC = 8.68 ± 1.45 µM versus
50 50
CQ IC50 = 32.87 ± 2.51 µM) cells, respectively. This result suggests that the cytoprotective
function of REVꢀERBβ in the context of CQꢀinduced cell death is not limited to BTꢀ474, but it is
preserved in different human tissue tumor cells.
To validate this hypothesis, we determine the cytotoxicity of CQ in different human tissue
cancer cells in which REVꢀERBβ was inhibited by genetic means.
We first determined whether, as previously shown in breast cancer BTꢀ474 cells (De Mei et al.,
2014), shRNAꢀmediated REVꢀERBβ knockdown in breast SKꢀBRꢀ3, liver HEPꢀG2, and prostate
LNCaP cancer cells resulted in the transcriptional deꢀrepression of clock genes. Consistent with
the role of REVꢀERBβ as a repressor of clock gene expression, REVꢀERBβ silencing
significantly enhanced the expression of BMAL1 in all the cells tested (Figure 4a).
We further validated that genetic inhibition of REVꢀERBβ in SKꢀBRꢀ3, HEPꢀG2 and LNCaP
compromises viability in the context of a CQꢀmediated autophagy blockade. Thus, cells were
transfected with plasmids coꢀexpressing GFP and shRNAs against REVꢀERBβ or a nonꢀ
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silencing control. In order to determine whether there was coꢀoperatively between CQ and REVꢀ
ERBβ targeting, cells were treated 24 h postꢀtransfection with a dose of CQ that reduced the
viability of nonꢀtransfected cells by ~25%. After a further 48 h, GFPꢀpositive cells were counted
and expressed as percentage of control.
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Consistent with a cytoprotective function of REVꢀERBβ toward CQꢀmediated cell death, the
percentage of GFPꢀpositive cells was significantly lower in REVꢀERBβꢀsilenced cells upon CQ
treatment (Figure 4b).
Notably, CQ cytotoxicity was most effective in HEPꢀG2 cells, and this correlated with greater
depression of REVꢀERBβ transcriptional targets upon REVꢀERBβ silencing (Figure 4). This
result is in line with the observation that the dual REVꢀERB and autophagy inhibitor, 30, is more
cytotoxic than CQ in HEPꢀG2 than in either SKꢀBRꢀ3 or LNCaP cells (the ratio between the IC50
of 30 and CQ is about 10 in HEPꢀG2 and about 3 in SKꢀBRꢀ3 and LNCaP).
We finally verified that compound 30 (which was more cytotoxic than CQ in all the cancer cell
lines) inhibited both REVꢀERBꢀmediated transcriptional repression and autophagy in SKꢀBRꢀ3,
HEPꢀG2 and LNCaP cells. Consistent with the results in BTꢀ474 (see above), BMAL1 was
depressed following treatment with 30 in all the cells tested (Figure 5a). To further validate that
30 blocks autophagy, we transduced these cells with a chimeric protein in which an acidꢀ
sensitive GFP and an acidꢀinsensitive RFP were fused to LC3. As expected from an autophagy
1
6
blockade at the late stage, the number of both GFP and RFP fluorescent dots increased after
treatment with 30 (Figure 5b and c).
CONCLUSIONS
In summary, we have identified a class of secondary diphenyl cycloalkylamines, exemplified
by 1 and 30 as the first dual REVꢀERBβ/autophagy inhibitors with in vitro cytotoxicity against a
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panel of human cancer cell lines. The preliminary SAR study around 1 facilitated the elucidation
of some crucial chemical and structural features associated with cancer cell cytotoxicity. Our
studies confirmed that the secondary cycloalkyl amine functionality is critical for maintaining
anticancer activity.
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We also found that the movement of a fluorine atom at the paraꢀ position of the A phenyl ring,
(
compound 18) or the replacement of Nꢀmethyl piperazine with an Nꢀmethyl piperidine group
compound 28) enhanced both REVꢀERBβꢀinhibitory and cytotoxic activity. Furthermore, the
(
combination of the optimal chemical and structural moieties identified in these two compounds
produced a dual REVꢀERBβ and autophagy inhibitor, 30. The in vitro anticancer activity of this
compound was superior to that of the clinically relevant autophagy inhibitor, CQ, in a panel of
tumor cell lines.
7
Different cancer cells present a diverse sensitivity to CQꢀrelated cytotoxicity. For instance,
CQ was shown to markedly decrease the proliferation of several prostate ductal adenocarcinoma
PDAC cells (IC ~ 10 µM), but had minimal effects on breast and lung cancer cells (IC > 100
5
0
50
17
µM). This differential sensitivity is clinically relevant, since the high micromolar CQ
concentrations that are required to block autophagy in vitro are not consistently achieved in
patients.
One way to overcome this issue may be the development of improved lysosomotropic agents
that reduce the dose of CQ that is required to achieve complete autophagy blockade. Indeed, a
class of dimeric CQ compounds, exemplified by N1ꢀ(7ꢀchloroquinolinꢀ4ꢀyl)ꢀN2ꢀ(2ꢀ(7ꢀ
1
8
chloroquinolinꢀ4ꢀylamino)ethyl)ꢀN2ꢀmethylethaneꢀ1,2ꢀdiamine (Lys01) (Figure 1), showed
increased lysosomotropy and more potent antitumor activity as a single agent compared with
CQ.
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Nonetheless, because both the lysosome and autophagosome have a homeostatic role in nonꢀ
cancer cells, the use of potent lysosomotropic agents may generate unwanted side effects in
1
9
healthy organs, such as brain, kidney, heart, and liver. Consequently, the identification of a
class of dual REVꢀERBβ/autophagy inhibitors, which have greater cytotoxicity than CQ but do
not potently inhibit autophagy, may provide a strategy for development of novel anticancer
agents with an improved therapeutic index.
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0
The only other REVꢀERB antagonist identified to date is (±)ꢀethyl 2ꢀ(5ꢀ
2
0
methylsulfanylthiopheneꢀ2ꢀcarbonyl)ꢀ3,4ꢀdihydroꢀ1Hꢀisoquinolineꢀ3ꢀcarboxylate
(SR8278);
this compound has unfavorable pharmacokinetic properties that preclude a robust in vivo
pharmacological evaluation. A critical next step, therefore, is the in vivo investigation of
antitumor activity and potential side effects associated with our dual inhibitors. This will be
crucial in order to determine the major strengths and liabilities of this class of compounds.
