Antitubercular activity of 1,2,3-triazolyl fatty acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.09.086

A collection of 1,2,3-triazoles unsaturated fatty acid mimics were efficiently synthesized by click chemistry. The 1,4-disubstituted analogs prepared covered different alkyl chain lengths and triazole positions. The compounds were subsequently tested against Mycobacterium tuberculosis, being most of them active with some of the analogs displaying activity at micromolar concentration. The most potent member of the series has the triazole moiety on the C-2 position with a carbon chain of eight or ten carbon atoms. The 1,5-isomers of the most active analog were significantly less active than the original isomer. The activity of the selected hit was assayed on several clinical MTB multi-drug resistant strains providing the same MIC.

Full text of DOI:10.1016/j.ejmech.2016.09.086

Accepted Manuscript
Antitubercular activity of 1,2,3-triazolyl fatty acid derivatives
Diego G. Ghiano, Agustina de la Iglesia, Nina Liu, Peter J. Tonge, Héctor R.
Morbidoni, Guillermo R. Labadie
PII:
S0223-5234(16)30822-4
10.1016/j.ejmech.2016.09.086
EJMECH 8952
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2016
Revised Date: 24 September 2016
Accepted Date: 26 September 2016
Please cite this article as: D.G. Ghiano, A. de la Iglesia, N. Liu, P.J. Tonge, H.R. Morbidoni, G.R.
Labadie, Antitubercular activity of 1,2,3-triazolyl fatty acid derivatives, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.086.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Antitubercular activity of 1,2,3-triazolyl fatty acid derivatives
Diego G. Ghiano,a Agustina de la Iglesia,b Nina Liu,c Peter J. Tonge,c Héctor R. Morbidoni,d,e and
Guillermo R. Labadie.a,f*
a Instituto de Química Rosario, UNR, CONICET, Suipacha 531, S2002LRK, Rosario, ARGENTINA.
b Cátedra de Microbiología, Virología y Parasitología, Facultad de Ciencias Médicas,
Universidad Nacional de Rosario, Santa Fe 3100,S2002KTR, Rosario, ARGENTINA.
c Institute of Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook
University, Stony Brook, NY 11794, USA.
d Consejo de Investigaciones, Universidad Nacional de Rosario, ARGENTINA.
e Laboratorio de Microbiología Molecular. Facultad de Ciencias Médicas, Universidad Nacional de
Rosario, Santa Fe 3100, S2002KTR, Rosario, ARGENTINA.
f Departamento de Química Orgánica, Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, ARGENTINA.
*Corresponding author
E-mail: labadie@iquir-conicet.gov.ar
Phone number: +54-341-4370477 # 108
FAX number: +54-341-4370477 # 112
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Highlights
A collection of different 1,2,3-triazolyl fatty acids derivatives were prepared.
The most active compounds displayed a M. tuberculosis MIC below 2 M.
Compound 11 had the same MIC towards a set of clinically isolated resistant strains.
µ
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Abstract
A collection of 1,2,3-triazoles unsaturated fatty acid mimics were efficiently synthesized by click
chemistry. The 1,4-disubstituted analogs prepared covered different alkyl chain lengths and
triazole positions. The compounds were subsequently tested against Mycobacterium tuberculosis,
being most of them active with some of the analogs displaying activity at micromolar
concentration. The most potent member of the series has the triazole moiety on the C-2 position
with a carbon chain of eight or ten carbon atoms. The 1,5-isomers of the most active analog were
significantly less active than the original isomer. The activity of the selected hit was assayed on
several clinical MTB multi-drug resistant strains providing the same MIC.
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1. Introduction
Tuberculosis (TB) has become the most prevalent disease in the world today, ever more than any
other time in human history. This disease caused by Mycobacterium tuberculosis (MTB) has
plagued mankind for centuries being the greatest single infectious cause of mortality worldwide,
killing roughly two million people annually.[1] The treatment of multidrug resistant tuberculosis
(MDR-TB) patients thus requires the administration of second line drugs like amikacin, kanamycin,
capreomycin, cycloserine, para-aminosalicylic acid, ethionamide, and fluoroquinolones.
