Design, synthesis and biological evaluation of N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylamines as novel monoamine oxidase B inhibitors

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Design, synthesis and biological evaluation of N-methyl-N-[(1,2,3-

triazol-4-yl)alkyl]propargylamines as novel monoamine oxidase B

inhibitors

Ornella Di Pietro ^a, Nelson Alencar ^b, Gerard Esteban ^c, Elisabet Viayna ^a, Natalia Szałaj ^a, Javier Vázquez ^b,

Jordi Juárez-Jiménez ^b, Irene Sola ^a, Belén Pérez ^d, Montse Solé ^c, Mercedes Unzeta ^c,

b,

Diego Muñoz-Torrero ^a,, F. Javier Luque

⇑

^aLaboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia and Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Joan XXIII 27-31,

E-08028 Barcelona, Spain

^bDepartment of Nutrition, Food Science and Gastronomy, Faculty of Pharmacy and Institute of Biomedicine (IBUB), University of Barcelona, Av. Prat de la Riba 171,

E-08921 Santa Coloma de Gramenet, Spain

^cDepartament de Bioquímica i Biologia Molecular, Facultat de Medicina and Institut de Neurociències, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain

^dDepartament de Farmacologia, Terapéutica i Toxicologia, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

Different azides and alkynes have been coupled via Cu-catalyzed 1,3-dipolar Huisgen cycloaddition to

afford a novel family of N₁- and C₅-substituted 1,2,3-triazole derivatives that feature the propargylamine

group typical of irreversible MAO-B inhibitors at the C4-side chain of the triazole ring. All the synthesized

compounds were evaluated against human MAO-A and MAO-B. Structure–activity relationships and

molecular modeling were utilized to gain insight into the structural and chemical features that enhance

the binding afﬁnity and selectivity between the two enzyme isoforms. Several lead compounds, in terms

of potency (submicromolar to low micromolar range), MAO-B selective recognition, and brain permeabil-

Received 9 June 2016

Revised 22 June 2016

Accepted 23 June 2016

Available online xxxx

Keywords:

Click-chemistry

Monoamine oxidase B

Alzheimer’s disease

Irreversible inhibition

ity, were identiﬁed. One of these leads (MAO-B IC₅₀of 3.54 lM, selectivity MAO-A/MAO-B index of 27.7)

was further subjected to reversibility and time-dependence inhibition studies, which disclosed a slow

and irreversible inhibition of human MAO-B. Overall, the results support the suitability of the 4-triazoly-

lalkyl propargylamine scaffold for exploring the design of multipotent anti-Alzheimer compounds

endowed with irreversible MAO-B inhibitory activity.

1. Introduction

and donepezil),^12–14and an N-methyl-

D-aspartate receptor antago-

nist (memantine),¹⁵primarily exert palliative effects. On the other

hand, the failure in clinical trials of a number of drug candidates

designed against targets mainly involved in b-amyloid biology

has shifted drug discovery efforts toward the compounds hitting

less explored biological targets alone or in combination with other

key targets, i.e., the so-called multi-target-directed ligands

(MTDLs).^16–18

Alzheimer’s disease (AD) is one of the most relevant age-related

neurodegenerative disorders currently representing the fourth

leading cause of death and afﬂicting over 46.8 million people

worldwide.^1,2Its clinical manifestation is mainly reﬂected in a pro-

gressive loss of memory and cognitive functions, often in associa-

tion with behavioral disturbances and depression.³Its complex

and multifaceted etiopathology, which involves massive loss of

cholinergic neurons,⁴oxidative stress,^5–7metal dyshomeostasis,⁸

excitotoxicity,⁹neuroﬁbrillary tangles and b-amyloid aggregate

deposition,^10,11has precluded so far the discovery of effective dis-

ease-modifying drugs. Currently approved drugs, namely, three

acetylcholinesterase (AChE) inhibitors (rivastigmine, galantamine,

In this context, monoamine oxidase (MAO, E.C.1.4.3.4) has

emerged as a promising target because of the neuroprotective

properties exerted by their inhibitors.^2,19–21MAO is a ﬂavin ade-

nine dinucleotide (FAD)-containing enzyme that catalyzes the

degradation of biogenic and xenobiotic amines. Two isoforms,

namely MAO-A and MAO-B, have been characterized by their

amino acid sequence, tissue distribution, substrate speciﬁcity and

inhibitor sensitivity.^22–24MAO-A, preferentially degrading sero-

tonin, adrenaline and noradrenaline, is irreversibly inhibited by

⇑

Corresponding authors.

E-mail addresses: dmunoztorrero@ub.edu (D. Muñoz-Torrero), fjluque@ub.edu

(F.J. Luque).

http://dx.doi.org/10.1016/j.bmc.2016.06.045

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

2

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

clorgyline, whereas MAO-B, speciﬁcally responsible for the oxida-

tive deamination of phenylethylamine and benzylamine, is irre-

versibly inhibited by (R)-(ꢀ)-deprenyl (selegiline) (Fig. 1). These

trends reﬂect structural differences in the binding sites as revealed

by high-resolution X-ray structures.^25–29In particular, a key struc-

tural feature in shaping the substrate cavity is the replacement of

the pair Phe208/Ile335 in MAO-A by Ile199/Tyr326 in MAO-B,

leading to the distinction between ‘substrate’ and ‘entrance’ sites

in MAO-B. The replacement of Ile180/Asn181 and Val210 in

MAO-A by Leu17/Cys172 and Thr201 in MAO-B are additional dif-

ferences in the binding sites, which may modulate the selective

inhibition by certain MAO inhibitors.³⁰

The neuroprotection exerted by MAO inhibitors may stem not

only from the increased amine neurotransmission, but also from

preventing the formation of neurotoxic species, which may lead

to neuronal damage,^31,32and from the anti-apoptotic properties

of the propargylamine group present in some MAO inhibitors.^33,34

Interestingly, the levels of MAO-B increase with age and its activity

is elevated in AD patients, which results in increased brain levels of

neurotoxic free radicals.²⁰In this context, the development of

MAO-B-inhibitor-based MTDLs emerges as a promising strategy

for the design of neuroprotective agents with potential disease-

modifying activity towards AD and other neurodegenerative disor-

ders, such as Parkinson disease.^35,36

Most efforts have been addressed toward the design of MTDLs

targeting AChE and/or butyrylcholinesterase (BuChE) and MAO. A

successful example is ladostigil (Fig. 1), a dual inhibitor of MAO-

B and AChE that combines the carbamate moiety of the AChE inhi-

bitor rivastigmine with the indolamine moiety of the selective

MAO-B inhibitor rasagiline, and shows neuroprotective and anti-

apoptotic activities.^37,38A novel series of MAO/ChE inhibitors that

combine the N-benzylpiperidine moiety of the AChE inhibitor

donepezil with the indolyl propargylamine of the potent MAO-B

inhibitor PF9601N has been reported.³⁹Within this series, the

most promising MDTL compound (ASS234, 6 in Fig. 1) also showed

anti-aggregating activity on b-amyloid, antioxidant and antiapop-

totic behavior, and crossed easily the blood–brain barrier (BBB).⁴⁰

Other strategies have relied on the hybridization of coumarins with

either N-benzyl-N-alkyloxy groups⁴¹or tacrine,⁴²leading to multi-

potent inhibitors of both MAO and ChEs, or alternatively have pur-

sued the development of MTDLs targeting both MAO inhibition and

additional activities, such as metal chelation.^43,44Also, natural

products endowed with MAO and ChEs inhibitory activities have

been recently reported.⁴⁵

on the suitability of an efﬁcient synthetic approach, which should

afford the fusion or linkage of chemical scaffolds while minimizing

drastic alterations in the activity against the multiple targets. In

this context, the Cu(I)-catalyzed azide–alkyne cycloaddition

(CuAAC) enables the synthesis of a virtually unlimited source of

ligands containing the 1,4-disubstituted 1,2,3-triazole core.^51–54

The CuAAC reaction between libraries of azides and alkynes featur-

ing different pharmacophoric moieties has been used for the

synthesis of triazole-linked hybrid compounds as MTDLs.^55–65

Azide–alkyne cycloaddition reactions have also been used for the

synthesis of high-afﬁnity multisite enzyme inhibitors inside the

biological target, in the absence of copper catalysis, i.e., the so-

called in situ click chemistry.^66–70In these compounds the triazole

ring is used not only as a non-hydrolyzable, non-oxidizable, and

non-reducible robust linker between the pharmacophoric moi-

eties, but also provides favorable physicochemical properties and

potential interactions with the biological target.⁷¹

In this context, as the ﬁrst step of a program directed to the syn-

thesis of MAO-B-inhibitor-based anti-Alzheimer MTDLs, here we

have explored the CuAAC-mediated synthesis of a series of 1,2,3-

triazole derivatives, featuring the propargylamine group of typical

irreversible MAO-B inhibitors in the side chain at position 4, while

the overall hydrophobicity has been modulated through different

lipophilic substituents at positions 1 and 5 (Scheme 1). To deter-

mine the therapeutic potential of the target compounds and their

usefulness as the MAO-B pharmacophoric moiety of novel families

of MTDLs, we have assessed the in vitro inhibitory activity of the

novel compounds against human MAO-B and MAO-A. Molecular

modeling studies have been performed to rationalize the differ-

ences in inhibitory potency and selectivity between the MAO iso-

forms, and the mechanism of action of these compounds has

been studied by reversibility and time-dependence inhibition stud-

ies. Finally, the brain penetration of the novel compounds has been

examined through the widely used parallel artiﬁcial membrane

permeability assay (PAMPA-BBB).

2. Results and discussion

2.1. Design and synthesis of the target N-methyl-N-[(1,2,3-

triazol-4-yl)alkyl]propargylamines

We initially planned the synthesis of the 1-ethyltriazolylmethyl

and 1-ethyltriazolylethyl propargylamines 31 and 32 (Scheme 2),

unsubstituted at position

5 of the triazole ring, and their

Although the development of MAO-inhibitor-based MTDLs is

very attractive, it is challenged by the need to keep a good balance

among potencies against multiple targets and optimal ADME-T

properties.^46–50Furthermore, the success of this strategy depends

5-methyl-substituted analogues 51 and 52, as the early simple pro-

totypes to validate the suitability of the CuAAC-assisted synthetic

methodology to deliver 1,4-disubstituted and 1,4,5-trisubstituted

1,2,3-triazole scaffolds featuring the propargylamine group of

Figure 1. Chemical structures of MAO inhibitors.

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

3

X

N

_0,1CN

R⁵

R¹

CuSO₄

X

NHBoc

N

or

N

+

R¹N₃

R¹= Me, Et, Ph-CH₂-, Ph-(CH₂)₂-, Ph

⁵= H, Me, Et, n-Pr, n-Bu

R

X = -CH₂-, -(CH₂)₂-, m or p-xylenediyl,

p-methylphenethyl

Scheme 1. General structure of the target N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylamines.

Scheme 2. Reagents and conditions: (i) CuSO₄ꢁ5H₂O, ascorbic acid, Na₂CO₃, H₂O/DMF, rt, overnight, 49–78% yield; (ii) MeI, NaH, THF, rt, 1.5–15 h, 81–97% yield; (iii) H₃PO₄,

CH₂Cl₂, rt, 1.5–4 h, 38–98% (25–30), 67–100% (44–50); (iv) propargyl bromide, Cs₂CO₃, acetone, rt for 2–15 h, or 0 °C for 2–2.5 h, or 0 °C for 30 min and then rt for 3 h, 22–89%

(31–36), 53–95% (51–57); (v) R⁵-Hal, n-BuLi, THF, ꢀ78 °C, 1 h, then ꢀ78 °C ? rt, and rt, 2 h, 43–98%.

irreversible MAO inhibitors. These compounds are quite polar

(LogP of 0.43, 0.84, 0.65, and 1.06, respectively)⁷²due to the pres-

ence of the triazole ring. However, the substrate cavity of MAO is

highly hydrophobic, and accordingly MAO inhibitors are usually

more lipophilic molecules (LogP in the range 2–4 for compounds

depicted in Fig. 1). Thus, to counterbalance the high polarity con-

ferred by the triazole ring, we envisaged a second generation of

derivatives that featured more lipophilic benzyl or phenethyl sub-

stituents at position 1 (i.e., compounds 33 and 34, respectively,

Scheme 2) or at position 5 of the triazole ring (i.e., the 1-methyl-

5-butyl-substituted compound 54). We also planned the synthesis

of derivatives of the 1-benzyl-substituted compound 33 bearing

different alkyl groups at position 5. However, the moderate acidity

and ethyl, propyl, or butyl substituents at position 5. Finally, we

also explored the isomerization of the benzene ring of compound

33 from the substituent at position 1 of the triazole ring to the side

chain at position 4, i.e., between the triazole ring and the propargy-

lamine moiety (compounds 82 and 83, Scheme 3).

After evaluation of MAO-A and MAO-B inhibitory activity of the

ﬁrst and second generation compounds, 33 emerged as the most

selective MAO-B inhibitor, with a one-digit micromolar potency

against MAO-B, 27-fold higher than against MAO-A, whereas com-

pound 83 turned out to be the most potent inhibitor of both MAO

isoforms, with submicromolar potencies but essentially without

any selectivity (see below). In light of these results and on the basis

of molecular modeling studies, we inferred that a total number of

three methylenes might afford the optimal length between the ter-

minal aromatic ring and the propargylamine nitrogen atom to con-

fer selectivity towards MAO-B. To check this hypothesis, as a third

generation we planned the synthesis of compound 36 (Scheme 2),

an isomeric derivative of 33, and compound 84 (Scheme 3), with a

of the a-nitrogen benzylic protons of the substituent at position 1

of the triazole ring interfered with the alkylation of the position 5

(see below). To avoid these synthetic issues, we envisaged the syn-

thesis of compounds 55, 56, and 57 (Scheme 2), bearing a phenyl

group instead of a benzyl group at position 1 of the triazole ring

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

4

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

Scheme 3. Reagents and conditions: (i) trimethylsilylacetylene, THF, n-BuLi, ꢀ78 °C, 30 min, then, ZnBr₂, THF, ꢀ78 °C ? 0 °C, then, 58, 59, or 60, Cl₂Pd(PPh₃)₂, rt, overnight,

44–83% yield; (ii) AgOTf, CH₂Cl₂/MeOH/H₂O, rt, overnight, 63–77% yield; (iii) methyl azide, CuSO₄ꢁ5H₂O, ascorbic acid, Na₂CO₃, H₂O/DMF, rt, overnight, 89–95% yield; (iv)

LiAlH₄, THF, reﬂux, 2 h: 70 (49%), 71 (80%), or H₂, Raney-Ni, 7 N NH₃in MeOH: 72 (quantitative); (v) (Boc)₂O, THF, rt, 3 h, quantitative yield; (vi) MeI, NaH, THF, 60 °C, 4 h: 76

(84%), 77 (80%); (vii) LiAlH₄, THF, 0 °C, then 75, reﬂux, 72 h: 81 (44% yield); (viii) H₃PO₄, CH₂Cl₂, rt, 1.5–3 h: 79 (quantitative), 80 (96%); (ix) propargyl bromide, Cs₂CO₃,

acetone, 0 °C for 3.5 h or 0 °C for 30 min and then rt for 3 h, 31–65% yield.

total number of three methylenes from the terminal aromatic ring

(phenyl in 36, triazole in 84) to the propargylamine nitrogen atom.

For comparison purposes, we also envisaged the synthesis of com-

pound 35 (Scheme 2), the shorter homologue of 33 with a total

number of two methylenes in the tether.

excellent yields (90–97%). However, when we subjected the 1-ben-

zyl-substituted intermediate 19 to the same alkylation reaction

conditions with methyl iodide as the alkylating agent, we did not

obtain the expected 1-benzyl-5-methyl-substituted derivative

but the 1-(

in 79% yield, resulting from the double alkylation at the position

5 of the triazole ring and at the moderately acidic -nitrogen ben-

a-methylbenzyl)-5-methyl-substituted derivative 39

The synthesis of the target compounds was undertaken through

synthetic sequences involving as the key step a CuAAC reaction^73–

⁷⁶between alkyl or phenyl azides and alkynes bearing an amino or

a cyano group to enable the subsequent installation of the propar-

gylamine moiety (Scheme 1). The synthesis of the 5-unsubstituted

triazolylmethyl and triazolylethyl propargylamines 31–36 was car-

ried out through a four-step synthetic sequence, starting from the

copper-catalyzed Huisgen 1,3-dipolar cycloaddition of the known

N-Boc-protected amines 7 and 8 with ethyl, benzyl, and phenethyl

azide, generated in situ by reaction of NaN₃with the corresponding

alkyl halide,⁷⁷which afforded the 1-substituted triazole derivatives

9–16 in moderate to good yields (49–78%, Scheme 2). Subsequent

methylation of the N-Boc-protected aliphatic nitrogen atom of

compounds 9–16 with MeI in anhydrous THF, in the presence of

NaH, followed by acidic deprotection of the resulting compounds

17–24 afforded the secondary amines 25–30 in general in excellent

yields. The ﬁnal propargylation of amines 25–30 on reaction with

propargyl bromide in acetone in the presence of Cs₂CO₃led to

propargylamines 31–36 in 22–89% yield (Scheme 2). Thus, the tar-

get compounds 31–36 were obtained in 5–58% overall yield with-

out the need of any column chromatography puriﬁcation, with the

sole exception of the last step for 31, 35 and 36.

a

zylic position of the substituent at position 1 (Scheme 2). Treat-

ment of intermediate 19 with ethyl bromide or n-butyl bromide

as the alkylating agents and n-BuLi or the more hindered t-BuLi

as the base yielded inseparable mixtures of 5-monoalkylated and

a,5-dialkylated products. To avoid double alkylation, we prepared

the 1-phenyl-substituted intermediate 22, which was alkylated

with ethyl bromide, n-propyl bromide, and n-butyl bromide using

n-BuLi as the base, to afford the expected 5-alkyl-substituted

derivatives 41, 42, and 43 in 98%, 96%, and 43% yield, respectively

(Scheme 2). The acidic N-Boc deprotection of 5-alkyl-substituted

compounds 37–43 afforded the secondary amines 44–50 in good

yields (67% to quantitative yield). Propargylation of amines

44–50 had to be performed by monitoring the reaction course at

different reaction temperatures and times to avoid the formation

of dipropargylated products. For example, reaction of amine 44

with propargyl bromide in the presence of Cs₂CO₃at rt overnight

yielded the dipropargylated derivative as the major reaction pro-

duct (92% yield), whereas on reaction at 0 °C for 2 h only the

desired monopropargylated product, 51, was obtained, in 62%

yield. Thus, by selecting the most appropriate reaction tempera-

ture and time, the target 5-alkyl-substituted triazolylmethyl and

triazolylethyl propargylamines 51–57 were synthesized (19–44%

overall yield over the ﬁve-step sequence).

For the synthesis of the 5-alkyl-substituted triazolylmethyl and

triazolylethyl propargylamines 51, 52, and 54, apart from the inter-

mediates 17 and 18, the triazole derivative 21 was also prepared in

good yield using the same CuAAC-N-methylation protocol

(Scheme 2). Alkylation of compounds 17, 18, and 21 with the

appropriate alkyl halide using n-BuLi as the base afforded the cor-

responding 5-alkylated compounds 37, 38, and 40, respectively, in

The synthesis of the triazolyl-m-xylyl and triazolyl-p-xylyl

propargylamines 82 and 83 and the triazolyl-p-methylphenethyl

propargylamine 84 was carried out through the seven–eight-step

sequences depicted in Scheme 3. First, the alkynes 64–66,

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

5

Table 1

necessary for the CuAAC, were prepared by an initial Negishi cou-

pling⁷⁷between benzyl bromides 58–60 and trimethylsily-

lacetylene in the presence of n-BuLi and ZnBr₂, under Pd(0)

catalysis, followed by desilylation of the protected alkyne deriva-

tives 61–63 with AgOTf in a mixture of CH₂Cl₂/MeOH/H₂O,⁷⁸and

silica gel column chromatography puriﬁcation. The CuAAC of

methyl azide to alkynes 64–66 afforded the corresponding triazole

derivatives 67–69 in excellent yield (89–95%). LiAlH₄reduction of

the nitrile group of 67 and 68 afforded the corresponding amines

70 and 71 in 49% and 80% yield, respectively. However, the same

reaction conditions failed to provide the amine derived from nitrile

69, which could be efﬁciently reduced to amine 72 by hydrogena-

tion using Raney-Ni as the catalyst. N-Boc protection of the pri-

mary amino group of 70–72 afforded the intermediates 73–75.

The N-Boc-protected amines 73 and 74 were converted into the

target propargylamines 82 and 83 in good yields through the stan-

dard N-methylation–acidic deprotection–propargylation protocol,

without the need of any additional column chromatography

puriﬁcation (Scheme 3). Because different attempts to methylate

the N-Boc-protected amine 75 were fruitless, this compound was

alternatively converted into the target propargylamine 84 by direct

conversion into the secondary amine 81 upon LiAlH₄reduction

followed by propargylation (Scheme 3).

MAO-A and MAO-B inhibitory activities, selectivity indices, and BBB permeability

c

Compound

hrMAO-A^a

IC₅₀M)

hrMAO-B^a

IC₅₀M)

SI^b

P_e(10^ꢀ6cm s^ꢀ1

)

(

l

(l

1st generation

31

32

51

52

134.7 21.8

553.5 83.6

267.4 30.8

91.2 13.6

248.6 94.5

373.9 119

104.7 27.1

237.6 51.8

0.5

1.5

2.6

0.4

nd^d

2nd generation

33

34

53

54

55

56

57

82

83

97.1 30.1

56.1 8.7

47.0 6.9

<300

7.5 1.1

26.1 4.0

17.3 4.5

2.1 0.3

0.5 0.1

3.5 0.4

27.7

0.3

0.4

<3.2

0.04

0.2

0.1

0.03

0.9

15.6 1.5 (CNS+)

14.8 0.9 (CNS+)

20.0 0.7 (CNS+)

29.9 0.8 (CNS+)

17.0 1.9 (CNS+)

17.2 0.6 (CNS+)

21.4 1.3 (CNS+)

15.6 1.5 (CNS+)

14.8 0.9 (CNS+)

172.8 47.8

134.2 25.5

94.7 16.3

213.4 54.0

156.8 31.9

155.5 25.0

65.0 15.4

0.6 0.1

3rd generation

35

36

84

>500

27.4 0.1

129 42.6

11.2 2.1

5.2 0.5

>3.9

>44.6

5.3

2.3 0.7 (CNS )

0.6 0.1 (CNSꢀ)

4.5 0.5 (CNS )

Selegiline

Clorgyline

2

0.1

0.018 0.003

65.5 7.1

111.1

0.0003

0.020 0.003

a

In vitro inhibitory activities against hr-MAO-A and hr-MAO-B. Experiments

All the compounds to be subjected to biological evaluation were

transformed into the corresponding hydrochloride salts and were

chemically characterized through IR, ¹H and ¹³C NMR spectra,

and HRMS.

performed with Amplex Red ﬂuorimetric method in 96-well plates. Data are the

mean of duplicate experiments SEM.

b

SI = IC₅₀(MAO-A)/IC₅₀(MAO-B).

Permeability values from the PAMPA-BBB assay. Values are expressed as the

c

mean SD of three independent experiments.

d

Not determined.

2.2. MAO inhibitory activity of the target compounds

The novel N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylami-

nes 31–36, 51–57, and 82–84 were evaluated in vitro against

human recombinant MAO-A and MAO-B (hrMAO-A and hrMAO-

B). Selegiline and clorgyline were also used as reference com-

pounds. The corresponding IC₅₀values and selectivity indices (SI;

deﬁned as the ratio IC₅₀(MAO-A)/IC₅₀(MAO-B)) are shown in

Table 1. The novel compounds turned out to be in most cases mod-

erate to weak MAO inhibitors, with 2–3-digit micromolar IC₅₀val-

ues, with the exceptions of compounds 33, 55, 82 and 83, which

exhibited submicromolar or low micromolar potencies against

one or both MAO isoforms.

following the strategy used in our previous studies,³⁹the propargy-

lamine nitrogen atom was imposed to overly the position of the

corresponding atom in the X-ray structures of covalently-bound

selegiline and rasagiline (PDB entries 2BYB and 2BK4).^28,79By

imposing this restraint, we may explore the arrangement of the

compound after covalent linkage of the propargylamine unit to

the FAD cofactor. Figure 2 shows that 33 superposes well with

the structures of the irreversible inhibitor 6 (PDB entry 4CRT),⁸⁰

and the reversible ligand 1,4-diphenyl-2-butene (PDB entry

1OJ9),²⁶which spans the substrate binding cavity. In particular, it

The ﬁrst generation compounds 31, 32, 51, and 52 were weak

inhibitors and showed no selectivity. These trends were not unex-

pected due to the large polarity of the triazole ring and the small

size of these fragment-like compounds. Furthermore, molecular

modeling studies pointed out the feasibility of these compounds

to adopt an arrangement compatible with the formation of the

covalent linkage with the cofactor, but also revealed their inability

to ﬁll the substrate cavity, allowing for distinct orientations in the

pocket, and the lack of complementarity between the polar triazole

moiety and the hydrophobic environment of the cavity. Accord-

ingly, several strategies were explored to modulate both the

molecular size and hydrophobicity of the triazole derivatives.