EXPERIMENTAL SECTION
Chemistry
Solvents and reagents were obtained from commercial suppliers and were used without further
purification. For simplicity, abbreviations were indicated as follows: acetic acid (AcOH),
acetonitrile (CH CN), ammonium acetate (NH OAc), 1ꢀ[Bis(dimethylamino)methylene]ꢀ1Hꢀ
3
4
1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate (HATU), chloroform (CHCl3),
cyclohexane (Cy), dichloromethane (DCM), diethyl ether (Et O), dimethylsulfoxide (DMSO),
2
N,Nꢀdimethylformamide (DMF), ethyl acetate (EtOAc), Nꢀethyldiisopropylamine (DIEA),
hydrochloric acid (HCl), methanol (MeOH), potassium carbonate (K CO ), retention time (Rt),
2
3
room temperature (rt), sodium sulphate (Na SO ) sodium triacetoxyborohydride (NaBH(OAc) ),
2
4 ,
3
tetrahydrofuran (THF), thin layer chromatography (TLC), triethylamine (Et N), water (H O).
3
2
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Automated column chromatography purifications were run using a Teledyne ISCO apparatus
®
(
CombiFlash Rf) with preꢀpacked silica gel columns of different sizes (from 4 g until 24 g).
Flash column chromatography was performed manually on preꢀpacked silica cartridges (from 2 g
up to 10 g) from Biotage. In both cases, mixtures of increasing polarity of Cy and EtOAc or
DCM and MeOH were used as eluents. Preparative TLC were performed using MachereyꢀNagel
preꢀcoated 0.05 mm TLC plates (SIL Gꢀ50 UV ). NMR experiments were run on a Bruker
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56
57
58
59
60
2
54
1
13
Avance III 400 system (400.13 MHz for H, and 100.62 MHz for C), equipped with a BBI
probe and Zꢀgradients. Spectra were acquired at 300 K, using deuterated dimethylsulfoxide
(
DMSOꢀd ), deuterated chloroform (CDCl ) or deuterated water (D O) as solvents. Chemical
6 3 2
1
13
shifts (δ) for H and C spectra were recorded in parts per million (ppm) using the residual nonꢀ
1
13
deuterated solvent as the internal standard (for DMSOꢀd : 2.50 ppm, H; 39.52 ppm, C; for
6
1
13
1
CDCl : 7.26 ppm, H and 77.16 ppm, C; for D O: 4.79 ppm, H). Data are reported as follows:
3
2
chemical shift (sorted in descending order), multiplicity (indicated as: s, singlet; bs, broad signal,
d, doublet; dd, double doublet; ddd, double double doublet; dt, double triplet; ddq, double double
quartet; t, triplet; td; triplet doublet; q, quartet; p, pentet, m, multiplet and combinations thereof),
coupling constants (J) in Hertz (Hz) and integrated intensity. UPLC/MS analyses were run on a
Waters ACQUITY UPLC/MS system consisting of a Single Quadropole Detector Mass
Spectrometer (MS) equipped with an Electrospray (ES) Ionization interface and a Photodiode
Array (PDA) Detector. PDA range was 210ꢀ400 nm. Analyses were performed on an ACQUITY
UPLC BEH C18 column (50x2.1 mm ID, particle size 1.7 µm) with a VanGuard BEH C18 preꢀ
column (5x2.1 mm ID, particle size 1.7 µm). Mobile phase was 10 mM NH OAc in H O at pH 5,
4
2
adjusted with AcOH (A) and 10 mM NH OAc in CH CNꢀH O (95:5) at pH 5 (B). Electrospray
4
3
2
ionization in positive and negative mode was applied. Analyses were performed with method A
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or B. Method A: Gradient: 5 to 95% B over 3 min. Flow rate 0.5 mL/min. Temperature 40 °C.
Method B: Gradient: 50 to 100% B over 3 min. Flow rate 0.5 mL/min. Temperature 40 °C.
Purification by preparative HPLC/MS of compound 9 was run on a Waters Autopurification
system consisting of a 3100 Single Quadropole Mass Spectrometer equipped with an
Electrospray Ionization interface and a 2998 Photodiode Array Detector. HPLC system included
a 2747 Sample Manager, 2545 Binary Gradient Module, System Fluidic Organizer and 515
HPLC Pump. PDA range was 210ꢀ400 nm. Purifications were performed on a XBridgeTM Prep
C18 OBD column (100x19 mm ID, particle size 5 µm) with a XBridgeTM Prep C18 (10x 19
mm ID, particle size 5 µm) Guard Cartridge. Mobile phase was 10 mM NH OAc in CH CNꢀH O
0
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2
(95:5) at pH 5. Electrospray ionization in positive and negative mode was used. Microwave
®
heating was performed using Explorer ꢀ48 positions instrument (CEM). All final compounds
displayed ≥ 95% purity as determined by NMR and UPLC/MS analysis.
General Procedure for the Synthesis of Secondary Amines. A mixture of corresponding
amine (1.0 equiv.) and aldehyde (1.0 equiv.) in anhydrous DCM (0.06 M) was stirred for 10 min
at rt and then NaBH(OAc) (2.0 equiv.) was added. Stirring was continued until UPLC and TLC
3
analysis indicated the disappearance of the starting material. The reaction mixture was then
quenched with 10% aqueous K CO solution and the two phases were separated. The organic
2
3
layer was dried over Na SO . After evaporation of the solvent, the crude was purified by column
2
4
chromatography, eluting with Cy/EtOAc DCM/MeOH or DCM/(MeOH/Et N or NH ) as
3
3
indicated in each case.
-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-
4
yl)methyl]phenol trihydrochloride (1). 1 was prepared according to the general procedure for
the synthesis of secondary amines, starting from 32a (100.0 mg, 0.42 mmol), 31b (75.0 mg, 0.42
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mmol) and NaBH(OAc) (178.0 mg, 0.84 mmol) in anhydrous DCM (4.5 mL). The crude was
3
purified by column chromatography [DCM and MeOH/NH (19% MeOH and 1% NH in DCM),
3
3
from 95:5 to 40:60] to afford the free base of 1 as a colorless oil (137.0 mg, 82 %). UPLC/MS
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+
(
method A): Rt 1.58 min; MS (ES) C H FN O requires m/z 397, found m/z 398 [M+H] . The
2
4
32
3
free base of 1 (60.0 mg, 0.15 mmol) was then dissolved in DCM (1.5 mL) and 2.0 M HCl
solution in Et O (1.5 mL, 3.0 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 1
2
1
as a white powder. H NMR (400 MHz, DMSOꢀd ) δ 12.07 (bs, 1H), 10.71 (bs, 1H), 9.53 (s,
6
2H), 7.65 (t, J = 8.0 Hz, 1H), 7.56 – 7.47 (m, 1H), 7.41 (s, 1H), 7.37 – 7.28 (m, 2H), 7.23 (d, J =
7
.7 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 3.73 (t, J = 5.6 Hz, 2H), 3.71 – 3.22 (m, 10H), 2.79 (s, 3H),
1
3
2
.51 (m, 2H), 2.37 (dt, J = 12.4, 5.7 Hz, 2H), 2.02 – 1.83 (m, 2H), 1.67 – 1.48 (m, 2H). C NMR
(101 MHz, DMSOꢀd ) δ 160.7 (J = 250.4 Hz), 157.8, 136.1, 135.8, 133.1, 131.3, 130.2, 125.1,
6
1
24.9, 122.3, 116.7, 115.2, 70.4, 47.6, 47.4, 46.8, 41.9, 40.1, 34.4, 22.4.