Unfortunately, these drugs are less efficient and frequently more toxic requiring longer treatment
times. Another drawback is that the MDR-TB treatment can cost up to 100 times higher than the
basic six-month short-course chemotherapy regimen.[2] Estimates have shown that one-third of
the world’s population is infected with latent M. tuberculosis. That scenario has been increasingly
aggravated due to the resistance on the drugs routinely used to treat the disease. Last statistical
data published by WHO revealed the existence of a 3.5% of MDR strains worldwide and that rate
has not changed.[1] Extensively drug resistant strains known as XDR, are resistant to isoniazid and
rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs.[3] The
resurgence of TB among immune compromised individuals, such as those with diabetes necrosis
or those infected with HIV, is alarming due to the development of pathogenic synergy.[4] The
scenario has turned dramatic considering that over the last 40 years no new antitubercular drug
has been developed.[5] That complicated situation has prompted WHO to declare tuberculosis as
a global public health crisis.[1] This has created an urgent need to develop fast acting new
antitubercular drugs with diverse and unique structural features. Recently, things have turned
around with bedaquiline being approved by the U.S. Food and Drug Administration (FDA) under
the name Sirturo in 2012, and by the European Medicine Agency (EMA) in 2014 for the treatment
of MDR-TB patients.[6]
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Mycobacteria are surrounded by a thick waxy cell wall that is thought to impact the ability of many
common antibiotics to reach the mycobacterial cytoplasm. Key components of these cell walls are
mycolic acids, which are very long chain (C60-C90) α- branched chain fatty acids esterified to the
arabinogalactan component of the cell wall or to trehalose.[7] The mycobacterial mycolic acid
biosynthesis pathway (FAS-II) represents a validated and well exploited target for drug
discovery.[8] The most common FAS-II enzyme targets are the enoyl ACP reductase FabI (InhA),
that catalyze the last step in fatty acid elongation, and the ketoacyl-ACP synthases that catalyzes
the first step, the condensation of acetyl-CoA with malonyl-ACP. Isoniazid is the current front line
tuberculosis treatment regimen. This hydrazide inhibites InhA after its activation by KatG, resulting
in the formation of an adduct with NAD+. InhA inhibitors that do not require activation by KatG are
effective against most INH-resistant MTB.[9] Over the last 20 years many InhA inhibitors have
been reported being an attractive target to develop new antitubercular drugs.[10]
The discovery of TMC207, a first-in-class diarylquinoline compound with a novel mechanism of
action,[11] has trigger the research of heterocycles as antitubercular agents. That finding was
followed by different heterocycles scaffolds as promising structures to develop
chemotherapies.[12-14]
One strategy to introduces heterocycles into the molecular scaffold is the use of 1,2,3-triazoles.
That approach requires the incorporation of 1,2,3-triazoles into a drug candidate, as a key part of
the pharmacophore, or to link several molecular portions together.[15, 16] The emerging field of
click chemistry offers a unique approach to the synthesis of 1,2,3-triazole-containing molecules. It
becomes popular for medicinal chemistry because azides and alkynes are practically inert to most
biological and organic conditions, water, molecular oxygen, and the majority of common reaction
conditions in organic synthesis. Another key advantage that turns then in an attractive connection
unit is that they are stable to metabolic degradation and capable of hydrogen bonding, which can
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be favorable in the binding of biomolecular targets and can improve the solubility.[17] Over the
years, many examples of biologically active 1,2,3-triazole-containing molecules have been
reported, including antiparasite,[18, 19] antifungal, [20] anticancer,[21] antiviral,[22, 23] and
against many bacterial species.[24, 25] The synthesis of collections of 1,2,3-trizoles to develop new
chemical entities toward M. tuberculosis has not been an exception. Different authors have
reported new compounds that display promising in vitro activities against MTB.[26-36] (Figure 1)
Figure 1. Antitubercular 1,2,3-triazole containing products
On a previous exploratory report[37] we have designed a collection of compounds based on the
critical role displayed by fatty acid biosynthesis and metabolism and the renewed interest on
triazoles as antimycobacterial drugs. Inspired on the structures of the antitubercular compounds
8PP[38] and 2-alkynoic acid[39] we have prepared and screened a series of 1,4-disubtituted 1,2,3-
triazoles. (Figure 2)
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Figure 2. Strategy previously followed for library design. [37]
The antimycobacterial activity of that collection showed a clear tendency where the most active
compounds have alkyl chain and ethyl acetoxy as N1 and C4 triazole substituents, respectively.
