Development of inhibitors of receptor protein tyrosine phosphatase β/ζ (PTPRZ1) as candidates for CNS disorders

Article abstract of DOI:10.1016/j.ejmech.2017.11.080

A new series of blood-brain barrier permeable molecules designed to mimic the activity of Pleiotrophin in the CNS has been designed and synthesized. These compounds exert their action by interacting with the intracellular domain PD1 of the Protein Tyrosin

Full text of DOI:10.1016/j.ejmech.2017.11.080

Accepted Manuscript
Development of inhibitors of receptor protein tyrosine phosphatase β/ζ (PTPRZ1) as
candidates for CNS disorders
Miryam Pastor, Rosalía Fernández-Calle, Bruno Di Geronimo, Marta Vicente-
Rodríguez, José María Zapico, Esther Gramage, Claire Coderch, Carmen Pérez-
García, Amy W. Lasek, Leonor Puchades-Carrasco, Antonio Pineda-Lucena, Beatriz
de Pascual-Teresa, Gonzalo Herradón, Ana Ramos
PII:
S0223-5234(17)30983-2
10.1016/j.ejmech.2017.11.080
EJMECH 9955
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 September 2017
Revised Date: 10 November 2017
Accepted Date: 27 November 2017
Please cite this article as: M. Pastor, Rosalí. Fernández-Calle, B. Di Geronimo, M. Vicente-Rodríguez,
José.Marí. Zapico, E. Gramage, C. Coderch, C. Pérez-García, A.W. Lasek, L. Puchades-Carrasco, A.
Pineda-Lucena, B. de Pascual-Teresa, G. Herradón, A. Ramos, Development of inhibitors of receptor
protein tyrosine phosphatase β/ζ (PTPRZ1) as candidates for CNS disorders, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.080.
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ACCEPTED MANUSCRIPT
S
F3C
O
CF3
S
µ
10a: IC50 (PTPRZ1) = 0.1
M
N
H
F3C
CF3
S
S
µ
12b: IC50 (PTPRZ1) = 0.1
M
1

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Development of Inhibitors of Receptor Protein
Tyrosine Phosphatase β/ζ (PTPRZ1) as Candidates
for CNS disorders
Miryam Pastor,† Rosalía Fernández-Calle,§ Bruno Di Geronimo,† Marta Vicente-Rodríguez,§
José María Zapico,† Esther Gramage,§ Claire Coderch,† Carmen Pérez-García,§ Amy W. Lasek,#
Leonor Puchades-Carrasco,≠ Antonio Pineda-Lucena,≠ Beatriz de Pascual-Teresa,*,† Gonzalo
Herradón,*,§ Ana Ramos.*,†
† Departamento de Química y Bioquímica, Facultad de Farmacia, Universidad San Pablo-CEU,
CEU Universities, Urbanización Montepríncipe, 28925, Alcorcón, Madrid, Spain
§ Departamento de Ciencias Farmacéuticas y de la Salud, Facultad de Farmacia, Universidad San
Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28925, Alcorcón, Madrid, Spain
≠ Unidad Mixta en Metabolómica Clínica Instituto de Investigación Sanitaria La Fe – Centro de
Investigación Príncipe Felipe, Hospital Universitario y Politécnico La Fe, Avenida Fernando
Abril Martorell, 106, Torre A, 6-17, 46026-Valencia, Spain
# Department of Psychiatry, University of Illinois at Chicago,1601 West Taylor Street, Chicago,
IL 60612, USA.
Abbreviations1
1 EDCI, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, hydroxybenzotriazole;
MCPBA, meta-chloroperbenzoic acid; MK, Midkine; PAMPA-BBB, membrane permeability
assay for the blood-brain barrier; PTN, Pleiotrophin; PTPRZ1, Protein Tyrosine-Phosphatase
Receptor Z1; SDDB, strategic drug delivery to brain; TPSA, topological polar surface area.
1

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ABSTRACT
A new series of blood-brain barrier permeable molecules designed to mimic the activity of
Pleiotrophin in the CNS has been designed and synthesized. These compounds exert their action
by interacting with the intracellular domain PD1 of the Protein Tyrosine-Phosphatase Receptor
Z1 (PTPRZ1), and inhibiting its tyrosine phosphatase activity. The most potent compounds 10a
and 12b (IC50 = 0,1 µM) significantly increase the phosphorylation of key tyrosine residues of
PTPRZ1 substrates involved in neuronal survival and differentiation, and display protective
effects against amphetamine-induced toxicity. Docking and molecular dynamics experiments
have been used to analyze the binding mode and to explain the observed selectivity against
PTP1B. An In vivo experiment has demonstrated that 10a can cross the BBB, thus promoting the
possibility of moving forward these candidates for the development of drugs for the treatment of
CNS disorders, such as drug addiction and neurodegenerative diseases.
KEYWORDS. PTPRZ1; CNS disorders; Drug addiction; Molecular Dynamics; Synthesis
INTRODUCTION
Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that share over 50% identity in
amino acid sequence [1]. Both PTN and MK play important roles in development and repair of
the central nervous system (CNS) [2]. The PTN and MK genes are widely expressed at different
times in different cell types during development [3]. However, their expression levels are highly
restricted to a few cell types in adults [4]. Both PTN and MK are upregulated at sites of injury,
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inflammation and repair in different cells of the CNS [5]. It is important to note that both PTN
and MK regulate neuroinflammation depending on the inflammatory stimulus and the brain area
considered [6]. In addition, MK and PTN are expressed in senile plaques of patients with
Alzheimer’s disease [7] and PTN upregulation has been also observed in the substantia nigra of
Parkinson’s Disease (PD) patient [8]. Both cytokines are also upregulated in different brain areas
after administration of different drugs of abuse [9], suggesting PTN and MK signaling may be
critical in different steps of wound repair in neurotoxic and neurodegenerative processes.
Accordingly, in studies carried out in PTN knockout (PTN-/-) mice, it was shown that PTN
prevents amphetamine-induced dopaminergic injury in the nigrostriatal pathway [10]. In
concordance with these data, striatal overexpression of PTN in different mouse models of
dopaminergic injury exerts neuroprotective effects [11].
These cytokines also modulate addictive behaviors. MK and PTN contribute to the extinction
of cocaine- and amphetamine-seeking behaviors, respectively [10c, 12] PTN limits morphine
withdrawal syndrome [13] and both, PTN and MK, are potent regulators of behavioral effects
induced by ethanol [14]. Particularly, it has been demonstrated that PTN transgenic
overexpression in the brain blocks the rewarding effects of alcohol [14a, 14b]. Overall, the data
suggest that PTN and MK could be used for the treatment of neurodegenerative diseases, for
prevention of drugs of abuse-induced neurotoxicity and for the treatment of a wide variety of
drug addiction disorders. Like other proteins, PTN and MK exert suboptimal drug-like
properties/inability to cross the blood-brain barrier (BBB). Although strategic drug delivery to
the brain has been successfully used with other proteins [15], the risks in many occasions
outweigh the benefits. If possible, pharmacological modulation of the signaling pathways
triggered by PTN and MK may be preferred [9].
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Both PTN and MK bind to the Protein Tyrosine-Phosphatase Receptor Z1 (PTPRZ1; a.k.a.
(R)PTPβ or RPTPβ/ζ), induce its oligomerization and inactivate its phosphatase activity [16].
This leads to an increase in tyrosine phosphorylation of substrates critical for the effects of these
cytokines such as β-catenin16b, Fyn kinase [17] and Anaplastic Lymphoma Kinase (ALK) [18].
We hypothesize that PTN and MK actions on neurodegenerative diseases and drug addiction
disorders described above can be reproduced with rationally designed small molecule inhibitors
of PTPRZ1 [5a, 9].
PTPRZ1 inhibitors have been recently tested in tumor models [19]. However, these molecules
were unable to cross the cell membrane and required intracellular delivery by liposome carriers
precluding use for the treatment of CNS diseases. In the present work, we propose to modulate
the phosphatase activity of PTPRZ1 through the design and synthesis of small molecules that can
cross the BBB and mimic the actions of PTN and MK.
Many known phosphatase inhibitors contain multi-charged phosphate-mimicking components,
and show poor cellular uptake. Huang et al. [20] described, for the first time, one compound
carrying an uncharged phosphate mimic (compound 1, Table 1) with a moderate activity against
PTPRZ1 (IC50 = 3.5 µM), and a certain degree of selectivity against other related phosphatases.
Considering 1 as a hit compound, we have followed a classical medicinal hit to lead optimization
for the discovery of new PTPRZ1 inhibitors with a potential increased activity and selectivity
and, more importantly, capable of crossing the BBB.
RESULTS AND DISCUSSION
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Design Rationale
The structure of compound 1 and all the analogs synthesized in this work is depicted in
Schemes 1-3.
We have analyzed the effect of the substitution in the aromatic rings present in 1, and the
nature and length of the connecting linker. In compounds 5a-c, the ether linkage was substituted
by amides of different length (Scheme 1). Topological Polar Surface Area (TPSA) for these
sulfoxides and the possibility of establishing ten hydrogen bond acceptors (see table 1) predict a
low bioavailability for a successful CNS drug [21]. To analyze whether the sulfoxide group is
necessary for activity, and with the aim of improving pharmacokinetic properties, sulfides 4a-d,
and 10a-b (Scheme 2) were also biologically tested. Calculated logP values for these sulfides are
higher than the optimal 5 value. For this reason, sulfide 10c, where an aromatic ring was
substituted by a pyridine, and sulfides 12a-e (Scheme 3) carrying an amino linker, were also
selected for synthesis and biological evaluation.
Chemistry
The synthesis of compounds 4 and 5 is depicted in Scheme 1. Sulfides 4a and 4d were
synthesized by reaction of 4-((trifluoromethyl)thio) benzoyl chloride (2a) and the corresponding
amine 3. For the synthesis of amides 4b and 4c, a (EDCI) catalyzed coupling between 2-(4-
((trifluoromethyl)thio)phenyl)acetic acid (2b) and the corresponding amine 3 was followed.
Oxidation of 4a-c with m-chloroperbenzoic acid provided sulfones 5a-c.
Scheme 1. Synthetic route to 4 and 5a
5