4
-[[[1-(2-Fluorophenyl)cyclopentyl]-methyl-amino]methyl]-2-[(4-methylpiperazin-1-
yl)methyl] phenol trihydrochloride (2). A mixture of 31b (32.0 mg, 0.18 mmol) and 32a (42.0
mg, 0.18 mmol), in anhydrous DCM (3.0 mL) was stirred for 10 min at rt and then NaBH(OAc)3
(76.0 mg, 0.36 mmol) was added. Stirring was continued overnight at rt, then HCHO (240.0 µL,
0
.36 mmol) was added followed by an additional amount of NaBH(OAc) (76.0 mg, 0.36 mmol).
3
Stirring was continued until UPLC and TLC analysis indicated the disappearance of the starting
material. The reaction mixture was then quenched with 10% aqueous K CO solution and the two
2
3
phases were separated. The organic layer was dried over Na SO . After evaporation of the
2
4
solvent, the crude was purified by column chromatography [DCM and MeOH/Et N (19% MeOH
3
and 1% Et N in DCM), from 95:5 to 40:60] to afford the free base of 2 as a colorless oil (65.0
3
mg, 62%). UPLC/MS (method A): Rt 2.18 min; MS (ES) C H FN O requires m/z 411, found
25
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+
m/z 412 [M+H] . The free base (30.0 mg, 0.08 mmol) was then dissolved in 0.5 mL of DCM and
2
.0 M HCl solution in Et O (1.3 mL, 1.6 mmol, 20.0 equiv.) was added. Evaporation of solvents
2
1
afforded 2 as a white powder. H NMR (400 MHz, DMSOꢀd ) δ 12.36 – 11.48 (m, 2H), 10.78
6
0
1
2
3
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5
6
7
8
9
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(
bs, 2H), 7.87 (t, J = 7.5 Hz, 1H), 7.70 – 7.53 (m, 2H), 7.52 – 7.29 (m, 3H), 7.01 (d, J = 8.0 Hz,
1H), 4.63 (d, J = 12.3 Hz, 1H), 4.38 – 4.00 (m, 2H), 3.84 – 3.23 (m, 8H), 3.18 – 2.98 (m, 1H),
1
3
2
.97 – 2.71 (m, 3H), 2.71 – 2.57 (m, 3H), 2.52 – 2.42 (m, 3H), 1.87 (m, 2H), 1.36 (m, 2H). C
NMR (101 MHz, DMSOꢀd ) δ 160.7 (J = 250.4 Hz), 157.8, 138.3, 136.1, 135.3, 133.3, 133.1,
6
131.2, 125.9, 122.4, 118.1, 116.2, 70.4, 56.5, 56.4, 50.0, 47.9, 42.6, 36.9, 34.5, 21.6.
N-[1-(2-Fluorophenyl)cyclopentyl]-4-hydroxy-3-[(4-methylpiperazin-1-
yl)methyl]benzamide (3). To a stirred solution of 41 (40.0 mg, 0.16 mmol) in anhydrous DMF
(1.5 mL), HATU (67.0 mg, 0.17 mmol) was added and the resulting solution was stirred for 10
min at rt. Then, 31b (34.0 mg, 0.19 mmol) and DIEA (40.0 µL, 0.24 mmol) were sequentially
added. The reaction mixture was stirred at rt for 16 h and, then, quenched with brine (3.0 mL)
and extracted with EtOAc. The combined organic phase was dried over Na SO . After
2
4
evaporation of the solvent, the crude was purified by column chromatography [DCM/MeOH
(20% MeOH in DCM), from 100:0 to 50:50] to afford 3 as white off solid (43.0 mg, 65%).
UPLC/MS (method A): Rt 2.08 min; MS (ES) C H FNO requires m/z 411, found m/z 412
24
30
3
+
1
[
M+H] . H NMR (400 MHz, DMSOꢀd ) δ 8.12 (s, 1H), 7.64 (dd, J = 8.4, 2.3 Hz, 1H), 7.59 (d, J
6
= 2.2 Hz, 1H), 7.43 (td, J = 8.1, 1.8 Hz, 1H), 7.23 (tdd, J = 7.2, 5.0, 1.8 Hz, 1H), 7.14 – 6.98 (m,
2
H), 6.75 (d, J = 8.4 Hz, 1H), 3.66 (s, 2H), 3.51 – 2.96 (m, 8H), 2.68 – 2.56 (m, 2H), 2.28 (s,
1
3
3
H), 2.10 – 1.96 (m, 2H), 1.87 – 1.64 (m, 4H). C NMR (101 MHz, DMSOꢀd ) δ 165.8, 160.8 (J
6
=
246.4 Hz), 159.8, 129.5, 129.3, 128.9, 128.5, 128.3, 126.4, 123.5, 122.0, 116.1, 115.3, 64.9,
5
8.6, 54.6, 51.9, 45.4, 37.8, 23.01.
