Synthesis and in Vitro Anticancer Activity of the First Class of Dual

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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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Journal of Medicinal Chemistry

Synthesis and in Vitro Anticancer Activity of the First Class of Dual Inhibitors of REV-ERBβ and Autophagy Esther Torrente, §,‡ Chiara Parodi, §,‡ Luisa Ercolani, § Claudia De Mei, § Alessio Ferrari, § Rita 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, REV-ERB, REV-ERB antagonist.

1 ACS Paragon Plus Environment


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ABSTRACT: 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-(2fluorophenyl)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.

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 selfdegradation process by which cells consume their own macromolecules or organelles to survive starvation and stress.1 In cancer, the role of autophagy is complex: it may either inhibit or promote tumor growth, depending on the cellular context.2-4 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 2 ACS Paragon Plus Environment

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nutrient limitation, autophagy provides a mechanism to recycle intracellular carbon and nitrogen.2,4 In addition, recent studies reveal a direct role of autophagy-related proteins in regulating the stability and function of oncogenic factors, such as MCL15 and CMYC.6 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 (±)-N4(7-chloro-4-quinolyl)-N1,N1-diethyl-pentane-1,4-diamine (chloroquine, CQ, Figure 1), are currently being evaluated in numerous cancer clinical trials.7 Lysosomotropic agents block autophagy by affecting lysosomal function and preventing the complete maturation of autophagosomes.8 We recently reported that REV-ERBβ, 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 REV-ERBβ sensitizes cancer cells to cytotoxicity induced by CQ.9 We thus hypothesized that combined pharmacological inhibition of autophagy and REV-ERBβ may offer a novel anticancer approach. To pursue this idea, we screened an internal compound collection, and identified 4-[[[1-(2fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol,

1

(ARN5187) (Figure 1)9. Subsequent cell-based assays revealed that 1 has both lysosomotropic and REV-ERBβ-inhibitory activities.9 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 REV-ERBβ-mediated transcriptional repression. 1 and CQ have similar lysosomotropic activity and are equally effective with regard to autophagy inhibition. However, 1 was significantly more cytotoxic than CQ toward BT-474



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breast cancer cells, while it had negligible effects on non-cancer human mammary epithelial cell (HMEC) viability.9 In the present work, we describe structure–activity relationship (SAR) studies of 1 elucidating 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 REV-ERBβ, 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 REV-ERBβ antagonism and increased anticancer activity against BT-474 cells compared to 1. Combining the optimal chemical and structural moieties of 18 and 28 generated compound 30, which reduced both REV-ERBβ activity and BT-474 viability about 15-fold 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 REV-ERBβ confirmed that this nuclear receptor is required for tumor cell survival in the context of a CQ-mediated 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 diarylalkylamines by standard reductive amination reaction (Scheme 1),10 we exploited previously reported synthetic procedures for the preparation of differently substituted amines 31a-k (Scheme 2) and aldehydes 32a-j (Scheme 3), 33a-e (Scheme 4), and 34a-b (Scheme 5).1115

The cycloalkyl amines, 31a-h, were prepared through a three-step 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 (NaN3) in the presence of trifluoracetic acid (TFA), provided tertiary azide intermediates. Lithium aluminum hydride (LiAlH4) was used as a reducing agent to directly convert these intermediates into the corresponding amines 31a-h.11,12 Amines 31i-k are commercially available, and 31k can be also readily synthesized in a one-pot procedure by slight

modification of a previously reported method.13 Specifically,

methylmagnesium bromide is added to commercially available 2-fluorobenzenitrile in the presence of titanium tetraisoproxide (Ti(Oi-Pr)4), under microwave irradiation conditions (Scheme 2).13 The synthesis of 3-substituted-4-hydroxy-benzaldehydes 32a-j was performed by using a Mannich reaction14 between 4-hydroxy-benzaldehyde 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 three-step 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)3 to give esters 37a-f. A reduction-oxidation procedure, which employed LiAlH4 followed by manganese dioxide (MnO2), was used to convert 37a-d and 37f into aldehydes 33a-e (Scheme 4). Benzaldehydes 34a-b were prepared following a five-step synthetic procedure, consisting of a Wittig-Horner reaction between 38 and the appropriate commercially available cycloalkanone 39a-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 presence of triethylsilane (Et3SiH) and palladium on carbon (Pd/C),15 followed by a reductionoxidation procedure, by the use of LiAlH4 and MnO2 (Scheme 5). Compound 2 (Entry 2, Table 1) was prepared in a one-pot procedure consisting of a reductive amination reaction between amine 31b and aldehyde 32a (Schemes 2 and 3), in the presence of NaBH(OAc)3, followed by the addition of HCHO. 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 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid 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, BT-474, and normal human mammary epithelial cells (HMEC). BT-474


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were adopted as we previously used them to characterize the REV-ERBβ- and autophagyinhibitory activity of 1.9 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 BT-474, whereas both 2 and 3 had negligible effects on both BT-474 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 4-6 (Table 1). The removal of a cyclopentyl ring (compound 4) or the insertion of mono- or di-methyl substituents at the benzylic position (compounds 5 and 6) led to loss of anticancer activity (compounds with IC50 > 100 µM were considered inactive). 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 BT-474 cells (IC50 = 30.65 ± 2.06 µM) 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 sterically-hindered 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. 7 ACS Paragon Plus Environment


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We then evaluated the nature and position of different substituents on the A and B phenyl rings (Figure 2). To gain insights into the function of the ortho-F (R1) atom at the A phenyl ring, we assessed the activity of the des-fluoro 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 BT-474 cells. However, 16 had adverse toxic effects in HMEC (IC50 = 17.37± 2.31 µM). In marked contrast, the movement of the fluorine atom to the meta- and parapositions (17 and 18) significantly increased cytotoxicity in BT-474 (IC50 = 14.15 ± 2.81 µM and 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 meta-position 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 meta- or paraposition of the A phenyl ring appeared beneficial for in vitro anticancer activity. We then investigated the function of the OH group (R2) at the B phenyl ring by preparing the des-hydroxy analog 20 and the methoxy derivative 21 (Table 3). While 20 was equipotent to 1 in BT-474 cells (IC50 = 34.05 ± 3.40 µM), the methoxy analog 21 was inactive at the doses tested. Because the OH group at the B phenyl ring appeared dispensable for the biological activity, and considering the easier synthetic accessibility of des-hydroxy 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 meta-position of


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the substituents on the B phenyl ring, these analogs had no effect on the viability of BT-474 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 1-piperidine ring (26) did not affect the activity (IC50 = 35.62 ± 6.42 µM), the introduction of diethylamino- or morpholine- 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 BT-474 cells (IC50 = 39.96 ± 4.30 µM). As a further step, we replaced the N-methyl piperazine moiety with an N-methyl piperidine group to create analog 28; this compound exhibited increased cytotoxicity in BT-474 tumor cells (IC50 = 15.49 ± 0.74 µM) but was non-toxic in normal HMEC at the doses tested. As a last step 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 BT-474 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 (IC50 = 2.10 ± 0.19 µM) than the parent 18 and 28, had 15-fold higher activity than 1 in BT-474, and remained nontoxic 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, REV-ERBβ, or both. Indicating that these analogs had a lysosomotropic activity similar to 1, all compounds were equipotent in terms of reducing lysosomal pH in BT-474 cells (IC50 ≈10 µM, Figure 3A). 9 ACS Paragon Plus Environment