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Z
O
H2N
+
S
S
O
F3C
O
n
F3C
O
m
O
b
a
N
N
n
H
n
m
O
S
m
H
CF3
CF3
S
S
S
CF3
O
CF3
4a
: m=0; n=0
5a: m=0; n=0
5b: m=1; n=0
5c:
m=1; n=1
4b: m=1; n=0
2a: Z=Cl: m=0
2b:
3a: n = 0
3b:
4c
: m=1; n=1
4d: m=0; n=1
Z=OH; m=1
n = 1
a Reagents and conditions: (a) for 4a and 4d: K2CO3, THF; for 4c and 4b: HOBt, EDCI, DMF;
(b) MCPBA, DCM, 80 ºC, sealed tube.
Compounds 10 were synthesized by arylation of benzylic alcohols 8 and 9 with (4-
fluorophenyl)(trifluoromethyl)sulfane (6) or 1-fluoro-4-(trifluoromethylsulfonyl)benzene (7),
using sodium hydride as base (Scheme 2). Sulfones 7 and 1 were obtained by oxidation of 6 and
10a with m-chloroperbenzoic acid.
Scheme 2. Synthetic route to 1 and 10
O
O
S
S
a
F3C
F3C
F
F
X
6
7
O
O
S
F3C
F3C
b
a
HO
O
O
CF3
S
CF3
CF3
10a: X=S
10b: X=SO2
8
S
1
S
O
O
b
HO
S
F3C
N
O
9
10c
N
a
Reagents and conditions: (a) MCPBA, DCM, 80 ºC, sealed tube; (b) NaH, DMF and
compound 6 for 10a and 10c respectively, or compound 7 for 10b.
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Amines 12a-e were obtained by reductive amination of aldehydes 11 with amines 3 using
sodium triacetoxyborohydride as reductive agent (Scheme 3).
Scheme 3. Synthetic route to 12a-ea
O
N
NH2
n
a
n
H
H
F3C
F3C
+
S
R
S
SCF3
R
3a: n=0
3b: n=1
12a: n=0; R=SCF3
12b: n=1; R=SCF3
12c
: n=1; R=H
11a: R = SCF3
11b:
R = Cl
11c: R = H
12d: n=0; R=H
12e: n=0; R=Cl
N
F3CS
13
SCF3
a Reagents and conditions: (a) NaBH(OAc)3, CH3CO2H or CF3CO2H, DCM.
Reductive amination of 11a with 3b gave, together with the expected compound 12b, a tertiary
amine 13, which was also included for biological evaluation.
Biological Evaluation
As a preliminary screening, we tested the total levels of phosphorylation of tyrosine residues in
HeLa cells treated with different concentrations (0.1, 1.0 and 10.0 µM) of all 17 compounds
(Phospho-Tyr in Table 1). The compounds that induced at least a 10% increase of Phospho-Tyr
levels at any of the used concentrations were selected for further evaluation of inhibition of the
phosphatase activity of PTPRZ1 (Figure 1S, see supplementary material). The selected
compounds were 4a-d, 5b-c, 10a, 12a-b, 12d, and 13. A limitation of this approach is that HeLa
cells constitutively express other tyrosine phosphatases that may compensate for the inhibition of
PTPRZ1 induced by these compounds. Thus, it is possible that some of the inhibitors of PTPRZ1
that we designed and synthesized were discarded as false negatives. This may be the case of
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compound 1, the PTP1B inhibitor synthesized by Huang et al.20 that also exerted some inhibition
on PTPRZ1 (IC50 = 3.5 µM) and other phosphatases. However, we chose this strict selection
criterion to select the most potent inhibitors for the next phase.
Table 1. Values of calculated TPSA, logP/logD and results of the Phospho-Tyr and PTPRZ1
inhibition test at 1.0 µM
Comp. TPSA LogP /LogD Phospho-Tyr PTPRZ1 inhibition
at 1.0 µM
94.27 5.35
79.70 7.26
79.70 7.37
79.70 6.99
79.70 6.88
114.14 4.88
114.14 5.00
114.14 4.62
59.83 7.73
51.75 6.54
47.42 4.34
62.63 7.55
62.63 7.50/6.64
41.91 5.43/4.41
37.33 5.48
37.33 5.99
79.14 11.72/11.56
X
√
√
√
√
X
√
√
√
X
√
√
√
X
√
X
√
-
1
X
X
√
√
-
4a
4b
4c
4d
5a
√
X
√
-
5b
5c
10a
10b
10c
12a
12b
12c
12d
12e
13
X
X
√
√
X
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Next, we performed an enzymatic assay of PTPRZ1 using the selected 11 compounds
mentioned above. After optimizing the assay conditions, we performed a screening test to assess
the capacity of a single concentration (1.0 µM) of each of these compounds to induce at least a
10% inhibition of the phosphatase activity of PTPRZ1. Six compounds, 4c-d, 5b, 10a, 12b and
12d, met this criterion and underwent concentration-response studies to calculate the half-
maximal inhibitory concentration (IC50). Among the remaining compounds, we found that 10a,
12b and 4c exhibited the most potent inhibition, with a range of IC50 values between 0.1 – 0.8
µM (Table 2). In contrast with the results of Huang et al [20], it should be noted that compound
1 failed to inhibit PTPRZ1 phosphatase activity in our assays. These apparent discrepancies may
reflect significant methodological differences. First, while we have used a commercial PTPRZ1
protein, plasmids containing the PTPRZ1 active domain fragment were used in the previous
report to subclone the fragment for expression in insect cells [20]. Second, we have used a
different method of detection of inorganic phosphate in the PTPRZ1 enzymatic assay, the
phosphate sensor reagent that binds inorganic phosphate in a rapid, tight (Kd ~ 0.1 µM) and
more sensitive manner.
Table 2: IC50 Values (in µM) of PTPRZ1 and PTP1B inhibition.
Phosphatase
PTPRZ1
PTP1B
4c
4d
>100
-
5b
>10
-
10a
0.1
12b
0.1
0.7
12d
5.0
1.0
0.8
NDª
NDª
ª ND = Non Detected
We also assessed the inhibitor selectivity of all six compounds for PTP1B. We selected PTP1B
as the most prominent PTP currently investigated as a pharmacological target [22]. As shown in
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table 2, 10a and 12b strongly and selectively inhibit PTPRZ1. Compound 4c, although less
potent, displays a remarkable selectivity against PTP1B. To further study the selectivity of the
most potent compounds, we decided to test their effects on the phosphorylation levels of specific
substrates of PTPRZ1 in an in vitro biological assay. As relevant substrates of PTPRZ1, we
chose TrkA [23] and anaplastic lymphoma kinase (ALK) [18] because they are known to be
involved in the neuroprotective effects of PTN, the endogenous inhibitor of PTPRZ1 [9]. We
used neuroblastoma SH-SY5Y cells, which are known to express PTPRZ1 [24]. We stimulated
SH-SY5Y cells with different concentrations of 10a and 12b (1.0, 5.0 and 10.0 µM) for 20
minutes, and evaluated using western blots the phosphorylation of those specific tyrosine
residues in TrkA (Tyr490) and ALK (Tyr1278), which are involved in the activation of both
proteins. It has to be noted that previous evidence suggests that PTPRZ1 preferentially
dephosphorylates Y674 and/or Y675 [23]. However, we chose to study Y490 because
phosphorylation of this residue results in a cascade of molecular events and survival effects in
vitro that resembles the ones found in PTN-stimulated cells [10c]. We chose to study ALK
(Tyr1278) because treatment of SH-SY5Y cells with midkine (MK), the only other member of the
PTN family of cytokines that also binds PTPRZ1 and inhibits its phosphatase activity [25],
causes a significant increase in the phosphorylation of ALK (Tyr1278) in SH-SY5Y cells [26]. We
chose these concentrations of the inhibitors as relevant for the subsequent functional studies
described below. Western blots probed with anti-phospho-TrkA antibodies demonstrated that
steady state levels of tyrosine phosphorylation of TrkA increased 2-3-fold after treatment with
both 10a and 12b (Figure 1). Western blots probed with anti-phospho-ALK antibodies
demonstrated that both compounds caused a 2-fold increase in the phosphorylation of one
isoform of ALK (the 140 kDa protein [27]) (Figure 1).
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(b) TrkA
(c) ALK140
(a)
2.5
3
***
*
p-TrkA
2.0
**
TrkA
*
2
1
1.5
**
p-ALK140
1.0
0.5
0.0
ALK140
0
0.0 1.0 5.0 10.0
10a (µM)
0.0
1.0
5.0
10.0
0.0
1.0
5.0
10.0
10a (µM)
10a (µM)
(e) TrkA
(f) ALK140
(d)
2.5
2.0
1.5
1.0
0.5
2.0
****
p-TrkA
**
**
*
1.5
1.0
0.5
0.0
*
TrkA
p-ALK140
ALK140
0.0
0.0
1.0
5.0
10.0
0.0
1.0
5.0
10.0
0.0
1.0
5.0 10.0
12b (µM)
12b (µM)
12b (µM)
Figure 1. (a) Representative western blots showing 10a-induced increases in phosphorylated
TrkA (pTrkA) and the 140 kDa ALK isoform (pALK). Total TrkA and ALK western blots are
shown below each phosphorylated protein blot for comparison. (b–c) Quantification of western
blots using ImageJ. (d) Representative western blots showing 12b-induced increases in pTrkA
and pALK. Total. (e-f) Quantification of western blots using ImageJ. Data are presented as mean
± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to vehicle controls (0.0
µM).
The data clearly demonstrate that two of the inhibitors, 10a and 12b, significantly increase the