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4
-[[(2-Fluorophenyl)methylamino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol
trihydrochloride (4). 4 was prepared according to the general procedure for the synthesis of
secondary amines, starting from 32a (54.0 mg, 0.23 mmol), 31i (27.0 µL, 0.23 mmol) and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NaBH(OAc) (97.0 mg, 0.46 mmol) in anhydrous DCM (3.0 mL). The crude was purified by
3
column chromatography [DCM and MeOH/Et N (19% MeOH and 1% Et N in DCM), from 95:5
3
3
to 40:60] to afford the free base of 4 as a colorless oil (48.0 mg, 67%). UPLC/MS (method A): Rt
+
1
.47 min; MS (ES) C H FN O requires m/z 343, found m/z 344 [M+H] . The free base of 4
2
4
26
3
(48.0 mg, 0.13 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et O
2
(1.3 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 4 as a white
1
powder. H NMR (400 MHz, DMSOꢀd ) δ 11.68 (bs, 1H), 11.06 – 10.35 (bs, 1H), 9.70 (bs, 2H),
6
7.73 (td, J = 7.6, 1.7 Hz, 1H), 7.62 (s, 1H), 7.48 (ddq, J = 9.8, 5.2, 2.7, 1.9 Hz, 2H), 7.34 – 7.22
(m, 2H), 7.03 (d, J = 8.4 Hz, 1H), 4.16 (t, J = 5.4 Hz, 2H), 4.13 – 4.02 (m, 2H), 3.73 – 3.51 (m,
1
3
2
1
4
H), 3.50 – 3.26 (m, 8H), 2.74 (s, 3H). C NMR (101 MHz, DMSOꢀd ) δ 160.5 (J = 249.0 Hz),
6
57.9, 136.4, 136.3, 133.8, 132.7, 131.7, 125.4, 122.2, 119.5, 116.0, 115.8, 50.2, 50.1, 50.0, 48.1,
2.7, 42.4.
4-[[1-(2-Fluorophenyl)ethylamino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol
trihydrochloride (5). 5 was prepared according to the general procedure for the synthesis of
secondary amines, starting from 32a (50.0 mg, 0.21 mmol), 31j (50.0 mg, 0.21 mmol) and
NaBH(OAc) (119.0 mg, 0.56 mmol) in anhydrous DCM (3.0 mL). The crude was purified by
3
column chromatography [DCM and MeOH/Et N (19% MeOH and 1% Et N in DCM), from 95:5
3
3
to 40:60] to afford the free base of 5 as a colorless oil (68.0 mg, 90%). UPLC/MS (method A): Rt
+
1
.23 min; MS (ES) C H FN O requires m/z 357, found m/z 358 [M+H] . The free base of 5
2
1
28
3
(
68.0 mg, 0.19 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et O
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
1.9 mL, 3.80 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 5 as a white
1
powder. H NMR (400 MHz, DMSOꢀd ) δ 11.82 (bs, 1H), 10.78 (bs, 1H), 10.37 (s, 2H), 9.80
6
(bs, 2H), 8.07 – 7.89 (m, 1H), 7.59 (s, 1H), 7.55 – 7.39 (m, 2H), 7.39 – 7.18 (m, 2H), 7.05 (d, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.3 Hz, 1H), 4.68 – 4.49 (m, 1H), 4.40 – 4.13 (m, 2H), 4.06 – 3.88 (m, 2H), 3.86 – 3.73 (m, 2H),
1
3
3.73 – 3.54 (m, 4H), 3.53 – 3.34 (m, 1H), 2.80 (s, 3H), 1.65 (d, J = 6.5 Hz, 3H). C NMR (101
MHz, DMSOꢀd ) δ 160.5 (J = 248.5Hz), 157.9, 136.4, 133.7, 132.7, 131.6, 129.2, 125.8, 122.1,
6
1
16.2, 116.1, 53.9, 50.3, 49.9, 49.4, 48.5, 42.6, 19.3.
4-[[[1-(2-Fluorophenyl)-1-methyl-ethyl]amino]methyl]-2-[(4-methylpiperazin-1-
yl)methyl]phenol trihydrochloride (6). 6 was prepared according to the general procedure for
the synthesis of secondary amines, starting from 32a (54.0 mg, 0.23 mmol) 31k (45.0 mg, 0.23
mmol), Et N (64.0 µL, 0.46 mmol) and NaBH(OAc) (97.0 mg, 0.46 mmol) in anhydrous DCM
3
3
(3.0 mL). The crude was purified by column chromatography [DCM and MeOH/NH (19%
3
MeOH and 1% NH in DCM), from 95:5 to 40:60] to afford the free base of 6 as a colorless oil
3
(48.0 mg, 56%). UPLC/MS (method A): Rt 1.69 min; MS (ES) C H FN O requires m/z 371,
20 30 3
+
found m/z 372 [M+H] . The free base of 6 (48.0 mg, 0.13 mmol) was then dissolved in 1.5 ml of
DCM and 2.0 M HCl solution in Et O (1.3 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation
2
1
of solvents afforded 6 as a white powder. H NMR (400 MHz, DMSOꢀd ) δ 12.03 (bs, 1H),
6
1
0.75 (bs, 1H), 9.67 (bs, 2H), 7.63 (d, J = 1.0 Hz, 1H), 7.58 – 7.45 (m, 2H), 7.41 – 7.28 (m, 3H),
7.03 (d, J = 8.4 Hz, 1H), 4.53 – 3.98 (m, 2H), 3.76 (dd, J = 7.7, 4.3 Hz, 2H), 3.71 – 3.54 (m, 2H),
13
3
2
4
.52 – 3.30 (m, 6H), 2.80 (s, 3H), 1.86 (s, 6H). C NMR (101 MHz, DMSOꢀd ) δ 160.4 (J =
6
25.8 Hz), 158.0, 135.8, 133.1, 131.3, 128.4, 126.5, 125.2, 122.1, 116.5, 115.6, 61.3, 49.4, 47.7,
5.7, 45.5, 42.0, 25.4.
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4
-[[[1-(2-Fluorophenyl)cyclobutyl]amino]methyl]-2-[(4-methylpiperazin-1-
yl)methyl]phenol trihydrochloride (7). 7 was prepared according to the general procedure for
the synthesis of secondary amines, starting from 32a (61.0 mg, 0.26 mmol), 31a (45.0 mg, 0.26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mmol) and NaBH(OAc) (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude was
3
purified by column chromatography [DCM/MeOH (20% in DCM), from 100:0 to 0:100] to
afford the free base of 7 as a colorless oil (60.0 mg, 60%). UPLC/MS (method A): Rt 1.70 min;
+
MS (ES) C H FN O requires m/z 383, found m/z 384 [M+H] . The free base of 7 (50.0 mg,
2
3
30
3
0.13 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.65
1
mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 7 as a white powder. H
NMR (400 MHz, DMSOꢀd ) δ 11.95 (bs, 1H), 10.80 (bs, 1H), 9.95 (bs, 2H), 7.71 – 7.61 (m,
6
1H), 7.57 – 7.44 (m, 2H), 7.38 – 7.23 (m, 3H), 6.97 (d, J = 8.4 Hz, 1H), 4.37 – 4.07 (m, 2H),
3.80 – 3.65 (m, 2H), 3.65 – 3.53 (m, 2H), 3.53 – 3.19 (m, 6H), 2.99 – 2.84 (m, 2H), 2.80 (s, 3H),
1
3
2
.76 – 2.61 (m, 2H), 2.41 – 2.21 (m, 1H), 1.90 – 1.67 (m, 1H). C NMR (101 MHz, DMSOꢀd )
6
δ 161.2 (J = 248.0 Hz), 157.8, 136.3, 136.2, 133.6, 132.3, 131.4, 125.4, 124.8, 122.4, 116.6,
116.1, 62.6, 49.4, 47.7, 46.0, 31.6, 42.5, 31.6, 14.9, 14.8.