The ﬁrst approach involved the substitution of the ethyl group

at position 1 of the triazole ring of the initial prototypes by benzyl

and phenethyl groups, which led to increased inhibitory potencies.

Thus, the 1-phenethyl-derivative 34 was 2- and 10-fold more

potent MAO-A inhibitor than the 1-benzyl- and 1-ethyl-analogues

33 and 32, respectively. In contrast, for MAO-B inhibition, the opti-

mal substitution pattern involved the presence of a 1-benzyl

group, with compound 33 being 49- and 106-fold more potent

than the 1-phenethyl- and 1-ethyl-analogues 34 and 32,

respectively.

Figure 2. Representation of the docked pose of compound 33 (shown as green-

colored sticks) in the binding cavity of MAO-B (shown as gray cartoon). The surface

of the binding cavity is depicted as a gray isocontour. The structures of inhibitor 6

(PDB entry 4CRT; covalently-bound to FAD, also shown as gray-colored sticks) and

the reversible ligand 1,4-diphenyl-2-butene (PDB entry 1OJ9) are shown as white-

colored and yellow sticks, respectively.

This latter ﬁnding suggests that the size of compound 33 is bet-

ter suited to ﬁll the binding cavity in MAO-B, as conﬁrmed by the

results obtained from restrained docking calculations. Thus,

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

6

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

is worth noting that the benzene ring of 33 matches well the posi-

tion ﬁlled by the benzene unit in 1,4-diphenyl-2-butene. In turn,

docking of the phenethyl derivative 34 made it necessary to adopt

a folded conformation, reﬂecting some degree of internal strain

due to clashes within the cavity (data not shown).

An alternative strategy explored to modulate the hydrophobic-

ity distribution of triazole derivatives relied on the alkylation of

position 5 of the triazole ring (compounds 53–57). However, alky-

lation did not seem to have a clear effect on MAO inhibitory

potency, although this chemical modiﬁcation was generally bene-

ﬁcial for MAO-A selectivity for compounds bearing large sub-

Overall, compound 83 emerges as the most potent MAO inhibi-

tor of the series, with submicromolar potencies against both

MAO-A and MAO-B, and hence, essentially without selectivity,

whereas compounds 33, 36, and 84, with a total number of three

methylene groups in the tethers that connect their phenyl and tri-

azole rings with the propargylamine nitrogen atom, emerge as low

micromolar selective MAO-B inhibitors of interest as novel leads

for AD drug discovery.

2.3. Reversibility and time-dependent inhibition studies

stituents at position

1

(Ph-CH(Me)– or Ph), with selectivity

The triazole derivative 33, one the most promising compounds

of the series by virtue of its one-digit micromolar inhibitory

potency and MAO-B selectivity, was subjected to further studies

to gain insight into the type of inhibition of MAO-B. First, we inves-

tigated whether the propargylamino group present in 33 led to the

irreversible inhibition of the enzyme. As shown in Figure 4, the

reversibility assays conﬁrmed that 33 irreversibly inhibited MAO-

B, since the inhibition was not reverted after various cycles of con-

secutive centrifugations and washings with buffer. Furthermore,

this ﬁnding is in agreement with the time-dependent inhibition

of MAO-B resulting from incubation with 33, as shown in Figure 4.

indices in the range 0.04–0.4 (compounds 53, 55–57 in Table 1).

Finally, we also envisaged the isomerization of the benzene ring

of compound 33 from the substituent at position 1 of the triazole

ring to the side chain at position 4. This approach led to increased

MAO-A inhibitory potencies, with compounds 82 and 83 being 46-

and 183-fold more potent than 33, and also to increased MAO-B

inhibitory activity in the case of 83 (6-fold more potent than 33),

bearing a para-disubstituted benzene ring in the side chain at posi-

tion 4. Docking calculations of compound 83 also led to a binding

pose that ﬁlled the binding cavity of MAO-B (Fig. 3), while docking

of the derivative with a meta-disubstituted benzene (compound

82) revealed steric clashes in the substrate binding cavity (data

not shown), in agreement with the 100-fold ratio in the IC₅₀values

determined for these compounds against hrMAO-B (Table 1).

In agreement with the hypothesis that a total number of three

methylenes separating the terminal aromatic ring and the propar-

gylamine nitrogen atom afforded the optimal length to confer

selectivity towards MAO-B, third generation compounds 36 and

84 turned out to be selective for MAO-B versus MAO-A inhibition,

with IC₅₀values for MAO-B inhibition in the low micromolar range.

Compound 36 was found to be more selective MAO-B inhibitor

than its shorter homologue 35, and even more selective than its

isomer 33 with the same total number of methylenes in the tether

(27-fold more potent for MAO-B than for MAO-A inhibition;

Table 1). Also, in contrast with compounds 82 and 83, with the

inverse selectivity or with no selectivity, compound 84 was found

to be 5-fold more potent for MAO-B than for MAO-A inhibition,

albeit with a 9-fold lower potency than its shorter homologue 83.

Thus, after pre-incubation of hrMAO-B with a 10 lM concentration

of 33 for different times ranging from 5 to 360 min, the enzymatic

activity showed a slow time-dependent inhibition of MAO-B. These

ﬁndings mimic the behavior reported for the MTDL inhibitor 6,³⁹

and for the structurally related MAO-B inhibitor PFN9601.⁸¹As

previously noted for 6,³⁹these results suggest that the proper

accommodation of 33 in a conformation suitable for covalent

attachment to the FAD cofactor may require an a priori conforma-

tional rearrangement for the structural adaptation to the peculiar

shape of the MAO-B binding pocket.

2.4. Blood–brain barrier permeation assay

Because an essential feature of anti-neurodegenerative drug

candidates is the ability to cross the BBB, all the novel compounds

were subjected to the well-established PAMPA-BBB assay, as an

in vitro model of passive permeation.⁸²The in vitro permeability

(P_e) of the novel compounds through a lipid extract of porcine

brain was determined using a mixture of phosphate-buffered sal-

ine (PBS)/EtOH 70:30. Assay validation was carried out by compar-

ison of the experimental and reported permeability values of 14

commercial drugs (see Section 4), which provided a good linear

correlation: P_e(exp) = 1.003 P_e(lit) ꢀ 0.783 (R²= 0.93). Using this

equation and the limits established by Di et al. for BBB perme-

ation,⁸²the following ranges of permeability were established: P_e

(10^ꢀ6cm s^ꢀ1) > 5.18 for compounds with high BBB permeation

(CNS+); P_e(10^ꢀ6cm s^ꢀ1) < 2.06 for compounds with low BBB per-

meation (CNSꢀ); and 5.18 > P_e(10^ꢀ6cm s^ꢀ1) > 2.06 for compounds

with uncertain BBB permeation (CNS ). As shown in Table 1, the

tested compounds had generally P_evalues well above the threshold

for high BBB permeation, so that they were predicted to be able to

cross the BBB and reach their biological target in the CNS, although

unexpectedly the third generation compounds showed lower per-

meabilities than the rest of compounds, maybe as a consequence of

solubility issues.

3. Conclusion

The results reported in this study support the feasibility of the

CuAAC-mediated synthesis of 1,2,3-triazole derivatives featuring

the propargylamine group typical of irreversible MAO-A and

MAO-B inhibitors, leading to suitable templates for the design of

MAO-B inhibitor-based MTDLs. The synthetic procedures of the

Figure 3. Representation of the docked pose of compound 83 (shown as green-

colored sticks) in the binding cavity of MAO-B (shown as gray cartoon). The surface

of the binding cavity is depicted as a gray isocontour. The structures of inhibitor 6

(PDB entry 4CRT; covalently-bound to FAD, also shown as gray-colored sticks) and

the reversible ligand 1,4-diphenyl-2-butene (PDB entry 1OJ9) are shown as white-

colored and yellow sticks, respectively.

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

7

1000

800

600

400

200

0

110

100

90

80

70

60

50

40

30

20

10

0

control

L-deprenyl

33

-10

0

40

80

120 160 200 240 280 320 360

0

5

10

15

20

25

30

35

40

incubation (min)

Figure 4. Plot of the enzymatic activity (mFU/min) versus pre-incubation time (min) (left) and plot of the remaining enzymatic activity (as % of control sample) versus pre-

incubation time (right) in the study of reversibility of hrMAO-B inhibition by 33.

1,4-disubstituted or 1,4,5-trisubstituted 1,2,3-triazole scaffolds

involve 4–6-step sequences from the CuAAC of alkyl or phenyl

azides with an alkyne bearing an N-Boc-protected primary amino

or a cyano group as precursors of the propargylamine warhead.

Nevertheless, introduction of lipophilic substituents were neces-

sary to counterbalance the high polarity conferred by the triazole

ring and to modulate the inhibitory activity against the two MAO

isoforms. The triazolyl-p-xylyl propargylamine 83 has been found

to be a moderately potent nonselective MAO inhibitor, with submi-

cromolar potencies against both human MAO-A and MAO-B. On

the other hand, the isomeric 1-benzyltriazolylethyl propargy-

lamine 33 and 1-phenethyltriazolylmethyl propargylamine 36,

and the triazolyl-p-methylphenethyl propargylamine 84 have

emerged as moderately potent and selective MAO-B inhibitor with

low micromolar potencies and large selectivity indices, with an

appropriate hydrophilic–lipophilic balance (LogP values of 2.1,

1.9, and 2.4, respectively, i.e., in the range of classical MAO inhibi-

tors) and relatively low molecular weight (254–268). Further work

will be focused on reﬁning the MAO-B inhibitory potency of these

scaffolds by enabling the linkage to a second pharmacophoric moi-

ety to derive novel MTDLs, as well as to develop small-molecule

probes for MAO-B imaging in models of neurodegenerative

diseases.⁸³

silica gel 60 F₂₅₄(Merck, ref 1.05554), and spots were visualized

with UV light and 1% aqueous solution of KMnO₄. High resolution

mass spectra were carried out at the CCiTUB with a LC/MSD TOF

Agilent Technologies spectrometer. The analytical samples of all

of the compounds that were subjected to pharmacological evalua-

tion were dried at 65 °C/2 Torr (standard conditions).

4.1.1. N-(tert-Butoxycarbonyl)-N-[(1-ethyl-1H-1,2,3-triazol-4-yl)

methyl]amine 9

To a solution of bromoethane (0.53 mL, 774 mg, 7.10 mmol) in

H₂O/DMF 1:4 (50 mL), NaN₃(502 mg, 7.72 mmol), Na₂CO₃(2.05 g,

19.3 mmol), ascorbic acid (907 mg, 5.15 mmol), CuSO₄ꢁ5H₂O

(641 mg, 2.57 mmol) and N-Boc-propargylamine,

7 (1.00 g,

6.44 mmol) were added. The reaction mixture was stirred at rt

overnight, diluted with 20% aq NH₄OH (200 mL), treated with solid

EDTA (ca 5 g) and extracted with EtOAc (2 ꢂ 150 mL). The com-

bined organic extracts were washed with H₂O (3 ꢂ 100 mL), dried

over anhydrous Na₂SO₄, and concentrated under reduced pressure.

The resulting residue was washed with pentane (3 ꢂ 10 mL) and

dried under vacuum, to give compound 9 (1.14 g, 78% yield) as a

white solid; R_f0.57 (CH₂Cl₂/MeOH 9:1); mp 77–78 °C; IR (ATR)

m

3318 (NAH), 1685, 1675 (C@O) cm^{ꢀ1 1}H NMR (400 MHz, CDCl₃)

;

d 1.40 [s, 9H, C(CH₃)₃], 1.51 (t, J = 7.2 Hz, 3H, 1-CH₂–CH₃), 4.35

(q, J = 7.2 Hz, 2H, 1-CH₂–CH₃), 4.36 (s, 2H, 4-CH₂–NH), 5.21 (broad

s, 1H, NHBoc), 7.50 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CDCl₃) d

15.5 (CH₃, 1-CH₂–CH₃), 28.4 [3CH₃, C(CH₃)₃], 36.2 (CH₂, 4-CH₂–N),

45.3 (CH₂, 1-CH₂–CH₃), 79.7 [C, C(CH₃)₃], 121.3 (CH, C5), 145.6

(C, C4), 156.0 (C, NCOO); HRMS (ESI), calcd for (C₁₀H₁₈N₄O₂+ H⁺)

227.1503, found 227.1493.

4. Experimental part

4.1. Chemistry. General methods

Melting points were determined in open capillary tubes with a

MFB 595010M Gallenkamp melting point apparatus. 400 MHz

¹H/100.6 MHz ¹³C NMR spectra were recorded on a Varian Mercury

400 spectrometer at the Centres Cientíﬁcs i Tecnològics of the

University of Barcelona (CCiTUB). The chemical shifts are reported

in ppm (d scale) relative to solvent signals (CD₃OD at 3.31 and

49.0 ppm in the ¹H and ¹³C NMR spectra, respectively; CDCl₃at

7.26 and 77.16 ppm in the ¹H and ¹³C NMR spectra, respectively),

and coupling constants are reported in Hertz (Hz). Assignments

given for the NMR spectra of the new compounds have been car-

ried out by comparison with the NMR data of compounds 10, 25,

34, 37, 52, and 82, which in turn, were assigned on the basis of

DEPT, COSY ¹H/¹H (standard procedures), and COSY ¹H/¹³C (gHSQC

or gHMBC sequences) experiments. IR spectra were run on a Per-

kin–Elmer Spectrum RX I spectrophotometer. Absorption values

are expressed as wavenumbers (cm^ꢀ1); only signiﬁcant absorption

bands are given. Column chromatography was performed on silica

gel 60 AC.C (40–60 mesh, SDS, ref 2000027). Thin-layer chro-

matography was performed with aluminum-backed sheets with

4.1.2. N-(tert-Butoxycarbonyl)-N-[2-(1-ethyl-1H-1,2,3-triazol-4-

yl)ethyl]amine 10

It was prepared as described for 9. From bromoethane (0.65 mL,

949 mg, 8.71 mmol), NaN₃(618 mg, 9.51 mmol), Na₂CO₃(2.52 g,

23.8 mmol), ascorbic acid (1.12 g, 6.36 mmol), CuSO₄ꢁ5H₂O

(790 mg, 3.16 mmol), and N-Boc-3-butynylamine,

8 (1.34 g,

7.92 mmol), compound 10 (1.48 g, 78% yield) was obtained as a

white solid; R_f0.50 (CH₂Cl₂/MeOH 9:1); mp 78–80 °C; IR (ATR)

3341 (NAH), 1692 (C@O) cm^{ꢀ1 1}H NMR (400 MHz, CDCl₃) d 1.40

m

;

[s, 9H, C(CH₃)₃], 1.51 (t, J = 7.6 Hz, 3H, 1-CH₂–CH₃), 2.87 (t,

J = 6.4 Hz, 2H, 4-CH₂–CH₂–NH), 3.44 (dt, J = J⁰= 6.4 Hz, 2H, 4-CH₂–

CH₂–NH), 4.35 (q, J = 7.6 Hz, 2H, 1-CH₂–CH₃), 5.04 (broad s, 1H,

NHBoc), 7.35 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CDCl₃) d 15.6

(CH₃, 1-CH₂–CH₃), 26.3 (CH₂, 4-CH₂–CH₂–N), 28.5 [3CH₃,

C

(CH₃)₃], 39.9 (CH₂, 4-CH₂–CH₂–N), 45.2 (CH₂, 1-CH₂–CH₃), 79.2

[C, C(CH₃)₃], 120.8 (CH, C5), 145.5 (C, C4), 156.1 (C, NCOO); HRMS

(ESI), calcd for (C₁₁H₂₀N₄O₂+ H⁺) 241.1659, found 241.1654.

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

8

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

4.1.3. N-(tert-Butoxycarbonyl)-N-[2-(1-benzyl-1H-1,2,3-triazol-

4-yl)ethyl]amine 11

(400 MHz, CDCl₃) d 1.43 [s, 9H, C(CH₃)₃], 3.00 (t, J = 6.8 Hz, 2H, 4-

CH₂–CH₂–N), 3.55 (td, J = 6.8 Hz, J⁰= 6.0 Hz, 2H, 4-CH₂–CH₂–N),

5.02 (br s, 1H, NHBoc), 7.43 (tt, J = 7.4 Hz, J⁰= 1.2 Hz, 1H, Ph–C4-

H), 7.52 [m, 2H, Ph–C3(5)–H], 7.71 [ddd, J = 8.8 Hz, J⁰= 2.4 Hz,

J⁰⁰= 1.2 Hz, 2H, Ph–C2(6)–H], 7.81 (s, 1H, 5-H); ¹³C NMR

(100.6 MHz, CDCl₃) d 26.4 (CH₂, 4-CH₂–CH₂–N), 28.5 [3CH₃, C

(CH₃)₃], 39.9 (CH₂, 4-CH₂–CH₂–N), 79.4 [C, C(CH₃)₃], 119.8 (CH,

Ph–C4), 120.5 (CH, C5), 128.7 (2CH), 129.9 (2CH) [Ph–C2(6), Ph–

C3(5)], 137.2 (C, Ph–C1), 146.3 (C, C4), 156.1 (C, NCOO); HRMS

(ESI), calcd for (C₁₅H₂₀N₄O₂+ Na⁺) 311.1478, found 311.1476.

It was prepared as described for 9. From benzyl bromide

(0.75 mL, 1.08 g, 6.31 mmol), NaN₃(462 mg, 7.11 mmol), Na₂CO₃

(1.88 g, 17.7 mmol), ascorbic acid (834 mg, 4.74 mmol), CuSO₄ꢁ

5H₂O (590 mg, 2.36 mmol), and N-Boc-3-butynylamine, 8 (1.00 g,

5.91 mmol), compound 11 (1.27 g, 71% yield) was obtained as a

white solid; R_f0.58 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05);

mp 103–105 °C; IR (ATR) v 3407 (NAH), 1688 (C@O) cm^ꢀ^{1 1}H

;

NMR (400 MHz, CDCl₃) d 1.38 [s, 9H, C(CH₃)₃], 2.84 (t, J = 6.8 Hz,

2H, 4-CH₂–CH₂–NH), 3.41 (td, J = 6.8 Hz, J⁰= 6.0 Hz, 2H, 4-CH₂–

CH₂–NH), 5.04 (broad s, 1H, NHBoc), 5.46 (s, 2H, 1-CH₂–Ph), 7.22

[ddm, J = 7.6 Hz, J⁰= 2.0 Hz, 2H, Ph–C2(6)–H], 7.29–7.37 [complex

signal, 4H, 5-H, Ph–C3(5)–H, Ph–C4–H]; ¹³C NMR (100.6 MHz,

CDCl₃) d 26.3 (CH₂, 4-CH₂–CH₂N), 28.4 [3CH₃, C(CH₃)₃], 39.8 (CH₂,

4-CH₂–CH₂–N), 54.1 (CH₂, 1-CH₂–Ph), 79.2 [C, C(CH₃)₃], 121.4

(CH, C5), 128.1 (2CH), 128.8 (CH), 129.2 (2CH) (Ph–CH), 134.8

(C, Ph–C1), 145.9 (C, C4), 156.0 (C, NCOO); HRMS (ESI), calcd for

(C₁₆H₂₂N₄O₂+ H⁺) 303.1816, found 303.1813.

4.1.7. N-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]-N-(tert-

butoxycarbonyl)amine 15

It was prepared as described for 9. From benzyl bromide

(1.04 mL, 1.50 g, 8.74 mmol), NaN₃(622 mg, 9.57 mmol), Na₂CO₃

(2.53 g, 23.9 mmol), ascorbic acid (1.12 g, 6.36 mmol), CuSO₄ꢁ5H₂O

(796 mg, 3.19 mmol), and N-Boc-propargylamine,

7 (1.24 g,

7.99 mmol), compound 15 (1.68 g, 68% yield) was obtained as a

white solid; R_f0.62 (CH₂Cl₂/MeOH/NH₄OH 9.75:0.25:0.05); mp

97–99 °C; IR (ATR) v 3400, 3302, 3105, 3059 (NAH), 1684, 1679

4.1.4. N-(tert-Butoxycarbonyl)-N-[2-(1-phenethyl-1H-1,2,3-

triazol-4-yl)ethyl]amine 12

(C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.32 [s, 9H, C(CH₃)₃],

;

4.27 (d, J = 6.0 Hz, 2H, 4-CH₂–N), 5.06 (br s, 1H, NHBoc), 5.40 (s,

2H, 1-CH₂–Ph), 7.26 [m, 2H, Ph–C2(6)–H], 7.30–7.40 [complex sig-

nal, 3H, Ph–C3(5)–H, Ph–C4–H], 7.43 (s, 1H, 5-H); ¹³C NMR

(100.6 MHz, CDCl₃) d 28.4 [3CH₃, C(CH₃)₃], 36.2 (CH₂, 4-CH₂–N),

54.2 (CH₂, 1-CH₂–Ph), 79.8 [C, C(CH₃)₃], 121.9 (CH, C5), 128.2

(2CH), 128.8 (CH), 129.2 (2CH) (Ph–CH), 134.7 (C, Ph–C1), 146.0

(C, C4), 155.9 (C, NCOO); HRMS (ESI), calcd for (C₁₅H₂₀N₄O₂+ Na⁺)

311.1478, found 311.1487.

It was prepared as described for 9. From phenethyl bromide

(0.89 mL, 1.21 g, 6.52 mmol), NaN₃(462 mg, 7.11 mmol), Na₂CO₃

(1.88 g, 17.7 mmol), ascorbic acid (834 mg, 4.74 mmol), CuSO₄ꢁ

5H₂O (590 mg, 2.36 mmol), and N-Boc-3-butynylamine, 8 (1.00 g,

5.91 mmol), compound 12 (908 mg, 49% yield) was obtained as a

white solid; R_f0.52 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05);

mp 123–125 °C; IR (ATR) v 3392 (NAH), 1688 (C@O) cm^ꢀ^{1 1}H

;

NMR (400 MHz, CDCl₃) d 1.43 [s, 9H, C(CH₃)₃], 2.85 (t, J = 6.8 Hz,

2H, 4-CH₂–CH₂–N), 3.19 (t, J = 7.2 Hz 2H, 1-CH₂–CH₂–Ph), 3.42

(m, 2H, 4-CH₂–CH₂–N), 4.55 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph),

4.95 (br s, 1H, NHBoc), 7.09 [dm, J = 6.8 Hz, 2H, Ph–C2(6)–H],

7.22–7.32 [complex signal, 4H, 5-H, Ph–C3(5)–H, Ph–C4–H]; ¹³C

NMR (100.6 MHz, CDCl₃) d 26.2 (CH₂, 4-CH₂–CH₂–N), 28.5 [3CH₃,

C(CH₃)₃], 36.9 (CH₂, 1-CH₂–CH₂–Ph), 40.0 (CH₂, 4-CH₂–CH₂–N),

51.6 (CH₂, 1-CH₂–CH₂–Ph), 79.3 [C, C(CH₃)₃], 121.9 (CH, C5),

127.2 (CH), 128.8 (2CH), 128.9 (2CH) (Ph–CH), 137.2 (C, Ph–C1),

145.2 (C, C4), 156.1 (C, NCOO); HRMS (ESI), calcd for (C₁₇H₂₄N₄-

O₂+ H⁺) 317.1972, found 317.1968.

4.1.8. N-(tert-Butoxycarbonyl)-N-[(1-phenethyl-1H-1,2,3-

triazol-4-yl)methyl]amine 16

It was prepared as described for 9. From phenethyl bromide

(1.11 mL, 1.50 g, 8.13 mmol), NaN₃(574 mg, 8.83 mmol), Na₂CO₃

(2.34 g, 22.1 mmol), ascorbic acid (1.04 g, 5.91 mmol), CuSO₄ꢁ5H₂O

(735 mg, 2.94 mmol), and N-Boc-propargylamine,

7 (1.00 g,

6.44 mmol), compound 16 (1.43 g, 73% yield) was obtained as a

white solid; R_f0.57 (CH₂Cl₂/MeOH/NH₄OH 9.75:0.25:0.05); mp

98–100 °C; IR (ATR) v 3410, 3111, 3069, 3054 (NAH), 1684 (C@O)

cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.43 [s, 9H, C(CH₃)₃], 3.19 (t,

;

J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), 4.34 (d, J = 6.0 Hz, 2H, 4-CH₂–N),

4.56 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), 5.14 (br s, 1H, NHBoc), 7.10

[dm, J = 8.4 Hz, 2H, Ph–C2(6)–H], 7.21–7.32 [complex signal, 4H,

5-H, Ph–C3(5)–H, Ph–C4–H]; ¹³C NMR (100.6 MHz, CDCl₃) d 28.5

[3CH₃, C(CH₃)₃], 36.2 (CH₂, 4-CH₂–N), 36.8 (CH₂, 1-CH₂–CH₂–Ph),

51.7 (CH₂, 1-CH₂–CH₂–Ph), 79.7 [C, C(CH₃)₃], 122.2 (CH, C5), 127.2

(CH), 128.7 (2CH), 128.9 (2CH) (Ph–CH), 137.0 (C, Ph–C1), 145.3

(C, C4), 155.9 (C, NCOO); HRMS (ESI), calcd for (C₁₆H₂₂N₄O₂+ H⁺)

303.1816, found 303.1827.