phosphorylation of key tyrosine residues of the PTPRZ1 substrates TrkA and ALK, in agreement
with their IC50 values (Table 2). It is interesting to note that TrkA is the high affinity nerve
growth factor (NGF) receptor. NGF activates the kinase activity of TrkA by increasing the
phosphorylation of Y490 in TrkA, which is critical for NGF-induced survival and neuroprotective
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effects [28]. Increased phosphorylation of Y1278 in ALK is also involved in neuronal survival and
differentiation [9]. The data suggest that the ability of PTPRZ1 inhibitors to increase the
phosphorylation of the same residues in TrkA and ALK, by inhibiting the phosphatase activity of
PTPRZ1 on its substrates, will induce similar neuroprotective effects. Accordingly, the
endogenous inhibitor of the phosphatase activity of PTPRZ1, PTN, has been shown to prevent
amphetamine-induced neuronal injury in vitro and in vivo [10b, 10c]. To test the possibility that
PTPRZ1 inhibitors could protect cell cultures from amphetamine-induced toxicity, we used
catecholaminergic PC12 cells, which express readable levels of PTPRZ1 [29]. PC12 cells were
incubated for 24 hours with amphetamine (1 mM) and/or 10a or 12b (1.0 µM). Interestingly, 10a
significantly prevented amphetamine-induced loss of PC12 cell viability (Figure 2a) and the
same trend was observed with 12b (p = 0.06). These data demonstrate that PTPRZ1 inhibitors
mimic the protective effects of PTN against amphetamine-induced toxicity in PC12 cells [10c].
Figure 2. Effects of 10a and 12b on amphetamine-induced toxicity in PC12 cells. PC12 cells
cultured with media supplemented with amphetamine (Amph; 1 mM) and/or 10a (1.0
µM)
and/or 12b (1.0 M) for 24 hours. Cellular viability was assessed by the MTT test. Results are
µ
expressed as mean ± S.E.M. **p < 0.01, ***p < 0.001 vs. vehicle-treated (control) cells. ###p <
0.001 vs. Amph+10a-treated cells.
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In Vivo Permeability Study
To test the ability of the designed compounds to cross the BBB, compound 10a was selected to
carry out an in vivo experiment. Samples were obtained from mice sacrificed 1h post-gavage
with 60 mg/kg of 10a and analyzed by GC-MS. Extracted ion chromatograms (EIC) showed the
presence of a peak at the same retention time as a standard 10a solution, in both plasma and
brain samples, while this peak was not present in samples from untreated mice. A preliminary
quantification of the permeability has been carried out, showing that the estimated concentration
of 10a in brain samples is of 500-1000 ng/mL, while the concentration in plasma is 100-300
ng/mL. Moreover, we have recently shown that treatment with 10a completely blocked alcohol-
induced reward in mice [30] and modulation of the rewarding effects of drugs of abuse such
alcohol, requires direct actions in the brain.
Computational studies
Despite its biological interest, PTPRZ1 has not been thoroughly studied to date as a druggable
target. The first crystal structure obtained for PTPRZ1 was deposited as 5AWX.19 However,
inhibitors failed to crystalize along with the protein. To consider several conformations of the
target macromolecule, together with the available apo form (5AWX), a computational study was
carried out. Since no crystal structure was available for holo conformations of PTPRZ1,
homology models were built using Receptor-type Tyrosine-Protein Phosphatase Gamma
(PTPRG)19 as a template, based on the 73.61% of sequence homology between both
phosphatases [31]. PTPRZ1 belongs to the receptor-associated Class I PTP subfamily, which
contains a highly conserved phosphatase domain (PD1) and a cysteine in the active site [32]. The
general structure of the PD1 is constituted by an active site motif C(X)5R [33] and several
surrounding loops implicated in the catalytic cycle and substrate recognition process. The WPD-
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loop plays an acid-base role, carried out by the Trp-Pro-Asp motif and therefore is implicated in
the recycling of the active site. The pTyr-loop [34] is implicated in the recognition of the
phosphotyrosine substrate; and the Q-loop, has a relevant role in the activation of a key water
molecule during the catalytic cycle. The templates used for the homology modeling can be
classified according to the different conformations of the WPD-loop as follows: i) closed
conformation: when the phosphatase domain is bound to a substrate-like ligand [35] or a
competitive inhibitor (PDB code 3QCC) [35,36]; ii) a “superopen” conformation: when the
phosphatase is bound to an inhibitor that alters the overall conformation of the WPD-loop (PDB
code 3QCH) [35] by acting as a wedge between the WPD-loop and the rest of the protein; and
iii) open conformation: when the protein is in the apo form (PDB code 5AWX) [33,37]. Models
for PTPRZ1 were generated with the SWISS-MODEL web server [38]as described elsewhere
[31]. They were subsequently used for docking experiments carried out as described in the
experimental section, using compounds 4c, 4d, 5b, 10a, 12b and 12d, as ligands. The highest
docking scores for all compounds inside the different conformations of PTPRZ1 were obtained
for the “superopen” conformation. The general binding mode predicted for these compounds
implies the enclosing of the trifluoromethylthiobenzyl moiety within the hydrophobic pocket that
is accessible by the displacement of the WPD-loop. Among the amino acids that line the
hydrophobic pocket, the main van der Waals interactions are established between the
trifluoromethylthiobenzyl moiety and the aromatic side chains of Trp1989 and Phe1984, and the
aliphatic and cycloaliphatic side chains of Arg1939 and Pro1905, respectively. The rest of the
molecule, in all cases, interacts with the surroundings of the WPD-loop and the active site, with
no direct interaction with the catalytic site (Table S1 and Figures S2, see Supplementary
material).
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To assess the stability of the proposed binding modes, 10 ns molecular dynamics (MD)
simulations were carried out for all complexes. Binding energies were analyzed making use of
the fast and versatile MM-ISMSA program [39]. Results confirm the complexes stability in the
“superopen” conformation for all compounds bound to PTPRZ1 as can be seen by the stable
RMSD values (Table S2 and Figure S3, see Supplementary material).
Given their high selectivity and drug-like properties, the binding modes for 4c
, 10a and 12b
were intensively inspected, and the most populated conformations explored during 97% (4c
),
78% (10a) and 77% (12b) of the simulation time, respectively, were selected for the per-residue
energy decompositions (Figure 3). Results show that the previously described van der Waals
interactions established with the hydrophobic pocket in the initial docking binding models are
maintained during most of the simulation time for the three complexes and that those having a
higher energetic contribution are the π–π stacking and T-shaped interactions established between
the trifluoromethylthiobenzyl moiety of the ligands and the aromatic side chains of Phe1984 and
Trp1889. Furthermore, compound 4c interacts strongly with the side chains of Pro1900, Val1904 and
the aliphatic part of the side chain of Glu1980 of the WPD-loop. In addition, the amide moiety of
4c stablishes alternating hydrogen bonding interactions to either the side chain NH of Gln1981 or
the backbone carbonyls of Pro1905 and Glu1980. In the complex of 10a with PTPR1Z, the
hydrophobic interactions persist, whereas electrostatic interactions are weaker, since the
previously described hydrogen bonds cannot be established. This binding mode is shared by all
the other studied compounds in their most populated conformers except for 12b-PTPRZ1
complex, because of its protonated state. Compound 12b can stablish strong interactions between
the positively charged amine moiety and the side chains of Glu1980 and Asp1987 and with the
backbone carbonyl of Glu1906, all three located at the entrance of the hydrophobic pocket.
15