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In marked contrast, compounds 18, 28, and 30 were significantly more effective in relieving REV-ERB-mediated transcriptional repression compared to 1, as assessed by a REV-ERBβdependent luciferase-based reporter assay (Figure 3A).9 The ability of these compounds to antagonize this nuclear receptor correlated with their relative anticancer activity in BT-474 cells. Indeed, the most active compound (30) was at least one order of magnitude more potent than 1 in REV-ERBβ inhibition (IC50 = 1.34 ± 1.18 µM versus IC50 = 17.50 ± 1.08 µM toward REVERBβ, respectively). Together, these data indicate that the improved cytotoxicity of 18, 28 and 30 was related to their enhanced REV-ERBβ-inhibitory activity. A 4-h treatment of BT-474 cells with equimolar concentrations (10 µM) of 1, 18, 28, or 30 induced increases in the levels of LC3-II protein (a marker of autophagy) that correlated with the observed lysosomotropic and REV-ERBβ-inhibitory 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 de-repress the REV-ERBregulated 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 REV-ERBβ-inhibitory activity. When used as a single agent, CQ is only an efficient inhibitor of autophagy in cancer cells at high micromolar doses.7 Therefore we reasoned that the dual activity of our compounds (i.e., the combined inhibition of both autophagy and REV-ERBβ) 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 BT-474 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 line-dependent differences in the potency of 30, there was no apparent influence of ERBB2 expression on compound cytotoxicity. Indeed, 30 equally affected the viability of ERBB2-positive BT-474 and ERBB2-negative MCF-7 cells (IC50 = 2.10 ± 0.19 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 non-breast cancer cells, we compared the activity of 30 and CQ in both hepatocellular carcinoma HEP-G2 and prostatic adenocarcinoma LNCaP cells. This analysis showed that 30 was about 10- and 4- fold more potent than CQ against HEP-G2 (30 IC50 = 2.73 ± 1.23 µM versus CQ IC50 = 25 ± 3.51 µM) and LNCaP (30 IC50 = 8.68 ± 1.45 µM versus CQ IC50 = 32.87 ± 2.51 µM) cells, respectively. This result suggests that the cytoprotective function of REV-ERBβ in the context of CQ-induced cell death is not limited to BT-474, 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 REV-ERBβ was inhibited by genetic means. We first determined whether, as previously shown in breast cancer BT-474 cells (De Mei et al., 2014), shRNA-mediated REV-ERBβ knockdown in breast SK-BR-3, liver HEP-G2, and prostate LNCaP cancer cells resulted in the transcriptional de-repression of clock genes. Consistent with the role of REV-ERBβ as a repressor of clock gene expression, REV-ERBβ silencing significantly enhanced the expression of BMAL1 in all the cells tested (Figure 4a). We further validated that genetic inhibition of REV-ERBβ in SK-BR-3, HEP-G2 and LNCaP compromises viability in the context of a CQ-mediated autophagy blockade. Thus, cells were transfected with plasmids co-expressing GFP and shRNAs against REV-ERBβ or a non-



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silencing control. In order to determine whether there was co-operatively between CQ and REVERBβ targeting, cells were treated 24 h post-transfection with a dose of CQ that reduced the viability of non-transfected cells by ~25%. After a further 48 h, GFP-positive cells were counted and expressed as percentage of control. Consistent with a cytoprotective function of REV-ERBβ toward CQ-mediated cell death, the percentage of GFP-positive cells was significantly lower in REV-ERBβ-silenced cells upon CQ treatment (Figure 4b). Notably, CQ cytotoxicity was most effective in HEP-G2 cells, and this correlated with greater depression of REV-ERBβ transcriptional targets upon REV-ERBβ silencing (Figure 4). This result is in line with the observation that the dual REV-ERB and autophagy inhibitor, 30, is more cytotoxic than CQ in HEP-G2 than in either SK-BR-3 or LNCaP cells (the ratio between the IC50 of 30 and CQ is about 10 in HEP-G2 and about 3 in SK-BR-3 and LNCaP). We finally verified that compound 30 (which was more cytotoxic than CQ in all the cancer cell lines) inhibited both REV-ERB-mediated transcriptional repression and autophagy in SK-BR-3, HEP-G2 and LNCaP cells. Consistent with the results in BT-474 (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 acidsensitive GFP and an acid-insensitive RFP were fused to LC3. As expected from an autophagy blockade at the late stage,16 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 REV-ERBβ/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. We also found that the movement of a fluorine atom at the paraposition of the A phenyl ring, (compound 18) or the replacement of N-methyl piperazine with an N-methyl piperidine group (compound 28) enhanced both REV-ERBβ-inhibitory and cytotoxic activity. Furthermore, the combination of the optimal chemical and structural moieties identified in these two compounds produced a dual REV-ERBβ 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. Different cancer cells present a diverse sensitivity to CQ-related cytotoxicity.7 For instance, CQ was shown to markedly decrease the proliferation of several prostate ductal adenocarcinoma PDAC cells (IC50 ~ 10 µM), but had minimal effects on breast and lung cancer cells (IC50 > 100 µM).17 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 N1-(7-chloroquinolin-4-yl)-N2-(2-(7chloroquinolin-4-ylamino)ethyl)-N2-methylethane-1,2-diamine (Lys01)18 (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 noncancer cells, the use of potent lysosomotropic agents may generate unwanted side effects in healthy organs, such as brain, kidney, heart, and liver.19 Consequently, the identification of a class of dual REV-ERBβ/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. The

only

other

REV-ERB

antagonist

identified

to

date

is

(±)-ethyl

methylsulfanylthiophene-2-carbonyl)-3,4-dihydro-1H-isoquinoline-3-carboxylate

2-(5-

(SR8278);20

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 (CH3CN), ammonium acetate (NH4OAc), 1-[Bis(dimethylamino)methylene]-1H1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), chloroform (CHCl3), cyclohexane (Cy), dichloromethane (DCM), diethyl ether (Et2O), dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), ethyl acetate (EtOAc), N-ethyldiisopropylamine (DIEA), hydrochloric acid (HCl), methanol (MeOH), potassium carbonate (K2CO3), retention time (Rt), room temperature (rt), sodium sulphate (Na2SO4), sodium triacetoxyborohydride (NaBH(OAc)3), tetrahydrofuran (THF), thin layer chromatography (TLC), triethylamine (Et3N), water (H2O). 14 ACS Paragon Plus Environment

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Automated column chromatography purifications were run using a Teledyne ISCO apparatus (CombiFlash® Rf) with pre-packed silica gel columns of different sizes (from 4 g until 24 g). Flash column chromatography was performed manually on pre-packed 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 Macherey-Nagel pre-coated 0.05 mm TLC plates (SIL G-50 UV254). NMR experiments were run on a Bruker Avance III 400 system (400.13 MHz for 1H, and 100.62 MHz for

13

C), equipped with a BBI

probe and Z-gradients. Spectra were acquired at 300 K, using deuterated dimethylsulfoxide (DMSO-d6), deuterated chloroform (CDCl3) or deuterated water (D2O) as solvents. Chemical shifts (δ) for 1H and 13C spectra were recorded in parts per million (ppm) using the residual nondeuterated solvent as the internal standard (for DMSO-d6: 2.50 ppm, 1H; 39.52 ppm,

13

C; for

CDCl3: 7.26 ppm, 1H and 77.16 ppm, 13C; for D2O: 4.79 ppm, 1H). Data are reported as follows: 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 210-400 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 precolumn (5x2.1 mm ID, particle size 1.7 µm). Mobile phase was 10 mM NH4OAc in H2O at pH 5, adjusted with AcOH (A) and 10 mM NH4OAc in CH3CN-H2O (95:5) at pH 5 (B). Electrospray ionization in positive and negative mode was applied. Analyses were performed with method A 15 ACS Paragon Plus Environment