4-[[[1-(2-Fluorophenyl)cyclohexyl]amino]methyl]-2-[(4-methylpiperazin-1-
yl)methyl]phenol trihydrochloride (8). 8 was prepared according to the general procedure for
the synthesis of secondary amines, starting from 32a (70.0 mg, 0.42 mmol), 31c (58.0 mg, 0.42
mmol) and NaBH(OAc) (177.0 mg, 0.84 mmol) in anhydrous DCM (3.0 mL). The crude was
3
purified by column chromatography [DCM and MeOH/NH (19% MeOH and 1% NH in DCM),
3
3
from 95:5 to 40:60] to afford the free base of 8 as a colorless oil (110.0 mg, 63%). UPLC/MS
+
(
method A): Rt 1.67 min; MS (ES) C H FN O requires m/z 411, found m/z 412 [M+H] .The
25
34
3
free base of 8 (76.0 mg, 0.18 mmol) was then dissolved in 1.5 ml of DCM and 2.0 M HCl
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
solution in Et O (1.5 mL, 3.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 8
2
1
as a white powder. H NMR (400 MHz, DMSOꢀd ) δ 12.06 (bs, 1H), 10.71 (bs, 1H), 9.51 (s,
6
2
H), 7.78 (t, J = 8.1 Hz, 1H), 7.56 (td, J = 7.4, 4.1 Hz, 1H), 7.36 (ddd, J = 22.0, 14.6, 8.1 Hz,
H), 7.25 (dd, J = 8.6, 2.3 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 4.52 – 3.90 (bm, 4H), 3.80 – 3.50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
(m, 6H), 3.49 – 3.30 (bm, 2H), 2.84 (dd, J = 36.7, 8.2 Hz, 2H), 2.80 (bs, 3H) 2.12 (t, J = 12.2 Hz,
1
3
2
H), 1.77 (d, J = 13.1 Hz, 2H), 1.67 – 1.48 (m, 1H), 1.34 – 1.05 (m, 3H). C NMR (101 MHz,
DMSOꢀd ) δ 161.5 (J = 250.2 Hz), 157.6, 135.6, 133.1, 131.9, 131.6, 125.3, 122.4, 121.6, 116.9,
6
115.4, 69.4, 53.2, 49.3, 47.8, 44.4, 42.0, 32.8, 24.7, 21.9.
4-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-(piperazin-1-ylmethyl)-phenol (9). 9
was prepared according to the general procedure for the synthesis of secondary amines, starting
from 32e (118.0 mg, 0.36 mmol), 31b (66.0 mg, 0.36 mmol) and NaBH(OAc) (152.0 mg, 0.72
3
mmol) in anhydrous DCM (4.0 mL). The residue was then dissolved in 1,4ꢀdioxane (1.0 mL) and
4.0 M HCl solution in dioxane (0.5 mL) was added and the reaction mixture stirred at rt for 1h.
After evaporation of the solvents, the crude was purified by preparative HPLC/MS system to
afford 9 as white solid. UPLC/MS (method A): Rt 1.52 min; MS (ES) C H FN O requires m/z
2
3
30
3
+
1
383, found m/z 384 [M+H] . H NMR (400 MHz, DMSOꢀd ) δ 7.41 (td, J = 8.2, 1.9 Hz, 1H),
6
7
.30 (tdd, J = 7.5, 5.2, 1.8 Hz, 1H), 7.21 – 7.08 (m, 2H), 6.92 – 6.78 (m, 2H), 6.59 (d, J = 8.1 Hz,
H), 3.53 (s, 2H), 3.17 (bs, 2H), 2.69 (m, 4H), 2.35 (m, 4H), 2.20 – 2.07 (m, 2H), 1.88 – 1.74 (m,
1
1
3
2H), 1.70 – 1.55 (m, 4H), 1.36 – 1.16 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 161.8 (J =
6
2
49.5 Hz), 156.0, 136.0, 135.4, 133.1, 131.8, 129.6, 128.7, 127.8, 124.3, 116.1, 115.6, 68.2, 60.3,
3.7, 47.7, 45.7, 37.5, 23.1.
5
1-[4-[[5-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-hydroxyphenyl]-
methyl]piperazin-1-yl]ethanone dihydrochloride (10). 10 was prepared according to the
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general procedure for the synthesis of secondary amines, starting from 32b (60.0 mg, 0.23
mmol), 31b (41.0 mg, 0.23 mmol) and NaBH(OAc) (81.0 mg, 0.36 mmol) in anhydrous DCM
3
(
3.0 mL). The crude was purified by column chromatography (EtOAc/Cy/Et N, 4:1:0.1) to afford
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the free base of 10 as a colorless oil (50.0 mg, 50%). UPLC/MS (method A): Rt 1.88 min; MS
+
(ES) C H FN O requires m/z 425, found m/z 426 [M+H] . The free base of 10 (36.0 mg, 0.08
2
4
33
3
mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et O (1.0 mL, 1.6
2
1
mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 10 as a white powder. H NMR
(400 MHz, DMSOꢀd ) δ 10.83 (d, J = 11.3 Hz, 1H), 10.63 (s, 1H), 9.73 – 9.22 (m, 2H), 7.64 (t, J
6
=
7.9 Hz, 1H), 7.59 – 7.40 (m, 2H), 7.41 – 7.25 (m, 2H), 7.21 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.2
Hz, 1H), 4.40 (d, J = 13.7 Hz, 1H), 4.21 (bs, 2H), 3.97 (d, J = 13.1 Hz, 1H), 3.73 (t, J = 5.3 Hz,
2H), 3.55 (m, 1H), 3.45 – 3.20 (m, 2H), 3.21 – 2.97 (m, 2H), 2.88 (m, 1H), 2.54ꢀ2.46 (m, 2H),
13
2
.42 – 2.30 (m, 2H), 2.03 (s, 3H), 1.96 – 1.85 (m, 2H), 1.63 – 1.50 (m, 2H). C NMR (101
MHz, DMSOꢀd ) δ 168.9, 157.8 (J = 249.5 Hz), 157.3, 146.4, 136.0, 134.1, 133.2, 131.8, 130.4,
6
125.1, 122.3, 116.5, 115.5, 68.9, 53.3, 47.2, 42.4, 35.8, 37.5, 22.3, 21.1.