ACCEPTED MANUSCRIPT
Furthermore, compound 12b, due to its high flexibility, folds to establish an intramolecular π–π
interaction between the two trifluoromethylthiobenzyl moieties. In this folded conformation,
compound 12b establishes van der Waals interactions between the trifluoromethylthiobenzyl
moieties and the hydrophobic residues that line the hydrophobic pocket, some of which are
located in the WPD-loop (Figure S4, see Supplementary material).
With the aim of rationalizing the high selectivity displayed by 4c, 10a and 12b, the same
computational approach was carried out for PTP1B. The PTP1B structures were obtained from
the PDB in the closed and open conformation (PDB codes 1PTY and 3A5J, respectively) and a
homology model for the “superopen” conformation was built using as template PDB structure
3QCH. Docking calculations were carried out as described previously and, in this case, the
highest docking scores were obtained for all compounds bound to the closed conformation of
PTP1B (Table S1, see Supplementary material) where they share an overall similar binding
mode (Figure S5, see Supplementary material). Corresponding 10 ns MD simulations were
carried out and the binding modes were further analyzed. Lower affinity towards PTP1B were
found, as demonstrated by the unstable binding modes calculated through MM-ISMSA and the
shifting RMSD values, that are comparatively higher than the ones obtained for the PTPRZ1
complexes (Table S2 and Figure S3, see supplementary material). It is noteworthy that the only
stable binding modes in PTP1B, which have the most similar RMSD fluctuations if compared to
those of PTPRZ1 complexes, are shown by compounds 4d and 5b along with 4c. The other
compounds fluctuate during the MD simulation and slowly unbind from the original binding
mode, resulting in completely different conformations that can be extracted from the MD
simulations (Figure S6, see Supplementary material). The per-residue energy decompositions of
the complexes between PTP1B and 4c
,
10a and 12b were also carried out. The 4c-PTP1B
16