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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 210-400 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 NH4OAc in CH3CN-H2O (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)3 (2.0 equiv.) was added. Stirring was continued until UPLC and TLC analysis indicated the disappearance of the starting material. The reaction mixture was then quenched with 10% aqueous K2CO3solution and the two phases were separated. The organic layer was dried over Na2SO4. After evaporation of the solvent, the crude was purified by column chromatography, eluting with Cy/EtOAc DCM/MeOH or DCM/(MeOH/Et3N or NH3) as indicated in each case. 4-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1yl)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)3 (178.0 mg, 0.84 mmol) in anhydrous DCM (4.5 mL). The crude was purified by column chromatography [DCM and MeOH/NH3 (19% MeOH and 1% NH3 in DCM), from 95:5 to 40:60] to afford the free base of 1 as a colorless oil (137.0 mg, 82 %). UPLC/MS (method A): Rt 1.58 min; MS (ES) C24H32FN3O requires m/z 397, found m/z 398 [M+H]+. The 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 Et2O (1.5 mL, 3.0 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 1 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.07 (bs, 1H), 10.71 (bs, 1H), 9.53 (s, 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), 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). 13C NMR (101 MHz, DMSO-d6) δ 160.7 (J = 250.4 Hz), 157.8, 136.1, 135.8, 133.1, 131.3, 130.2, 125.1, 124.9, 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-1yl)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)3 (76.0 mg, 0.36 mmol). Stirring was continued until UPLC and TLC analysis indicated the disappearance of the starting material. The reaction mixture was then quenched with 10% aqueous K2CO3 solution and the two phases were separated. The organic layer was dried over Na2SO4. After evaporation of the solvent, the crude was purified by column chromatography [DCM and MeOH/Et3N (19% MeOH and 1% Et3N in DCM), from 95:5 to 40:60] to afford the free base of 2 as a colorless oil (65.0 mg, 62%). UPLC/MS (method A): Rt 2.18 min; MS (ES) C25H34FN3O requires m/z 411, found 17 ACS Paragon Plus Environment


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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 Et2O (1.3 mL, 1.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 2 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.36 – 11.48 (m, 2H), 10.78 (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), 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). 13C NMR (101 MHz, DMSO-d6) δ 160.7 (J = 250.4 Hz), 157.8, 138.3, 136.1, 135.3, 133.3, 133.1, 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-1yl)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 Na2SO4. After 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) C24H30FNO3 requires m/z 411, found m/z 412 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 7.64 (dd, J = 8.4, 2.3 Hz, 1H), 7.59 (d, J = 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, 2H), 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, 3H), 2.10 – 1.96 (m, 2H), 1.87 – 1.64 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 165.8, 160.8 (J = 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, 58.6, 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 NaBH(OAc)3 (97.0 mg, 0.46 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/Et3N (19% MeOH and 1% Et3N in DCM), from 95:5 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) C24H26FN3O requires m/z 343, found m/z 344 [M+H]+. The free base of 4 (48.0 mg, 0.13 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et2O (1.3 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 4 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.68 (bs, 1H), 11.06 – 10.35 (bs, 1H), 9.70 (bs, 2H), 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, 2H), 3.50 – 3.26 (m, 8H), 2.74 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.5 (J = 249.0 Hz), 157.9, 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, 42.7, 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)3 (119.0 mg, 0.56 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/Et3N (19% MeOH and 1% Et3N in DCM), from 95:5 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) C21H28FN3O requires m/z 357, found m/z 358 [M+H]+. The free base of 5 (68.0 mg, 0.19 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et2O 19 ACS Paragon Plus Environment


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(1.9 mL, 3.80 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 5 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.82 (bs, 1H), 10.78 (bs, 1H), 10.37 (s, 2H), 9.80 (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 = 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), 3.73 – 3.54 (m, 4H), 3.53 – 3.34 (m, 1H), 2.80 (s, 3H), 1.65 (d, J = 6.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.5 (J = 248.5Hz), 157.9, 136.4, 133.7, 132.7, 131.6, 129.2, 125.8, 122.1, 116.2, 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-1yl)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), Et3N (64.0 µL, 0.46 mmol) and NaBH(OAc)3 (97.0 mg, 0.46 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/NH3 (19% MeOH and 1% NH3 in DCM), from 95:5 to 40:60] to afford the free base of 6 as a colorless oil (48.0 mg, 56%). UPLC/MS (method A): Rt 1.69 min; MS (ES) C20H30FN3O requires m/z 371, 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 Et2O (1.3 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 6 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.03 (bs, 1H), 10.75 (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), 3.52 – 3.30 (m, 6H), 2.80 (s, 3H), 1.86 (s, 6H).

13

C NMR (101 MHz, DMSO-d6) δ 160.4 (J =

225.8 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, 45.7, 45.5, 42.0, 25.4.


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4-[[[1-(2-Fluorophenyl)cyclobutyl]amino]methyl]-2-[(4-methylpiperazin-1yl)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 mmol) and NaBH(OAc)3 (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude was 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) C23H30FN3O requires m/z 383, found m/z 384 [M+H]+. The free base of 7 (50.0 mg, 0.13 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.65 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 7 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.95 (bs, 1H), 10.80 (bs, 1H), 9.95 (bs, 2H), 7.71 – 7.61 (m, 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), 2.76 – 2.61 (m, 2H), 2.41 – 2.21 (m, 1H), 1.90 – 1.67 (m, 1H). 13C NMR (101 MHz, DMSO-d6) 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-1yl)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)3 (177.0 mg, 0.84 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/NH3 (19% MeOH and 1% NH3 in DCM), 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) C25H34FN3O requires m/z 411, found m/z 412 [M+H]+.The 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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solution in Et2O (1.5 mL, 3.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 8 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.06 (bs, 1H), 10.71 (bs, 1H), 9.51 (s, 2H), 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, 3H), 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 (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, 2H), 1.77 (d, J = 13.1 Hz, 2H), 1.67 – 1.48 (m, 1H), 1.34 – 1.05 (m, 3H). 13C NMR (101 MHz, DMSO-d6) 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, 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)3 (152.0 mg, 0.72 mmol) in anhydrous DCM (4.0 mL). The residue was then dissolved in 1,4-dioxane (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) C23H30FN3O requires m/z 383, found m/z 384 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.41 (td, J = 8.2, 1.9 Hz, 1H), 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, 1H), 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, 2H), 1.70 – 1.55 (m, 4H), 1.36 – 1.16 (m, 2H).

13

C NMR (101 MHz, DMSO-d6) δ 161.8 (J =

249.5 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, 53.7, 47.7, 45.7, 37.5, 23.1. 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)3 (81.0 mg, 0.36 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography (EtOAc/Cy/Et3N, 4:1:0.1) to afford the free base of 10 as a colorless oil (50.0 mg, 50%). UPLC/MS (method A): Rt 1.88 min; MS (ES) C24H33FN3O requires m/z 425, found m/z 426 [M+H]+. The free base of 10 (36.0 mg, 0.08 mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et2O (1.0 mL, 1.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 10 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.83 (d, J = 11.3 Hz, 1H), 10.63 (s, 1H), 9.73 – 9.22 (m, 2H), 7.64 (t, J = 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), 2.54-2.46 (m, 2H), 2.42 – 2.30 (m, 2H), 2.03 (s, 3H), 1.96 – 1.85 (m, 2H), 1.63 – 1.50 (m, 2H).

13

C NMR (101

MHz, DMSO-d6) δ 168.9, 157.8 (J = 249.5 Hz), 157.3, 146.4, 136.0, 134.1, 133.2, 131.8, 130.4, 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)3 (106.0 mg, 0.50 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/Et3N (19% MeOH and 1% Et3N in 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) C25H34FN3O requires m/z 411, found m/z 412 [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 Et2O (1.4 mL, 2.8 mmol, 20.0 equiv.) was added. Evaporation of solvents 23 ACS Paragon Plus Environment


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afforded 11 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.99 (bs, 1H), 10.74 (bs, 1H), 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 – 2.96 (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, 2H), 1.26 (t, J = 7.0 Hz, 3H).