4.1.5. N-(tert-Butoxycarbonyl)-N-[(1-methyl-1H-1,2,3-triazol-4-

yl)methyl]amine 13

It was prepared as described for 9. From iodomethane (0.44 mL,

1.00 g, 7.07 mmol), NaN₃(502 mg, 7.72 mmol), Na₂CO₃(2.05 g,

19.3 mmol), ascorbic acid (907 mg, 5.15 mmol), CuSO₄ꢁ5H₂O

(641 mg, 2.57 mmol), and N-Boc-propargylamine,

7 (1.00 g,

6.44 mmol), compound 13 (938 mg, 69% yield) was obtained as a

white solid; R_f0.87 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05);

mp 102–104 °C; IR (ATR) v 3379 (NAH), 1679 (C@O) cm^{ꢀ1 1}H

;

NMR (400 MHz, CDCl₃) d 1.41 [s, 9H, C(CH₃)₃], 4.05 (s, 3H, 1-

CH₃), 4.35 (d, J = 6.0 Hz, 2H, 4-CH₂–N), 5.17 (br s, 1H, NHBoc),

7.48 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CDCl₃) d: 28.5 [3CH₃, C

(CH₃)₃], 36.2 (CH₂, 4-CH₂–N), 36.7 (CH₃, 1-CH₃), 79.7 [C, C(CH₃)₃],

122.9 (CH, C5), 145.9 (C, C4), 156.0 (C, NCOO); HRMS (ESI), calcd

for (C₉H₁₆N₄O₂+ H⁺) 213.1346, found 213.1336.

4.1.9. N-(tert-Butoxycarbonyl)-N-[(1-ethyl-1H-1,2,3-triazol-4-yl)

methyl]-N-methylamine 17

A solution of 9 (634 mg, 2.80 mmol) in anhydrous THF (6.5 mL)

was cooled to 0 °C and treated with NaH (60% suspension in min-

eral oil, 246 mg, 6.15 mmol) and iodomethane (0.19 mL, 433 mg,

3.05 mmol). The reaction mixture was stirred at rt for 3 h, cooled

to 0 °C, diluted dropwise with satd aq NH₄Cl (40 mL), and extracted

with EtOAc (3 ꢂ 40 mL). The combined organic extracts were dried

over anhydrous Na₂SO₄and evaporated under reduced pressure.

The resulting residue was washed with pentane (3 ꢂ 10 mL) and

dried under vacuum, to give 17 (654 mg, 97% yield) as a colorless

4.1.6. N-(tert-Butoxycarbonyl)-N-[2-(1-phenyl-1H-1,2,3-triazol-

4-yl)ethyl]amine 14

It was prepared as described for 9. From phenyl azide (0.5 M

solution in tert-butyl methyl ether, 26.2 mL, 13.1 mmol), Na₂CO₃

(3.47 g, 32.7 mmol), ascorbic acid (1.54 g, 8.74 mmol), CuSO₄ꢁ5H₂-

O (1.09 mg, 4.36 mmol), and N-Boc-3-butynylamine, 8 (2.00 g,

11.8 mmol), compound 14 (1.89 g, 56% yield) was obtained as a

yellow solid; R_f0.90 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05); mp

oil; R_f0.64 (CH₂Cl₂/MeOH 9:1); IR (ATR)

m 1688, 1677 (C@O)

cm^ꢀ1; ¹H NMR (400 MHz, CDCl₃) d 1.40 [s, C(CH₃)₃minor rotamer],

1.42 [s, C(CH₃)₃major rotamer], 1.50 (t, J = 7.2 Hz, 1-CH₂–CH₃

minor rotamer), 1.51 (t, J = 7.2 Hz, 1-CH₂–CH₃major rotamer),

2.87 (s, 3H, N–CH₃), 4.39 (q, J = 7.2 Hz, 1-CH₂–CH₃minor rotamer),

127–129 °C; IR (ATR) v 3297 (NAH), 1712 (C@O) cm^ꢀ^{1 1}H NMR

;

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

9

4.40 (q, J = 7.2 Hz, 1-CH₂–CH₃major rotamer), 4.49 (s, 4-CH₂–N),

7.37 (br s, 5-H minor rotamer), 7.49 (s, 5-H major rotamer); HRMS

(ESI), calcd for (C₁₁H₂₀N₄O₂+ H⁺) 241.1659, found 241.1654.

4.82 mmol), stirring at rt for 1.5 h, compound 21 (945 mg, 95%

yield) was obtained as a yellowish oil; R_f0.31 (CH₂Cl₂/MeOH/50%

aq NH₄OH 9.5:0.5:0.05); IR (ATR) v 1686 (C@O) cm^ꢀ^{1 1}H NMR

;

(400 MHz, CDCl₃) d 1.45 [s, 9H, C(CH₃)₃], 2.89 (s, 3H, N–CH₃),

4.07 (s, 3H, 1-CH₃), 4.47 (s, 2H, 4-CH₂–N), 7.37 (br s), 7.49 (broad

s) (5-H); ¹³C NMR (100.6 MHz, CDCl₃) d 28.6 [3CH₃, C(CH₃)₃],

34.2 (br CH₃), 34.6 (br CH₃) (N–CH₃), 36.8 (CH₃, 1-CH₃), 43.8 (br

CH₂), 44.5 (br CH₂) (4-CH₂–N), 79.9 [br C, C(CH₃)₃], 122.6 (br CH),

123.5 (br CH) (C5), 145.3 (br C), 145.8 (br C) (C4), 155.3 (br C),

155.9 (br C) (NCOO); HRMS (ESI), calcd for (C₁₀H₁₈N₄O₂+ Na⁺)

249.1322; found 249.1323.

4.1.10. N-(tert-Butoxycarbonyl)-N-[2-(1-ethyl-1H-1,2,3-triazol-

4-yl)ethyl]-N-methylamine 18

It was prepared as described for 17. From compound 10 (1.70 g,

7.08 mmol), NaH (60% suspension in mineral oil, 424 mg,

10.6 mmol), and iodomethane (0.48 mL, 1.09 g, 7.71 mmol), stir-

ring at rt for 2 h, compound 18 (1.71 mg, 95% yield) was obtained

as a colorless oil; R_f0.58 (CH₂Cl₂/MeOH 9:1); IR (ATR)

m 1690

(C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.41 [s, 9H, C(CH₃)₃],

;

1.51 (t, J = 7.2 Hz, 3H, 1-CH₂–CH₃), 2.82 (s, 3H, N–CH₃), 2.93 (m,

2H, 4-CH₂–CH₂–N), 3.52 (br t, J = 6.8 Hz, 2H, 4-CH₂–CH₂–N), 4.35

(q, J = 7.2 Hz, 2H, 1-CH₂–CH₃), 7.28 (br s), 7.40 (br s) (5-H); ¹³C

NMR (100.6 MHz, CDCl₃) d 15.6 (CH₃, 1-CH₂–CH₃), 24.3 (CH₂),

24.7 (CH₂) (4-CH₂–CH₂–N), 28.5 [3CH₃, C(CH₃)₃], 34.4 (CH₃, N–

CH₃), 45.2 (CH₂, 1-CH₂–CH₃), 48.0 (CH₂), 48.9 (CH₂) (4-CH₂–CH₂–

N), 79.5 [C, C(CH₃)₃], 120.8 (CH, C5), 145.2 (br C, C4), 155.8 (br C,

NCOO); HRMS (ESI), calcd for (C₁₂H₂₂N₄O₂+ H⁺) 255.1816, found

255.1815.

4.1.14. N-(tert-Butoxycarbonyl)-N-methyl-N-[2-(1-phenyl-1H-

1,2,3-triazol-4-yl)ethyl]amine 22

It was prepared as described for 17. From compound 14 (1.82 g,

6.32 mmol), NaH (60% suspension in mineral oil, 0.76 g,

19.0 mmol), and iodomethane (1.96 mL, 4.47 g, 31.5 mmol), stir-

ring at rt overnight, compound 22 (1.82 g, 95% yield) was obtained

as a yellowish oil; R_f0.86 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05);

IR (ATR) v 1689, 1681 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d

;

1.42 [s, 9H, C(CH₃)₃], 2.88 (s, 3H, N–CH₃), 3.04 (m, 2H, 4-CH₂–

CH₂–N), 3.61 (m, 2H, 4-CH₂–CH₂–N), 7.43 (m, 1H, Ph–C4-H), 7.51

[m, 2H, Ph–C3(5)–H], 7.71 [dm, J = 8.8 Hz, 2H, Ph–C2(6)–H], 7.89

(br s, 1H, 5-H); ¹³C NMR (100.6 MHz, CDCl₃) d 24.3 (br CH₂), 24.6

(br CH₂) (4-CH₂–CH₂–N), 28.5 [3CH₃, C(CH₃)₃], 34.4 (CH₃, N–CH₃),

47.8 (br CH₂), 48.7 (br CH₂) (4-CH₂–CH₂–N), 79.6 [C, C(CH₃)₃],

119.8 (CH, Ph–C4), 120.5 (CH, C5), 128.6 (2CH), 129.8 (2CH) [Ph–

C2(6), Ph–C3(5)], 137.2 (C, Ph–C1), 145.9 (br C, C4), 155.8 (br C,

NCOO); HRMS (ESI), calcd for (C₁₆H₂₂N₄O₂+ H⁺) 303.1816, found

303.1809.

4.1.11. N-(tert-Butoxycarbonyl)-N-[2-(1-benzyl-1H-1,2,3-

triazol-4-yl)ethyl]-N-methylamine 19

It was prepared as described for 17. From compound 11 (3.67 g,

12.1 mmol), NaH (60% suspension in mineral oil, 0.73 g,

18.2 mmol), and iodomethane (0.83 mL, 1.89 g, 13.3 mmol), stir-

ring at rt for 3 h, compound 19 (3.64 g, 95% yield) was obtained

as

a

yellowish oil; R_f0.60 (CH₂Cl₂/MeOH/50% aq NH₄OH

9.5:0.5:0.05); IR (ATR) v 1687 (C@O) cm^ꢀ1

;

¹H NMR (400 MHz,

CDCl₃) d 1.37 [s, 9H, C(CH₃)₃], 2.78 (s, 3H, N–CH₃), 2.90 (m, 2H,

4-CH₂–CH₂–N), 3.48 (t, J = 6.8 Hz, 2H, 4-CH₂–CH₂–N), 5.45 (s, 2H,

1-CH₂-Ph), 7.20–7.37 (complex signal, 6H, 5-H, Ph–CH); ¹³C NMR

(100.6 MHz, CDCl₃) d 24.2 (CH₂), 24.7 (CH₂) (4-CH₂–CH₂–N), 28.4

[3CH₃, C(CH₃)₃], 34.4 [CH₃, N–CH₃), 48.0 (CH₂), 48.7 (CH₂) (4-

CH₂–CH₂–N), 54.1 (CH₂, 1-CH₂–Ph), 79.5 [C, C(CH₃)₃], 121.4 (br

CH, C5), 128.1 (2CH), 128.7 (CH), 129.1 (2CH) (Ph–CH), 134.9 (C,

Ph–C1), 145.6 (br C, C4), 155.7 (C, NCOO); HRMS (ESI), calcd for

(C₁₇H₂₄N₄O₂+ H⁺) 317.1972, found 317.1964.

4.1.15. N-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]-N-(tert-

butoxycarbonyl)-N-methylamine 23

It was prepared as described for 17. From compound 15 (1.60 g,

5.16 mmol), NaH (60% suspension in mineral oil, 670 mg,

16.7 mmol), and iodomethane (1.73 mL, 3.94 g, 27.8 mmol), stir-

ring at rt overnight, compound 23 (1.30 g, 83% yield) was obtained

as a brown solid; R_f0.68 (CH₂Cl₂/MeOH/NH₄OH 9.75:0.25:0.05);

mp 79–81 °C; IR (ATR) v 1679 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz,

;

CDCl₃) d 1.41 [br s, 9H, C(CH₃)₃], 2.89 (s, 3H, N–CH₃), 4.45 (s, 2H,

4-CH₂–N), 5.50 (s, 2H, 1-CH₂–Ph), 7.26 [br dd, J = 8.0 Hz, J⁰= 1.6 Hz,

2H, Ph–C2(6)–H], 7.34–7.40 [complex signal, 3H, Ph–C3(5)–H, Ph–

C4–H], 7.44 (br s, 1H, 5-H); ¹³C NMR (100.6 MHz, CDCl₃) d 28.4

[3CH₃, C(CH₃)₃], 34.5 (CH₃, N–CH₃), 43.8 (CH₂), 44.6 (CH₂) (4-

CH₂–N), 54.2 (CH₂, 1-CH₂–Ph), 79.8 [C, C(CH₃)₃], 121.6 (CH),

122.4 (CH) (C5), 128.1 (2CH), 128.8 (CH), 129.2 (2CH) (Ph–CH),

134.7 (C, Ph–C1), 145.4 (C), 145.6 (C) (C4), 155.4 (C), 155.8 (C)

(NCOO); HRMS (ESI), calcd for (C₁₆H₂₂N₄O₂+ Na⁺) 325.1635, found

325.1638.

4.1.12. N-(tert-Butoxycarbonyl)-N-methyl-N-[2-(1-phenethyl-

1H-1,2,3-triazol-4-yl)ethyl]amine 20

It was prepared as described for 17. From compound 12

(876 mg, 2.77 mmol), NaH (60% suspension in mineral oil,

167 mg, 4.17 mmol), and iodomethane (0.19 mL, 433 mg,

3.05 mmol), stirring at rt for 1.5 h, compound 20 (738 mg, 81%

yield) was obtained as a yellowish oil; R_f0.57 (CH₂Cl₂/MeOH/50%

aq NH₄OH 9.5:0.5:0.05); mp 58–60 °C; IR (ATR) v 1686 (C@O)

cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.42 [s, 9H, C(CH₃)₃], 2.80 (s,

;

3H, N–CH₃), 2.90 (m, 2H, 4-CH₂–CH₂–N), 3.18 (t, J = 7.2 Hz, 2H, 1-

CH₂–CH₂–Ph), 3.48 (m, 2H, 4-CH₂–CH₂–N), 4.54 (t, J = 7.2 Hz, 2H,

1-CH₂–CH₂–Ph), 7.03 (br s), 7.19 (br s) (5-H), 7.10 [dm, J = 8.0 Hz,

2H, Ph–C2(6)–H], 7.21–7.32 (complex signal, 3H, Ph–C3(5)–H,

Ph–C4–H]; ¹³C NMR (100.6 MHz, CDCl₃) d 24.2 (CH₂), 24.6 (CH₂)

(4-CH₂–CH₂–N), 28.5 (3CH₃, C(CH₃)₃], 34.5 (CH₃, N–CH₃), 36.9

(CH₂, 1-CH₂–CH₂–Ph), 48.1 (CH₂), 48.9 (CH₂) (4-CH₂–CH₂–N), 51.6

(CH₂, 1-CH₂–CH₂–Ph), 79.5 [C, C(CH₃)₃], 121.8 (CH, C5), 127.1

(CH), 128.7 (2CH), 128.9 (2CH) (Ph–CH), 137.2 (C, Ph–C1), 144.8

(br C, C4), 155.8 (C, NCOO); HRMS (ESI), calcd for (C₁₈H₂₆N₄O₂+ H⁺)

331.2129, found 331.2122.

4.1.16. N-(tert-Butoxycarbonyl)-N-methyl-N-[(1-phenetyl-1H-

1,2,3-triazol-4-yl)methyl]amine 24

It was prepared as described for 17. From compound 16 (1.27 g,

4.20 mmol), NaH (60% suspension in mineral oil, 505 mg,

12.6 mmol), and iodomethane (1.31 mL, 2.99 g, 21.0 mmol), stir-

ring at rt overnight, compound 24 (1.07 g, 81% yield) was obtained

as a brown oil; R_f0.59 (CH₂Cl₂/MeOH/NH₄OH 9.75:0.25:0.05); IR

(ATR) v 1687 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.43 [s,

;

9H, C(CH₃)₃], 2.84 (s, 3H, N–CH₃), 3.19 (t, J = 7.2 Hz, 2H, 1-CH₂–

CH₂–Ph), 4.43 (s, 2H, 4-CH₂–N), 4.56 (m, 2H, 1-CH₂–CH₂–Ph),

7.08 [dm, J = 8.4 Hz, 2H, Ph–C2(6)–H], 7.20–7.32 [complex signal,

4H, 5-H, Ph–C3(5)–H, Ph–C4–H]; ¹³C NMR (100.6 MHz, CDCl₃) d

28.2 [CH₃, C(CH₃)₃minor rotamer], 28.3 [CH₃, C(CH₃)₃major rota-

mer], 33.9 (CH₃, N–CH₃minor rotamer), 34.2 (CH₃, N–CH₃major

rotamer), 36.5 (CH₂, 1-CH₂–CH₂–Ph minor rotamer), 36.6 (CH₂, 1-

CH₂–CH₂–Ph major rotamer), 43.5 (CH₂, 4-CH₂–N major rotamer),

4.1.13. N-(tert-Butoxycarbonyl)-N-methyl-N-[(1-methyl-1H-

1,2,3-triazol-4-yl)methyl]amine 21

It was prepared as described for 17. From compound 13

(928 mg, 4.38 mmol), NaH (60% suspension in mineral oil,

263 mg, 6.57 mmol), and iodomethane (0.30 mL, 684 mg,

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

10

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

44.2 (CH₂, 4-CH₂–N minor rotamer), 51.4 (CH₂, 1-CH₂–CH₂–Ph

minor rotamer), 51.5 (CH₂, 1-CH₂–CH₂–Ph major rotamer), 79.6

[br C, C(CH₃)₃], 121.9 (CH, C5 minor rotamer), 122.6 (CH, C5 major

rotamer), 126.8 (CH, Ph–C4 minor rotamer), 127.0 (CH, Ph–C4

major rotamer), 128.4 (CH), 128.5 (CH), 128.7 (CH) [Ph–C2(6),

Ph–C3(5)], 136.8 (C, Ph–C1 minor rotamer), 136.9 (C, Ph–C1 major

rotamer), 144.4 (br C, C4), 155.2 (C, NCOO minor rotamer), 155.7

(C, NCOO major rotamer); HRMS (ESI), calcd for (C₁₇H₂₄N₄O₂+ H⁺)

317.1972, found 317.1979.

130.15 (CH), 130.20 (2CH) (Ph–CH), 135.3 (C, Ph–C1), 142.6 (C,

C4); HRMS (ESI), calcd for (C₁₂H₁₆N₄+ H⁺) 217.1448, found

217.1442.

4.1.20. N-Methyl-N-[2-(1-phenethyl-1H-1,2,3-triazol-4-yl)ethyl]

amine 28

It was prepared as described for 25. From 20 (693 mg,

2.10 mmol) and H₃PO₄(85% purity, 3.61 mL, 31.3 mmol), amine

28 (472 mg, 98% yield) was obtained as a yellow oil; R_f0.26 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

4.1.17. N-[(1-Ethyl-1H-1,2,3-triazol-4-yl)methyl]-N-

methylamine 25

28ꢁHCl: beige solid, mp 160–162 °C; IR (ATR) v 3500–2400 (max

at 3413, 3354, 3126, 3092, 2954, 2783, 2703, 2444, ⁺NAH and

To a solution of 17 (411 mg, 1.71 mmol) in CH₂Cl₂(6 mL), H₃PO₄

(85% purity, 2.96 mL, 25.7 mmol) was added. The reaction mixture

was stirred at rt for 2 h, cooled to 0 °C, diluted with H₂O (15 mL),

alkalinized dropwise to pH = 14 with 5 N NaOH, and extracted with

CH₂Cl₂(2 ꢂ 30 mL). The combined organic extracts were dried over

anhydrous Na₂SO₄and evaporated under reduced pressure to give

amine 25 (205 mg, 86% yield) as a colorless oil; R_f0.07 (CH₂Cl₂/

MeOH/50% aq NH₄OH 9:1:0.05).

CAH) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 2.75 (s, 3H, N–CH₃),

;

3.25 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), overimposed in part 3.29

(t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.37 (t, J = 7.2 Hz, 2H, 4-CH₂–

CH₂–N), 4.80 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), 4.99 (s, ⁺NH₂),

7.18–7.30 (complex signal, 5H, Ph–CH), 8.30 (s, 1H, 5-H); ¹³C

NMR (100.6 MHz, CD₃OD) d 22.1 (CH₂, 4-CH₂–CH₂–N), 33.8 (CH₃,

N–CH₃), 36.7 (CH₂, 1-CH₂–CH₂–Ph), 48.4 (CH₂, 4-CH₂–CH₂–N),

54.6 (CH₂, 1-CH₂–CH₂–Ph), 127.5 (CH, C5), 128.2 (CH), 129.8

(4CH) (Ph–CH), 137.9 (C, Ph–C1), 141.6 (C, C4); HRMS (ESI), calcd

for (C₁₃H₁₈N₄+ H⁺) 231.1604; found 231.1604.

A solution of amine 25 (25 mg, 0.18 mmol) in CH₂Cl₂(4 mL) was

ﬁltered through a PTFE ﬁlter (0.2 lm), treated with a HCl/MeOH

solution (0.75 N, 0.71 mL, 0.53 mmol), and evaporated under

reduced pressure. The resulting residue was washed with pentane

(3 ꢂ 2 mL) to give, after drying under standard conditions, 25ꢁHCl

4.1.21. N-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]-N-

methylamine 29

It was prepared as described for 25. From 23 (723 mg,

2.39 mmol) and H₃PO₄(85% purity, 4.14 mL, 35.9 mmol), amine

29 (420 mg, 87% yield) was obtained as a brown oil; R_f0.14 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9.75:0.25:0.05).

(28 mg) as a beige sticky solid; IR (ATR)

m

3500–2500 (max at

3386, 3126, 3080, 3028, 2981, 2935, 2756, 2695, ⁺NAH and CAH)

cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 1.55 (t, J = 7.2 Hz, 3H, 1-CH₂–

;

CH₃), 2.76 (s, 3H, N–CH₃), 4.35 (s, 2H, 4-CH₂–N), 4.50 (q,

J = 7.2 Hz, 2H, 1-CH₂–CH₃), 4.96 (s, ⁺NH₂), 8.22 (s, 1H, 5-H); ¹³C

NMR (100.6 MHz, CD₃OD) d 15.8 (CH₃, 1-CH₂–CH₃), 33.1 (CH₃, N–

CH₃), 44.2 (CH₂, 4-CH₂–N), 46.8 (CH₂, 1-CH₂–CH₃), 126.2 (CH,

C5), 139.3 (C, C4); HRMS (ESI), calcd for (C₆H₁₂N₄+ H⁺) 141.1135,

found 141.1133.

29ꢁHCl: yellow solid, mp 218–219 °C; IR (ATR) v 3500–2400

(max at 3126, 3059, 3018, 2981, 2940, 2852, 2770, 2697, ⁺NAH

and CAH) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 2.74 (s, 3H, N–

;

CH₃), 4.33 (s, 2H, 4-CH₂–N), 4.87 (s, ⁺NH₂), 5.64 (s, 2H, 1-CH₂–

Ph), 7.31–7.40 (complex signal, 5H, Ph–CH), 8.19 (s, 1H, 5-H); ¹³C

NMR (100.6 MHz, CD₃OD) d 33.1 (CH₃, N–CH₃), 44.2 (CH₂, 4-CH₂–

N), 55.0 (CH₂, 1-CH₂–Ph), 126.5 (CH, C5), 129.3 (2CH), 129.7 (CH),

130.0 (2CH) (Ph–CH), 136.6 (C, Ph–C1), 139.8 (C, C4); HRMS (ESI),

calcd for (C₁₁H₁₄N₄+ H⁺) 203.1291, found 203.1284.

4.1.18. N-[2-(1-Ethyl-1H-1,2,3-triazol-4-yl)ethyl]-N-

methylamine 26

It was prepared as described for 25. From 18 (263 mg,

1.03 mmol) and H₃PO₄(85% purity, 1.80 mL, 15.6 mmol), and stir-

ring at rt for 4 h, amine 26 (143 mg, 90% yield) was obtained as a

colorless oil; R_f0.07 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

4.1.22. N-Methyl-N-[(1-phenethyl-1H-1,2,3-triazol-4-yl)methyl]

amine 30

26ꢁHCl: beige sticky solid, IR (ATR)

m

3500–2400 (max at 3395,

It was prepared as described for 25. From 24 (983 mg,

3.11 mmol) and H₃PO₄(85% purity, 5.37 mL, 46.6 mmol), amine

30 (258 mg, 38% yield) was obtained as a colorless oil; R_f0.16 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9.75:0.25:0.05).