ACCEPTED MANUSCRIPT
complex showed worse energy interactions compared to those found for PTPRZ1. The main
interactions established by compound 4c are with Tyr46, Asp48 and Val49 from the described P-
loop, Phe182 from the WPD-loop, Ser216 and Ala217 from the active site, and Gln262 from the Q-
loop (Figure S7, see Supplementary material). Meanwhile, compound 10a in complex with
PTP1B, presented no stable binding mode during the simulations, which resulted in an
unbounding from the active site, clearly observed by the rising RMSD values after 1.4 ns of the
MD simulation and the reduced per-residue interactions of the binding. The complex of 12b and
PTP1B rendered no stable binding within the active site during the simulation. Nevertheless,
after ~ 2.5 ns 12b moves away from the active site and establishes a strong hydrogen bonding
interaction between the protonated amine and the side chain of the Gln262 present in the Q-loop,
which is maintained throughout the rest of the simulation (Figures S4 and S7, Supplementary
material).
These results clearly account for the lower affinity towards PTP1B displayed by compounds
4c, 10a and 12b, and most importantly, they are in complete agreement with the remarkable
PTPRZ1/PTP1B selectivity profile described above.
17

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Figure 3. Proposed binding modes of 4c
,
10a and 12b inside PTPRZ1. (Left) PyMOL stick and
cartoon representation of the proposed binding modes for 4c, 10a and 12b inside PTPRZ1.
Structures shown correspond to the most populated conformations along the MD simulations in
the PTPRZ1 superopen conformation. 4c 10a and 12b are shown in green, orange and light pink
sticks, respectively. Residues involved in interactions are shown as sticks. The protein is colored
,
18

ACCEPTED MANUSCRIPT
cyan and the active site, WPD-loop and Q-loop are highlighted in green, magenta and grey,
respectively. (Right) Per-residue energy decomposition of each complex with PTPRZ1. Orange,
green and pale magenta bars represent the van der Waals contributions and deep green, red and
magenta bars represent electrostatic binding energies for 4c, 10a and 12b.
CONCLUSION
PTN has a protective role in the CNS through its interaction with PTPRZ1. The interaction of
PTN with PTPRZ1 inactivates its intrinsic tyrosine phosphatase activity, increasing the
phosphorylation level of substrates which are crucial to prevent neurodegenerative and drug
addiction disorders.
In this work, we have designed and synthesized a series of small molecules capable of
mimicking the activity of PTN, by interacting with the active site of the intracellular domain PD1
of PTPRZ1.
The most active compounds 10a and 12b (IC50 = 0,1 µM) are selective against PTP1B, and
significantly increase the phosphorylation of key tyrosine residues of TrkA and ALK, two
PTPRZ1 substrates involved in neuronal survival and differentiation. Moreover, both compounds
mimic the PTN protective effects against amphetamine-induced toxicity in PC12 cells.
Docking and MD experiments have allowed to propose the binding mode of all these
compounds inside PTPRZ1 and assess the stability of the corresponding complexes along the
simulation time. More importantly, these methods have allowed to explain the remarkable
PTPRZ1/PTP1B selectivity displayed by 4c and 10a
.
19

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We have demonstrated that 10a can cross the BBB and, therefore, we propose this compound
as a promising candidate for the development of new drugs for the treatment of CNS disorders,
such as addictive and neurodegenerative diseases.
EXPERIMENTAL SECTION
Chemistry
Materials. All reagents and solvents were obtained from different commercial sources (Sigma-
Aldrich, Fluka, Acros Organics, Alfa Aesar or Scharlab) and used without further purification.
Tetrahydrofuran and dichloromethane were dried by the solvent purification system Technical
Bulletin AL-258.
General Methods. Reaction progress was monitored using analytical thin-layer
chromatography (TLC) on Merck silica gel 60 F-254 plate. Visualization was achieved by UV
light (254 nm). Flash chromatography was performed with Scharlau silica gel 60 (0.04-0.06 mm)
1
packing. H and 13C spectra were recorded on a Bruker Advance III 400 MHz instrument.
Signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). Chemical
shifts (
δ) are expressed in parts per million relative to solvent resonance as the internal standard.
Coupling constants (
J
) are in hertz (Hz). Elemental analyses were performed by the
Microanalytical Service Laboratory of the Universidad Complutense (Madrid) on a LECO
CHNS-932 apparatus. Analyses indicated by the symbols of the elements or functions were
within ± 0.4 % of the theoretical values. Melting points were determined on a Stuart Scientific
(BIBBY) melting point apparatus. Mass spectrometry was performed by the CEMBIO Analytical
Service Laboratory of the Universidad CEU San Pablo on a MS/IT Esquire 3000 Bruker
20

ACCEPTED MANUSCRIPT
Daltonics apparatus. All final products were analyzed for purity by reverse-phase high-
performance liquid chromatography (HPLC) on Agilent Technologies 1260 Infinity II apparatus.
HPLC methods used for each compound shown in Supplementary material.
1-((Trifluoromethyl)sulfonyl)-4-((4-((trifluoromethyl)sulfonyl)benzyl)oxy)benzene (1). To
a solution of 10a (153 mg, 0.39 mmol) in dry DCM (10 mL) was added MCPBA (688 mg, 3.98
mmol). The reaction mixture was stirred at 80 ºC for 48 h in a sealed tube. The crude was
washed with NaOH 1M, brine and dried (MgSO4). The drying agent was filtered off and the
solvent was evaporated. The crude was purified by column chromatography on silica gel using
hexane-EtOAc as eluent (8 : 2 v/v) to afford 120 mg (67%) of
1 as a white solid. Mp. 139-141
ºC. 1H NMR (400 MHz, CDCl3):
δ
8.11 (d,
J = 8.2 Hz, 2H, ArH), 8.02 (d, J = 8.9 Hz, 2H, ArH),
7.76 (d,
MHz, CDCl3):
J = 8.2 Hz, 2H, ArH), 7.21 (d, J
= 8.9 Hz, 2H, ArH), 5.33 (s, 2H, -CH2). 13C NMR (100
δ
164.6, 144.9, 133.5, 131.4, 131.4 (“q”, J = 2 .0 Hz), 128.3, 123.4 (q, J = 2.0
Hz), 112.0 (q, JCF = 325.8 Hz), 112.0 (q, JCF = 325.8 Hz), 116.1, 69.3. HPLC Purity >98%, tR=
4.436, Method 1. Anal. C15H10F6O5S2 (C, H, F, O, S).
4-((Trifluoromethyl)thio)-N-(4-((trifluoromethyl)thio)phenyl)benzamide (4a). To
a
solution of K2CO3 (174 mg, 1.26 mmol) in 4 mL of dry THF at 0 ºC were subsequently added
11a (100 mg, 0.42 mmol) and aniline 3a (128 mg, 0.66 mmol). The resulting mixture was stirred
at RT under argon till completion was seen by TLC (20 h). The solvent of the reaction was
evaporated and the residue was suspended in H2O. The aqueous layer was extracted with EtOAc,
and the combined organic layers were washed with 1M HCl and brine, dried (MgSO4), filtered
1
and evaporated to give 4a as a white solid (31 mg, 19%). Mp. 162-164 ºC (hexane/EtOAc). H
NMR (400 MHz, CDCl3):
Hz, 2H, ArH), 7.73 (d,
δ
7.91 (d,
J
= 8.4 Hz, 2H, ArH), 7.87 (s, 1H, -NH), 7.80 (d,
J = 8.4
J
= 8.8 Hz, 2H, ArH), 7.68 (d, J
= 8.8 Hz, 2H, ArH). 13C NMR (100
21