Based on those results we wanted to expand that study, preparing a new library to look into the
relationship between the triazole position and the activity of the previously found
antimycobacterial products. On the initial study only a series of analogs with C4 alkyl substituents
of 3, 5 and 8 carbon atom chain were reported. In this work, we wanted to increase the chain
length up to 24 carbon atoms. (Figure 3)
Figure 3. Structural diversity explored in this work.
Additionally, the general structure of the most active compounds resembles unsaturated fatty
acids, being the triazole as isoster of the triple bond present on alkynoic acids. Based on that
assumption, the position of the triazole on the carbon chain was not explored before, requiring
the synthesis of analogs with different carbon length moving the triazole along the chain. Also, the
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connectivity of the 1,2,3-triazole was investigated by adding analogs with the nitrogen connected
to the chain close to C1. (Figure 3) To determine the structure-activity relationship in the present
study, the activity of novel 1,4-disubstituted 1,2,3-triazole derivatives were evaluated against M.
tuberculosis H37Rv as well as their in vitro cytotoxicity.
2. Results and Discussion
2.1. Chemistry
A set of new analogs were designed to evaluate the structural requirements for the activity. Based
on structure of the unsaturated fatty acid substrate for InhA, three different factors were
evaluated: the chain length, the triazole position and the functional group on the head. That
hypothesis has been experimentally validated on other scaffolds,[40, 41] but not for unsaturated
fatty acids. Molecular modeling studies were performed to corroborate that the replacement on
the unsaturated fatty ester by 1,2,3-triazoles displays an equivalency to an allylic group (Figure 4).
In the case of unsaturated Z-fatty ester there is some difference on the geometry compared with
the 1,4-disubtituted 1,2,3- triazole, with an angle of 155.4° and a distance of 5.04 Å for the
substituents of the heterocycle. Those values are close to the 179.0° measured on the E-double
bond substituents having a shorter distance of 3.92 Å between them. For the 1,5 disubtituted-
1,2,3- triazole the angle is 76.2° and the distance is 3.18 Å, being very close to the 75.4° and the
3.18 Å on the Z double bond. These results suggested that 1,5-disubstituted 1,2,3-triazole
perfectly mimics the topology of a Z-double bond, meanwhile the 1,4- disubstituted regioisomer
share similar topology but longer than a double bond, being equivalent to fatty acids one carbon
longer.
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Figure 4. Comparison between unsaturated fatty esters and 1,2,3-triazole regioisomers
Compounds holding ethyl and methyl carboxylate, free carboxylic acid, and alcohols were
prepared covering fatty acid chain length from C8 to C20. The position of the 1,2,3-triazole was
also moved from C2 to C7. To prepare the collection, the necessary azides were synthesized by
direct substitution with sodium azide in DMF starting from commercially available
ω-bromo
ethylesters of fatty acids from 2 to 5 carbon length. With these azides in hand, a series of 1,2,3-
triazole derivatives were synthesized using commercial 1-alkynes from different carbon length.
(Scheme 1) On the other side, starting from methyl propiolate, propiolic acid and propargyl
alcohol a series of new products were prepared by Cu(I) catalyzed 1,3-dipolar cycloaddition with
different aliphatic azides.