13

C NMR (101 MHz, DMSO-d6) δ 160.7 (J = 250.4 Hz), 157.8,

136.1, 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, 46.87, 36.1, 22.1, 8.5. 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)3 (186.0 mg, 0.88 mmol) in anhydrous DCM (6.0 mL). The crude was 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) C30H36FN3O requires m/z 473, found m/z 474 [M+H]+. The free base of 12 (60.0 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, DMSO-d6) δ 12.22 (bs, 1H), 10.68 (bs, 1H), 9.47 (bs, 2H), 7.73 – 7.57 (m,

3H), 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), 2.55 – 2.50 (m, 2H), 2.42 – 2.31 (m, 2H), 2.02 – 1.80 (m, 2H), 1.65 – 1.50 (m, 2H).

13

C NMR

(101 MHz, DMSO-d6) δ 161.2 (J = 249.5 Hz), 157.8, 136.2, 133.6, 132.2, 131.8, 131.4, 130.9, 129.2, 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-1yl)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, 0.26 mmol) and NaBH(OAc)3 (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude 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) C26H36FN3O requires m/z 425, found m/z 426 [M+H]+. The free base of 13 (81.0 mg, 0.20 mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et2O (2.0 mL, 4.0 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 13 as a white powder. 1H NMR (400 MHz, D2O) δ 7.46 – 7.34 (bs, 2H), 7.21 (t, J = 7.6 Hz, 1H), 7.17 – 7.05 (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), 1.63 (dt, J = 13.8, 5.2 Hz, 2H), 1.27 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, D2O) δ 160.4 (J = 249.4 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, 49.5, 48.6, 47.5, 44.8, 35.7, 21.9, 15.9. 4-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-phenylpiperazin-1yl)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)3 (106.0 mg, 0.50 mmol) in anhydrous DCM (3.0 mL). The crude 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) C29H34FN3O 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 Et2O (1.3 mL, 2.6 mmol, 20.0 equiv.) 25 ACS Paragon Plus Environment


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was added. Evaporation of solvents afforded 14 as a white powder. 1H NMR (400 MHz, DMSOd6) 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, 2H), 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 (m, 2H).

13

C NMR (101 MHz, DMSO-d6) δ 162.3 (J = 248.5 Hz), 157.9, 150.0, 136.7, 134.0,

133.5, 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, 36.3, 22.7. 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)3 (102.0 mg, 0.48 mmol) in anhydrous DCM (3.0 mL). The crude was purified by 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) C24H33FN3O requires m/z 379, found m/z 380 [M+H]+. The free base (57.0 mg, 0.15 mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et2O (1.5 mL, 3.0 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 15 as a white powder. 1H NMR (400 MHz, DMSO-d6) 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 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, 12H), 2.79 (s, 3H), 2.45 – 2.30 (m, 4H), 1.85 (m, 2H), 1.65 – 1.37 (m, 2H). 13C NMR (101 MHz, DMSO-d6) 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, 47.6, 47.4, 46.6, 41.9, 35.24, 21.8. 4-[[[1-(2-Methoxyphenyl)-cyclopentyl]amino]-methyl]-2-[(4-methylpiperazin-1yl)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)3 (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/NH3 (19% MeOH and 1% NH3 in 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) C25H35N3O2 requires m/z 409, found m/z 410 [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 Et2O (0.3 mL, 1.2 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 16 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.91 (bs, 1H), 11.07 – 10.42 (bs, 1H), 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), 7.12 (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, 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, 4H), 1.94 – 1.76 (m, 2H), 1.63 – 1.43 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 157.1, 156.3, 136.10, 135.5, 133.44, 131.13, 128.28, 124.4, 122.1, 121.24, 112.33, 116.02, 70.6, 56.0, 53.6, 49.9, 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)3 (80.0 mg, 0.38 mmol) in anhydrous DCM (2.5 mL). The crude 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) C24H32FN3 requires m/z 397, found m/z 398 [M+H]+. The free base of 17 (50.0 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 17 as a white powder. 1H NMR



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(400 MHz, DMSO-d6) δ 11.82 (bs, 1H), 10.72 (bs, 1H), 9.92 (bs, 2H), 7.74 – 7.50 (m, 4H), 7.43 (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). 13C NMR (101 MHz, DMSO-d6) δ 160.4, 156.3, 140.0, 136.3, 133.67, 132.0, 131.60, 124.01, 122.0, 116.1, 116.0, 115.2, 70.1, 50.4, 49.7, 47.6, 46.7, 42.6, 36.01, 22.4. 1-(4-Fluorophenyl)-N-[[3-[(4-methylpiperazin-1-yl)methyl]phenyl]methyl]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)3 (80.0 mg, 0.38 mmol) in anhydrous DCM (2.5 mL). The crude 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) C24H32FN3 requires m/z 397, found m/z 398 [M+H]+. The free base of 18 (53.0 mg, 0.13 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.65 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 18 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.85 (bs, 1H), 10.71 (bs, 1H), 9.85 (s, 2H), 7.83 (dd, J = 8.7, 5.2 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 – 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), 1.57 – 1.41 (m, 2H).

13

C NMR (101 MHz, DMSO-d6) δ 162.5 (J = 248.5 Hz), 157.5, 135.4,

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. 4-[1-(3-Methoxyphenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1yl)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)3 (76.0 mg, 0.36 mmol) in anhydrous DCM (3.0 mL). The crude 28 ACS Paragon Plus Environment

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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) C25H35N3O requires m/z 409, found m/z 410 [M+H]+. The free base of 19 (50.0 mg, 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, DMSO-d6) δ 11.74 (s, 1H), 10.76 (s, 1H), 9.76 (m, 2H), 7.47 – 7.34 (m,

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 – 3.25 (m, 2H), 2.82 (s, 3H), 2.44 – 2.25 (m, 4H), 1.85 (m, 2H), 1.52 (m, 2H).

13

C NMR (101

MHz, DMSO-d6) δ 160.1, 157.8, 139.7, 136.0, 134.0, 130.7, 126.4, 122.5, 119.8, 115.0, 113.7, 113.0, 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)3 (144.0 mg, 0.68 mmol) in anhydrous DCM (3.0 mL). The crude 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%). UPLC-MS (method generic A): Rt 1.77 min; MS (ES) C24H32FN3 requires m/z 381, found m/z 382 [M+H]+. The free base of 20 (50.0 mg, 0.13 mmol) was then dissolved in 0.5 mL of DCM and 2.0 M HCl solution in Et2O (1.2 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 20 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.18 (bs, 2H), 9.74 (bs, 2H), 7.65 (m, 2H), 7.51 (t, J = 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 = 7.4, 6.8 Hz, 2H), 1.60 (dt, J = 11.1, 5.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.2 (J = 29 ACS Paragon Plus Environment


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248.5 Hz), 132.4, 132.1, 131.9, 130.8, 128.9, 125.5, 124.8, 117.1, 70.1, 49.2, 48.4, 48.4, 47.9, 42.6, 36.3, 22.6. 1-(2-Fluorophenyl)-N-[[4-methoxy-3-[(4-methylpiperazin-1-yl)methyl]-phenyl]methyl] cyclopentanamine trihydrochloride (21). 21 was prepared according to the general procedure for the synthesis of secondary amines, starting from 33d (38.0 mg, 0.15 mmol), 31b (28.0 mg, 0.15 mmol) and NaBH(OAc)3 (64.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/Et3N (19% MeOH and 1% Et3N in DCM), from 95:5 to 40:60] to afford 21 as a colorless oil (40 mg, 65%). UPLC/MS (method A): Rt 1.70 min; MS (ES) C25H34FN3O requires m/z 411, found m/z 412 [M+H]+. The free base of 21 (50.0 mg, 0.12 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et2O (1.2 mL, 2.4 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 21 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.07 (bs, 2H), 9.65 (bs, 2H), 7.66 (t, J = 7.8 Hz, 1H), 7.50 (m, 2H), 7.46 – 7.38 (m, 1H), 7.37 – 7.26 (m, 2H), 7.07 (d, J = 8.4 Hz, 1H), 4.21 (bm, 2H), 3.84 (s, 3H), 3.77 (m, 2H), 3.72 (m, 8H), 2.79 (bs, 3H), 2.60 – 2.45 (m, 2H), 2.37 (m, 2H), 2.09 – 1.77 (m, 2H), 1.69 – 1.41 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.4 (J = 248.5 Hz), 158.8, 136.6, 135.4, 133.4, 131.7, 130.1, 125.0, 124.6, 123.8, 116.5, 110.9, 55.9, 69.8, 49.3, 49.2, 47.2, 46.8, 42.0, 35.7, 22.2. 1-(2-Fluorophenyl)-N-[[2-[(4-methylpiperazin-1yl)methyl]phenyl]methyl]cyclopentanamine

trihydrochloride

(22).