+

3116, 3064, 2976, 2936, 2752, 2691, 2446, NAH and CAH) cm^ꢀ1

;

¹H NMR (400 MHz, CD₃OD) d 1.54 (t, J = 7.2 Hz, 3H, 1-CH₂–CH₃),

2.76 (s, 3H, N–CH₃), 3.17 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.37 (t,

J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 4.48 (q, J = 7.2 Hz, 2H, 1-CH₂–CH₃),

4.87 (s, ⁺NH₂), 8.07 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD) d

15.6 (CH₃, 1-CH₂–CH₃), 22.9 (CH₂, 4-CH₂–CH₂–N), 33.7 (CH₃, N–

CH₃), 47.1 (CH₂, 1-CH₂–CH₃), 49.3 (CH₂, 4-CH₂–CH₂–N), 124.6

(CH, C5), 143.3 (C, C4); HRMS (ESI), calcd for (C₇H₁₄N₄+ H⁺)

155.1291, found 155.1297.

30ꢁHCl: yellow solid, mp 173–175 °C; IR (ATR) v 3500–2400

(max at 3111, 3069, 3023, 2981, 2925, 2764, 2749, 2692, 2599,

2542, ⁺NAH and CAH) cm^{ꢀ1 1}H NMR (400 MHz, CD₃OD) d 2.67

;

(s, 3H, N–CH₃), 3.24 (t, J = 7.2 Hz, 2H, 1CH₂–CH₂–Ph), 4.26 (s, 2H,

4-CH₂–N), 4.71 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), 4.87 (s, ⁺NH₂),

7.15 [dm, J = 8.4 Hz, 2H, Ph–C2(6)–H], 7.16–7.29 [complex signal,

3H, Ph–C3(5)–H, Ph–C4–H], 7.94 (s, 1H, 5-H); ¹³C NMR

(100.6 MHz, CD₃OD) d 32.9 (CH₃, N–CH₃), 37.3 (CH₂, 1-CH₂–CH₂–

Ph), 44.1 (CH₂, 4-CH₂–N), 52.7 (CH₂, 1-CH₂–CH₂–Ph), 126.8 (CH,

C5), 128.0 (CH), 129.7 (2CH), 129.8 (2CH) (Ph–CH), 138.6 (C, Ph–

C1), 139.0 (C, C4); HRMS (ESI), calcd for (C₁₂H₁₆N₄+ H⁺)

217.1448, found 217.1443.

4.1.19. N-[2-(1-Benzyl-1H-1,2,3-triazol-4-yl)ethyl]-N-

methylamine 27

It was prepared as described for 25. From 19 (1.12 g,

3.54 mmol) and H₃PO₄(85% purity, 6.08 mL, 52.7 mmol), amine

27 (721 mg, 94% yield) was obtained as a yellow oil; R_f0.23 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

27ꢁHCl: beige solid, mp 189–190 °C; IR (ATR) v 3600–2400 (max

at 3554, 3064, 3013, 2927, 2868, 2735, 2685, 2447, CAH, ⁺NAH and

CAH) cm^ꢀ1; ¹H NMR (400 MHz, CD₃OD) d 2.75 (s, 3H, N–CH₃), 3.24

(t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.38 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–

4.1.23. N-[(1-Ethyl-1H-1,2,3-triazol-4-yl)methyl]-N-methyl-N-

propargylamine 31

A solution of amine 25 (85 mg, 0.61 mmol) in anhydrous ace-

tone (20 mL) was cooled to 0 °C and treated with Cs₂CO₃

(199 mg, 0.61 mmol) and propargyl bromide (80% solution in

toluene, 0.09 mL, 0.61 mmol). The reaction mixture was stirred at

rt overnight, then ﬁltered, and evaporated under reduced pressure

to give an oily residue (161 mg), which was puriﬁed through

+

N), 4.98 (s, NH₂), 5.72 (s, 2H, 1-CH₂–Ph), 7.35–7.46 (complex sig-

nal, 5H, Ph–CH), 8.30 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD) d

22.4 (CH₂, 4-CH₂–CH₂–N), 33.7 (CH₃, N–CH₃), 48.6 (CH₂, 4-CH₂–

CH₂–N), 56.5 (CH₂, 1-CH₂-Ph), 126.6 (CH, C5), 129.8 (2CH),

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

11

column chromatography (40–60

l

m silica gel, CH₂Cl₂/MeOH/50%

yellow oil, without the need of chromatographic puriﬁcation; R_f

0.75 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

aq NH₄OH mixtures, gradient elution). On elution with CH₂Cl₂/

MeOH/50% aq NH₄OH 98:2:0.2, propargylamine 31 (97 mg, 89%

yield) was isolated as a colorless oil; R_f0.33 (CH₂Cl₂/MeOH/50%

aq NH₄OH 9:1:0.05).

34ꢁHCl: brown solid, mp 157–159 °C; IR (ATR) v 3500–2300

(max at 3471, 3369, 3203, 3094, 3059, 3023, 2925, 2842, 2584,

2532, ⁺NAH, „CAH, and CAH), 2126 (C„C) cm^ꢀ1

;

¹H NMR

31ꢁHCl: beige sticky solid, IR (ATR)

3205, 3126, 2987, 2941, 2568, 2477, 2381, ⁺NAH, „CAH, and

CAH), 2125 (C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 1.56 (t,

m

3500–2300 (max at 3412,

(400 MHz, CD₃OD) d 3.03 (s, 3H, N–CH₃), 3.27 (t, J = 7.2 Hz, 2H, 1-

CH₂–CH₂–Ph), 3.32 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.42 (t,

J = 2.4 Hz, 1H, propargyl CH), 3.61 (m, 2H, 4-CH₂–CH₂–N), 4.23 (d,

J = 2.4 Hz, 2H, propargyl CH₂), 4.78 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–

Ph), 4.98 (s, ⁺NH), 7.16–7.30 (complex signal, 5H, Ph–CH), 8.22 (s,

1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD) d 20.8 (CH₂, 4-CH₂–CH₂–

N), 36.8 (CH₂, 1-CH₂–CH₂–Ph), 40.7 (CH₃, N–CH₃), 46.4 (CH₂,

propargyl CH₂), 54.3 (CH₂, 1-CH₂–CH₂–Ph), 54.6 (CH₂, 4-CH₂–

CH₂–N), 72.6 (C, propargyl C), 81.8 (CH, propargyl CH), 127.1 (CH,

C5), 128.2 (CH), 129.8 (4CH) (Ph–CH), 138.0 (C, Ph–C1), 141.5 (C,

C4); HRMS (ESI), calcd for (C₁₆H₂₀N₄+ H⁺) 269.1761; found

269.1758.

;

J = 7.6 Hz, 3H, 1-CH₂–CH₃), 2.94 (s, 3H, N–CH₃), 3.39 (t, J = 2.4 Hz,

1H, propargyl CH), 4.12 (d, J = 2.4 Hz, 2H, propargyl CH₂), 4.50 (q,

J = 7.6 Hz, 2H, 1-CH₂–CH₃), 4.54 (s, 2H, 4-CH₂–N), 4.87 (s, ⁺NH),

8.26 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD) d 15.7 (CH₃, 1-

CH₂–CH₃), 40.3 (CH₃, N–CH₃), 45.6 (CH₂, propargyl CH₂), 46.8

(CH₂, 1-CH₂–CH₃), 50.4 (CH₂, 4-CH₂–N), 73.3 (C, propargyl C),

81.2 (CH, propargyl CH), 127.4 (CH, C5), 137.8 (C, C4); HRMS

(ESI), calcd for (C₉H₁₄N₄+ H⁺) 179.1291, found 179.1289.

4.1.24. N-[2-(1-Ethyl-1H-1,2,3-triazol-4-yl)ethyl]-N-methyl-N-

propargylamine 32

4.1.27. N-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]-N-methyl-N-

propargylamine 35

It was prepared as described for 31. From amine 29 (420 mg,

2.08 mmol), Cs₂CO₃(676 mg, 2.07 mmol), and propargyl bromide

(80% solution in toluene, 0.31 mL, 2.08 mmol), and stirring at rt

for 3 h, an oily residue (451 mg) was obtained and subjected to

It was prepared as described for 31. From amine 26 (299 mg,

1.94 mmol), Cs₂CO₃(630 mg, 1.93 mmol), and propargyl bromide

(80% solution in toluene, 0.29 mL, 1.95 mmol), and stirring at 0 °C

for 2.5 h, propargylamine 32 (220 mg, 59% yield) was obtained as

a yellow oil, without the need of chromatographic puriﬁcation; R_f

0.55 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

puriﬁcation through column chromatography (40–60 lm silica

32ꢁHCl: yellow sticky solid, IR (ATR)

3191, 2937, 2521, 2434, 2359, ⁺NH, „CAH, and CAH), 2121 (C„C)

cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 1.53 (t, J = 7.2 Hz, 3H, 1-CH₂–

m

3500–2300 (max at 3417,

gel, CH₂Cl₂/MeOH mixtures, gradient elution). On elution

with CH₂Cl₂/MeOH 98:2, propargylamine 35 (195 mg, 39%

yield) was isolated as a colorless oil; R_f0.48 (CH₂Cl₂/MeOH

9.75:0.25).

;

CH₃), 3.05 (s, 3H, N–CH₃), 3.24 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N),

3.41 (t, J = 2.8 Hz, 1H, propargyl CH), 3.62 (br t, J = 7.2 Hz, 2H, 4-

CH₂–CH₂–N), 4.24 (d, J = 2.8 Hz, 2H, propargyl CH₂), 4.46 (q,

J = 7.2 Hz, 2H, 1-CH₂–CH₃), 4.86 (s, ⁺NH), 8.01 (s, 1H, 5-H); ¹³C

NMR (100.6 MHz, CD₃OD) d 15.7 (CH₃, 1-CH₂–CH₃), 21.6 (CH₂, 4-

CH₂–CH₂–N), 40.7 (CH₃, N–CH₃), 46.3 (CH₂, propargyl CH₂), 46.8

(CH₂, 1-CH₂–CH₃), 55.4 (CH₂, 4-CH₂–CH₂–N), 72.6 (C, propargyl

C), 81.6 (CH, propargyl CH), 124.2 (CH, C5), 143.1 (C, C4); HRMS

(ESI), calcd for (C₁₀H₁₆N₄+ H⁺) 193.1448, found 193.1440.

35ꢁHCl: pale brown solid, mp 121–123 °C; IR (ATR) v 3500–

2300 (max at 3193, 3126, 2976, 2961, 2692, 2578, ⁺NAH, „CAH,

and CAH), 2118 (C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 2.96

;

(s, 3H, N–CH₃), 3.40 (t, J = 2.8 Hz, 1H, propargyl CH), 4.15 (d,

J = 2.8 Hz, 2H, propargyl CH₂), 4.57 (s, 2H, 4-CH₂–N), 4.86 (s,

⁺NH), 5.66 (s, 2H, 1-CH₂–Ph), 7.32–7.41 (complex signal, 5H, Ph–

CH), 8.27 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD) d 40.2 (CH₃,

N–CH₃), 45.7 (CH₂, propargyl CH₂), 50.3 (CH₂, 4-CH₂–N), 55.1

(CH₂, 1-CH₂–Ph), 72.9 (C, propargyl C), 81.6 (CH, propargyl CH),

128.1 (CH, C5), 129.3 (2CH), 129.7 (CH), 130.1 (2CH) (Ph–CH),

136.5 (C, Ph–C1), 137.9 (C, C4); HRMS (ESI), calcd for

(C₁₄H₁₆N₄+ H⁺) 241.1448, found 241.1444.

4.1.25. N-[2-(1-Benzyl-1H-1,2,3-triazol-4-yl)ethyl]-N-methyl-N-

propargylamine 33

It was prepared as described for 31. From amine 27 (690 mg,

3.19 mmol), Cs₂CO₃(1.04 g, 3.19 mmol), and propargyl bromide

(80% solution in toluene, 0.47 mL, 3.16 mmol), and stirring at rt

for 2 h, propargylamine 33 (577 mg, 71% yield) was obtained as a

yellow oil, without the need of chromatographic puriﬁcation; R_f

0.48 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

4.1.28. N-Methyl-N-[(1-phenethyl-1H-1,2,3-triazol-4-yl)

methyl]-N-propargylamine 36

It was prepared as described for 31. From amine 30 (258 mg,

1.19 mmol), Cs₂CO₃(199 mg, 0.61 mmol), and propargyl bromide

(80% solution in toluene, 0.17 mL, 1.14 mmol), and stirring at rt

for 3 h, an oily residue (204 mg) was obtained and subjected to

33ꢁHCl: brown solid, mp 97–99 °C; IR (ATR) v 3500–2300 (max

at 3413, 3194, 2948, 2522, ⁺NAH, „CAH, and CAH), 2124 (C„C)

cm^ꢀ1

;

¹H NMR (400 MHz, CD₃OD) d 3.03 (s, 3H, N–CH₃), 3.31 (t,

puriﬁcation through column chromatography (40–60 lm silica

J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.39 (t, J = 2.4 Hz, 1H, propargyl

CH), 3.62 (m, 2H, 4-CH₂–CH₂–N), 4.23 (d, J = 2.4 Hz, 2H, propargyl

CH₂), 5.00 (s, ⁺NH), 5.68 (s, 2H, 1-CH₂–Ph), 7.33–7.43 (complex sig-

nal, 5H, Ph–CH), 8.23 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD) d

21.2 (CH₂, 4-CH₂–CH₂–N), 40.7 (CH₃, N–CH₃), 46.4 (CH₂, propargyl

CH₂), 54.8 (CH₂, 4-CH₂–CH₂–N), 56.1 (CH₂, 1-CH₂–Ph), 72.6 (C,

propargyl C), 81.7 (CH, propargyl CH), 126.1 (CH, C5), 129.6

(2CH), 130.0 (CH), 130.1 (2CH) (Ph–CH), 135.6 (C, Ph–C1), 142.6

(C, C4); HRMS (ESI), calcd for (C₁₅H₁₈N₄+ H⁺) 255.1604, found

255.1607.

gel, CH₂Cl₂/EtOAc mixtures, gradient elution). On elution with CH₂-

Cl₂/EtOAc 0:100, propargylamine 36 (63 mg, 22% yield) was iso-

lated as a colorless oil; R_f0.26 (EtOAc).

36ꢁHCl: beige sticky solid, IR (ATR) v 3500–2300 (max at 3400,

3271, 3209, 3023, 2945, 2501, ⁺NAH, „CAH, and CAH), 2124

(C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 2.88 (s, 3H, N–CH₃),

;

3.25 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), 3.42 (t, J = 2.4 Hz, 1H,

propargyl CH), 4.02 (br s, 2H, propargyl CH₂), 4.51 (s, 2H, 4-CH₂–

N), 4.73 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–Ph), 4.90 (s, ⁺NH), 7.14 [dm,

J = 8.4 Hz, 2H, Ph–C2(6)–H], 7.17–7.29 [complex signal, 3H, Ph–

C3(5)–H, Ph–C4-H), 8.02 (s, 1H, 5-H); ¹³C NMR (100.6 MHz, CD₃OD)

d 37.3 (CH₂, 1-CH₂–CH₂–Ph), 40.1 (CH₃, N–CH₃), 45.5 (CH₂, propar-

gyl CH₂), 50.2 (CH₂, 4-CH₂–N), 52.8 (CH₂, 1-CH₂–CH₂–Ph), 72.8 (C,

propargyl C), 81.7 (CH, propargyl CH), 128.0 (CH), 128.4 (CH) (C5,

Ph–C4), 129.7 (2CH), 129.9 (2CH) [Ph–C2(6), Ph–C3(5)], 137.0 (C,

Ph–C1), 138.6 (C, C4); HRMS (ESI), calcd for (C₁₅H₁₈N₄+ H⁺)

255.1604, found 255.1601.

4.1.26. N-Methyl-N-[2-(1-phenethyl-1H-1,2,3-triazol-4-yl)

ethyl]-N-propargylamine 34

It was prepared as described for 31. From amine 28 (384 mg,

1.67 mmol), Cs₂CO₃(543 mg, 1.67 mmol), and propargyl bromide

(80% solution in toluene, 0.25 mL, 1.68 mmol), and stirring at rt

for 2 h, propargylamine 34 (295 mg, 66% yield) was obtained as a

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

12

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

4.1.29. N-(tert-Butoxycarbonyl)-N-[(1-ethyl-5-methyl-1H-1,2,3-

triazol-4-yl)methyl]-N-methylamine 37

2.81 (s, 3H, N–CH₃), 3.93 (s, 3H, 1-CH₃), 4.49 (s, 2H, 4-CH₂–N);

HRMS (ESI), calcd for (C₁₄H₂₆N₄O₂+ H⁺) 283.2129; found 283.2123.

A solution of 17 (420 mg, 1.75 mmol) in anhydrous THF (30 mL)

was cooled to ꢀ78 °C and then treated with n-BuLi (2.5 M solution

in hexanes, 1.05 mL, 2.62 mmol) and iodomethane (0.43 mL,

980 mg, 6.91 mmol). The reaction mixture was stirred at ꢀ78 °C

for 1 h, then warmed to rt and stirred an additional 2 h. The result-

ing solution was diluted with H₂O (20 mL) and extracted with CH₂-

Cl₂(2 ꢂ 40 mL). The combined organic extracts were dried over

anhydrous Na₂SO₄and evaporated under reduced pressure. The

resulting residue was washed with pentane (3 ꢂ 10 mL) and dried

under vacuum, to give compound 37 (427 mg, 96% yield) as a col-

4.1.33. N-(tert-Butoxycarbonyl)-N-methyl-N-[2-(5-ethyl-1-

phenyl-1H-1,2,3-triazol-4-yl)ethyl]amine 41

It was prepared as described for 37. From 22 (542 mg,

1.79 mmol), n-BuLi (2.5 M solution in hexanes, 1.43 mL,

3.57 mmol) and bromoethane (0.53 mL, 774 mg, 7.10 mmol), com-

pound 41 (578 mg, 98% yield) was obtained as a yellowish oil; R_f

0.86 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05); IR (ATR) v 1688,

1683 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.05 (t, J = 7.6 Hz,

;

3H, 5-CH₂–CH₃), 1.45 [s, 9H, C(CH₃)₃], 2.70 (m, 2H, 5-CH₂–CH₃),

2.88 (s, 3H, N–CH₃), 2.93 (m, 2H, 4-CH₂–CH₂–N), 3.58 (t,

J = 7.6 Hz, 2H, 4-CH₂–CH₂–N), 7.42 [ddm, J = 8.0 Hz, J⁰= 2.4 Hz,

2H, Ph–C2(6)–H], 7.48–7.57 [complex signal, 3H, Ph–C3(5)–H,

Ph–C4–H]; HRMS (ESI), calcd for (C₁₈H₂₆N₄O₂+ H⁺) 331.2129;

found 331.2128.

orless oil; R_f0.57 (CH₂Cl₂/MeOH 9:1); IR (ATR)

m ;

1686 (C@O) cm^ꢀ1

¹H NMR (400 MHz, CDCl₃) d 1.46 [s, 9H, C(CH₃)₃], superimposed in

part 1.47 (t, J = 7.6 Hz, 3H, 1-CH₂–CH₃), 2.31 (s, 3H, 5-CH₃), 2.85 (s,

3H, N–CH₃), 4.26 (q, J = 7.6 Hz, 2H, 1-CH₂–CH₃), 4.48 (s, 2H, 4-CH₂–

N); ¹³C NMR (100.6 MHz, CDCl₃) d 7.8 (CH₃, 5-CH₃), 15.2 (CH₃, 1-

CH₂–CH₃), 28.5 [3CH₃, C(CH₃)₃], 34.0 (CH₃, N–CH₃), 42.7 (CH₂, 4-

CH₂–N), 43.1 (CH₂, 1-CH₂–CH₃), 79.8 [C, C(CH₃)₃], 130.5 (C, C5),

142.0 (C, C4), 156.0 (C, NCOO); HRMS (ESI), calcd for (C₁₂H₂₂N₄-

O₂+ H⁺) 255.1816, found 255.1811.

4.1.34. N-(tert-Butoxycarbonyl)-N-methyl-N-[2-(1-phenyl-5-

propyl-1H-1,2,3-triazol-4-yl)ethyl]amine 42

It was prepared as described for 37. From 22 (577 mg,

1.91 mmol), n-BuLi (2.5 M solution in hexanes, 1.53 mL,

3.82 mmol) and 1-bromopropane (0.69 mL, 934 mg, 7.60 mmol),

compound 42 (634 mg, 96% yield) was obtained as an orange oil;

R_f0.84 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05); IR (ATR) v 1692,

4.1.30. N-(tert-Butoxycarbonyl)-N-[2-(1-ethyl-5-methyl-1H-

1,2,3-triazol-4-yl)ethyl]-N-methylamine 38

It was prepared as described for 37. From 18 (942 mg,

3.71 mmol), n-BuLi (2.5 M solution in hexanes, 2.97 mL,

7.42 mmol) and iodomethane (0.92 mL, 2.10 g, 14.8 mmol), com-

pound 38 (895 mg, 90% yield) was obtained as a colorless oil; R_f

1682 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 0.82 (t, J = 7.6 Hz,

;

3H, 5-CH₂–CH₂–CH₃), superimposed in part 1.42 (tt, J = J⁰= 7.6 Hz,

2H, 5-CH₂–CH₂–CH₃), 1.46 [s, 9H, C(CH₃)₃], 2.63 (m, 2H, 5-CH₂–

CH₂–CH₃), 2.88 (s, 3H, N–CH₃), 2.93 (m, 2H, 4-CH₂–CH₂–N), 3.59

(t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 7.41 [ddm, J = 8.0 Hz, J⁰= 2.4 Hz,

2H, Ph–C2(6)–H], 7.48–7.56 [complex signal, 3H, Ph–C3(5)–H,

Ph–C4–H]; HRMS (ESI), calcd for (C₁₉H₂₈N₄O₂+ H⁺) 345.2285,

found 345.2286.

0.57 (CH₂Cl₂/MeOH 9:1); IR (ATR)

m ;

1685 (C@O) cm^{ꢀ1 1}H NMR

(400 MHz, CDCl₃) d 1.40 [s, 9H, C(CH₃)₃], 1.44 (t, J = 7.6 Hz, 3H, 1-

CH₂–CH₃), 2.20 (s, 3H, 5-CH₃), 2.77 (s, 3H, N–CH₃), 2.82 (m, 2H,

4-CH₂–CH₂–N), 3.43 (t, J = 7.2 Hz, 4-CH₂–CH₂–N), 4.23 (q,

J = 7.6 Hz, 2H, 1-CH₂–CH₃); ¹³C NMR (100.6 MHz, CDCl₃) d 7.7

(CH₃, 5-CH₃), 15.3 (CH₃, 1-CH₂–CH₃), 23.7 (br CH₂), 24.1 (br CH₂)

(4-CH₂–CH₂–N), 28.5 [3CH₃, C(CH₃)₃], 34.7 (br CH₃), 35.0 (br CH₃)

(N–CH₃), 43.0 (CH₂, 1-CH₂–CH₃), 48.7 (br CH₂), 49.2 (br CH₂) (4-

CH₂–CH₂–N), 79.4 [C, C(CH₃)₃], 129.2 (br C), 129.4 (br C) (C5),

142.2 (C, C4), 155.7 (C, NCOO); HRMS (ESI), calcd for (C₁₃H₂₄N₄-

O₂+ H⁺) 269.1972, found 269.1974.

4.1.35. N-(tert-Butoxycarbonyl)-N-methyl-N-[2-(5-butyl-1-

phenyl-1H-1,2,3-triazol-4-yl)ethyl]amine 43

It was prepared as described for 37. From 22 (590 mg,

1.95 mmol), n-BuLi (2.5 M solution in hexanes, 1.56 mL,

3.90 mmol) and 1-bromobutane (0.84 mL, 1.07 g, 7.82 mmol), an

oily residue (678 mg) was obtained. After column chromatography

4.1.31. N-(tert-Butoxycarbonyl)-N-methyl-N-{2-[5-methyl-1-(

methyl)benzyl-1H-1,2,3-triazol-4-yl]ethyl}amine 39

a-

puriﬁcation of the residue (40–60 lm silica gel, CH₂Cl₂/50% aq

NH₄OH 100:0.2), compound 43 (300 mg, 43% yield) was isolated

It was prepared as described for 37. From 19 (356 mg,

1.13 mmol), n-BuLi (2.5 M solution in hexanes, 0.90 mL,

2.25 mmol) and iodomethane (0.28 mL, 638 mg, 4.50 mmol), com-

pound 39 (306 mg, 79% yield) was obtained as a yellowish oil; IR

as a yellow oil; R_f0.82 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05);

IR (ATR) v 1692 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 0.79 (t,

;

J = 7.2 Hz, 3H, 5-CH₂–CH₂–CH₂–CH₃), 1.21 (tq, J = J⁰= 7.2 Hz, 2H,

5-CH₂–CH₂–CH₂–CH₃), 1.35 (m, 2H, 5-CH₂–CH₂–CH₂–CH₃), 1.45

[s, 9H, C(CH₃)₃], 2.65 (m, 2H, 5-CH₂–CH₂–CH₂–CH₃), 2.87 (s, 3H,

N–CH₃), 2.91 (m, 2H, 4-CH₂–CH₂–N), 3.59 (t, J = 7.6 Hz, 2H, 4-

CH₂–CH₂–N), 7.41 [ddm, J = 8.0 Hz, J⁰= 2.4 Hz, 2H, Ph–C2(6)–H],

7.47–7.56 [complex signal, 3H, Ph–C3(5)–H, Ph–C4–H]; HRMS

(ESI), calcd for (C₂₀H₃₀N₄O₂+ H⁺) 359.2442, found 359.2438.