ACCEPTED MANUSCRIPT
MHz, CDCl3):
δ
164.8, 140.2, 137.8, 136.7, 136.4, 129.6(q, JCF = 308.3 Hz), 129.4 (q, JCF =
308.3 Hz), 129.3 (q,
J = 2.0 Hz), 128.2, 120.8, 119.9(q, J = 2.0 Hz). HPLC Purity >98%, tR=
4.734, Method 5. LC/MS (ESI) m/z 398.0 [M+H]+. Anal. C15H9F6NOS2 (C, H, F, N, O, S).
N-2-Bis(4-((trifluoromethyl)thio)phenyl)acetamide (4b). To a solution of acid 2b (200 mg,
0.64 mmol) in 2.5 mL of DMF were added HOBt (104 mg, 0.77 mmol), EDCI (139 mg, 0.90
mmol) and the corresponding aniline 3a (247 mg, 1.28 mmol). The resulting mixture was stirred
at RT till completion was seen by TLC (72 h). The mixture was then diluted with EtOAc and
washed with NaHCO3 saturated solution, NH4Cl and brine. The organic layer was dried
(MgSO4), filtered and evaporated to give 4b as a white solid (208 mg, 78%). Mp. 163-165 ºC
(hexane/EtOAc). 1H NMR (400 MHz, CDCl3):
δ
7.69 (d,
= 7.9 Hz, 2H, ArH), 7.18 (s, 1H, -NH),
168.3, 140.1, 137.6, 137.1, 130.7, 129.6 (q,
= 2.0 Hz), 120.4, 119.5 (q, = 2.0 Hz), 44.5.
J = 7.9 Hz, 2H, ArH), 7.59 (d, J = 8.5
Hz, 2H, ArH), 7.54 (d,
J = 8.5 Hz, 2H, ArH), 7.41 (d, J
3.78 (s, 2H, -CH2). 13C NMR (100 MHz, CDCl3):
δ
JCF = 308 Hz), 129.6 (q, JCF = 308 Hz), 124.1 (q,
J
J
HPLC Purity >98%, tR= 3.690, Method 5. LC/MS (ESI) m/z 412.00 [M+H]+. Anal.
C16H11F6NOS2 (C, H, F, N, O, S).
N-(4-((Trifluoromethyl)thio)benzyl)-2-(4-((trifluoromethyl)thio)phenyl)acetamide
(4c).
To a solution of 2b (200 mg, 0.64 mmol) in 2.5 mL of DMF were added HOBt (104 mg, 0.77
mmol), EDCI (139 mg, 0.90 mmol) and amine 3b (199 mg, 0.96 mmol). The resulting mixture
was stirred at RT till completion was seen by TLC (72 h). The mixture was then diluted with
EtOAc and washed with NaHCO3 saturated solution, NH4Cl and brine. The organic layer was
dried (MgSO4), filtered and evaporated to give 4c as a white solid (105 mg, 39%). Mp. 124-126
ºC (hexane/EtOAc)
.
1H NMR (400 MHz, CDCl3):
δ
7.65 (d,
J
= 8.1 Hz, 2H, ArH), 7.59 (d,
J =
8.1 Hz, 2H, ArH), 7.35 (d,
J
= 8.2 Hz, 2H, ArH), 7.25 (d,
J = 8.1 Hz, 2H, ArH), 5.75 (s, 1H, -
22

ACCEPTED MANUSCRIPT
NH), 4.47 (d,
J
= 6.0 Hz, 2H, -CH2-NH-), 3.65 (s, 2H, -CH2CO). 13C NMR (100 MHz, CDCl3)
δ
170.1, 141.3, 137.9, 137.0, 136.8, 130.6, 129.6 (q, JCF = 308.2 Hz), 129.6 (q, JCF = 308.2 Hz),
128.6, 123.7 (q,
J = 2.1 Hz), 123.6 (d, J = 2.1 Hz), 43.2, 43.1. HPLC Purity >97%, tR= 3.073,
Method 6. LC/MS (ESI) m/z 425.9 [M+H]+. Anal. C17H13F6NOS2 (C, H, F, N, O, S).
4-((Trifluoromethyl)thio)-N-(4-((trifluoromethyl)thio)benzyl)benzamide (4d). To
a
solution of K2CO3 (87 mg, 0.63 mmol) in 2 mL of dry THF at 0 ºC were subsequently added
chloride 2a (50 mg, 0.21 mmol) and amine 3b (68 mg, 0.33 mmol). The resulting mixture was
stirred at RT under argon till completion was seen by TLC (20 h). The solvent of the reaction
was evaporated and the residue was suspended in H2O. The aqueous layer was extracted with
EtOAc, and the combined organic layers were washed with 1M HCl and brine, dried (MgSO4),
filtered and evaporated. To give 4d as a white solid (119.5 mg, 34%). Mp. 117-119 ºC
(hexane/EtOAc). 1H NMR (400 MHz, CDCl3):
δ
7.84 (d,
J
= 8.3 Hz, 2H, ArH), 7.74 (d,
J = 8.3
Hz, 2H, ArH), 7.65 (d,
= 5.9 Hz, 2H, -CH2). 13C NMR (100 MHz, CDCl3)
J = 8.1 Hz, 2H, ArH), 7.41 (d, J = 8.1 Hz, 2H, ArH), 6.48 (s, 1H, -NH),
4.70 (d,
J
δ
166.5, 141.2, 136.9, 136.3, 129.7
= 2.0 Hz), 128.1, 123.8 (q, J=
(q, JCF = 308.3 Hz), 129.4 (q, JCF = 308.3 Hz), 128.9, 128.7 (q,
J
2.0 Hz), 43.7. HPLC Purity >98%, tR= 4.534, Method 5. LC/MS (ESI) m/z 412.00 [M+H]+.
Anal. C16H11F6NOS2 (C, H, F, N, O, S).
4-((Trifluoromethyl)sulfonyl)-
N-(4-((trifluoromethyl)sulfonyl)phenyl)benzamide
(5a). To a solution of 4a (65 mg, 0.16 mmol) in dry DCM (10 mL) was added MCPBA (276
mg, 1.6 mmol). The resulting mixture was stirred at 100 ºC in a sealed tube until the reaction was
completed (TLC) (72 h). The reaction mixture was washed with NaOH 2M, NaCl sat. and H2O.
The organic layer was then dried (MgSO4), filtered and evaporated to give 5a as a white solid
(40.6 mg, 55%). Mp. 194-196 ºC (hexane/EtOAc). δ 8.23 (d, J =
1H NMR (400 MHz, CDCl3):
23