22

was

prepared

according to the general procedure for the synthesis of secondary amines, starting from 33a (56.0 mg, 0.26 mmol), 31b (46.0 mg, 0.26 mmol) and NaBH(OAc)3 (110.0 mg, 0.52 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM/MeOH (20% in DCM), from 100:0 to 0:100] to afford the free base of 22 as a colorless oil (52.0 mg,


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55%). UPLC/MS (method A): Rt 2.05 min; MS (ES) C24H32FN3 requires m/z 381, found m/z 382 [M+H]+. The free base of 22 (52.0 mg, 0.13 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.65 mL, 2.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 22 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.86 – 11.06 (bs, 1H), 10.17 – 9.24 (bs, 2H), 7.70 (t, J = 8.1 Hz, 1H), 7.60 – 7.43 (m, 2H), 7.37 (q, J = 7.0 Hz, 2H), 7.33 – 7.20 (m, 2H), 7.07 (d, J = 7.6 Hz, 1H), 4.07 (s, 2H), 3.87 – 3.25 (m, 8H), 3.36 – 3.00 (m, 2H), 2.77 (s, 3H), 2.62 – 2.36 (m, 4H), 2.11 – 1.80 (m, 2H), 1.75 – 1.42 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.9 (J = 249.0 Hz), 132.6, 132.2, 132.1, 131.7, 130.8, 130.7, 129.8, 129.4, 125.1, 124.7, 117.1, 70.7, 66.7, 53.2, 47.6, 45.3, 42.3, 40.2, 23.0. 1-(2-Fluorophenyl)-N-[[4-[(4-methylpiperazin-1yl)methyl]phenyl]methyl]cyclopentanamine

trihydrochloride

(23).

23

was

prepared

according to the general procedure for the synthesis of secondary amines, starting from 33e (43.0 mg, 0.19 mmol), 31b (35.0 mg, 0.19 mmol) and NaBH(OAc)3 (80.0 mg, 0.38 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM/MeOH (20% in DCM), from 100:0 to 0:100] to afford the free base of 23 as a colorless oil (52.0 mg, 72%). UPLC/MS (method A): Rt 2.06 min; MS (ES) C24H32FN3 requires m/z 381, found m/z 382 [M+H]+. The free base of 23 (50.0 mg, 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 23 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 12.07 (bs, 1H), 9.69 (bs, 2H), 7.67 – 7.53 (m, 3H), 7.48 (tdd, J = 7.3, 5.0, 1.6 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.34 – 7.19 (m, 2H), 4.28 (s, 2H), 3.88 (t, J = 5.7 Hz, 2H), 3.65 – 3.55 (m, 2H), 3.54 – 3.20 (m, 6H), 2.77 (s, 3H), 2.60 – 2.55 (m, 2H), 2.40 -2.32 (m, 2H), 2.04 – 1.79 (m, 2H), 1.71 – 1.47 (m, 2H).



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13

C NMR (101 MHz, DMSO-d6) δ 161.0 (J = 249.2 Hz), 132.6, 132.3, 131.9, 131.7, 131.2,

129.2, 125.5, 124.7, 117.0, 49.9, 47.5, 46.5, 42.6, 36.31, 22.7. 2-(Diethylaminomethyl)-4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]phenol trihidrochloride (24). 24 was prepared according to the general procedure for the synthesis of secondary amines, starting from 32i (45.0 mg, 0.22 mmol), 31b (39.0 mg, 0.22 mmol) and NaBH(OAc)3 (93.0 mg, 0.44 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM and MeOH/NH3 (19% MeOH and 1% NH3 in DCM), from 95:5 to 100:0] to afford the free base of 24 as a colorless oil (42.0 mg, 52%). UPLC/MS (method A): Rt 1.55 min; MS (ES) C23H31FN2O requires m/z 370, found m/z 371 [M+H]+. The free base of 24 (42.0 mg, 0.12 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et2O (1.2 mL, 2.4 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 24 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 9.65 (s, 1H), 9.48 (s, 2H), 7.70 – 7.59 (m, 1H), 7.57 – 7.41 (m, 2H), 7.36 – 7.25 (m, 2H), 7.19 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 4.14 (d, J = 4.7 Hz, 2H), 3.74 (t, J = 5.6 Hz, 2H), 3.08 (s, 4H), 2.70 – 2.41 (m, 2H), 2.41 – 2.23 (m, 2H), 2.07 – 1.77 (m, 2H), 1.72 – 1.47 (m, 2H), 1.27 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 160.2 (J = 250.4 Hz), 156.3, 135.3, 134.7, 132.6, 132.2, 131.6, 130.0, 124.7, 120.9, 116.2, 115.2, 69.8, 49.0, 46.9, 45.9, 35.5, 22.0, 8.36. 4-[[[1-(2-Fluorophenyl)tetrahydropyran-4-yl]amino]methyl]-2-[(4-methylpiperazin-1yl)methyl]phenol trihydrochloride (25). 25 was prepared according to the general procedure for the synthesis of secondary amines, starting from 32i (73.0 mg, 0.34mmol), 31b (75.0 mg, 0.34 mmol) and NaBH(OAc)3 (144.0 mg, 0.68 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography (Cy/EtOAc from 95:5 to 40:60) to afford the free base of 25 as colorless oil (78.0 mg, 60%). UPLC/MS (method A): Rt 1.94 min; MS (ES)


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C23H29FN2O2 requires m/z 384, found m/z 385 [M+H]+. The free base of 25 (76.0 mg, 0.19 mmol) was then dissolved in 1.5 mL of DCM and 2.0 M HCl solution in Et2O (1.9 mL, 3.8 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 25 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.44 (s, 2H), 7.70 – 7.60 (m, 1H), 7.58 – 7.48 (m, 1H), 7.46 (s, 1H), 7.37 – 7.28 (m, 2H), 7.22 (dd, J = 8.4, 2.2 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.32 – 4.09 (m, 2H), 4.04 – 3.88 (m, 2H), 3.87 – 3.62 (m, 4H), 3.33 – 3.21 (m, 4H), 3.11 (s, 2H), 2.45 – 2.28 (m, 2H), 1.92 (tq, J = 12.7, 6.7, 5.4 Hz, 2H), 1.73 – 1.44 (m, 2H).