2-[(4-Ethylpiperazin-1-yl)methyl]-4-[[[1-(2-fluorophenyl)cyclopentyl]amino]-
methyl]phenol trihydrochloride (11). 11 was prepared according to the general procedure for
the synthesis of secondary amines, starting from 32d (62.0 mg, 0.25 mmol), 31b (45.0 mg, 0.25
mmol) and NaBH(OAc) (106.0 mg, 0.50 mmol) in anhydrous DCM (3.0 mL). The crude was
3
purified by column chromatography [DCM and MeOH/Et N (19% MeOH and 1% Et N in
3
3
DCM), from 95:5 to 40:60] to afford the free base of 11 as a colorless oil (57.0 mg, 56%).
UPLC/MS (method A): Rt 1.63 min; MS (ES) C H FN O requires m/z 411, found m/z 412
25 34
3
+
[
M+H] . The free base of 11 (57.0 mg, 0.14 mmol) was then dissolved in 0.5 mL of DCM and
2
.0 M HCl solution in Et O (1.4 mL, 2.8 mmol, 20.0 equiv.) was added. Evaporation of solvents
2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
afforded 11 as a white powder. H NMR (400 MHz, DMSOꢀd ) δ 11.99 (bs, 1H), 10.74 (bs, 1H),
6
9
.50 (bs, 2H), 7.65 (t, J = 8.0 Hz, 1H), 7.51 (m, 1H), 7.44 (m, 1H), 7.38 – 7.27 (m, 2H), 7.24 (d,
J = 8.2 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 4.38 – 4.10 (m, 2H), 3.73 (bs, 2H), 3.65 (s, 8H), 3.28 –
.96 (m, 2H), 2.55 – 2.50 (m, 2H), 2.43 – 2.31 (m, 2H), 1.96 – 1.85 (m, 2H), 1.66 – 1.50 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
3
2H), 1.26 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, DMSOꢀd ) δ 160.7 (J = 250.4 Hz), 157.8,
6
1
36.1, 135.8, 133.0, 131.3, 130.0, 128.1, 124.8, 122.3, 116.4, 115.0, 67.1, 50.3, 48.1, 47.1, 47.0,
6.87, 36.1, 22.1, 8.5.
4
2-[(4-Benzylpiperazin-1-yl)methyl]-4-[[[1-(2-fluorophenyl)cyclopentyl]amino]-
methyl]phenol trihydrochloride (12). 12 was prepared according to the general procedure for
the synthesis of secondary amines, starting from 32f (136.0 mg, 0.44 mmol), 31b (78.0 mg, 0.44
mmol) and NaBH(OAc) (186.0 mg, 0.88 mmol) in anhydrous DCM (6.0 mL). The crude was
3
purified by column chromatography [DCM/MeOH (20% MeOH in DCM), from 95:5 to 60:40]
to afford the free base of 12 as a white solid (64.0 mg, 31%). UPLC/MS (method A): Rt 2.84
+
min; MS (ES) C H FN O requires m/z 473, found m/z 474 [M+H] . The free base of 12 (60.0
30
36
3
mg, 0.12 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.6
mL, 2.4 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 12 as a white powder.
1
H NMR (400 MHz, DMSOꢀd ) δ 12.22 (bs, 1H), 10.68 (bs, 1H), 9.47 (bs, 2H), 7.73 – 7.57 (m,
6
3
H), 7.57 – 7.48 (m, 1H), 7.48 – 7.36 (m, 4H), 7.37 – 7.27 (m, 2H), 7.23 (dd, J = 8.5, 2.2 Hz,
1H), 6.94 (d, J = 8.4 Hz, 1H), 4.52 – 4.03 (m, 2H), 3.78 – 3.67 (m, 2H), 3.67 – 3.13 (m, 10H),
13
2
.55 – 2.50 (m, 2H), 2.42 – 2.31 (m, 2H), 2.02 – 1.80 (m, 2H), 1.65 – 1.50 (m, 2H). C NMR
(
101 MHz, DMSOꢀd ) δ 161.2 (J = 249.5 Hz), 157.8, 136.2, 133.6, 132.2, 131.8, 131.4, 130.9,
6
1
29.2, 129.1, 125.5, 124.7, 122.3, 117.0, 116.2, 48.1, 48.0, 47.8, 47.7, 46.6, 36.3, 22.8.
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4
-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-isopropylpiperazin-1-
yl)methyl]phenol trihydrochloride (13). 13 was prepared according to the general procedure
for the synthesis of secondary amines, starting from 32g (70.0 mg, 0.26 mmol), 31b (42.0 mg,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
.26 mmol) and NaBH(OAc) (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude
3
was purified by column chromatography [DCM/MeOH (20% MeOH in DCM), from 95:5 to
60:40] to afford the free base of 13 as a colorless oil (81.0 mg, 74%). UPLC/MS (method A): Rt
+
1.80 min; MS (ES) C H FN O requires m/z 425, found m/z 426 [M+H] . The free base of 13
26
36
3
(81.0 mg, 0.20 mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et O
2
(2.0 mL, 4.0 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 13 as a white
1
powder. H NMR (400 MHz, D O) δ 7.46 – 7.34 (bs, 2H), 7.21 (t, J = 7.6 Hz, 1H), 7.17 – 7.05
2
(m, 2H), 7.03 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 4.26 (s, 2H), 3.89 (s, 2H), 3.73 – 3.25
(m, 10H), 2.43 (dt, J = 12.0, 5.6 Hz, 2H), 2.17 (dt, J = 14.4, 7.3 Hz, 2H), 1.89 – 1.72 (m, 2H),
13
1
2
4
.63 (dt, J = 13.8, 5.2 Hz, 2H), 1.27 (d, J = 6.6 Hz, 6H). C NMR (101 MHz, D O) δ 160.4 (J =
2
49.4 Hz), 156.6, 134.5, 134.2, 133.6, 132.2, 129.0, 124.9, 122.7, 116.6, 116.3, 115.0, 68.2, 55.4,
9.5, 48.6, 47.5, 44.8, 35.7, 21.9, 15.9.