ACCEPTED MANUSCRIPT
8.4 Hz, 2H, ArH), 8.17 (d,
J
= 8.4 Hz, 2H, ArH), 8.08 (d,
J
= 8.8 Hz, 2H, ArH), 8.00 (d,
J = 8.8
Hz, 2H, ArH). 13C NMR (100 MHz, DMSO)
δ
165.0, 147.0, 142.3, 132.4, 132.2, 131.2, 130.2,
123.0, 120.9, 119.5 (“q”, J = 324.11), 119.4 (“q”, J = 326.40) . HPLC Purity >97%, tR= 4.277,
Method 5. LC/MS (ESI) m/z 461.9 [M+H]+. Anal. C15H9F6NO5S2 (C, H, F, N, O, S).
N,2-bis(4-((trifluoromethyl)sulfonyl)phenyl)acetamide (5b). To a solution of 4b (102 mg,
0.248 mmol) in dry DCM (10 mL) was added MCPBA (428 mg, 2.48 mmol). The resulting
mixture was stirred at 100 ºC in a sealed tube until the reaction was completed (TLC) (24 h). The
reaction mixture was washed with NaOH 2M, NaCl sat. and H2O. The organic layer was then
dried (MgSO4), filtered and evaporated to give 5b as a white solid (80 mg, 68%). Mp. 171-173
1
ºC (hexane/EtOAc). H NMR (400 MHz, CDCl3):
δ
8.07 (d,
= 8.3 Hz, 2H, ArH), 7.57 (bs, 1H, -
167.6, 144.9, 143.0, 132.6, 131.5,
J = 8.3 Hz, 2H, ArH), 8.00 (d, J =
8.9 Hz, 2H, ArH), 7.83 (d,
J = 8.9 Hz, 2H, ArH), 7.67 (d, J
NH), 3.97 (s, 2H, -CH2).; 13C NMR (100 MHz, CDCl3)
δ
131.1, 130.8, 125.9,119.9, 119.9 (q, JCF = 325.7 Hz), 119.8 (q, JCF = 325.7 Hz), 44.4. HPLC
Purity >97%, tR= 5.674, Method 3. LC/MS (ESI) m/z 475.9 [M+H]+. Anal. C16H11F6NO5S2 (C,
H, F, N, O, S).
N-(4-((trifluoromethyl)sulfonyl)benzyl)-2-(4-((trifluoromethyl)sulfonyl)phenyl)acetamide
(5c). To a solution of 4c (42 mg, 0.1 mmol) in dry DCM (10 mL) was added MCPBA (260 mg,
1.5 mmol). The resulting mixture was stirred at 100 ºC in a sealed tube until the reaction was
completed (TLC) (48 h). The reaction mixture was washed with NaOH 2M, NaCl sat. and H2O.
The organic layer was then dried (MgSO4), filtered and evaporated to give 5c as a white solid (33
mg, 67%). Mp. 164-170 ºC (hexane/EtOAc). 1H NMR (400 MHz, CDCl3):
δ 8.03 (d, J = 8.0 Hz,
2H, ArH), 7.99 (d,
J
= 8.0 Hz, 2H, ArH), 7.63 (d,
J = 7.9 Hz, 2H, ArH), 7.54 (d, J = 7.9 Hz, 2H,
ArH), 6.03 (bs, 1H, -NH), 4.60 (d,
J
= 6.1 Hz, 2H, -CH2NH), 3.77 (s, 2H, -CH2CO). 13C NMR
24

ACCEPTED MANUSCRIPT
(100 MHz, CDCl3)
δ
169.1, 147.7, 140.03, 131.4, 131.0, 130.5, 130.47, 128.95, 119.9 (q, JCF =
323.7 Hz), 43.5, 43.3. HPLC Purity 95%, tR= 4.549, Method 3. LC/MS (ESI) m/z 489.9
[M+H]+. Anal. C17H13F6NO5S2 (C, H, F, N, O, S).
1-Fluoro-4-((trifluoromethyl)sulfonyl)benzene (7). To a solution of 6 (100 mg, 0.51 mmol)
in dry DCM (10 mL) MCPBA (440 mg, 2.55 mmol) was added and the mixture was heated at 80
ºC in a sealed tube for 6 h. The reaction crude was diluted with DCM (10 mL) and the organic
layer was washed with NaOH 1 M, brine and dried (MgSO4). The drying agent was filtered off
and the solvent was removed at vacuum to give
in the next reaction without further purification.
7 as a light-orange oil. The compound was used
(Trifluoromethyl)(4-((4-((trifluoromethyl)thio)benzyl)oxy)phenyl)sulfane (10a). To a
solution of (303 mg, 1.46 mmol) in 5 mL of dry DMF, was added NaH (60%) (42 mg, 1.75
mmol) at 0 ºC under an argon atmosphere. After stirring for 15 min, compound (0.22 mL, 1.53
8
6
mmol) was added and the mixture was stirred at RT. The reaction was monitored with TLC until
completion (24 h). The solvent was evaporated, the residue was solved in EtOAc, washed with
brine and H2O, dried (MgSO4), filtered and evaporated. The crude was purified by column
chromatography on silica gel using hexane-EtOAc as eluent (9.75 : 0.25 v/v) to afford 10a as a
white solid (350 mg, 62%). Mp. 114-116 ºC. 1H NMR (400 MHz, CDCl3):
δ
7.69 (d,
= 8.2 Hz, 2H, ArH), 6.99 (d, = 8.8 Hz, 2H,
160.8, 139.5, 138.5, 136.8, 129.8 (q,
J = 8.2 Hz,
2H, ArH), 7.59 (d,
J
= 8.8 Hz, 2H, ArH), 7.48 (d,
J
J
13
ArH), 5.12 (s, 2H, -CH2). C-NMR (100 MHz, CDCl3):
δ
JCF = 308 Hz), 129.7 (q, JCF = 308 Hz), 128.3, 124.4 (q, J = 2.1 Hz), 115.9, 115.8 (q, J = 2.1
Hz), 69.4. HPLC Purity >98% tR = 5.215, Method 1. Anal. C15H10F6OS2 (C, H, F, O, S).
(Trifluoromethyl)(4-((4-((trifluoromethyl)sulfonyl)phenoxy)methyl)phenyl)sulfane (10b)
To a solution of (106 mg, 0.51 mmol) in 5 mL of dry DMF, was added NaH (60%) (15 mg,
,
.
8
25