13

C NMR (101 MHz,

DMSO-d6) δ 160.1 (J = 250.5 Hz), 157.6, 136.4, 135.9, 133.6, 133.0, 131.6, 130.1, 124.9, 122.1, 116.4, 115.4, 70.1, 62.7, 53.4, 50.3, 47.1, 35.6, 22.1. 4-[[[1-(2-Fluorophenyl)cyclopentyl]amino]methyl]-2-(1-piperidylmethyl)phenol dihydrochloride (26). 26 was prepared according to the general procedure for the synthesis of secondary amines, starting from 32h (80.0 mg, 0.36 mmol), 31b (68.0 mg, 0.36 mmol) and NaBH(OAc)3 (152.0 mg, 0.72 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [Cy/EtOAc, from 90:10 to 0:100] to afford the free base of 26 as a colorless oil (45.0 mg, 33%). UPLC/MS (method A): Rt 1.85 min; MS (ES) C24H31FN2O requires m/z 382, found m/z 383 [M+H]+. The free base of 26 (40.0 mg, 0.10 mmol) was then dissolved in 1.0 mL of DCM and 2.0 M HCl solution in Et2O (1.0 mL, 2.0 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 26 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 10.16 (s, 1H), 9.54 (s, 2H), 7.72 – 7.61 (m, 1H), 7.58 – 7.45 (m, 2H), 7.38 – 7.25 (m, 2H), 7.21 (dd, J = 8.4, 2.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.13 (d, J = 4.4 Hz, 2H), 3.84 – 3.63 (m, 2H), 3.47 – 3.18 (m, 4H), 3.03 – 2.75 (m, 2H), 2.51 (dt, J = 3.7, 1.9 Hz, 2H), 2.37 (dd, J = 13.2, 6.4 Hz, 2H), 1.92 (d, J = 8.2 Hz, 2H), 1.79 (d, J = 12.9 Hz, 3H), 1.70 – 1.49 (m, 3H). 13C



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NMR (101 MHz, DMSO-d6) δ 162.3 (J = 249.5 Hz), 157.6, 136.6, 133.7, 133.4, 132.3, 130.7, 125.5, 125.0, 122.0, 116.9, 116.0, 53.9, 52.1, 53.6, 52.0, 47.7, 36.4, 22.8, 22.6. 1-(2-Fluorophenyl)-N-[[3-(1-piperidylmethyl)phenyl]methyl]cyclopentanamine dihydrochloride (27). 27 was prepared according to the general procedure for the synthesis of secondary amines, starting from 33b (44.0 mg, 0.24 mmol), 31b (50.0 mg, 0.24 mmol) and NaBH(OAc)3 (102.0 mg, 0.48 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM/MeOH, from 100:0 to 0:100] to afford the free base of 27 as a colorless oil (50.0 mg, 57%). UPLC/MS (method A): Rt 2.12 min; MS (ES) C24H31FN2 requires m/z 366, found m/z 367 [M+H]+. The free base of 27 (50.0 mg, 0.14 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.7 mL, 2.8 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 27 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.71 (d, J = 8.7 Hz, 2H), 7.65 (s, 2H), 7.58 (s, 1H), 7.57 – 7.47 (m, 1H), 7.46 – 7.37 (m, 2H), 7.35 – 7.23 (m, 2H), 4.20 (d, J = 5.1 Hz, 2H), 3.86 (t, J = 5.5 Hz, 2H), 3.33 – 3.15 (m, 2H), 2.96 – 2.74 (m, 2H), 2.62 – 2.47 (m, 2H), 2.46 – 2.28 (m, 2H), 2.06 – 1.50 (m, 9H), 1.47 – 1.15 (m, 1H).

13

C NMR (101 MHz, DMSO-d6) δ 160.8 (J = 250.5 Hz), 134.2, 132.5,

132.4, 132.1, 131.7, 130.8, 128.8, 125.4, 124.6, 117.0, 70.8, 59.2, 52.8, 48.0, 36.4, 23.0, 22.8, 22.3. 1-(2-Fluorophenyl)-N-[[3-[1-methyl-4-piperidyl)methyl]phenyl]methyl]cyclopentanamide dihydrochloride (28). 28 was prepared according to the general procedure for the synthesis of secondary amines, starting from 34a (60.0 mg, 0.23 mmol), 31b (50.0 mg, 0.23 mmol) and NaBH(OAc)3 (97.0 mg, 0.46 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM/MeOH (20% MeOH in DCM), from 90:10 to 0:100] to afford the free base of 28 a colorless oil (60.0 mg, 57%). UPLC/MS (method A): Rt 2.04 min; MS (ES)


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C25H33FN2 requires m/z 380, found m/z 381 [M+H]+. The free base of 28 (30.0 mg, 0.08 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.4 mL, 1.6 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 28 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (bs, 1H), 9.52 (bs, 2H), 7.62 (td, J = 8.5, 8.1, 1.9 Hz, 1H), 7.56 – 7.48 (m, 1H), 7.42 – 7.23 (m, 3H), 7.21 – 7.14 (m, 2H), 7.11 (s, 1H), 3.83 (t, J = 5.1 Hz, 2H), 3.42 – 3.42 (m, 2H), 2.94 – 2.73 (m, 2H), 2.68 (d, J = 4.8 Hz, 2H), 2.56 – 2.45 (m, 5H), 2.37 (dt, J = 12.4, 5.6 Hz, 2H), 1.92 (td, J = 11.9, 9.5, 5.5 Hz, 2H), 1.80 – 1.64 (m, 3H), 1.64 – 1.41 (m, 4H). 13

C NMR (101 MHz, DMSO-d6) δ 160.1 (J = 250.5 Hz), 139.3, 131.8, 130.9, 130.4, 129.9,

129.0, 127.9, 127.7, 124.8, 123.9, 116.4, 69.8, 53.5, 47.4, 42.5, 41.3, 35.6, 34.11, 28.8, 22.1. 4-[[[1-(2-Fluorophenyl)tetrahydropyran-4-yl]amino]methyl]-2-[(4-methylpiperazin-1yl)methyl]phenol trihydrochloride [1:1, Cis and trans stereoisomers] (29). 29 was prepared according to the general procedure for the synthesis of secondary amines, starting from 34b (20.0 mg, 0.11 mmol), 31b (25.0 mg, 0.11 mmol) and NaBH(OAc)3 (50.0 mg, 0.22 mmol) in anhydrous DCM (2.5 mL). The crude was purified by column chromatography (Cy/EtOAc from 100:0 to 30:70) to afford the free base of 29 as 1:1 mixture of cis:trans stereoisomers, as colorless oil (31.0 mg, 75%). UPLC/MS (method B): Rt 3.05 min; MS (ES) C26H34FN requires m/z 379, found m/z 380 [M+H]+. The free base of 29 (31.0 mg, 0.08 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.05 mL, 0.16 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 29 as a white powder as mixture 50:50 of cis:trans isomers. 1H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 2H), 7.64 – 7.56 (m, 1H), 7.56 – 7.47 (m, 1H), 7.35 – 7.28 (m, 2H), 7.28 – 7.20 (m, 1H), 7.16 – 7.10 (m, 2H), 7.07 (d, J = 7.3 Hz, 1H), 3.82 (dd, J = 7.2, 4.7 Hz, 2H), 2.52 – 2.29 (m, 7H), 2.01 – 1.82 (m, 2H), 1.73 – 1.51 (m, 5H), 1.49 – 1.20 (m, 5H), 1.04 – 0.64 (m, 5H).

13

C NMR (101 MHz, DMSO-d6) δ 160.8 (J = 246.4 35

ACS Paragon Plus Environment


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Hz), 141.7, 132.4, 131.2, 130.9, 130.6, 129.8, 128.4, 125.4, 127.9, 117.1, 70.6, 48.1, 43.7, 39.2, 36.4, 35.2, 32.90, 32.8, 28.2, 24.3, 22.6, 22.8, 20.6. 1-(4-Fluorophenyl)-N-[[3-[(1-methyl-4-piperidyl)methyl]phenyl]methyl] cyclopentanamine (30). 30 was prepared according to the general procedure for the synthesis of secondary amines, starting from 34a (36.0 mg, 0.16 mmol), 31e (30.0 mg, 0.16 mmol) and NaBH(OAc)3 (68.0 mg, 0.32 mmol) in anhydrous DCM (3.0 mL). The crude was purified by column chromatography [DCM/MeOH (20% MeOH in DCM), from 100:0 to 0:100] to afford the free base of 30 a colorless oil (35.0 mg, 57%). UPLC/MS (method A): Rt 2.31 min; MS (ES) C25H33FN2 requires m/z 380, found m/z 381 [M+H]+. The free base of 30 (30.0 mg, 0.09 mmol) was then dissolved in 0.5 mL of dioxane and 4.0 M HCl solution in dioxane (0.45 mL, 1.8 mmol, 20.0 equiv.) was added. Evaporation of solvents afforded 30 as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.88 (s, 2H), 7.81 (m, 2H), 7.41 – 7.26 (m, 3H), 7.24 – 7.13 (m, 3H), 3.73 – 3.62 (m, 2H), 3.42 – 3.25 (m, 2H), 2.93 – 2.72 (m, 3H), 2.68 (d, J = 4.6 Hz, 3H), 2.50 – 2.46 (m, 1H), 2.44 – 2.28 (m, 4H), 1.96 – 1.79 (m, 2H), 1.83 – 1.63 (m, 3H), 1.65 – 1.35 (m, 4H), 1.32 – 1.17 (m, 1H).