(ATR) v 1686, 1681 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d

;

1.41 [s, 9H, C(CH₃)₃], 2.019 [d, J = 7.2 Hz, 3H, 1-CH(CH₃)–Ph],

2.023 (s, 3H, 5-CH₃), 2.76 (s, 3H, N–CH₃), 2.81 (m, 2H, 4-CH₂–

CH₂–N), 3.47 (m, 2H, 4-CH₂–CH₂–N), 5.49 [q, J = 7.2 Hz, 1H, 1-CH

(CH₃)–Ph], 7.14 [br d, J = 7.2 Hz, Ph–C2(6)–H], 7.24–7.34 [complex

signal, 3H, Ph–C3(5)–H, Ph–C4–H]; HRMS (ESI), calcd for (C₁₉H₂₈

-

N₄O₂+ H⁺) 345.2285, found 345.2285.

4.1.36. N-[(1-Ethyl-5-methyl-1H-1,2,3-triazol-4-yl)methyl]-N-

methylamine 44

4.1.32. N-(tert-Butoxycarbonyl)-N-[(5-butyl-1-methyl-1H-1,2,3-

triazol-4-yl)methyl]-N-methylamine 40

It was prepared as described for 25. From 37 (370 mg,

1.46 mmol) and H₃PO₄(85% purity, 2.52 mL, 21.9 mmol), and stir-

ring at rt for 4 h, amine 44 (205 mg, 91% yield) was obtained as a

colorless oil; R_f0.11 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

It was prepared as described for 37. From 21 (945 mg,

4.18 mmol), n-BuLi (2.5 M solution in hexanes, 3.36 mL,

8.40 mmol) and 1-bromobutane (1.81 mL, 2.31 g, 16.9 mmol),

compound 40 (1.14 g, 97% yield) was obtained as a yellowish oil;

R_f0.87 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05); IR (ATR) v

44ꢁHCl: beige sticky solid, IR (ATR)

m 3500–2400 (max at 3390,

+

2925, 2868, 2837, 2757, 2697, 2546, 2440, NAH and CAH) cm^ꢀ1

;

¹H NMR (400 MHz, CD₃OD) d 1.48 (t, J = 7.2 Hz, 3H, 1-CH₂–CH₃),

2.41 (s, 3H, 5-CH₃), 2.76 (s, 3H, N–CH₃), 4.28 (s, 2H, 4-CH₂–N),

4.38 (q, J = 7.2 Hz, 2H, 1-CH₂–CH₃), 4.87 (s, ⁺NH₂); ¹³C NMR

(100.6 MHz, CD₃OD) d 7.7 (CH₃, 5-5-CH₃), 15.2 (CH₃, 1-CH₂–CH₃),

33.1 (CH₃, N–CH₃), 43.6 (CH₂), 44.4 (CH₂) (1-CH₂–CH₃, 4-CH₂–N),

1689 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 0.92 (t, J = 7.2 Hz,

;

3H, 5-CH₂–CH₂–CH₂–CH₃), 1.35 (tq, J = J⁰= 7.2 Hz, 2H, 5-CH₂–

CH₂–CH₂–CH₃), superimposed 1.40–1.52 (2H, 5-CH₂–CH₂–CH₂–

CH₃), 1.44 [s, 9H, C(CH₃)₃], 2.69 (m, 2H, 5-CH₂–CH₂–CH₂–CH₃),

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

13

134.7 (C, C5), 136.8 (C, C4); HRMS (ESI), calcd for (C₇H₁₄N₄+ H⁺)

155.1291, found 155.1288.

through column chromatography (40–60

l

m silica gel, CH₂Cl₂/

MeOH/50% aq NH₄OH mixtures, gradient elution). On elution with

CH₂Cl₂/MeOH/50% aq NH₄OH 98:2:0.2 to 96:4:0.2, amine 48

(322 mg, 89% yield) was obtained as a yellowish oil; R_f0.51 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

4.1.37. N-[2-(1-Ethyl-5-methyl-1H-1,2,3-triazol-4-yl)ethyl]-N-

methylamine 45

It was prepared as described for 25. From 38 (890 mg,

3.32 mmol) and H₃PO₄(85% purity, 5.70 mL, 49.4 mmol), and

stirring at rt for 1.5 h, amine 45 (495 mg, 89% yield) was

obtained as a colorless oil; R_f0.10 (CH₂Cl₂/MeOH/50% aq NH₄OH

9:1:0.05).

48ꢁHCl: yellow sticky solid, IR (ATR) v 3400–2300 (max at 3380,

2964, 2930, 2873, 2741, 2434, 2351, ⁺NAH and CAH) cm^{ꢀ1 1}H

;

NMR (400 MHz, CD₃OD) d 1.05 (t, J = 7.6 Hz, 3H, 5-CH₂–CH₃),

superimposed in part 2.80 (q, J = 7.6 Hz, 2H, 5-CH₂–CH₃), 2.81 (s,

3H, N–CH₃), 3.20 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.45 (t,

J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 4.89 (s, ⁺NH₂), 7.53 [ddm,

J = 7.6 Hz, J⁰= 2.0 Hz, 2H, Ph–C2(6)–H], 7.62–7.67 [complex signal,

3H, Ph–C3(5)–H, Ph–C4–H]; ¹³C NMR (100.6 MHz, CD₃OD) d 13.4

(CH₃, 5-CH₂–CH₃), 16.9 (CH₂, 5-CH₂–CH₃), 22.5 (CH₂, 4-CH₂–CH₂–

N), 33.9 (CH₃, N–CH₃), 49.3 (CH₂, 4-CH₂–CH₂–N), 126.8 (2CH),

130.9 (2CH), 131.4 (CH) (Ph–CH), 137.5 (C, Ph–C1), 139.1 (C, C5),

140.5 (C, C4); HRMS (ESI), calcd for (C₁₃H₁₈N₄+ H⁺) 231.1604;

found 231.1607.

45ꢁHCl: beige sticky solid, IR (ATR)

m

3500–2300 (max at 3374,

2981, 2937, 2746, 2692, 2449, 2359, ⁺NAH and CAH st) cm^ꢀ1

;

¹H

NMR (400 MHz, CD₃OD) d 1.49 (t, J = 7.2 Hz, 3H, 1-CH₂–CH₃), 2.38

(s, 3H, 5-CH₃), 2.76 (s, 3H, N–CH₃), 3.09 (t, J = 7.2 Hz, 2H, 4-CH₂–

CH₂–N), 3.34 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 4.40 (q, J = 7.2 Hz,

2H, 1-CH₂–CH₃), 4.86 (s, ⁺NH₂); ¹³C NMR (100.6 MHz, CD₃OD) d

7.6 (CH₃, 5-CH₃), 15.0 (CH₃, 1-CH₂–CH₃), 22.2 (CH₂, 4-CH₂–CH₂–

N), 33.7 (CH₃, N–CH₃), 44.9 (CH₂, 1-CH₂–CH₃), 49.0 (4-CH₂–CH₂–

N), 133.4 (C, C5), 140.1 (C, C4); HRMS (ESI), calcd for

(C₈H₁₆N₄+ H⁺) 169.1448, found 169.1452.

4.1.41. N-Methyl-N-[2-(1-phenyl-5-propyl-1H-1,2,3-triazol-4-yl)

ethyl]amine 49

It was prepared as described for 25. From 42 (573 mg,

1.66 mmol) and H₃PO₄(85% purity, 2.85 mL, 24.7 mmol), and stir-

ring at rt for 3 h, an orange oil (564 mg) was obtained and puriﬁed

4.1.38. N-Methyl-N-{2-[5-methyl-1-(a-methyl)benzyl-1H-1,2,3-

triazol-4-yl]ethyl}amine 46

It was prepared as described for 25. From 39 (306 mg,

0.89 mmol) and H₃PO₄(85% purity, 1.60 mL, 13.9 mmol), amine

46 (215 mg, quantitative yield) was obtained as a yellow oil; R_f

0.33 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

through column chromatography (40–60 lm silica gel, CH₂Cl₂/

MeOH/50% aq NH₄OH mixtures, gradient elution). On elution with

CH₂Cl₂/MeOH/50% aq NH₄OH 99:1:0.2 to 95:5:0.2, amine 49

(270 mg, 67% yield) was obtained as a yellowish oil; R_f0.52 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

46ꢁHCl: yellow sticky solid, IR (ATR) v 3400–2400 (max at 3373,

2950, 2709, 2434, ⁺NAH and CAH) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃-

;

OD) d 2.01 [d, J = 7.2 Hz, 3H, 1-CH(CH₃)–Ph], 2.30 (s, 3H, 5-CH₃),

2.76 (s, 3H, N–CH₃), 3.19 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 3.36 (t,

J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 4.94 (s, ⁺NH₂), 5.93 (q, J = 7.2 Hz,

1H, 1-CH(CH₃)–Ph], 7.30 [dm, J = 8.4 Hz, 2H, Ph–C2(6)–H], 7.36–

7.42 [complex signal, 3H, Ph–C3(5)-H, Ph–C4–H]; ¹³C NMR

(100.6 MHz, CD₃OD) d 8.2 (CH₃, 5-CH₃), 21.7 [CH₃, 1-CH(CH₃)–

Ph], 21.9 (CH₂, 4-CH₂–CH₂–N), 33.7 (CH₃, N–CH₃), 53.8 (CH₂, 4-

CH₂–CH₂–N), 61.5 [CH, 1-CH(CH₃)–Ph], 127.5 (2CH), 129.7 (CH),

130.2 (2CH) (Ph–CH), 135.6 (C, Ph–C1), 139.3 (C, C5), 140.5 (C,

C4); HRMS (ESI), calcd for (C₁₄H₂₀N₄+ H⁺) 245.1761, found

245.1762.

49ꢁHCl: yellow sticky solid, IR (ATR) v 3400–2400 (max at 3350,

2960, 2935, 2863, 2721, 2430, ⁺NAH and CAH) cm^{ꢀ1 1}H NMR

;

(400 MHz, CD₃OD) d 0.81 (t, J = 7.6 Hz, 3H, 5-CH₂–CH₂–CH₃), 1.42

(tq, J = J⁰= 7.6 Hz, 2H 5-CH₂–CH₂–CH₃), 2.74 (t, J = 7.6 Hz, 2H, 5-

CH₂–CH₂–CH₃), 2.80 (s, 3H, N–CH₃), 3.16 (t, J = 7.2 Hz, 2H, 4-CH₂–

CH₂–N), 3.43 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 4.85 (s, ⁺NH₂),

7.48–7.65 (complex signal, 5H, Ph–CH); ¹³C NMR (100.6 MHz, CD₃-

OD) d 13.9 (CH₃, 5-CH₂–CH₂–CH₃), 22.6 (CH₂, 4-CH₂–CH₂–N), 22.9

(CH₂, 5-CH₂–CH₂–CH₃), 25.2 (CH₂, 5-CH₂–CH₂–CH₃), 33.8 (CH₃,

N–CH₃), 49.4 (CH₂, 4-CH₂–CH₂–N) 126.8 (2CH), 130.9 (2CH),

131.3 (CH) (Ph–CH), 137.4 (C, Ph–C1), 137.7 (C, C5), 141.3 (C,

C4); HRMS (ESI), calcd for (C₁₄H₂₀N₄+ H⁺) 245.1761, found

245.1761.

4.1.39. N-[(5-Butyl-1-methyl-1H-1,2,3-triazol-4-yl)methyl]-N-

methylamine 47

It was prepared as described for 25. From 40 (954 mg,

3.38 mmol) and H₃PO₄(85% purity, 5.84 mL, 50.7 mmol), amine

47 (620 mg, quantitative yield) was obtained as a yellow oil; R_f

0.06 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

4.1.42. N-Methyl-N-[2-(5-butyl-1-phenyl-1H-1,2,3-triazol-4-yl)

ethyl]amine 50

It was prepared as described for 25. From 43 (265 mg,

0.74 mmol) and H₃PO₄(85% purity, 1.27 mL, 11.0 mmol), and

stirring at rt for 3 h, amine 50 (175 mg, 92% yield) was obtained

as a yellowish oil; R_f0.54 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

50ꢁHCl: yellow oil, IR (ATR) v 3400–2400 (max at 3389, 2954,

47ꢁHCl: beige sticky solid, IR (ATR) v 3400–2500 (max at 3372,

2957, 2930, 2863, 2711, ⁺NAH and CAH) cm^ꢀ1; ¹H NMR (400 MHz,

CD₃OD) d 0.97 (t, J = 7.2 Hz, 3H, 5-CH₂–CH₂–CH₂–CH₃), 1.42 (tq,

J = J⁰= 7.2 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 1.58 (tt, J = 7.6 Hz,

J⁰= 7.2 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 2.79 (s, 3H, N–CH₃), 2.87 (t,

J = 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 4.05 (s, 3H, 1-CH₃), 4.31 (s,

2H, 4-CH₂–N), 5.06 (s, ⁺NH₂); ¹³C NMR (100.6 MHz, CD₃OD) d

14.0 (CH₃, 5-CH₂–CH₂–CH₂–CH₃), 22.9 (CH₂), 23.4 (CH₂) (5-CH₂–

CH₂–CH₂–CH₃), 31.6 (CH₂, 5-CH₂–CH₂–CH₂–CH₃), 33.3 (CH₃, N–

CH₃), 35.5 (CH₃, 1-CH₃), 43.5 (CH₂, 4-CH₂–N), 136.5 (C, C5), 139.3

(C, C4); HRMS (ESI), calcd for (C₉H₁₈N₄+ H⁺) 183.1604, found

183.1599.

2919, 2868, 2755, 2444, ⁺NAH and CAH) cm^ꢀ1

;

¹H NMR

(400 MHz, CD₃OD) d 0.80 (t, J = 7.6 Hz, 3H, 5-CH₂–CH₂–CH₂–CH₃),

1.22 (tq, J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 1.38 (tt,

J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 2.77 (t, J = 7.6 Hz, 2H, 5-

CH₂–CH₂–CH₂–CH₃), 2.81 (s, 3H, N–CH₃), 3.15 (t, J = 7.2 Hz, 2H, 4-

CH₂–CH₂–N), 3.44 (t, J = 7.2 Hz, 2H, 4-CH₂–CH₂–N), 4.85 (s, ⁺NH₂),

7.51 [dm, J = 8.0 Hz, 2H, Ph–C2(6)–H], 7.61–7.66 [complex signal,

3H, Ph–C3(5)–H, Ph–C4–H]; ¹³C NMR (100.6 MHz, CD₃OD) d 13.8

(CH₃, 5-CH₂–CH₂–CH₂–CH₃), 22.6 (CH₂, 4-CH₂–CH₂–N), 23.0

(CH₂), 23.2 (CH₂) (5-CH₂–CH₂–CH₂–CH₃), 31.6 (CH₂, 5-CH₂–CH₂–

CH₂–CH₃), 33.8 (CH₃, N–CH₃), 49.4 (CH₂, 4-CH₂–CH₂–N), 126.8

(2CH), 130.9 (2CH), 131.3 (CH) (Ph–CH), 137.5 (C, Ph–C1), 137.7

(C, C5), 141.2 (C, C4); HRMS (ESI), calcd for (C₁₅H₂₂N₄+ H⁺)

259.1917, found 259.1922.

4.1.40. N-Methyl-N-[2-(5-ethyl-1-phenyl-1H-1,2,3-triazol-4-yl)

ethyl]amine 48

It was prepared as described for 25. From 41 (517 mg,

1.57 mmol) and H₃PO₄(85% purity, 2.68 mL, 23.2 mmol), and stir-

ring at rt for 3 h, an orange oil (435 mg) was obtained and puriﬁed

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

14

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

4.1.43. N-[(1-Ethyl-5-methyl-1H-1,2,3-triazol-4-yl)methyl]-N-

methyl-N-propargylamine 51

(CH₂, 4-CH₂–CH₂–N), 72.5 (C, propargyl C), 81.9 (CH, propargyl

CH), 136.3 (C, C5), 137.8 (C, C4); HRMS (ESI), calcd for

(C₁₁H₁₈N₄+ H⁺) 207.1604, found 207.1609.

It was prepared as described for 31. From amine 44 (128 mg,

0.83 mmol), Cs₂CO₃(270 mg, 0.83 mmol), and propargyl bromide

(80% solution in toluene, 0.12 mL, 0.81 mmol), and stirring at 0 °C

for 2 h, propargylamine 51 (96 mg, 62% yield) was obtained as a

yellow oil, without the need of chromatographic puriﬁcation; R_f

0.43 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

4.1.45. N-Methyl-N-{2-[5-methyl-1-(a-methyl)benzyl-1H-1,2,3-

triazol-4-yl]ethyl}-N-propargylamine 53

It was prepared as described for 31. From amine 46 (234 mg,

0.93 mmol), Cs₂CO₃(0.30 g, 0.92 mmol), and propargyl bromide

(80% solution in toluene, 0.10 mL, 0.67 mmol), and stirring at 0 °C

for 30 min and at rt for an additional 3 h, an orange oily residue

(263 mg) was obtained and puriﬁed through column chromatogra-

51ꢁHCl: yellow sticky solid, IR (ATR)

3197, 2938, 2496, 2352, ⁺NAH, „CAH, and CAH), 2123 (C„C)

cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 1.50 (t, J = 7.2 Hz, 3H, 1-CH₂–

m 3500–2300 (max at 3386,

;

CH₃), 2.45 (s, 3H, 5-CH₃), 3.00 (s, 3H, N–CH₃), 3.44 (t, J = 2.4 Hz,

1H, propargyl CH), 4.17 (d, J = 2.4 Hz, 2H, propargyl CH₂), 4.39 (q,

J = 7.2 Hz, 2H, 1-CH₂–CH₃), 4.52 (s, 2H, 4-CH₂–N), 4.84 (s, ⁺NH);

¹³C NMR (100.6 MHz, CD₃OD) d 8.0 (CH₃, 5-CH₃), 15.2 (CH₃, 1-

CH₂–CH₃), 40.3 (CH₃, N–CH₃), 44.5 (CH₂, 1-CH₂–CH₃), 45.6 (CH₂,

propargyl CH₂), 49.8 (CH₂, 4-CH₂–N), 73.1 (C, propargyl C), 81.5

(CH, propargyl CH), 135.1 (C, C5), 136.3 (C, C4); HRMS (ESI), calcd

for (C₁₀H₁₆N₄+ H⁺) 193.1448, found 193.1450.

Note: From amine 44 (117 mg, 0.76 mmol), Cs₂CO₃(362 mg,

1.11 mmol), and propargyl bromide (80% solution in toluene,

0.16 mL, 1.08 mmol), and stirring at rt overnight, an oily residue

was obtained and puriﬁed through column chromatography (40–

phy (40–60 lm silica gel, CH₂Cl₂/MeOH/50% aq NH₄OH mixtures,

gradient elution). On elution with CH₂Cl₂/MeOH/50% aq NH₄OH

99.97:0.03:0.2, propargylamine 53 (100 mg, 53% yield) was

obtained as a yellow oil; R_f0.45 (CH₂Cl₂/MeOH/50% aq NH₄OH

9:1:0.05).

53ꢁHCl: beige sticky solid, IR (ATR) v 3400–2300 (max at 3386,

3209, 2936, 2351, ⁺NAH, „CAH, and CAH), 2118 (C„C) cm^ꢀ^{1 1}H

;

NMR (400 MHz, CD₃OD) d 2.01 [d, J = 6.8 Hz, 3H, 1-CH(CH₃)–Ph],

2.26 (s, 3H, 5-CH₃), 3.06 (s, 3H, N–CH₃), 3.22 (t, J = 8.0 Hz, 2H, 4-

CH₂–CH₂–N), 3.42 (t, J = 2.4 Hz, 1H, propargyl CH), 3.59 (t,

J = 8.0 Hz, 2H, 4-CH₂–CH₂–N), 4.25 (d, J = 2.4 Hz, 2H, propargyl

CH₂), 4.89 (s, ⁺NH), 5.86 [q, J = 6.8 Hz, 1H, 1-CH(CH₃)–Ph], 7.24–

7.40 (complex signal, 5H, Ph–CH); ¹³C NMR (100.6 MHz, CD₃OD)

d 8.1 (CH₃, 5-CH₃), 20.5 (CH₂, 4-CH₂–CH₂–N), 22.0 [CH₃, 1-CH

(CH₃)–Ph], 40.7 (CH₃, N–CH₃), 46.3 (CH₂, propargyl CH₂), 54.7

(CH₂, 4-CH₂–CH₂–N), 61.0 [CH, 1-CH(CH₃)–Ph], 72.6 (C, propargyl

C), 81.7 (CH, propargyl CH), 127.4 (2CH), 129.6 (CH), 130.2 (2CH)

(Ph–CH), 134.5 (C, Ph–C1), 139.6 (C, C5), 141.1 (C, C4); HRMS

(ESI), calcd for (C₁₇H₂₂N₄+ H⁺) 283.1917, found 283.1923.

60 lm silica gel, CH₂Cl₂/MeOH/50% aq NH₄OH mixtures, gradient

elution). On elution with CH₂Cl₂/MeOH/50% aq NH₄OH 99:1:0.2

to 98:2:0.2, propargylamine 51 (15 mg, 10% yield) was isolated

as a colorless oil. On elution with CH₂Cl₂/MeOH/50% aq NH₄OH

98:2:0.2, slightly impure N-[(1-ethyl-5-methyl-1H-1,2,3-triazol-

4-yl)methyl]-N-methyl-N,N-dipropargylammonium

bromide

(223 mg) was isolated as a brown oil; R_f0. 04 (CH₂Cl₂/MeOH/50%

aq NH₄OH 9:1:0.05).

A solution of the dipropargylated byproduct (223 mg) in CH₂Cl₂

(20 mL) was ﬁltered through a PTFE ﬁlter (0.2 lm) and evaporated

4.1.46. N-[(5-Butyl-1-methyl-1H-1,2,3-triazol-4-yl)methyl]-N-

methyl-N-propargylamine 54

under reduced pressure. The resulting residue was washed with

pentane (3 ꢂ 2 mL) to give, after drying under standard conditions,

the analytical sample of the dipropargylated byproduct (213 mg)

It was prepared as described for 31. From amine 47 (529 mg,

2.90 mmol), Cs₂CO₃(946 mg, 2.90 mmol), and propargyl bromide

(80% solution in toluene, 0.44 mL, 2.96 mmol), and stirring at rt

for 3.5 h, propargylamine 54 (418 mg, 65% yield) was obtained as

a yellow oil, without the need of chromatographic puriﬁcation; R_f

0.29 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

as a brownish oil; IR (ATR)

m ;

3207 („CAH), 2124 (C„C st) cm^ꢀ1

¹H NMR (400 MHz, CDCl₃) d 1.53 (t, J = 7.6 Hz, 3H, 1-CH₂–CH₃),

2.73 (s, 3H, 5-CH₃), 2.85 (t, J = 2.4 Hz, 2H, 2 propargyl CH), 3.42

(s, 3H, N–CH₃), 4.34 (q, J = 7.6 Hz, 2H, 1-CH₂–CH₃), 4.83 (dd,

J = 16.0 Hz, J⁰= 2.4 Hz, 2H), 4.91 (dd, J = 16.0 Hz, J⁰= 2.4 Hz, 2H) (2

propargyl CH₂), 5.34 (s, 2H, 4-CH₂–N); ¹³C NMR (100.6 MHz, CDCl₃)

d 8.8 (CH₃, 5-CH₃), 14.8 (CH₃, 1-CH₂–CH₃), 43.6 (CH₂, 1-CH₂–CH₃),

47.2 (CH₃, N–CH₃), 51.8 (2CH₂, 2 propargyl CH₂), 55.7 (CH₂, 4-

CH₂–N), 70.9 (2C, 2 propargyl C), 82.2 (2CH, 2 propargyl CH),

132.5 (C, C5), 136.5 (C, C4); HRMS (ESI), calcd for (C₁₃H₁₉N⁺₄)

231.1604, found 231.1613.

54ꢁHCl: brown sticky solid, IR (ATR) v 3500–2400 (max at 3411,

3219, 2955, 2930, 2863, 2491, ⁺NAH, „CAH, and CAH), 2121

(C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 0.98 (t, J = 7.6 Hz, 3H,

;

5-CH₂–CH₂–CH₂–CH₃), 1.43 (tq, J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–

CH₂–CH₃), 1.59 (tt, J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 2.88

(t, J = 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 3.03 (s, 3H, N–CH₃), 3.47

(t, J = 2.4 Hz, 1H, propargyl CH), 4.06 (s, 3H, 1-CH₃), 4.22 (d,

J = 2.4 Hz, 2H, propargyl CH₂), 4.55 (s, 2H, 4-CH₂–N), 4.92 (s,

⁺NH); ¹³C NMR (100.6 MHz, CD₃OD) d 14.1 (CH₃, 5-CH₂–CH₂–

CH₂–CH₃), 23.0 (CH₂), 23.4 (CH₂) (5-CH₂–CH₂–CH₂–CH₃), 31.6

(CH₂, 5-CH₂–CH₂–CH₂–CH₃), 35.5 (CH₃, 1-CH₃), 40.5 (CH₃, N–

CH₃), 45.6 (CH₂, propargyl CH₂), 49.5 (CH₂, 4-CH₂–N), 73.2 (C,

propargyl C), 81.6 (CH, propargyl CH), 134.9 (C, C5), 140.5 (C,

C4); HRMS (ESI), calcd for (C₁₂H₂₀N₄+ H⁺) 221.1761, found

221.1762.