ACCEPTED MANUSCRIPT
0.61 mmol) at 0 ºC under an argon atmosphere. After stirring for 15 min, fluorine derivative
7
(116 mg, 0.51 mmol) was added, the mixture was stirred at RT and the reaction was monitored
with TLC until completion (2 h). The reaction mixture was poured into a mixture of HCl (1M) /
H2O (1:1) and then extracted with DCM. The organic layer was dried (MgSO4), filtered and
evaporated. The crude was purified by column chromatography on silica gel using hexane-DCM
as eluent (9:1 v/v) to afford 10b as a white solid (121 mg, 31%). Mp. 110-112 ºC. 1H NMR (400
MHz, CDCl3):
δ
7.98 (d,
J = 8.9 Hz, 2H, ArH), 7.72 (d, J = 8.1 Hz, 2H, ArH), 7.49 (d, J = 8.1
Hz, 2H, ArH), 7.18 (d,
J
= 8.9 Hz, 2H, ArH), 5.22 (s, 2H, -CH2). 13C NMR (100 MHz, CDCl3):
δ
164.8, 138.2, 136.7, 133.4, 129.5 (q, JCF = 308 Hz), 128.3, 124.7 (q,
J
= 2.0 Hz), 122.7 (q,
J
=
2.0 Hz), 119.9 (q, JCF = 308 Hz), 115.9, 69.8. HPLC Purity >97%, tR = 7.296, Method 2. Anal.
C15H10F6O3S2 (C, H, F, O, S).
4-((4-((Trifluoromethyl)thio)phenoxy)methyl)pyridine (10c). To a solution of
1.02 mmol) in 5 mL of dry DMF, was added NaH (60%) (29 mg, 1.23 mmol) at 0 ºC under an
argon atmosphere. After stirring for 15 min, derivative (0.15 mL, 1.02 mmol) was added, the
9 (111 mg,
6
mixture was stirred at RT until the reaction was completed (TLC) (72 h). The reaction mixture
was poured into a mixture of H2O/Ice and the solution was taken to pH=7 with NaHCO3
saturated solution. The precipitate was then filtered and washed with H2O. The crude was
purified by column chromatography on silica gel using hexane-EtOAc as eluent (2: 8 v/v) to
afford 10c as a white solid (66 mg, 23%). Mp. 97-99 ºC. 1H NMR (400 MHz, CDCl3):
δ
8.63 (d,
J
= 4.6 Hz, 2H, ArH), 7.59 (d,
J
= 8.7 Hz, 2H, ArH), 7.34 (d,
J
= 5.2 Hz, 2H, ArH), 6.98 (d,
J =
8.7 Hz, 2H, ArH), 5.11 (s, 2H,-CH2). 13C NMR (100 MHz, CDCl3):
δ
160.4, 150.2, 145.3, 138.4,
129.6 (q, JCF = 308.2 Hz), 121.4, 115.9 (q,
J = 2.0 Hz), 115.7, 68.3. HPLC Purity >98%, tR =
4.674, Method 3. LC/MS (ESI) m/z 286.0 [M+H]+ . Anal. C13H10F3NOS (C, H, F, N, O, S).
26

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4-((Trifluoromethyl)thio)-N-(4-((trifluoromethyl)thio)benzyl)aniline (12a). To a solution
of aldehyde 11a (200 mg, 1.04 mmol) and aniline 3a (214 mg, 1.04 mmol) in dry DCM (10 mL)
were added, under argon, NaBH(OAc)3 (438 mg, 2.07 mmol) and CH3COOH (59 µL). The
resulting solution was stirred at RT until the reaction was completed (TLC) (6 h). The reaction
mixture was neutralized with NaOH 1M aqueous solution, and the organic phase was washed
with brine and H2O, dried (MgSO4), filter and evaporated. Column chromatography on silica gel
using as eluent hexane-EtOAc (9.5 : 0.5 v/v) gave 12a as a white solid (207 mg, 52%). Mp. 87-
1
89 ºC. H NMR (400 MHz, CDCl3):
δ
7.65 (d,
J = 8.1 Hz, 2H, ArH), 7.43 (d, J = 8.8 Hz, 2H,
ArH), 7.41 (d,
(100 MHz, CDCl3):
Hz), 128.5, 123.7 (q,
5.010, Method 4. Anal. C15H11F6NS2 (C, H, F, N, S).
Bis(4-((trifluoromethyl)thio)benzyl)amine hydrochloride
J = 8.1 Hz, 2H, ArH), 6.61 (d, J
= 8.8 Hz, 2H, ArH), 4.42 (s, 2H, -CH2). 13C NMR
δ
149.9, 141.9, 138.5, 137.0, 130.0 (q, JCF = 308.1 Hz), 129.8 (q, JCF = 308.1
= 1.8 Hz), 113.6, 111.0 (q, = 1.8 Hz), 47.6. HPLC Purity >98%, tR=
J
J
(12b)
&
tris(4-
((trifluoromethyl)thio)benzyl)amine (13). To a solution of aldehyde 11a (237 mg, 1.15 mmol)
and amine 3b (0.18 mL, 1.15 mmol) in dry DCM (10 mL) were added, under argon,
NaBH(OAc)3 (1459 mg, 6.88 mmol) and CH3COOH (66 µL). The resulting solution was stirred
at RT until the reaction was completed (TLC) (72 h). The reaction mixture was neutralized with
NaOH 1M aqueous solution, and the organic phase was washed with brine and H2O, dried
(MgSO4), filter and evaporated. Column chromatography on silica gel using hexane-EtOAc
(gradient from 9.25:0.75 to pure EtOAc) as eluent, gave amines 13 (40 mg, 6%) and 12b (80 mg,
17%).
For 13: 1H NMR (400 MHz, CDCl3)
δ 7.62 (d, J = 7.9 Hz, 6H, ArH), 7.45 (d, J = 7.9 Hz, 6H,
ArH), 3.61 (s, 6H, 3x -CH2). Bubbling HCl(g) through an ethereal solution of the compound and
27

ACCEPTED MANUSCRIPT
isolation by filtration gave the corresponding hydrochloride (84%). Mp. 115-117 ºC. HPLC
Purity >94%, tR= 6.346, Method 5. LC/MS (ESI) m/z 588 [M+H]+. Anal. C24H19ClF9NS3 (C, H,
Cl, F, N, S).
1
For 12b: H NMR (400 MHz, CDCl3):
δ
7.59 (d,
J
= 7.9 Hz, 4H, ArH), 7.42 (d,
J
= 7.9 Hz,
13
4H, ArH); 3.60 (s, 4H, 2 x -CH2). C NMR (100 MHz, CDCl3):
δ
142.1, 135.5, 128.6 (q, JCF
=
309.0 Hz), 128.1, 121.8 (q, JCF= 2.0 Hz), 51.5.
Bubbling HCl(g) through an ethereal solution of the compound and isolation by filtration gave
the corresponding hydrochloride (87%). Mp. 197-199 ºC. HPLC Purity >98%, tR= 5.108,
Method 5. LC/MS (ESI) m/z 398.00 [M+H]+. Anal
-benzyl-1-(4-((trifluoromethyl)thio)phenyl)methanamine (12c). To a solution of aldehyde
. C16H14ClF6NS2 (C, H, Cl, F, N, S)..
N
11c (0.048 mL, 0.483 mmol) and amine 3b (0.08 mL, 0.48 mmol) in dry DCM (5 mL) were
added, under argon, NaBH(OAc)3 (204 mg, 0.97 mmol) and CF3COOH (96 µL). The resulting
solution was stirred at RT until the reaction was completed (TLC) (48 h). The reaction mixture
was neutralized with NaOH 1M aqueous solution, and the organic phase was washed with brine
and H2O, dried (MgSO4), filtered and evaporated. Column chromatography on silica gel using as
eluent hexane-DCM (gradient from 4:6 v/v to 1:1 v/v) gave 12c (50 mg, 34%)
.
1H NMR (400
= 8.1 Hz, 2H), 7.35-7.27 (m, 5H), 3.85 (s,
143.5, 139.9, 136.6, 129.8 (q, JCF = 307.0 Hz),
J = 2.0 Hz), 53.3, 52.5. Bubbling HCl(g) through an
MHz, CDCl3):
δ 7.62 (d, J = 8.1 Hz, 2H), 7.42 (d, J
2H) 3.82 (s, 2H). 13C NMR (100 MHz, CDCl3):
δ
129.3, 128.6, 128.3, 127.3, 122.8 (q,
ethereal solution of the compound and isolation by filtration gave the corresponding
hydrochloride (53%). Mp. 244-246 ºC. HPLC Purity >97%, tR= 3.020, Method 4. LC/MS (ESI)
m/z 298.00 [M+H]+. Anal. C15H15ClF3NS (C, H, Cl, F, N, S).
28