13

C NMR (101 MHz, DMSO-d6) δ 162.5 (J = 245.0 Hz), 140.2,

134.3, 131.5, 130.5, 128.7, 128.3, 116.1, 71.4, 53.8, 47.71, 43.1, 40.2, 35.9, 34.7, 28.8, 22.4. Biological evaluation Cell lines. BT-474 (HTB-20), MDA-MB-361 (HT-B27), SK-BR-3 (HTB-30), MCF-7 (HTB22), HEK-293 (CRL-1573), and HEP-G2 (HB-8065) cells were acquired from the American Type Culture Collection and the National Collection of Type Cultures (ATCC). LNCaP (89110211) cells were acquired from Sigma-Aldrich. BT-474 cells were grown in DMEM High Glucose (4.5g/l D-Glucose) containing 4 mM L-glutamine, 10% FBS and 0.25 mM sodium pyruvate. HEK-293, MCF-7 and HEP-G2 cells were grown in DMEM High Glucose containing


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4 mM L-glutamine, 10% FBS and 0.5 mM sodium pyruvate. MDA-MB-361 and LNCaP cells were grown in RPMI-1640 containing 4 mM L-glutamine and 10% FBS. SK-BR-3 cells were grown in McCoy’s medium containing 10% FBS. Lysosomotropy and cytotoxicity assays. Cells were seeded in 96-well plates at 2000 cells/100 µl/well in culture medium and incubated overnight. For lysosomotropy assays, 24 h post-treatment cells lysosomal staining was measured as described previously.8 For cytotoxicity assays, 72 h post-treatment the relative percentage of cell number was evaluated with the CyQUANT kit (Invitrogen, Carlsbad, USA), setting vehicle-treated sample as 100%. This method was adopted because it is independent of cellular metabolic activity, which may be affected by both REV-ERB and autophagy inhibition. Seven 3-fold serial dilutions of a 100 µM concentration of compounds were used to obtain Log(inhibitor)-versus-response curves using GraphPad-Prism Software (San Diego, CA-USA). IC50 values deriving from at least 6 independent experiments were used to obtain mean IC50 ± SEM values shown in Tables 1-6. Because in our experimental condition CQ affected cell viability of BT-474, MDA-MB-361 and SK-BR-3 cells only at high micromolar concentration, seven 3-fold dilutions of a 900 µM concentration were used to obtain IC50 values shown in Table 6. REV-ERB luciferase assay.9 A reporter vector that contains two repetitions of the REV-ERBresponsive element consensus (5'-AGA ATG TAG GTC ATC TAG AAT GTA GGT CA-3') driving the expression of Cypridina luciferase gene was co-transfected with a plasmid expressing REV-ERBβ in HEK-293 cells. The following day, cells were treated with the compounds and after 24 h luciferase activities were measured according to the manufacturer’s instructions. A vector with an SV40 promoter-driven Gaussia luciferase was used for normalization.



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Quantitative RT-PCR. RNA sample preparation and relative transcript expression levels were assessed as described previously.9 GAPDH transcript was used for normalization. Primer sequences were: BMAL1 (5’-CCA GAG GCC CCT AAC TCC TC-3’ and 5’-TGG TCT GCC ATT GGA TGA TCT-3’, forward and reverse, respectively); HRTP (5’-GTT ATG GCG ACC CGC AG-3’ and 5’-ACC CTT TCC AAA TCC TCA GC-3’, forward and reverse, respectively); GAPDH (5’-AAG GTG AAG GTC GGA GTC AA-3’ and 5’-AAT GAA GGG GTC ATT GAT GG-3’, forward and reverse, respectively). shRNA. Sequence information for shRNAs used in the silencing experiments has been described previously.9. Fugene (Roche) transfection reagents was used for shRNA, according to the manufacturer’s instructions. For transcription expression analysis, forty eight hours posttransfection, GFP-positive cells were sorted by FACS and processed for qRT-PCR analysis to evaluate the expression of the REV-ERB-regulated clock gene, BMAL1. For CQ-sensitization experiments, twenty four hours post-transfection, cells were treated with a dose of CQ reducing the viability of non-transfected cells of about 25% and GFP-positive cells were counted at 48 h post-treatment and expressed as percentage of control. Fluorescent analysis of autophagy inhibition. Cells were transduced with a chimeric protein in which an acid-sensitive GFP and an acid-insensitive RFP were fused to LC3. Forty eight hours post-transduction, compound 30 or DMSO (vehicle) were added to the medium. After 4 hours the number of GFP and RFP fluorescent dots was assessed by fluorescent microscopy. Immunoblot analysis. Protein samples were extracted in RIPA buffer as described previously.9 LC3B and GAPDH levels were analyzed with anti-LC3B (Cell signaling, 3868) and anti-GADPH (Invitrogen, 398600) specific antibodies. Immunoblot experiments were performed in TBS-T buffer containing 5% bovine serum albumin (BSA). Anti-LC3B and anti-GAPDH


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antibodies were diluted 1:500 and 1:50000, respectively. Complementary HPR-conjugated secondary antibodies were diluted 1:20000. Upon reaction with ECL Western Blotting Detection Reagent (Euroclone), chemiluminescent signals were acquired with a LAS-4000 luminescent image analyzer (Fujifilm) and optical density of specific band signal was calculated with Photoshop image analysis software (Adobe) to obtain data presented in Figure 3B as LC3B/GAPDH signal ratio. Statistical analysis. Log(inhibitor)-versus-response curves, one-way ANOVA with Dunnett’s post-test and two-tails-t-test were performed using GraphPad-Prism Software (San Diego, CAUSA).



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ASSOCIATED CONTENT Supporting Information. Detailed experimental procedures, analytical and spectroscopic data of all intermediates for the synthesis of compounds 1-30. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *B.G.: [email protected]; phone, +39 01071781299; fax, +3901071781228. *R.S.: [email protected]; phone: +3901071781233; fax, +3901071781228. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. NOTES Esther Torrente, Rita Scarpelli and Benedetto Grimaldi are co-inventors in a patent that includes compounds here disclosed owned by Istituto Italiano di Tecnologia (IIT). The remaining authors declare no conflict of interest.

ACKNOWLEDGMENT We thank Dr. Mark Wade for his precious scientific comments. We also thank Dr. Tiziano Bandiera, Dr Ana Guijarro and Dr. Jonathan Hardman for discussions, and Ennio Albanesi, Carla Lo Vecchio, Silvia Venzano, Sine Mandrup Bertozzi and Luca Goldoni for their technical 40 ACS Paragon Plus Environment

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support. Dr Luisa Ercolani has been supported by an interdepartmental post-doctoral fellowship from the Fondazione Istituto Italiano di Tecnologia (IIT). ABBREVIATIONS HER2, Human epidermal growth factor receptor-2; ER, Estrogen receptor; CQ, chloroquine; PDAC, prostate ductal adenocarcinoma cells; HMEC, human mammary epithelial cells; LC3, Microtubule-associated protein 1A/1B-light chain 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; BMAL1, Brain And Muscle ARNT-Like 1; REV-ERBβ, reverse orientation c-erbA gene, variant beta. REFERENCES 1.