4-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-phenylpiperazin-1-
yl)methyl]phenol trihydrochloride (14). 14 was prepared according to the general procedure
for the synthesis of secondary amines, starting from 32c (74.1 mg, 0.25 mmol), 31b (45.0 mg,
0.25 mmol) and NaBH(OAc) (106.0 mg, 0.50 mmol) in anhydrous DCM (3.0 mL). The crude
3
was purified by column chromatography (Cy/EtOAc, from 100:0 to 60:40) to afford the free base
of 14 as colorless oil (65.0 mg, 57%). UPLC/MS (method A): Rt 3.24 min; MS (ES) C H FN O
29 34
3
+
requires m/z 459, found m/z 460 [M+H] . The free base of 14 (60.0 mg, 0.13 mmol) was then
dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et O (1.3 mL, 2.6 mmol, 20.0 equiv.)
2
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
was added. Evaporation of solvents afforded 14 as a white powder. H NMR (400 MHz, DMSOꢀ
d6) δ 10.71 (m, 2H), 9.53 (bs, 2H), 7.65 (t, J = 7.9 Hz, 1H), 7.52 (m, 2H), 7.38 – 7.19 (m, 5H),
6
.98 (m, 3H), 6.86 (t, J = 7.2 Hz, 1H), 4.27 (bs, 2H), 3.89 – 3.65 (m, 4H), 3.44 (d, J = 9.4 Hz,
H), 3.17 (m, 4H), 2.55 – 2.50 (m, 2H), 2.41 – 2.34 (m, 2H), 2.00 – 1.85 (m, 2H), 1.67 – 1.53
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
13
(m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 162.3 (J = 248.5 Hz), 157.9, 150.0, 136.7, 134.0,
6
1
33.5, 132.33, 130.7, 129.4, 129.2, 125.7, 120.9, 117.1, 116.4, 116.2, 70.1, 53.5, 50.8, 47.8, 45.6,
6.3, 22.7.
3
2-[(4-Methylpiperazin-1-yl)methyl]-4-[[(1-phenylcyclopentyl)amino]methyl]-phenol
trihydrochloride (15). 15 was prepared according to the general procedure for the synthesis of
secondary amines, starting from 32a (58.0 mg, 0.24 mmol), 31h (40.0 mg, 0.24 mmol) and
NaBH(OAc) (102.0 mg, 0.48 mmol) in anhydrous DCM (3.0 mL). The crude was purified by
3
column chromatography [DCM/MeOH (20% MeOH in DCM), from 95:5 to 40:60] to afford the
free base of 15 as a colorless oil (57.0 mg, 63%). UPLC/MS (method A): Rt 1.51 min; MS (ES)
+
C H FN O requires m/z 379, found m/z 380 [M+H] . The free base (57.0 mg, 0.15 mmol) was
24
33
3
then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et O (1.5 mL, 3.0 mmol, 20.0
2
1
equiv.) was added. Evaporation of solvents afforded 15 as a white powder. H NMR (400 MHz,
DMSOꢀd ) δ 12.32 – 11.61 (bs, 1H), 10.72 (bs, 1H), 9.81 (bs, 2H), 7.77 (m, 3H), 7.51 (t, J = 7.2
6
Hz, 2H), 7.47 – 7.33 (m, 2H), 7.24 (d, J = 8.1 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 3.80 – 3.13 (m,
1
3
12H), 2.79 (s, 3H), 2.45 – 2.30 (m, 4H), 1.85 (m, 2H), 1.65 – 1.37 (m, 2H). C NMR (101 MHz,
DMSOꢀd ) δ 157.8, 135.8, 133.1, 130.0, 127.0, 128.5, 128.2, 126.8, 121.4, 115.4, 70.1, 49.7,
6
47.6, 47.4, 46.6, 41.9, 35.24, 21.8.
4
-[[[1-(2-Methoxyphenyl)-cyclopentyl]amino]-methyl]-2-[(4-methylpiperazin-1-
yl)methyl]phenol trihydrochloride (16). 16 was prepared according to the general procedure
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for the synthesis of secondary amines, starting from 32a (61.0 mg, 0.26 mmol), 31f (50.0 mg,
0
.26 mmol) and NaBH(OAc) (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude
3
was purified by column chromatography [DCM and MeOH/NH (19% MeOH and 1% NH in
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DCM), from 95:5 to 40:60] to afford the free base of 16 as a colorless oil (29.0 mg, 27%).
UPLC/MS (method A): Rt 1.66 min; MS (ES) C H N O requires m/z 409, found m/z 410
25
35
3
2
+
[
M+H] . The free base of 16 (26.0 mg, 0.06 mmol) was then dissolved in 1.5 ml and 2.0 M HCl
solution in Et O (0.3 mL, 1.2 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 16
2
1
as a white powder. H NMR (400 MHz, DMSOꢀd ) δ 11.91 (bs, 1H), 11.07 – 10.42 (bs, 1H),
6
9
.31 – 8.94 (bs, 2H), 7.46 – 7.37 (m, 2H), 7.33 (d, J = 7.4 Hz, 1H), 7.17 (dd, J = 8.4, 2.2 Hz, 1H),
.12 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 6.97 (dd, J = 8.5, 3.0 Hz, 1H), 4.35 – 4.14 (m,
7
4H), 3.92 (s, 3H), 3.73 – 3.54 (m, 4H), 3.54 – 3.26 (m, 4H), 2.90 – 2.70 (m, 3H), 2.41 – 2.27 (m,
13
4
1
4
H), 1.94 – 1.76 (m, 2H), 1.63 – 1.43 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 157.1, 156.3,
6
36.10, 135.5, 133.44, 131.13, 128.28, 124.4, 122.1, 121.24, 112.33, 116.02, 70.6, 56.0, 53.6,
9.9, 47.8, 42.6, 35.6, 23.3.