4.1.44. N-[2-(1-Ethyl-5-methyl-1H-1,2,3-triazol-4-yl)ethyl]-N-

methyl-N-propargylamine 52

It was prepared as described for 31. From amine 45 (490 mg,

2.91 mmol), Cs₂CO₃(952 mg, 2.92 mmol), and propargyl bromide

(80% solution in toluene, 0.43 mL, 2.89 mmol), and stirring at 0 °C

for 2.5 h, propargylamine 52 (319 mg, 54% yield) was obtained as

a yellow oil, without the need of chromatographic puriﬁcation; R_f

0.59 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

4.1.47. N-Methyl-N-[2-(5-ethyl-1-phenyl-1H-1,2,3-triazol-4-yl)

ethyl]-N-propargylamine 55

52ꢁHCl: yellow sticky solid, IR (ATR)

3181, 2981, 2932, 2583, 2518, 2461, 2408, 2366, ⁺NAH, „CAH,

and CAH), 2123 (C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 1.56

m 3500–2300 (max at 3406,

It was prepared as described for 31. From amine 48 (260 mg,

1.13 mmol), Cs₂CO₃(0.37 g, 1.14 mmol), and propargyl bromide

(80% solution in toluene, 0.13 mL, 0.87 mmol), and stirring at 0 °C

for 30 min and at rt for an additional 3 h, an orange oil (333 mg)

was obtained and puriﬁed through column chromatography (40–

;

(t, J = 7.2 Hz, 3H, 1-CH₂–CH₃), 2.50 (s, 3H, 5-CH₃), 3.08 (s, 3H, N–

CH₃), 3.35 (t, J = 7.6 Hz, 2H, 4-CH₂–CH₂–N), 3.45 (t, J = 2.8 Hz, 1H,

propargyl CH), 3.63 (t, J = 7.6 Hz, 2H, 4-CH₂–CH₂–N), 4.28 (d,

J = 2.8 Hz, 2H, propargyl CH₂), 4.51 (q, J = 7.2 Hz, 2H, 1-CH₂–CH₃),

4.94 (s, ⁺NH); ¹³C NMR (100.6 MHz, CD₃OD) d 8.0 (CH₃, 5-CH₃),

14.4 (CH₃, 1-CH₂–CH₃), 20.0 (CH₂, 4-CH₂–CH₂–N), 40.7 (CH₃, N–

CH₃), 46.3 (CH₂, propargyl CH₂), 46.4 (CH₂, 1-CH₂–CH₃), 54.0

60 lm silica gel, CH₂Cl₂/MeOH/50% aq NH₄OH mixtures, gradient

elution). On elution with CH₂Cl₂/MeOH/50% aq NH₄OH

99.9:0.1:0.2, propargylamine 55 (222 mg, 95% yield) was obtained

as a yellow oil; R_f0.59 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

15

55ꢁHCl: yellow sticky solid, IR (ATR) v 3400–2300 (max at 3395,

3193, 3059, 2958, 2925, 2573, 2518, 2459, 2397, ⁺NAH, „CAH,

CH₂–CH₂–CH₂–CH₃), 31.6 (CH₂, 5-CH₂–CH₂–CH₂–CH₃), 40.9 (CH₃,

N–CH₃), 46.3 (CH₂, propargyl CH₂), 55.3 (CH₂, 4-CH₂–CH₂–N),

72.7 (C, propargyl C), 81.7 (CH, propargyl CH), 126.8 (2CH), 130.9

(2CH), 131.4 (CH) (Ph–CH), 137.69 (C), 137.72 (C) (C5, Ph–C1),

140.6 (C, C4); HRMS (ESI), calcd for (C₁₈H₂₄N₄+ H⁺) 297.2074,

found 297.2085.

and CAH), 2123 (C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 1.06

;

(t, J = 7.6 Hz, 3H, 5-CH₂–CH₃), 2.81 (q, J = 7.6 Hz, 2H, 5-CH₂–CH₃),

3.11 (s, 3H, N–CH₃), 3.27 (t, J = 7.6 Hz, 2H, 4-CH₂–CH₂–N), 3.45 (t,

J = 2.8 Hz, 1H, propargyl CH), 3.70 (m, 2H, 4-CH₂–CH₂–N), 4.31 (d,

J = 2.8 Hz, 2H, propargyl CH₂), 4.88 (s, ⁺NH), 7.53 [ddm,

J = J⁰= 7.6 Hz, 2H, Ph–C2(6)–H], 7.62–7.67 [complex signal, 3H,

Ph–C3(5)–H, Ph–C4–H]; ¹³C NMR (100.6 MHz, CD₃OD) d 13.4

(CH₃, 5-CH₂–CH₃), 16.9 (CH₂, 5-CH₂–CH₃), 21.1 (CH₂, 4-CH₂–CH₂–

N), 40.8 (CH₃, N–CH₃), 46.3 (CH₂, propargyl CH₂), 55.3 (CH₂, 4-

CH₂–CH₂–N), 72.7 (C, propargyl C), 81.7 (CH, propargyl CH),

126.7 (2CH), 130.9 (2CH), 131.4 (CH) (Ph–CH), 137.6 (C, Ph–C1),

139.1 (C, C5), 140.2 (C, C4); HRMS (ESI), calcd for (C₁₆H₂₀N₄+ H⁺)

269.1761; found 269.1766.

4.1.50. 3-[3-(Trimethylsilyl)-2-propynyl]benzonitrile 61

A

solution of trimethylsilylacetylene (1.59 mL, 1.11 g,

11.3 mmol) in anhydrous THF (22 mL) was cooled to ꢀ78 °C, trea-

ted dropwise with n-BuLi (2.5 M solution in hexanes, 4.59 mL,

11.5 mmol), and stirred at ꢀ78 °C for 30 min. A solution of ZnBr₂

(2.59 g, 11.5 mmol) in anhydrous THF (8 mL) was then added and

the resulting mixture was warmed to 0 °C. 3-Bromomethylben-

zonitrile, 58 (1.50 g, 7.65 mmol), and Pd(PPh₃)₂Cl₂(290 mg,

0.41 mmol) were added and the reaction mixture was stirred at

rt overnight. The reaction mixture was cooled to 0 °C, treated with

1 N HCl (20 mL), and extracted with EtOAc (2 ꢂ 30 mL). The com-

bined organic extracts were washed with satd aq NaHCO₃

(20 mL), dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure to give a residue (2.02 g), which was puriﬁed

4.1.48. N-Methyl-N-[2-(1-phenyl-5-propyl-1H-1,2,3-triazol-4-yl)

ethyl]-N-propargylamine 56

It was prepared as described for 31. From amine 49 (168 mg,

0.69 mmol), Cs₂CO₃(0.22 g, 0.68 mmol), and propargyl bromide

(80% solution in toluene, 0.07 mL, 0.47 mmol), and stirring at 0 °C

for 30 min and at rt for an additional 3 h, an oily residue

(278 mg) was obtained and puriﬁed through column chromatogra-

through column chromatography (40–60 lm silica gel, hexane/

EtOAc mixtures, gradient elution). On elution with hexane/EtOAc

phy (40–60

l

m silica gel, CH₂Cl₂/MeOH/50% aq NH₄OH mixtures,

95:5, compound 61 (1.03 g, 63% yield) was isolated as a colorless

gradient elution). On elution with CH₂Cl₂/MeOH/50% aq NH₄OH

98:2:0.2, propargylamine 56 (117 mg, 88% yield) was obtained as

a yellow oil; R_f0.84 (CH₂Cl₂/MeOH/50% aq NH₄OH 9:1:0.05).

56ꢁHCl: orange sticky solid, IR (ATR) v 3500–2300 (max at 3400,

3197, 2958, 2930, 2873, 2354, ⁺NAH, „CAH, and CAH), 2121

oil; R_f0.73 (hexane/EtOAc 8:2); 2230 (C„N), 2178 (C„C) cm^ꢀ1

;

¹H NMR (400 MHz, CDCl₃) d 0.20 [s, 9H, Si(CH₃)₃], 3.68 (s, 2H, 1⁰-

H₂), 7.42 (dd, J = J⁰= 7.6 Hz, 1H, 5-H), 7.53 (d, J = 7.6 Hz, 1H), 7.57

(d, J = 7.6 Hz, 1H) (4-H, 6-H), 7.65 (br s, 1H, 2-H); ¹³C NMR

(100.6 MHz, CDCl₃) d 0.2 [3CH₃, Si(CH₃)₃], 26.0 (CH₂, C1⁰), 88.6 (C,

C3⁰), 102.4 (C, C2⁰), 112.7 (C, C1), 118.9 (C, CN), 129.4 (CH), 130.6

(CH), 131.6 (CH), 132.5 (CH) (phenylene CH), 138.1 (C, C3); HRMS

(ESI), calcd for (C₁₃H₁₅NSi + H⁺) 214.1047, found 214.1048.

(C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 0.83 (t, J = 7.6 Hz, 3H,

;

5-CH₂–CH₂–CH₃), 1.44 (tq, J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–CH₃),

2.77 (t, J = 7.6 Hz, 2H, 5-CH₂–CH₂–CH₃), 3.11 (s, 3H, N–CH₃), 3.22

(t, J = 7.6 Hz, 2H, 4-CH₂–CH₂–N), 3.45 (t, J = 2.4 Hz, 1H, propargyl

CH), 3.70 (m, 2H, 4-CH₂–CH₂–N), 4.31 (d, J = 2.4 Hz, 2H, propargyl

CH₂), 4.85 (s, ⁺NH), 7.52 (m, 2H), 7.62–7.66 (complex signal, 3H)

(Ph–CH); ¹³C NMR (100.6 MHz, CD₃OD) d 13.9 (CH₃, 5-CH₂–CH₂–

CH₃), 21.1 (CH₂, 4-CH₂–CH₂–N), 22.8 (CH₂, 5-CH₂–CH₂–CH₃), 25.2

(CH₂, 5-CH₂–CH₂–CH₃), 40.9 (CH₃, N–CH₃), 46.3 (CH₂, propargyl

CH₂), 55.3 (CH₂, 4-CH₂–CH₂–N), 72.7 (C, propargyl C), 81.7 (CH,

propargyl CH), 126.8 (2CH), 130.9 (2CH), 131.4 (CH) (Ph–CH),

137.6 (C), 137.7 (C) (C5, Ph–C1), 140.7 (C, C4); HRMS (ESI), calcd

for (C₁₇H₂₂N₄+ H⁺) 283.1917, found 283.1923.

4.1.51. 4-[3-(Trimethylsilyl)-2-propynyl]benzonitrile 62

It was prepared as described for 61. From trimethylsily-

lacetylene (1.06 mL, 737 mg, 7.50 mmol), n-BuLi (2.5 M solution

in hexanes, 3.06 mL, 7.65 mmol), ZnBr₂(1.72 g, 7.64 mmol), 4-bro-

momethylbenzonitrile, 59 (1.00 g, 5.10 mmol), and Pd(PPh₃)₂Cl₂

(183 mg, 0.26 mmol), a residue (1.38 g) was obtained and puriﬁed

through column chromatography (40–60 lm silica gel, hexane/

EtOAc mixtures, gradient elution). On elution with hexane/EtOAc

90:10, compound 62 (900 mg, 83% yield) was isolated as a colorless

oil; R_f0.77 (hexane/EtOAc 8:2); IR (ATR) v 2230 (C„N), 2178

4.1.49. N-Methyl-N-[2-(5-butyl-1-phenyl-1H-1,2,3-triazol-4-yl)

ethyl]-N-propargylamine 57

(C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 0.19 [s, 9H, Si(CH₃)₃],

;

3.70 (s, 2H, 1⁰-H₂), 7.46 [dm, J = 8.4 Hz, 2H, 3(5)–H], 7.61 [dm,

J = 8.4 Hz, 2H, 2(6)-H]; ¹³C NMR (100.6 MHz, CDCl₃) d 0.1 [3CH₃,

Si(CH₃)₃], 26.5 (CH₂, C1⁰), 88.6 (C, C3⁰), 102.3 (C, C2⁰), 110.8 (C,

C1), 119.0 (C, CN), 128.8 [2CH, C3(5)], 132.5 [2CH, C2(6)], 142.1

(C, C4); HRMS (ESI), calcd for (C₁₃H₁₅NSi + H⁺) 214.1047; found

214.1048.

It was prepared as described for 31. From amine 50 (157 mg,

0.61 mmol), Cs₂CO₃(0.22 g, 0.68 mmol), and propargyl bromide

(80% solution in toluene, 0.07 mL, 0.47 mmol), and stirring at 0 °C

for 30 min and at rt for an additional 3 h, an oily residue

(172 mg) was obtained and puriﬁed through column chromatogra-

phy (40–60 lm silica gel, CH₂Cl₂/MeOH/50% aq NH₄OH mixtures,

gradient elution). On elution with CH₂Cl₂/MeOH/50% aq NH₄OH

100:0:0.2 to 99:1:0.2, propargylamine 57 (164 mg, 91% yield)

was obtained as a yellow oil; R_f0.86 (CH₂Cl₂/MeOH/50% aq NH₄OH

9:1:0.05).

4.1.52. 4-[3-(Trimethylsilyl)-2-propynyl]phenylacetonitrile 63

It was prepared as described for 61. From trimethylsily-

lacetylene (5.20 mL, 3.61 g, 36.8 mmol), n-BuLi (2.5 M solution in

hexanes, 15.0 mL, 37.5 mmol), ZnBr₂(8.46 g, 37.6 mmol), 4-(bro-

momethyl)phenylacetonitrile, 60 (5.26 g, 25.0 mmol), and Pd

(PPh₃)₂Cl₂(940 mg, 1.34 mmol), a residue (7.12 g) was obtained

57ꢁHCl: yellow sticky solid, IR (ATR) v 3500–2300 (max at 3426,

3209, 2962, 2929, 2869, 2352, ⁺NAH, „CAH, and CAH), 2120

(C„C) cm^ꢀ1

;

¹H NMR (400 MHz, CD₃OD) d 0.80 (t, J = 7.6 Hz, 3H,

and puriﬁed through column chromatography (40–60 lm silica

5-CH₂–CH₂–CH₂–CH₃), 1.23 (tq, J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–

CH₂–CH₃), 1.39 (tt, J = J⁰= 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 2.78

(t, J = 7.6 Hz, 2H, 5-CH₂–CH₂–CH₂–CH₃), 3.11 (s, 3H, N–CH₃), 3.22

(t, J = 7.6 Hz, 2H, 4-CH₂–CH₂–N), 3.46 (t, J = 2.4 Hz, 1H, propargyl

CH), 3.69 (m, 2H, 4-CH₂–CH₂–N), 4.30 (d, J = 2.4 Hz, 2H, propargyl

CH₂), 4.85 (s, ⁺NH), 7.50 (m, 2H), 7.62–7.66 (complex signal, 2H)

(Ph–CH); ¹³C NMR (100.6 MHz, CD₃OD) d 13.8 (CH₃, 5-CH₂–CH₂–

CH₂–CH₃), 21.1 (CH₂, 4-CH₂–CH₂–N), 23.1 (CH₂), 23.2 (CH₂) (5-

gel, hexane/EtOAc mixtures, gradient elution). On elution with

hexane/EtOAc 95:5, compound 63 (2.50 g, 44% yield) was isolated

as a yellow oil; R_f0.50 (hexane/EtOAc 8:2); IR (ATR) v 2344 (C„N),

2144 (C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 0.19 [s, 9H, Si

;

(CH₃)₃], 3.65 (s, 2H, 1⁰-H₂), 3.73 (s, 2H, 1-CH₂–CN), 7.28 (br d,

J = 8.4 Hz, 2H), 7.36 (br d, J = 8.4 Hz, 2H) (Ph–CH); ¹³C NMR

(100.6 MHz, CDCl₃) d 0.03 [3CH₃, Si(CH₃)₃], 23.3 (CH₂, 1-CH₂–CN),

25.8 (CH₂, C1⁰), 87.3 (C, C3⁰), 103.6 (C, C2⁰), 117.8 (C, CN), 128.1

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

16

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

(2CH), 128.6 (2CH) (Ph–CH), 128.2 (C, Ph–C1), 136.5 (C, Ph–C4);

1⁰-CH₂), 112.7 (C, C1), 118.8 (C, CN), 122.6 (CH, C5⁰), 129.5 (CH),

130.4 (CH), 132.2 (CH), 133.4 (CH) (phenylene CH), 140.7 (C, C3),

146.1 (C, C4⁰); HRMS (ESI), calcd for (C₁₁H₁₀N₄+ H⁺) 199.0978,

found 199.0980.

HRMS (ESI), calcd for (C₁₄H₁₇NSi + H⁺) 228.1203; found 228.1203.

4.1.53. 3-(2-Propynyl)benzonitrile 64

To a solution of 61 (1.60 g, 7.51 mmol) in CH₂Cl₂(70 mL), a

solution of AgOTf (193 mg, 0.75 mmol) in MeOH/H₂O 11:1

(50 mL) was added. The reaction mixture was stirred at rt for 7 h,

treated with an additional amount of AgOTf (386 mg, 1.50 mmol),

and stirred at rt overnight. The resulting mixture was diluted with

satd aq NH₄Cl (50 mL) and extracted with CH₂Cl₂(3 ꢂ 50 mL). The

combined organic extracts were washed with H₂O (50 mL), dried

over anhydrous Na₂SO₄, and evaporated under reduced pressure

to give a crude (944 mg), which was puriﬁed through column chro-

4.1.57. 4-[(1-Methyl-1H-1,2,3-triazol-4-yl)methyl]benzonitrile 68

It was prepared as described for 9. From iodomethane (0.17 mL,

0.38 g, 2.70 mmol), NaN₃(191 mg, 2.94 mmol), Na₂CO₃(779 mg,

7.35 mmol), ascorbic acid (350 mg, 1.99 mmol), CuSO₄ꢁ5H₂O

(250 mg, 1.00 mmol), and alkyne 65 (346 mg, 2.45 mmol), com-

pound 68 (459 mg, 95% yield) was obtained as a white solid; R_f

0.63 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05); mp 116–118 °C;

IR (ATR) v 2227 (C„N) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 4.05

;

matography (40–60

lm silica gel, hexane/EtOAc mixtures, gradient

(s, 3H, 1⁰-CH₃), 4.12 (br s, 2H, 4-CH₂), 7.23 (s, 1H, 5⁰-H), 7.36 [dm,

J = 8.4 Hz, 2H, 3(5)-H], 7.57 [dm, J = 8.4 Hz, 2H, 2(6)-H]; ¹³C NMR

(100.6 MHz, CDCl₃) d 32.3 (CH₂, 4-CH₂), 36.8 (CH₃, 1⁰-CH₃), 110.6

(C, C1), 118.9 (C, CN), 122.6 (CH, C5⁰), 129.6 [2CH, C3(5)], 132.5

[2CH, C2(6)], 144.8 (C, C4), 146.1 (C, C4⁰); HRMS (ESI), calcd for

(C₁₁H₁₀N₄+ H⁺) 199.0978, found 199.0978.

elution). On elution with hexane/EtOAc 95:5, alkyne 64 (690 mg,

65% yield) was isolated as a white solid; R_f0.72 (hexane/EtOAc

8:2), mp 63–65 °C; IR (ATR) v 3247 („CAH), 2231 (C„N, C„C)

cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 2.26 (t, J = 2.8 Hz, 1H, 3⁰-H),

;

3.64 (d, J = 2.8 Hz, 2H, 1⁰-H₂), 7.43 (dd, J = J⁰= 8.0 Hz, 1H, 5-H),

7.54 (dm, J = 8.0 Hz, 1H), 7.58 (dm, J = 8.0 Hz, 1H) (4-H, 6-H), 7.68

13

(m, 1H, 2-H); C NMR (100.6 MHz, CDCl₃) d 24.6 (CH₂, C1⁰), 71.9

4.1.58. 4-[(1-Methyl-1H-1,2,3-triazol-4-yl)methyl]

phenylacetonitrile 69

(CH, C3⁰), 80.3 (C, C2⁰), 112.8 (C, C1), 118.8 (C, CN), 129.4 (CH),

130.7 (CH), 131.5 (CH), 132.5 (CH) (phenylene CH), 137.7 (C, C3);

HRMS (ESI), calcd for (C₁₀H₇N + H⁺) 142.0651, found 142.0653.

It was prepared as described for 9. From iodomethane (0.58 mL,

1.32 g, 9.32 mmol), NaN₃(660 mg, 10.2 mmol), Na₂CO₃(2.69 g,

25.4 mmol), ascorbic acid (1.19 g, 6.76 mmol), CuSO₄ꢁ5H₂O

(840 mg, 3.36 mmol), and alkyne 66 (1.31 g, 8.45 mmol), compound

69 (1.60 g, 89% yield) was obtained as a yellowish solid; R_f0.93 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05); mp 86–88 °C; IR (ATR) v

2241 (C„N) cm^ꢀ1; ¹H NMR (400 MHz, CDCl₃) d 3.71 (s, 2H, 1-CH₂–

CN), 4.02 (s, 3H, 1⁰-CH₃), 4.06 (s, 2H, 4-CH₂), 7.21 (s, 1H, 5⁰-H),

7.23–7.30 (complex signal, 4H, Ph–CH); ¹³C NMR (100.6 MHz,

CDCl₃) d 23.1 (CH₂, 1-CH₂–CN), 31.6 (CH₂, 4-CH₂), 36.4 (CH₃, 1⁰-

CH₃), 117.8 (C, CN), 122.3 (CH, C5⁰), 128.0 (C, Ph–C1), 128.1 (2CH),

129.2 (2CH) (Ph–CH), 139.0 (C, Ph–C4), 147.1 (C, C4⁰); HRMS (ESI),

calcd for (C₁₂H₁₂N₄+ H⁺) 213.1135, found 213.1135.

4.1.54. 4-(2-Propynyl)benzonitrile 65

It was prepared as described for 64. From 62 (642 mg,

3.01 mmol) and AgOTf [(80 mg, 0.31 mmol) ꢂ 2],

(548 mg) was obtained and puriﬁed through column chromatogra-

phy (40–60 m silica gel, hexane/EtOAc mixtures, gradient elu-

a

crude

l

tion). On elution with hexane/EtOAc 80:20, alkyne 65 (346 mg,

81% yield) was isolated as a white solid; R_f0.76 (hexane/EtOAc

8:2), mp 87–89 °C; IR (ATR) v 3254 („CAH), 2230 (C„N, C„C)

cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 2.25 (t, J = 2.8 Hz, 1H, 3⁰-H),

;

3.67 (d, J = 2.8 Hz, 2H, 1⁰-H₂), 7.48 [dm, J = 8.8 Hz, 2H, 3(5)-H],

7.62 [dm, J = 8.8 Hz, 2H, 2(6)-H]; ¹³C NMR (100.6 MHz, CDCl₃) d

25.2 (CH₂, C1⁰), 71.9 (CH, C3⁰), 80.2 (C, C2⁰), 111.0 (C, C1), 118.9

(C, CN), 128.8 [2CH, C3(5)], 132.5 [2CH, C2(6)], 141.7 (C, C4); HRMS

(ESI), calcd for (C₁₀H₇N + H⁺) 142.0651, found 142.0647.

4.1.59. 3-[(1-Methyl-1H-1,2,3-triazol-4-yl)methyl]benzylamine

70

A solution of nitrile 67 (739 mg, 3.73 mmol) in anhydrous THF

(30 mL) was cooled to 0 °C and treated dropwise with LiAlH₄

(4 M solution in Et₂O, 2.98 mL, 11.9 mmol). The reaction mixture

was stirred under reﬂux for 2 h, cooled to 0 °C, treated portionwise

with 1 N NaOH (100 mL), diluted with H₂O (100 mL), and extracted

with EtOAc (3 ꢂ 100 mL). The combined organic extracts were

dried over anhydrous Na₂SO₄and concentrated under reduced

pressure to afford amine 70 (368 mg, 49% yield) as a yellow oil;

R_f0.28 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

4.1.55. 4-(2-Propynyl)phenylacetonitrile 66

It was prepared as described for 64. From 63 (2.50 g,

11.0 mmol) and AgOTf (280 mg, 1.09 mmol) ꢂ 2], a crude (1.79 g)

was obtained and puriﬁed through column chromatography (40–

60 lm silica gel, hexane/EtOAc mixtures, gradient elution). On elu-

tion with hexane/EtOAc 95:5, alkyne 66 (1.31 g, 77% yield) was iso-

lated as a white solid; R_f0.41 (hexane/EtOAc 8:2), mp 58–59 °C; IR

(ATR) v 3262 („CAH), 2254 (C„N, C„C) cm^ꢀ1; ¹H NMR (400 MHz,

CDCl₃) d 2.19 (t, J = 2.8 Hz, 1H, 3⁰-H), 3.57 (br s, 2H, 1⁰-H₂), 3.69 (s,

2H, 1-CH₂–CN), 7.27 (br d, J = 8.0 Hz, 2H), 7.36 (br d, J = 8.0 Hz, 2H)

(Ph–CH); ¹³C NMR (100.6 MHz, CDCl₃) d 23.2 (CH₂, 1-CH₂–CN), 24.4

(CH₂, C1⁰), 70.8 (CH, C3⁰), 81.4 (C, C2⁰), 117.8 (C, CN), 128.1 (2CH),

128.6 (2CH) (Ph–CH), 128.3 (C, Ph–C1), 136.1 (C, Ph–C4); HRMS

(ESI), calcd for (C₁₁H₉N + H⁺) 156.0808, found 156.0808.