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10. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D., Reductive amination of aldehydes and ketones with sodium triacetoxyborohydride. Studies on direct and indirect reductive amination procedures(1). J. Org. Chem. 1996, 61, 3849-3862. 11. Tagad, H. D.; Hamada, Y.; Nguyen, J. T.; Hidaka, K.; Hamada, T.; Sohma, Y.; Kimura, T.; Kiso, Y., Structure-guided design and synthesis of P1' position 1-phenylcycloalkylaminederived pentapeptidic BACE1 inhibitors. Bioorg. Med. Chem. 2011, 19, 5238-5246. 12. Thurkauf, A.; de Costa, B.; Yamaguchi, S.; Mattson, M. V.; Jacobson, A. E.; Rice, K. C.; Rogawski, M. A., Synthesis and anticonvulsant activity of 1-phenylcyclohexylamine analogues. J. Med. Chem. 1990, 33, 1452-1458. 13. Wang, R.; Gregg, B. T.; Zhang, W.; Golden, K. C.; Quinn, J. F.; Cui, P.; Tymoshenko, D. O., Rapid Ti(Oi-Pr)4 facilitated synthesis of α,α,α-trisubstituted primary amines by the addition of Grignard reagents to nitriles under microwave heating conditions. Tetrahedron Lett. 2009, 50, 7070-7073. 14. Cromwell, N. H., The reactions of unsaturated ketones and derivatives with amino compounds; amino ketones. Chem. Rev. 1946, 38, 83-137. 15. Mirza-Aghayan, M.; Boukherroub, R.; Bolourtchian, M., A mild and efficient palladium– triethylsilane system for reduction of olefins and carbon–carbon double bond isomerization. Appl. Organomet. Chem. 2006, 20, 214-219. 16. Zhou, C.; Zhong, W.; Zhou, J.; Sheng, F.; Fang, Z.; Wei, Y.; Chen, Y.; Deng, X.; Xia, B.; Lin, J., Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-



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mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells. Autophagy 2012, 8, 1215-1226. 17. Yang, S.; Wang, X.; Contino, G.; Liesa, M.; Sahin, E.; Ying, H.; Bause, A.; Li, Y.; Stommel, J. M.; Dell'antonio, G.; Mautner, J.; Tonon, G.; Haigis, M.; Shirihai, O. S.; Doglioni, C.; Bardeesy, N.; Kimmelman, A. C., Pancreatic cancers require autophagy for tumor growth. Genes. Dev. 2011, 25, 717-729. 18. McAfee, Q.; Zhang, Z.; Samanta, A.; Levi, S. M.; Ma, X. H.; Piao, S.; Lynch, J. P.; Uehara, T.; Sepulveda, A. R.; Davis, L. E.; Winkler, J. D.; Amaravadi, R. K., Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency. Proc. Natl. Acad. Sci. U S A 2012, 109, 8253-8258. 19. Kimura, T.; Takabatake, Y.; Takahashi, A.; Isaka, Y., Chloroquine in cancer therapy: a double-edged sword of autophagy. Cancer Res. 2013, 73, 3-7. 20. Kojetin, D.; Wang, Y.; Kamenecka, T. M.; Burris, T. P., Identification of SR8278, a synthetic antagonist of the nuclear heme receptor REV-ERB. ACS Chem. Biol. 2011, 6, 131-134.


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Figure 1. Chemical structures of chloroquine (CQ) and 1.



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Figure 2. Planned chemical variations of 1 scaffold.


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Figure 3. (a) Compounds lysosomotropy (white bars) and REV-ERBβ (black bars) antagonism were evaluated by LysoTracker fluorescence- and luciferase REV-ERB-responsive reporterbased assays, respectively, as detailed in Experimental Section. Mean IC50 ± SEM obtained from concentration response plots of indicated compound is shown. N ≥ 6, *P 100

Compound

R

a

Concentration response plots of indicated compound cytotoxicity in breast cancer BT-474 and normal HMEC cells were used to calculate the concentration producing a 50% reduction of cell number compared to vehicle treatment (IC50), as described in Experimental Section. Shown as mean IC50 ± SEM (n ≥ 6). bCompounds with IC50 > 100 µM were considered inactive against the tested cells. cNot Assayed.


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Table 2. Role of R substituent on the piperazine ring in cancer cell cytotoxicity.

Compound

R

BT-474a

HMECa

9

H

31.85 ± 4.97

>100b

10

CH3CO

>100b

NAc

11

CH3CH2

39.32 ± 2.89

>100

12

PhCH2

34.72 ± 4.43

>100

13

(CH3)2CH

33.34 ± 5.65

>100

14

Ph

37.79 ± 2.63

>100

a

Concentration response plots of indicated compound cytotoxicity in breast cancer BT-474 and normal HMEC cells were used to calculate the concentration producing a 50% reduction of cell number compared to vehicle treatment (IC50), as described in Experimental Section. Shown as mean ± SEM (n ≥ 6). bCompounds with IC50 > 100 µM were considered inactive against the tested cells. cNot Assayed.



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Table 3. Role of nature and position of R1 and R2 substituents in cancer cell cytotoxicity.

Compound

R1

R2

BT-474a

HMECa

15

H

OH

27.50 ± 0.71

> 100b

16

o-OCH3

OH

31.05 ± 2.58

17.37± 2.31

17

m-F

OH

14.15 ± 2.81

> 100

18

p-F

OH

9.41 ± 0.62

> 100

19

m-OCH3

OH

11.82 ± 5.23

13.11 ± 3.42

20

o-F

H

34.05 ± 3.40

> 100

21

o-F

OCH3

> 100

NAc

a



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Table 4. Influence of relative position of the substituents on the B phenyl ring in cancer cytotoxicity.

Compound

position

BT-474a

HMECa

22

ortho

> 100b

NAc

23

para

> 100

NA

a




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Table 5. Influence of heterocyclic moiety in cancer cytotoxicity.

Compound

R1

R2

24

o-F

25

R3

BT-474a

HMECa

OH

> 100b

NAc

o-F

OH

> 100

NA

26

o-F

OH

35.62 ± 6.42

> 100

27

o-F

H

39.96 ± 4.30

> 100

28

o-F

H

15.49 ± 0.74

> 100

29

o-F

H

>100

NA

30

p-F

H

2.10 ±019

> 100

a

Concentration response plots of indicated compound cytotoxicity in breast cancer BT-474 and normal HMEC cells were used to calculate the concentration producing a 50% reduction of cell 62 ACS Paragon Plus Environment

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number compared to vehicle treatment (IC50), as described in Experimental Section. Shown as mean ± SEM (n ≥ 6). bCompounds with IC50 > 100 µM were considered inactive against the tested cells. cNot Assayed.



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Table 6. Improved cytotoxicity of dual autophagy-REV-ERBβ inhibitor 30, compared to the clinically relevant singular autophagy inhibitor, CQ against tumor cell lines.

Compound

BT-474a,b

MDA-MB361a,b

SK-BR3a,c

MCF-7a,d

HEP-G2a

LNCaPa

30

2.10 ± 0.19

30.63 ± 5.98

16.02 ± 0.60

2.19 ± 0.46

2.73 ± 1.23

8.68 ± 1.45

CQ

123 ± 13.67

97± 7.43

50.37 ± 7.43

23.43 ± 5.32

25 ± 3.51

32.87 ± 2.5

a

Concentration response plots of 30 and CQ cytotoxicity in the indicated tumor cell lines were used to calculate the concentration producing a 50% reduction of cell number compared to vehicle treatment (IC50), as described in Experimental Section. Shown as mean ± SEM (n ≥ 6). N.D. = not determined. bERBB2- and ER-positive breast cancer cells. cERBB2-positive breast cancer cells. dER-positive breast cancer cells.


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Table of Content graphic


Synthesis and in Vitro Anticancer Activity of the First Class of Dual

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