1-(3-Fluorophenyl)-N-[[3-[(4-methylpiperazin-1-yl)methyl]phenyl]methyl]-
cyclopentanamine trihydrochloride (17). 17 was prepared according to the general procedure
for the synthesis of secondary amines, starting from 32a (46.0 mg, 0.19 mmol), 31d (35.0 mg,
0
.19 mmol) and NaBH(OAc) (80.0 mg, 0.38 mmol) in anhydrous DCM (2.5 mL). The crude
3
was purified by column chromatography [DCM/MeOH (20% in DCM), from 95:5 to 50:50] to
afford the free base of 17 as a colorless oil (50.0 mg, 67%). UPLC/MS (method A): Rt 1.70 min;
+
MS (ES) C H FN requires m/z 397, found m/z 398 [M+H] . The free base of 17 (50.0 mg, 0.12
24 32
3
mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.6 mL, 2.4
1
mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 17 as a white powder. H NMR
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2
2
2
2
2
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3
3
3
3
3
3
3
3
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4
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4
4
4
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4
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5
5
5
5
5
5
5
6
(
400 MHz, DMSOꢀd ) δ 11.82 (bs, 1H), 10.72 (bs, 1H), 9.92 (bs, 2H), 7.74 – 7.50 (m, 4H), 7.43
6
(s, 1H), 7.34 – 7.20 (m, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.38 – 3.98 (m, 2H), 3.78 – 3.50 (m, 8H),
3
.51 – 3.22 (m, 2H), 2.81 (s, 3H), 2.47 – 2.30 (m, 4H), 2.01 – 1.78 (m, 2H), 1.64 – 1.40 (m, 2H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3C NMR (101 MHz, DMSOꢀd ) δ 160.4, 156.3, 140.0, 136.3, 133.67, 132.0, 131.60, 124.01,
6
122.0, 116.1, 116.0, 115.2, 70.1, 50.4, 49.7, 47.6, 46.7, 42.6, 36.01, 22.4.
-(4-Fluorophenyl)-N-[[3-[(4-methylpiperazin-1-yl)methyl]phenyl]methyl]-
1
cyclopentanamine trihydrochloride (18). 18 was prepared according to the general procedure
for the synthesis of secondary amines, starting from 32a (46.0 mg, 0.19 mmol), 31e (35.0 mg,
0
.19 mmol) and NaBH(OAc) (80.0 mg, 0.38 mmol) in anhydrous DCM (2.5 mL). The crude
3
was purified by column chromatography [DCM/MeOH (20% in DCM), from 95:5 to 50:50] to
afford the free base of 18 as a colorless oil (53.0 mg, 69%). UPLC/MS (method A): Rt 1.66 min;
+
MS (ES) C H FN requires m/z 397, found m/z 398 [M+H] . The free base of 18 (53.0 mg, 0.13
24
32
3
mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.65 mL,
1
2
.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 18 as a white powder. H
NMR (400 MHz, DMSOꢀd ) δ 11.85 (bs, 1H), 10.71 (bs, 1H), 9.85 (s, 2H), 7.83 (dd, J = 8.7, 5.2
6
Hz, 2H), 7.41 (s, 1H), 7.33 (t, J = 8.7 Hz, 2H), 7.29 – 7.20 (m, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.50
–
1
3.97 (m, 2H), 3.79 – 3.50 (m, 10H), 2.81 (s, 3H), 2.48 – 2.29 (m, 4H), 1.94 – 1.78 (m, 2H),
1
3
.57 – 1.41 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 162.5 (J = 248.5 Hz), 157.5, 135.4,
6
133.8, 133.1, 130.4, 129.8, 122.6, 115.4, 115.3, 71.1, 50.1, 49.5, 46.7, 47.3, 42.0, 35.4, 21.7.
-[1-(3-Methoxyphenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-
4
yl)methyl]phenol trihydrochloride (19). 19 was prepared according to the general procedure
for the synthesis of secondary amines, starting from 32a (42.0 mg, 0.18 mmol), 31g (35.0 mg,
0
.18 mmol) and NaBH(OAc) (76.0 mg, 0.36 mmol) in anhydrous DCM (3.0 mL). The crude
3
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was purified by column chromatography [DCM/MeOH (20% in DCM), from 100:0 to 0:100] to
afford the free base of 19 as a colorless oil (50.0 mg, 68%). UPLC/MS (method A): Rt 1.63 min;
+
MS (ES) C H N O requires m/z 409, found m/z 410 [M+H] . The free base of 19 (50.0 mg,
25
35
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
.12 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.60
mL, 2.4 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 19 as a white powder.
1
H NMR (400 MHz, DMSOꢀd ) δ 11.74 (s, 1H), 10.76 (s, 1H), 9.76 (m, 2H), 7.47 – 7.34 (m,
6
3H), 7.26 (m, 2H), 7.05 – 6.93 (m, 2H), 4.46 – 3.99 (m, 2H), 3.82 (s, 3H), 3.61 (m, 8H), 3.50 –
1
3
3.25 (m, 2H), 2.82 (s, 3H), 2.44 – 2.25 (m, 4H), 1.85 (m, 2H), 1.52 (m, 2H). C NMR (101
MHz, DMSOꢀd ) δ 160.1, 157.8, 139.7, 136.0, 134.0, 130.7, 126.4, 122.5, 119.8, 115.0, 113.7,
6
1
13.0, 71.5, 56.0, 50.0, 47.7, 47.3, 42.5, 35.8, 22.8.
1-(2-Fluorophenyl)-N-[[3-[(4-methylpiperazin-1-yl)methyl]phenyl]methyl]-
cyclopentanamine trihydrochloride (20). 20 was prepared according to the general procedure
for the synthesis of secondary amines, starting from 33c (89.0 mg, 0.40 mmol), 31b (72.0 mg,
0
.40 mmol) and NaBH(OAc) (144.0 mg, 0.68 mmol) in anhydrous DCM (3.0 mL). The crude
3
was purified by column chromatography [DCM/MeOH (20% in DCM), from 95:5 to 50:50] to
afford the free base of 20 as a colorless oil (76.0 mg, 50%). UPLCꢀMS (method generic A): Rt
+
1
.77 min; MS (ES) C H FN requires m/z 381, found m/z 382 [M+H] . The free base of 20
2
4
32
3
(50.0 mg, 0.13 mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et O
2
(1.2 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 20 as a white
1
powder. H NMR (400 MHz, DMSOꢀd ) δ 12.18 (bs, 2H), 9.74 (bs, 2H), 7.65 (m, 2H), 7.51 (t, J
6
=
6.6 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.36 – 7.20 (m, 2H), 3.97 – 3.72 (m, 2H), 3.70 – 3.11 (m,
10H), 2.80 (s, 3H), 2.58 (dd, J = 14.1, 6.9 Hz, 2H), 2.38 (dt, J = 12.1, 5.3 Hz, 2H), 1.93 (q, J =
13
7.4, 6.8 Hz, 2H), 1.60 (dt, J = 11.1, 5.4 Hz, 2H). C NMR (101 MHz, DMSOꢀd ) δ 161.2 (J =
6
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