70ꢁHCl: yellowish solid, mp 119–121 °C; IR (ATR) v 3400–2300

(max at 3303, 3077, 2908, 2870, 2356, 2322, ⁺NAH and CAH)

cm^{ꢀ1 1}H NMR (400 MHz, CD₃OD) d 4.14 (s, 2H, 3-CH₂), 4.296 (s,

;

3H, 1⁰-CH₃), 4.302 (s, 2H, 1-CH₂–N), 4.95 (s, ⁺NH₃), 7.38–7.52 (com-

plex signal, 4H, phenylene CH), 8.39 (s, 1H, 5⁰-H); ¹³C NMR

(100.6 MHz, CD₃OD) d 30.2 (CH₂, 3-CH₂), 39.8 (CH₃, 1⁰-CH₃), 44.1

(CH₂, 1-CH₂–N), 129.1 (CH), 129.2 (CH), 130.7 (2CH), 131.0 (CH)

(phenylene CH, C5⁰), 135.3 (C), 138.2 (C) (C1, C3), 144.9 (C, C4⁰);

HRMS (ESI), calcd for (C₁₁H₁₄N₄+ H⁺) 203.1291, found 203.1297.

4.1.56. 3-[(1-Methyl-1H-1,2,3-triazol-4-yl)methyl]benzonitrile 67

It was prepared as described for 9. From iodomethane (0.30 mL,

0.68 g, 4.82 mmol), NaN₃(342 mg, 5.26 mmol), Na₂CO₃(1.39 g,

13.1 mmol), ascorbic acid (616 mg, 3.50 mmol), CuSO₄ꢁ5H₂O

(436 mg, 1.75 mmol), and alkyne 64 (618 mg, 4.38 mmol), com-

pound 67 (823 mg, 95% yield) was obtained as a yellowish solid;

R_f0.64 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05); mp 85–87 °C;

4.1.60. 4-[(1-Methyl-1H-1,2,3-triazol-4-yl)methyl]benzylamine

71

It was prepared as described for 70. From nitrile 68 (310 mg,

1.56 mmol) and LiAlH₄(4 M solution in Et₂O, 1.18 mL, 4.72 mmol),

amine 71 (254 mg, 81% yield) was obtained as a yellow oil; R_f0.27

(CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

IR (ATR) v 2229 (C„N) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 4.05

;

(s, 3H, 1⁰-CH₃), 4.09 (br s, 2H, 3-CH₂), 7.25 (s, 1H, 5⁰-H), 7.39 (dd,

J = J⁰= 8.0 Hz, 1H, 5-H), 7.48–7.53 (complex signal, 3H, 2-H, 4-H,

6-H); ¹³C NMR (100.6 MHz, CDCl₃) d 31.7 (CH₃, 3-CH₃), 36.8 (CH₂,

71ꢁHCl: yellowish sticky solid, IR (ATR) v 3400–2500 (max at

3386, 2931, 2810, 2536, ⁺NAH and CAH) cm^ꢀ1

;

¹H NMR

(400 MHz, CD₃OD) d 4.11 (s, 2H, 4-CH₂), 4.22 (s, 3H, 1⁰-CH₃), 4.90

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

17

(s, 1-CH₂–N, ⁺NH₃), 7.38 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H)

4.1.64. N-(tert-Butoxycarbonyl)-4-[(1-methyl-1H-1,2,3-triazol-

4-yl)methyl]phenethylamine 75

(phenylene CH), 8.16 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD)

d 30.6 (CH₂, 4-CH₂), 38.9 (CH₃, 1⁰-CH₃), 43.9 (CH₂, 1-CH₂–N),

127.7 (CH, C5⁰), 130.6 (2CH), 130.7 (2CH) (phenylene CH), 133.4

(C), 139.3 (C) (C1, C4), 145.8 (C, C4⁰); HRMS (ESI), calcd for

(C₁₁H₁₄N₄+ H⁺) 203.1291, found 203.1292.

It was prepared as described for 73. From 72 (1.65 g,

7.63 mmol) and di-tert-butyl dicarbonate (1.67 g, 7.65 mmol), 75

(2.34 g, quantitative yield) was obtained as a solid; R_f0.91 (CH₂Cl₂/

MeOH/50% aq NH₄OH 9.5:0.5:0.05); mp 92–94 °C; IR (ATR) v 3358

(NAH), 1677 (C@O) cm^ꢀ1; ¹H NMR (400 MHz, CDCl₃) d 1.42 [s, 9H,

C(CH₃)₃], 2.76 (t, J = 6.8 Hz, 2H, 1-CH₂–CH₂–N), 3.35 (dt,

J = J⁰= 6.8 Hz, 2H, 1-CH₂–CH₂–N), 4.02 (s, 3H, 1⁰-CH₃), 4.05 (s, 2H,

4-CH₂), 4.54 (br s, 1H, NHBoc), 7.12 (br d, J = 8.0 Hz, 2H), 7.19 (br

d, J = 8.0 Hz, 2H) (Ph–CH), 7.16 (s, 1H, 5⁰-H); ¹³C NMR

(100.6 MHz, CDCl₃) d 28.4 [3CH₃, C(CH₃)₃], 31.8 (CH₂, 4-CH₂),

35.8 (CH₂, 1-CH₂–CH₂–N), 36.5 (CH₃, 1⁰-CH₃), 41.8 (CH₂, 1-CH₂–

CH₂–N), 79.2 [C, C(CH₃)₃], 122.3 (CH, C5⁰), 128.9 (2CH), 129.0

(2CH) (Ph–CH), 137.16 (C), 137.23 (C) (Ph–C1, Ph–C4), 147.9 (C,

C4⁰), 155.8 (C, NCOO); HRMS (ESI), calcd for (C₁₇H₂₄N₄O₂+ H⁺)

317.1972, found 317.1982.

4.1.61. 4-[(1-Methyl-1H-1,2,3-triazol-4-yl)methyl]

phenethylamine 72

A suspension of nitrile 69 (1.60 g, 7.54 mmol) and Raney-Ni

(2.02 g, wet) in 7 N methanolic NH₃(32 mL) was hydrogenated

by bubbling H₂overnight. The resulting mixture was ﬁltered and

the cake was washed with MeOH (2 ꢂ 15 mL). The combined ﬁl-

trates were concentrated under reduced pressure to afford amine

72 (1.70 g, quantitative yield) as an oily residue; R_f0.17 (CH₂Cl₂/

MeOH/50% aq NH₄OH 9.5:0.5:0.05).

72ꢁHCl: yellow solid, mp 158–160 °C; IR (ATR) v 3400–2500

(max at 3390, 3331, 3254, 2994, 2970, 2727, 2627, 2573, ⁺NAH

and CAH) cm^ꢀ1

;

¹H NMR (400 MHz, CD₃OD) d 2.93 (t, J = 7.6 Hz,

4.1.65. N-(tert-Butoxycarbonyl)-N-methyl-3-[(1-methyl-1H-

1,2,3-triazol-4-yl)methyl]benzylamine 76

2H, 1-CH₂–CH₂–N), 3.15 (t, J = 7.6 Hz, 2H, 1-CH₂–CH₂–N), 4.04 (s,

2H, 4-CH₂), 4.07 (s, 3H, 1⁰-CH₃), 4.86 (s, ⁺NH₃), 7.18–7.27 (complex

signal, 4H, Ph–CH), 7.73 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD)

d 31.9 (CH₂, 4-CH₂), 34.1 (CH₂, 1-CH₂–CH₂–N), 37.2 (CH₃, 1⁰-CH₃),

42.0 (CH₂, 1-CH₂–CH₂–N), 125.0 (CH, C5⁰), 130.1 (2CH), 130.3

(2CH) (Ph–CH), 136.1 (C, Ph–C1), 139.3 (C, Ph–C4), 148.1 (C, C4⁰);

HRMS (ESI), calcd for (C₁₂H₁₆N₄+ H⁺) 217.1448, found 217.1450.

It was prepared as described for 17. From compound 73 (480 mg,

1.59 mmol), NaH (60% suspension in mineral oil, 96 mg,

2.40 mmol), and iodomethane (0.11 mL, 251 mg, 1.77 mmol), stir-

ring at 60 °C for 2 h, treating again with NaH (60% suspension in

mineral oil, 96 mg, 2.40 mmol), and iodomethane (0.11 mL,

251 mg, 1.77 mmol) and stirring at 60 °C for an additional 2 h, com-

pound 76 (422 mg, 84% yield) was obtained as an orange oil; R_f0.85

(CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05); IR (ATR) v 1686 (C@O)

cm^ꢀ1; ¹H NMR (400 MHz, CDCl₃) d 1.48 [br s, 9H, C(CH₃)₃], 2.82 (br

s, 3H, N–CH₃), 4.04 (s, 3H, 1⁰-CH₃), 4.08 (s, 2H, 3-CH₂), 4.40 (br s, 2H,

1-CH₂–N), 7.08–7.19 (complex signal), 7.25–7.30 (complex signal)

(5H, phenylene CH, 5⁰-H); ¹³C NMR (100.6 MHz, CDCl₃) d 28.6

[3CH₃, C(CH₃)₃], 32.3 (CH₂, 3-CH₂), 34.1 (CH₃, N–CH₃), 36.7 (CH₃,

1⁰-CH₃), 52.0 (br CH₂), 52.7 (br CH₂) (1-CH₂–N), 79.8 [C, C(CH₃)],

122.5 (CH, C5⁰), 125.6 (br CH), 125.8 (br CH), 127.7 (CH), 127.8 (br

CH), 128.9 (CH) (phenylene CH), 138.6 (C), 139.5 (C) (C1, C3),

4.1.62. N-(tert-Butoxycarbonyl)-3-[(1-methyl-1H-1,2,3-triazol-

4-yl)methyl]benzylamine 73

To a solution of 70 (352 mg, 1.74 mmol) in anhydrous THF

(6 mL), a solution of di-tert-butyl dicarbonate (380 mg, 1.74 mmol)

in anhydrous THF (2 mL) was added at 0 °C. The reaction mixture

was stirred at rt for 3 h and evaporated under reduced pressure.

The resulting residue was washed with pentane (3 ꢂ 10 mL) and

dried under vacuum, to afford 73 (520 mg, quantitative yield) as

a yellow oil; R_f0.85 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05);

IR (ATR) v 3341 (NAH), 1697 (C@O) cm^ꢀ1

;

¹H NMR (400 MHz,

147.9 (br C, C4⁰), 156.4 (br C, NCOO); HRMS (ESI), calcd for (C₁₇H₂₄

N₄O₂+ H⁺) 317.1972, found 317.1970.

-

CDCl₃) d 1.55 [s, 9H, C(CH₃)₃], 4.12 (s, 3H, 1⁰-CH₃), 4.16 (s, 2H, 3-

CH₂), 4.38 (d, J = 5.6 Hz, 2H, 1-CH₂–N), 4.97 (br s, 1H, NHBoc),

7.22–7.27 (complex signal, 3H), 7.33–7.38 (overlapped signal,

2H) (phenylene CH, 5⁰-H); ¹³C NMR (100.6 MHz, CDCl₃) d 28.5

[3CH₃, C(CH₃)₃], 32.3 (CH₂, 3-CH₂), 36.7 (CH₃, 1⁰-CH₃), 44.7 (CH₂,

1-CH₂–N), 79.6 [C, C(CH₃)₃], 122.6 (CH, C5⁰), 125.7 (CH), 127.8

(2CH), 129.0 (CH) (phenylene CH), 139.4 (C), 139.6 (C) (C1, C3),

147.8 (C, C4⁰), 156.0 (C, NCOO); HRMS (ESI), calcd for (C₁₆H₂₂N₄-

O₂+ H⁺) 303.1816, found 303.1812.

4.1.66. N-(tert-Butoxycarbonyl)-N-methyl-4-[(1-methyl-1H-

1,2,3-triazol-4-yl)methyl]benzylamine 77

It was prepared as described for 17. From compound 74

(140 mg, 0.46 mmol), NaH (60% suspension in mineral oil, 28 mg,

0.70 mmol), and iodomethane (0.03 mL, 68 mg, 0.48 mmol), stir-

ring at 60 °C for 2 h, treating again with NaH (60% suspension in

mineral oil, 28 mg, 0.70 mmol), and iodomethane (0.03 mL,

68 mg, 0.48 mmol) and stirring at 60 °C for an additional 2 h, com-

pound 77 (120 mg, 83% yield) was obtained as a brown oil; R_f0.56

(CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05); IR (ATR) v 1686 (C@O)

cm^ꢀ1; ¹H NMR (400 MHz, CDCl₃) d 1.46 [br s, 9H, C(CH₃)₃], 2.79 (br

s, 3H, N–CH₃), 4.01 (s, 3H, 1⁰-CH₃), 4.05 (s, 2H, 4-CH₂), 4.37 (br s,

2H, 1-CH₂–N), 7.14 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H) (phe-

nylene CH), 7.20 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CDCl₃) d 28.6

[3CH₃, C(CH₃)₃], 32.0 (CH₂, 4-CH₂), 34.0 (CH₃, N–CH₃), 36.7 (CH₃, 1⁰-

CH₃), 51.7 (br CH₂), 52.4 (br CH₂) (1-CH₂–N), 79.8 [C, C(CH₃)₃],

122.5 (CH, C5⁰), 127.8 (br CH), 128.0 (br CH), 128.9 (CH), 129.0

(CH) (phenylene CH), 136.4 (C), 138.2 (C) (C1, C4), 148.0 (C, C4⁰),

155.9 (br C, NCOO); HRMS (ESI), calcd for (C₁₇H₂₄N₄O₂+ H⁺)

317.1972, found 317.1965.

4.1.63. N-(tert-Butoxycarbonyl)-4-[(1-methyl-1H-1,2,3-triazol-

4-yl)methyl]benzylamine 74

It was prepared as described for 73. From 71 (90 mg,

0.45 mmol) and di-tert-butyl dicarbonate (98 mg, 0.45 mmol), 74

(133 mg, quantitative yield) was obtained as a beige solid; R_f

0.78 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

The analytical sample of 74 was obtained by ﬁltration of a

dichloromethane solution through a PTFE ﬁlter (0.2 lm), evapora-

tion of the ﬁltrate under reduced pressure and washing of the

resulting solid with pentane (3 ꢂ 2 mL); IR (ATR) v 3382 (NAH),

1690 (C@O) cm^ꢀ^{1 1}H NMR (400 MHz, CDCl₃) d 1.44 [s, 9H, C

;

(CH₃)₃], 4.00 (s, 3H, 1⁰-CH₃), 4.04 (s, 2H, 4-CH₂), 4.26 (d,

J = 5.6 Hz, 2H, 1-CH₂–N), 4.86 (br s, 1H, NHBoc), 7.13 (s, 1H, 5⁰-

H), 7.20 (complex signal, 4H, phenylene CH); ¹³C NMR

(100.6 MHz, CDCl₃) d 28.5 [3CH₃, C(CH₃)₃], 32.0 (CH₂, 4-CH₂),

36.7 (CH₃, 1⁰-CH₃), 44.4 (CH₂, 1-CH₂–N), 79.6 [C, C(CH₃)₃], 122.5

(CH, C5⁰), 127.8 (2CH), 129.0 (2CH) (phenylene CH), 137.3 (C),

138.3 (C) (C1, C4), 148.0 (C, C4⁰), 156.0 (C, NCOO); HRMS (ESI),

calcd for (C₁₆H₂₂N₄O₂+ H⁺) 303.1816, found 303.1817.

4.1.67. N-Methyl-3-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]

benzylamine 79

It was prepared as described for 25. From 76 (384 mg,

1.21 mmol) and H₃PO₄(85% purity, 2.15 mL, 18.6 mmol), amine

79 (242 mg, quantitative yield) was obtained as an orange oil; R_f

0.30 (CH₂Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

18

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

79ꢁHCl: sticky solid, IR (ATR) v 3400–2400 (max at 3374, 3116,

3074, 2956, 2778, 2699, 2542, 2417, ⁺NAH and CAH) cm^{ꢀ1 1}H

1H, propargyl CH), 4.08 (br s, 2H, propargyl CH₂), 4.26 (s, 3H, 1⁰-

;

CH₃), 4.30 (s, 2H, 3-CH₂), 4.47 (br signal, 2H, 1-CH₂–N), 5.04 (s,

⁺NH), 7.48–7.56 (complex signal, 3H, 4-H, 5-H, 6-H), 7.61 (br s,

1H, 2-H), 8.33 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD) d 30.5

(CH₂, 3-CH₂), 39.3 (CH₃, 1⁰-CH₃), 40.1 (CH₃, N–CH₃), 45.4 (CH₂,

propargyl CH₂), 59.4 (CH₂, 1-CH₂–N), 72.8 (C, propargyl C), 81.9

(CH, propargyl CH), 128.4 (CH, C5⁰), 131.0 (CH), 131.2 (CH + C),

131.8 (CH), 132.6 (CH) (C1, phenylene CH), 139.2 (C, C3), 145.2

(C, C4⁰); HRMS (ESI), calcd for (C₁₅H₁₈N₄+ H⁺) 255.1604, found

255.1600.

NMR (400 MHz, CD₃OD) d 2.73 (s, 3H, N–CH₃), 4.21 (s, 2H, 3-

CH₂), 4.32 (s, 3H, 1⁰-CH₃), 4.33 (s, 2H, 1-CH₂–N), 4.99 (s, ⁺NH₂),

7.42–7.52 (complex signal, 3H, 4-H, 5-H, 6-H), 7.55 (br s, 1H, 2-

H), 8.47 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD) d 30.0 (CH₂,

3-CH₂), 33.2 (CH₃, N–CH₃), 40.0 (CH₃, 1⁰-CH₃), 53.3 (CH₂, 1-CH₂–

N), 129.5 (CH), 130.3 (CH), 131.1 (CH), 131.2 (CH), 131.5 (CH) (phe-

nylene CH, C5⁰), 133.6 (C, C1), 138.0 (C, C3), 144.5 (C, C4⁰); HRMS

(ESI), calcd for (C₁₂H₁₆N₄+ H⁺): 217.1448, found 217.1446.

4.1.68. N-Methyl-4-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]

benzylamine 80

4.1.71. N-Methyl-N-{4-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]

benzyl}-N-propargylamine 83

It was prepared as described for 25. From 77 (237 mg,

0.75 mmol) and H₃PO₄(85% purity, 1.29 mL, 11.2 mmol), amine

80 (156 mg, 96% yield) was obtained as an orange oil; R_f0.24 (CH₂-

Cl₂/MeOH/50% aq NH₄OH 9.5:0.5:0.05).

It was prepared as described for 31. From amine 80 (131 mg,

0.61 mmol), Cs₂CO₃(0.20 g, 0.61 mmol), and propargyl bromide

(80% solution in toluene, 0.07 mL, 0.47 mmol), and stirring at 0 °C

for 3.5 h, an orange oily residue (122 mg) was obtained and puri-

80ꢁHCl: sticky solid, IR (ATR) v 3400–2400 (max at 3339, 3085,

ﬁed through column chromatography (40–60 lm silica gel, CH₂-

2931, 2744, 2708, 2547, 2415, ⁺NAH and CAH) cm^ꢀ1

;

¹H NMR

Cl₂/50% aq NH₄OH 100:0.2), to afford propargylamine 83 (48 mg,

40% yield) as a yellow oil; R_f0.67 (CH₂Cl₂/MeOH/50% aq NH₄OH

9:1:0.05).

(400 MHz, CD₃OD) d 2.71 (s, 3H, N–CH₃), 4.19 (s, 2H, 4-CH₂), 4.23

(s, 3H, 1⁰-CH₃), 4.24 (s, 2H, 1-CH₂–N), 4.90 (s, ⁺NH₂), 7.42 (d,

J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H) (phenylene CH), 8.20 (s, 1H,

5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD) d 30.6 (CH₂, 4-CH₂), 33.1

(CH₃, N–CH₃), 39.0 (CH₃, 1⁰-CH₃), 53.1 (CH₂, 1-CH₂–N), 127.9 (CH,

C5⁰), 130.7 (2CH), 131.62 (2CH) (phenylene CH), 131.56 (C, C1),

139.7 (C, C4), 145.6 (C, C4⁰); HRMS (ESI), calcd for

(C₁₂H₁₆N₄+ H⁺) 217.1448, found 217.1448.

83ꢁHCl: mp 156–158 °C; IR (ATR) v 3400–2400 (max at 3178,

3095, 2923, 2692, 2501, 2418, ⁺NAH, „CAH, and CAH), 2120

(C„C) cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 2.92 (s, 3H, N–CH₃),

;

3.47 (t, J = 2.8 Hz, 1H, propargyl CH), 4.05 (br signal, 2H, propargyl

CH₂), 4.19 (s, 3H, 1⁰-CH₃), 4.22 (s, 2H, 4-CH₂), 4.41 (br signal, 2H, 1-

CH₂–N), 4.86 (s, ⁺NH), 7.44 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H)

(phenylene CH), 8.10 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD) d

30.6 (CH₂, 4-CH₂), 39.0 (CH₃, 1⁰-CH₃), 40.1 (CH₃, N–CH₃), 45.4 (CH₂,

propargyl CH₂), 59.3 (CH₂, 1-CH₂–N), 72.8 (C, propargyl C), 81.9

(CH, propargyl CH), 128.0 (CH, C5⁰), 129.4 (C, C1), 130.9 (2CH),

132.9 (2CH) (phenylene CH), 140.5 (C, C4), 145.5 (C, C4⁰); HRMS

(ESI), calcd for (C₁₅H₁₈N₄+ H⁺) 255.1604, found 255.1599.

4.1.69. N-Methyl-4-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]

phenethylamine 81

To a suspension of LiAlH₄(118 mg, 3.11 mmol) in dry THF

(4 mL), a solution of 75 (165 mg, 0.52 mmol) in dry THF (4 mL)

was added dropwise at 0 °C. The reaction mixture was stirred at

reﬂux for 72 h, cooled to 0 °C, diluted with 1 N NaOH (20 mL),

and extracted with CH₂Cl₂(3 ꢂ 15 mL). The combined organic

extracts were dried over anhydrous Na₂SO₄, ﬁltered, and concen-

trated under reduced pressure to afford an oily residue (169 mg),

4.1.72. N-Methyl-N-{4-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]

phenethyl}-N-propargylamine 84

It was prepared as described for 31. From amine 81 (30 mg,

0.13 mmol), Cs₂CO₃(42 mg, 0.13 mmol), and propargyl bromide

(80% solution in toluene, 0.02 mL, 0.13 mmol), and stirring at 0 °C

for 30 min and at rt for an additional 3 h, an oily residue (48 mg)

was obtained and puriﬁed through column chromatography (40–

which was puriﬁed through column chromatography (40–60 lm

silica gel, CH₂Cl₂/MeOH/50% aq NH₄OH mixtures, gradient elution).

On elution with CH₂Cl₂/MeOH/50% aq NH₄OH 95:5:0.2, amine 81

(53 mg, 44% yield) was isolated as a yellow oil; R_f0.16 (CH₂Cl₂/

MeOH/50% aq NH₄OH 9:1:0.05).

60 lm silica gel, CH₂Cl₂), to afford propargylamine 84 (20 mg,

81ꢁHCl: yellowish solid, mp 149–151 °C; IR (ATR) v 3400–2400

57% yield) as a yellow oil; R_f0.55 (CH₂Cl₂/MeOH/50% aq NH₄OH

9:1:0.05).

(max at 3395, 3118, 2938, 2771, 2450, ⁺NAH and CAH) cm^{ꢀ1 1}H

;

NMR (400 MHz, CD₃OD) d 2.71 (s, 3H, N–CH₃), 2.97 (t, J = 7.2 Hz,

2H, 1-CH₂–CH₂–N), 3.23 (t, J = 7.2 Hz, 2H, 1-CH₂–CH₂–N), 4.06 (s,

2H, 4-CH₂), 4.09 (s, 3H, 1⁰-CH₃), 4.86 (s, ⁺NH₂), 7.24 (dm,

J = 8.8 Hz, 2H), 7.27 (dm, J = 8.8 Hz, 2H) (Ph–CH), 7.81 (s, 1H, 5⁰-

H); ¹³C NMR (100.6 MHz, CD₃OD) d 31.7 (CH₂, 4-CH₂), 32.9 (CH₂,

1-CH₂–CH₂–N), 33.7 (CH₃, N–CH₃), 37.5 (CH₃, 1⁰-CH₃), 51.4 (CH₂,

1-CH₂–CH₂–N), 125.5 (CH, C5⁰), 130.1 (2CH), 130.4 (2CH) (Ph–

CH), 136.0 (C), 139.0 (C) (Ph–C1, Ph–C4), 147.7 (C, C4⁰); HRMS

(ESI), calcd for (C₁₃H₁₈N₄+ H+) 231.1604, found 231.1605.

84ꢁHCl: sticky solid; IR (ATR) v 3500–2400 (max at 3413, 3358,

3213, 3068, 2944, 2496, ⁺NAH, „CAH, and CAH), 2144 (C„C)

cm^ꢀ^{1 1}H NMR (400 MHz, CD₃OD) d 3.02 (s, 3H, N–CH₃), 3.06 (t,

;

J = 6.8 Hz, 2H, 1-CH₂–CH₂–N), 3.41 (m, 1H, propargyl CH), 3.48

(br signal, 2H, 1-CH₂–CH₂–N), 4.09 (s, 2H, propargyl CH₂), 4.12 (s,

3H, 1⁰-CH₃), 4.18 (s, 2H, 4-CH₂), 4.86 (s, ⁺NH), 7.28 (complex signal,

4H, Ph–CH), 7.89 (s, 1H, 5⁰-H); ¹³C NMR (100.6 MHz, CD₃OD) d 31.1

(CH₂), 31.4 (CH₂) (4-CH₂, 1-CH₂–CH₂–N), 37.9 (CH₃, 1⁰-CH₃), 40.7

(CH₃, N–CH₃), 46.3 (CH₂, propargyl CH₂), 57.5 (CH₂, 1-CH₂–CH₂–

N), 72.8 (C, propargyl C), 81.5 (CH, propargyl CH), 126.1 (CH, C5⁰),

130.3 (2CH), 130.4 (2CH) (Ph–CH), 135.7 (C), 138.7 (C) (Ph–C1,

Ph–C4), 147.3 (C, C4⁰); HRMS (ESI), calcd for (C₁₆H₂₀N₄+ H⁺)

269.1761, found 269.1760.

4.1.70. N-Methyl-N-{3-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]

benzyl}-N-propargylamine 82

It was prepared as described for 31. From amine 79 (236 mg,

1.09 mmol), Cs₂CO₃(354 mg, 1.09 mmol), and propargyl bromide

(80% solution in toluene, 0.16 mL, 1.08 mmol), and stirring at 0 °C

for 30 min and at rt for an additional 3 h, propargylamine 82

(180 mg, 66% yield) was obtained as a yellow oil, without the need

of chromatographic puriﬁcation; R_f0.66 (CH₂Cl₂/MeOH/50% aq

NH₄OH 9.5:0.5:0.05).

4.2. Biological assays

4.2.1. Inhibition of hrMAO-A and hrMAO-B

The activity of hrMAO-A and hrMAO-B (Sigma–Aldrich, Madrid,

Spain) was determined using Amplex UltraRed ﬂuorometric cou-

pled method.⁸⁴For both MAO isoforms, tyramine hydrochloride

was used as substrate and enzymatic assays were performed in

96-well black opaque microplates (OptiPlate-96F, PerkinElmer) in

82ꢁHCl: sticky solid; IR (ATR) v 3400–2400 (max at 3389, 3191,

+

3023, 2930, 2491, NAH, „CAH, and CAH), 2122 (C„C) cm^ꢀ1

;

¹H

NMR (400 MHz, CD₃OD) d 2.94 (s, 3H, N–CH₃), 3.47 (t, J = 2.8 Hz,

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

19

Table 2

a ﬁnal volume of 200 lL. Serial dilutions of each inhibitor were

Reported and experimental permeability values (P_e10^ꢀ6cm s^ꢀ1) of 14 commercial

drugs used for the PAMPA-BBB assay validation

pre-incubated with either 0.36 U/mL hrMAO A or 0.0675 U/mL

hrMAO B for 30 min at 37 °C. Enzymatic reactions were started

upon the addition of 100 lL of a mixture solution containing

Compound

Literature value^a

Experimental value^b

1 mM tyramine, 0.04 U/mL horseradish peroxidase (HRP) and

25 mM Amplex UltraRed^Òreagent in 0.25 mM sodium phosphate

(pH 7.4) as ﬁnal concentrations. The ﬂuorescence production of

resoruﬁn was measured for at least 30 min at 530/590 nm in a

spectrophotometric plate reader (FluoStar OPTIMA, BM G Labtech).

The enzymatic activity in the absence of compound was used to

determine 100% enzyme activity. The potential capacity of the

compounds to interfere with the ﬂuorescence generated in the

reaction was determined by adding the compounds to solutions

in the absence of MAO. From dose–response curves, IC₅₀values

were accordingly calculated using the GraphPad ‘PRISM’ software

(version 5.0). Data are expressed as mean SEM of at least three

different experiments performed in duplicate. Clorgyline and R-

deprenyl (Sigma–Aldrich) were used as reference compounds.

Cimetidine

Lomeﬂoxacin

Norﬂoxacin

Oﬂoxacin

Hydrocortisone

Piroxicam

0.0

0.1

0.8

1.9

2.5

5.3

5.1

13

8.8

9.3

12

17

16

0.70 0.03

0.78 0.04

0.90 0.02

0.98 0.06

1.40 0.05

1.93 0.11

6.50 0.05

6.70 0.10

12.3 0.1

13.8 0.3

16.8 0.1

17.8 0.1

24.0 1.4

28.1 1.6

Clonidine

Corticosterone

Imipramine

Promazine

Progesterone

Desipramine

Testosterone

Verapamil

a

Taken from Ref. 82.

Values are expressed as the mean SD of three independent experiments.

b

order to mimic the orientation after irreversible linkage with the

FAD. Each compound was subjected to 100 docking runs. Whereas

the protein was kept rigid, Glide accounts for the conformational

ﬂexibility of the ligand around rotatable bonds during docking cal-

culations. The output docking modes were analyzed by visual

inspection in conjunction with the docking scores.

4.2.2. Reversibility of hrMAO-B inhibition

To study the reversibility of the inhibition of hrMAO-B by com-

pound 33, 100-fold enzyme concentration was inhibited by

200 nM L-deprenyl or 50 lM 33 (10-fold IC₅₀values) for 1 h at

37 °C. Following pre-incubation times, enzyme solution was

rapidly diluted (100-fold) in a mix solution containing 1 mM

p-tyramine, HRP and Amplex UltraRed^Òreagent in 50 mM

phosphate buffer pH 7.4. Next, hrMAO-B activity was followed at

530/590 nm for 40 min.

Acknowledgements

We thank the ﬁnancial support from Ministerio de Economía y

Competitividad (SAF2014-57094-R) and the Generalitat de Catalu-

nya (GC; 2014SGR52 and 2014SGR1189). We are grateful to the

Consorci de Serveis Universitaris de Catalunya for computational

resources. F.J.L. acknowledges the support from ICREA Academia.

Fellowships from GC to O.D.P., N.A., E.V., J.J.-J. and I.S. are gratefully

acknowledged.

4.2.3. Time-dependent inhibition of hrMAO-B

To investigate the time-dependent inhibition of hrMAO-B activ-

ity by compound 33, the enzyme was inhibited by 10 lM 33 for at

different pre-incubation times (0–360 min). At the end of each pre-

incubation, activity was measured as previously described and

plotted as percentage of control samples versus pre-incubation

time.

Supplementary data

4.2.4. Determination of brain permeability: PAMPA-BBB assay

The in vitro permeability (P_e) of the novel 1,2,3-triazole-based

compounds and fourteen known drugs (Table 2) through lipid

extract of porcine brain membrane was determined by using a par-

allel artiﬁcial membrane permeation assay⁸²using a mixture PBS/

EtOH 70:30. Assay validation was made by comparison of the

experimental P_evalues of the known drugs with their reported val-

ues, which showed a good correlation: P_e(exp) = 1.003 P_e(lit) ꢀ

0.783 (R²= 0.93). From this equation and the limits established

by Di et al. for BBB permeation,⁸²three ranges of permeability were

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2016.06.045.

References and notes

1. Prince, M.; Wimo, A.; Guerchet, M.; Ali, G.-C.; Wu, Y.-T.; Prina, M. World

Alzheimer Report 2015. The Global Impact of Dementia. An Analysis of Prevalence,

Incidence, Cost & Trends; Alzheimer’s Disease International: London, 2015.

http://www.alz.co.uk.

2. Bolea, I.; Gella, A.; Unzeta, M. J. Neural Transm. 2013 , 120 , 893.

3. Guzior, N.; Wieckowska, A.; Panek, D.; Malawska, B. Curr. Med. Chem. 2015 , 22 ,

373.

4. Perry, E. K.; Perry, R. H.; Blessed, G.; Tomlinson, B. E. Lancet 1977 , 1 , 189.

5. Coyle, J. T.; Puttfarcken, P. Science 1993 , 262 , 689.

6. Perry, G.; Raina, A. K.; Nunomura, A.; Wataya, T.; Sayre, L. M.; Smith, M. A. Free

Radical Biol. Med. 2000 , 28 , 831.

established: compounds of high BBB permeation (CNS+): P_e(10^ꢀ6

-

cm s^ꢀ1) > 5.18; compounds of low BBB permeation (CNSꢀ): P_e

(10^ꢀ6cm s^ꢀ1) < 2.06; compounds of uncertain BBB permeation

(CNS ): and 5.18 > P_e(10^ꢀ6cm s^ꢀ1) > 2.06.

7. Gella, A.; Durany, N. Cell Adhes. Migr. 2009 , 3 , 88.

8. Huang, X.; Moir, R. D.; Tanzi, R. E.; Bush, A. I.; Rogers, J. T. Ann. N.Y. Acad. Sci.

2004 , 1012 , 153.

4.3. Docking studies

9. Mishizen-Eberz, A. J.; Rissman, R. A.; Carter, T. L.; Ikonomovic, M. D.; Wolfe, B.

B.; Armstrong, D. M. Neurobiol. Dis. 2004 , 15 , 80.

10. Goedert, M.; Spillantini, M. G.; Jakes, R.; Rutherford, D.; Crowther, R. A. Neuron

1989 , 3 , 519.

11. Glenner, G. G.; Murphy, M. A. J. Neurol. Sci. 1989 , 94 , 1.

12. Birks, J.; Harvey, R. Cochrane Database Syst. Rev. 2006 , 1 CD001190.

13. Birks, J.; Grimley, E. J.; Iakovidou, V.; Tsolaki, M. Cochrane Database Syst. Rev.

2000 , 4 CD001191.

14. Loy, C.; Schneider, L. Cochrane Database Syst. Rev. 2004 , 4 CD001747.

15. Areosa, S. A.; McShane, R.; Sheriff, F. Cochrane Database Syst. Rev. 2005 , 4

CD003154.

16. Chen, X.; Decker, M. Curr. Med. Chem. 2013 , 20 , 1673.

17. Galdeano, C.; Viayna, E.; Arroyo, P.; Bidon-Chanal, A.; Blas, J. R.; Muñoz-

Torrero, D.; Luque, F. J. Curr. Pharm. Des. 2010 , 16 , 2818.

18. Matinez, A. Emerging Drugs and Targets for Alzheimer’s Disease. Vol. 1. Beta-

Amyloid, Tau Protein and Glucose Metabolism ; RSC, 2010.

The binding mode of selected 1,2,3-triazole derivatives was

explored by means of docking calculations carried out with Glide,

using an empirical scoring function calibrated on the basis of pro-

tein–ligand complexes.⁸⁵Docking was performed using the MAO-B

X-ray structure of the complex with inhibitor 6 (PDB entry 4CRT)⁸⁰

and deprenyl (PDB entry 2BYB),²⁸but no signiﬁcant differences

were found for the best ligand poses. The protocol deﬁned in our

previous studies was adopted here.³⁹Brieﬂy, the docking volume

was deﬁned as the space covered by the binding cavity in MAO-

B. Suitable restraints were introduced to keep the amine nitrogen

of the propargylamine unit close to the position found for the cor-

responding atom in deprenyl and rasagiline (PDB entry 2BK4)⁷⁹in

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

20

O. Di Pietro et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

19. Thomas, T. Neurobiol. Aging 2000 , 21 , 343.

49. Dias, K. S. T.; Viegas, C., Jr. Curr. Neuropharmacol. 2014 , 12 , 239.

50. Prati, F.; Uliassi, E.; Bolognesi, M. L. MedChemComm 2014 , 5 , 853.

51. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.

2002 , 41 , 2596.

20. Riederer, P.; Danyelczyk, W.; Grunblat, E. Neurotoxicology 2004 , 25 , 271.

21. Kristal, B. S.; Conway, A. D.; Brown, A. M.; Jain, J. C.; Ulluci, P. A.; Li, S. W.; Burke,

W. J. Free Radical Biol. Med. 2001 , 30 , 924.

22. Edmondson, D. E.; Binda, C.; Wang, J.; Upadhyay, A. K.; Mattevi, A. Biochemistry

2009 , 48 , 4220.

23. Tripton, K. F.; Boyce, S.; O’Sullivan, J.; Davey, G. P.; Healy, J. Curr. Med. Chem.

2004 , 11 , 1965.

24. Chen, K.; Holschneider, D. P.; Wu, W.; Rebrin, I.; Shih, J. C. J. Biol. Chem. 2004 ,

279 , 39645.

25. Binda, C.; Newton-Vinson, P.; Hubálek, F.; Edmonson, D. E.; Mattevi, A. Nat.

Struct. Biol. 2002 , 9 , 22.

26. Binda, C.; Li, M.; Hubálek, F.; Restelli, N.; Edmonson, D. E.; Mattevi, A. Proc. Natl.

Acad. Sci. U.S.A. 2003 , 100 , 9750.

27. Binda, C.; Hubálek, F.; Li, M.; Herzig, Y.; Sterling, J.; Edmonson, D. E.; Mattevi, A.

J. Med. Chem. 2004 , 47 , 1767.

52. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002 , 67 , 3057.

53. Struthers, H.; Mindt, T. L.; Schibli, R. Dalton Trans. 2010 , 39 , 675.

54. Schulze, B.; Schubert, U. S. Chem. Soc. Rev. 2014 , 43 , 2522.

55. Jiaranaikulwanitch, J.; Govitrapong, P.; Fokin, V. V.; Vajragupta, O. Molecules

2012 , 17 , 8312.

56. Lenhart, J. A.; Ling, X.; Gandhi, R.; Guo, T. L.; Gerk, P. M.; Brunzell, D. H.; Zhang,

S. J. Med. Chem. 2010 , 53 , 6198.

57. Ramprasad, J.; Nayak, N.; Dalimba, U.; Yogeeswari, P.; Sriram, D. Bioorg. Med.

Chem. Lett. 2015 , 25 , 4169.

58. Mishra, N. M.; Briers, Y.; Lamberigts, C.; Steenackers, H.; Robijns, S.; Landuyt,

B.; Vanderleyden, J.; Schoofs, L.; Lavigne, R.; Luyten, W.; Van der Eycken, E. V.

Org. Biomol. Chem. 2015 , 13 , 7477.

28. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmonson, D. E.; Mattevi, A. Proc.

Natl. Acad. Sci. U.S.A. 2005 , 102 , 12684.

29. Son, S. Y.; Ma, J.; Kondou, Y.; Yoshimura, M.; Yamashita, E.; Tsukihara, T. Proc.

Natl. Acad. Sci. U.S.A. 2008 , 105 , 5739.

59. Ma, L.-Y.; Pang, L.-P.; Wang, B.; Zhang, M.; Hu, B.; Xue, D.-Q.; Shao, K.-P.; Zhang,

B.-L.; Liu, Y.; Zhang, E.; Liu, H.-M. Eur. J. Med. Chem. 2014 , 86 , 368.

60. Fabbrizzi, P.; Bianchini, F.; Menchi, G.; Raspanti, S.; Guarna, A.; Trabocchi, A.

Tetrahedron 2014 , 70 , 5439.

30. Juárez-Jiménez, J.; Mendes, E.; Galdeano, C.; Martins, C.; Silva, D. B.; Marco-

Contelles, J.; do Carmo Carreiras, M.; Luque, F. J.; Ramsay, R. R. Biochim. Biophys.

Acta 2014 , 1844 , 389.

31. Song, M. S.; Matveychuk, D.; MacKenzie, E. M.; Duchcherer, M.; Mousseau, D.

D.; Baker, G. B. Prog. Neuropsychopharmacol. Biol. Psychiatry 2013 , 44 , 118.

32. Burke, W. J.; Li, S. W.; Chung, H. D.; Ruggiero, D. A.; Kristal, B. S.; Johnson, E. M.;

Lampe, P.; Kumar, V. B.; Franko, M.; Williams, E. A.; Zahm, D. S. Neurotoxicology

2004 , 25 , 101.

61. Brabez, N.; Saunders, K.; Nguyen, K. L.; Jayasundera, T.; Weber, C.; Lynch, R. M.;

Chassaing, G.; Lavielle, S.; Hruby, V. J. ACS Med. Chem. Lett. 2013 , 4 , 98.

62. Liu, K.; Gandhi, R.; Chen, J.; Zhang, S. ACS Med. Chem. Lett. 2012 , 3 , 942.

63. Howell, L. A.; Bowater, R. A.; O’Connell, M. A.; Reszka, A. P.; Neidle, S.; Searcey,

M. ChemMedChem 2012 , 7 , 792.

64. Ouberai, M.; Brannstrom, K.; Vestling, M.; Olofsson, A.; Dumy, P.; Chierici, S.;

Garcia, J. Org. Biomol. Chem. 2011 , 9 , 1140.

65. Ronco, C.; Carletti, E.; Colletier, J.-P.; Weik, M.; Nachon, F.; Jean, L.; Renard, P.-Y.

33. Naoi, M.; Maruyama, W.; Yi, H.; Akao, Y.; Yamaoka, Y.; Shamoto-Nagai, M. J.

Neural Transm. Suppl. 2007 , 72 , 121.

34. Weinreb, O.; Amit, T.; Bar-Am, O.; Sagi, Y.; Mandel, S.; Youdim, M. B. J. Neural

Transm. Suppl. 2006 , 70 , 457.

ChemMedChem 2012 , 7 , 400.

66. Lewis, W. G.; Green, L. K.; Grynszpan, F.; Radi c ´, Z .; Carlier, P. R.; Taylor, P.; Finn,

M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002 , 41 , 1053.

67. Krasi n ´ ski, A.; Radi c ´, Z .; Manetsch, R.; Raushel, J.; Taylor, P.; Sharpless, K. B.;

Kolb, H. C. J. Am. Chem. Soc. 2005 , 127 , 6686.

68. Whiting, M.; Muldoon, J.; Lin, Y.-C.; Silverman, S. M.; Lindstrom, V.; Olson, A. J.;

Kolb, H. C.; Finn, M. G.; Sharpless, K. B.; Elder, J. H.; Fokin, V. V. Angew. Chem.,

Int. Ed. 2006 , 45 , 1435.

69. Sharpless, K. B.; Manetsch, R. Expert Opin. Drug Disc. 2006 , 1 , 525.

70. Oueis, E.; Santoni, G.; Ronco, C.; Syzgantseva, O.; Tognetti, V.; Joubert, L.;

Romieu, A.; Weik, M.; Jean, L.; Sabot, C.; Nachon, F.; Renard, P.-Y. Org. Biomol.

Chem. 2014 , 12 , 156.

71. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003 , 8 , 1128.

72. LogP values estimated with www.molinspiration.com.

73. Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010 , 39 , 1302.

74. Díez-González, S. Catal. Sci. Technol. 2011 , 1 , 166.

75. Mendal, M.; Tornøe, C. W. Chem. Rev. 2008 , 108 , 2952.

76. Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org. Chem. 2006 , 2006 , 51.

77. Shi, Z.-J.; Yu, D.-G. Compr. Inorg. Chem. II 2013 , 47.

78. Orsini, A.; Vitérisi, A.; Bodlenner, A.; Weibel, J.-M.; Pale, P. Tetrahedron Lett.

2005 , 46 , 2259.

79. Hubálek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi, A.; Castagnoli, N.; Edmondson,

D. E. J. Biol. Chem. 2005 , 280 , 15761.

80. Esteban, G.; Allan, J.; Samadi, A.; Mattevi, A.; Unzeta, M.; Marco-Contelles, J.;

Binda, C.; Ramsay, R. R. Biochim. Biophys. Acta 2014 , 1844 , 1104.

81. Pérez, V.; Marco, J. L.; Fernández-Alvarez, E.; Unzeta, M. Br. J. Pharmacol. 1999 ,

127 , 869.

35. Yáñez, M.; Viña, D. Curr. Top. Med. Chem. 2013 , 13 , 1692.

36. Jo, S.; Yarishkin, O.; Hwang, Y. J.; Chun, Y. E.; Park, M.; Woo, D. H.; Bae, J. Y.;

Kim, T.; Lee, J.; Chun, H.; Park, H. J.; Lee, D. Y.; Hong, J.; Kim, H. Y.; Oh, S.-J.; Park,

S. J.; Lee, H.; Yoon, B.-E.; Kim, Y. S.; Jeong, Y.; Shim, I.; Bae, Y. C.; Cho, J.; Kowall,

N. W.; Ryu, H.; Hwang, E.; Kim, D.; Lee, C. J. Nat. Med. 2014 , 20 , 886.

37. Weinreb, O.; Amit, T.; Bar-Am, O.; Youdim, M. B. Curr. Drug Targets 2012 , 13 ,

483.

38. Sterling, J.; Herzig, Y.; Goren, T.; Finkelstein, N.; Lerner, D.; Goldenberg, W.;

Mikcolczi, I.; Molnar, S.; Rantal, F.; Tamas, T.; Toth, G.; Zagyva, A.; Zekany, A.;

Finberg, J.; Lavian, G.; Gross, A.; Friedman, R.; Razin, M.; Huang, W.; Krais, B.;

Chorev, M.; Youdim, M. B.; Weinstock, M. J. Med. Chem. 2002 , 54 , 5260.

39. Bolea, I.; Juárez-Jiménez, J.; de los Ríos, C.; Chioua, M.; Pouplana, R.; Luque, F. J.;

Unzeta, M. J. Med. Chem. 2011 , 54 , 8251.

40. Bolea, I.; Gella, A.; Monjas, I.; Perez, C.; Rodríguez-Franco, M. I.; Marco-

Contelles, J.; Samadi, A.; Unzeta, M. Curr. Alzheimer Res. 2013 , 9 , 797.

41. Farina, R.; Pisani, L.; Catto, M.; Nicolotti, O.; Gadaleta, D.; Denora, N.; Soto-

Otero, R.; Mendez-Alvarez, E.; Passos, C. S.; Muncipinto, G.; Altomare, C. D.;

Nurisso, A.; Carrupt, P.-A.; Carotti, A. J. Med. Chem. 2015 , 58 , 5561.

42. Xie, S. S.; Wang, X.; Jiang, N.; Yu, W.; Wang, K. D. G.; Lan, J. S.; Li, Z. R.; Kong, L.

Y. Eur. J. Med. Chem. 2015 , 95 , 153.

43. Zheng, H.; Youdim, M. B. H.; Fridkin, M. J. Med. Chem. 2009 , 52 , 4095.

44. Zheng, H.; Youdim, M. B.; Fridkin, M. ACS Chem. Biol. 2010 , 5 , 603.

45. Passos, C. S.; Simões-Pires, C. A.; Nurisso, A.; Soldi, T. C.; Kato, L.; de Oliveira, C.

M.; de Faria, E. O.; Marcourt, L.; Gottfried, C.; Carrupt, P.-A.; Henriques, A. T.

Phytochemistry 2013 , 86 , 8.

82. Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. Eur. J. Med. Chem. 2003 ,

38 , 223.

46. Pisani, L.; Farina, R.; Soto-Otero, R.; Denora, N.; Mangiatordi, G.; Nicolotti, O.;

Mendez-Alvarez, E.; Cosimo, A.; Catto, M.; Carotti, A. Molecules 2016 , 21 , 362.

47. Tonelli, M.; Catto, M.; Tasso, B.; Novelli, F.; Canu, C.; Iusco, G.; Pisani, L.; Stradis,

A. D.; Denora, N.; Sparatore, A.; Boido, V.; Carotti, A.; Sparatore, F.

ChemMedChem 2015 , 10 , 1040.

83. Li, L.; Zhang, C.-W.; Ge, J.; Qian, L.; Chai, B.-H.; Zhu, Q.; Lee, J.-S.; Lim, K.-L.; Yao,

S. Q. Angew. Chem., Int. Ed. 2015 , 54 , 10821.

84. Zhou, M.; Panchu-Voloshina, N. Anal. Biochem. 1997 , 253 , 169.

85. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;

Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006 , 49 , 6177.

48. Domínguez, J. L.; Fernández-Nieto, F.; Castro, M.; Catto, M.; Paleo, M. R.; Porto,

S.; Sardina, F. J.; Brea, J. M.; Carotti, A.; Villaverde, M. C.; Sussman, F. J. Chem. Inf.

Model. 2015 , 55 , 135.

Please cite this article in press as: Di Pietro, O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.045

Article Doi

Design, synthesis and biological evaluation of N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylamines as novel monoamine oxidase B inhibitors

DOI: 10.1016/j.bmc.2016.06.045

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2016.06.045

Full text of DOI:10.1016/j.bmc.2016.06.045

Products guided by the article

R&D Labs maybe for 933719-82-5

Relevant to this article

Hot Product