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N
N
X
b)
R1
R3
R2
+
N
n
R1= COOH,
COOMe,
CH2OH
R2
R2= C8H17
X=Br
X=N3
1-8
a)
C10H21
C13H27
N
N
N
O
n= 1 to 5
R2
b)
R1
X
EtOOC
a)
+
R1= C3H7
C5H11
OEt
n
n
X=Br
9-25
C8H17
X=N3
C10H21
C13H27
C15H31
a) NaN3, DMF, 50 oC, 18h;
b) CuSO4, sodium ascorbate, H2O : t-BuOH (1:1) room temp.
Scheme 1. Synthesis of Fatty acids 1,2,3-triazoles mimics.
All the reactions were conducted in a parallel solution synthesis fashion setup under copper(II)
sulphate catalytic conditions in water:t-BuOH (1:1) using sodium ascorbate as reductant. The
collection of triazolyl fatty esters 1-25 was obtained with an 80 % average yield after purification
(Table 1). The products were characterized by 1D-, 2D- NMR experiments, IR and HRMS, displaying
a 1,4-substitution pattern as was expected based our previous experience[18, 37] and by several
other reports. [42, 43]
2.2. Biological evaluation
2.2.1. In vitro activity against M. tuberculosis
The prepared collection was assayed in vitro against M. tuberculosis H37rv obtaining the MIC by
serial micro dilution colorimetric assay using MTT as viability indicator.[44, 45] The results are
presented in the Table 1 and are organized based on the chain length equivalence, the substituent
on C1 and the 1,2,3-triazole position. The results are presented following the chain length and the
position of the 1,2,3-triazole. From the left to the right the chain length increases and from the top
to the bottom the position of the 1,2,3-triazole moves away from C1.
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The analysis of the activity on Table 1 showed that thirteen of the new prepared compounds have
MIC below 60
µM, if the three previously reported analogs are added (9-11) the number increases
to sixteen that is a 65 % of the collection. Twelve compounds displayed MICs of 25
µM or less with
only four analogs above that value. The elevated number of active analogs of the library revealed
the privileged character of the structure prepared.
The structure-activity relationship analysis indicates the importance of the chain length. In fact,
analogs equivalent to fatty acids equivalency between twelve to fifteen carbon atoms are the
most active. In that region there are ten compounds (4, 5, 7, 11, 12, 15, 17, 20, 22, 24) with
activities below 25
are esters, with the exception of acids 4 and 5 (MIC 14 and 20
insensitive to the heterocycle position, as 7, 15 and 24 (MIC 23, 22 and 22
µ
M that is 71% of the total prepared. The majority of the most active analogs
M), also the activity seems to be
M respectively)
µ
µ
displayed almost the same MIC having the 1,2,3-triazole on C2, C4 orC7 position. The other point
of diversity incorporated was the functional group on C1, which consists of alcohols, acids and
esters. From those, the alcohols seem to be the less active of the series, followed by the acids;
being the esters which performed best. It is important to point out that acids were difficult to
dissolve, preventing the study of longer analogs, even with the interesting profile they displayed.
An attractive series included analogs 9 to 14, esters having the 1,2,3-triazole on C3 and different
chain length that included 11, the most active compounds of our previous work.[37] These analogs
included the previously reported 9-11 and the longer analogs 12 to 14. This series allowed us to
conclude that the activity is heavily dependent on the chain length, with a minimum MIC for the
analog T12 (C19, MIC 1.7
µM) that is slightly more active than 11 (C17, MIC 1.9
µM). 13 (C22, MIC
296 M) and 14 (C24, MIC 17
µ
µM) , were 174 and 10 times less active than 11, respectively.
As a main conclusion it is clear that the combination of the triazole position on C3 (compounds
with n=1, Figure 1) with a chain length between 17 and 19 increases the antimycobacterial activity
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at least ten times. It has been reported[46] that fatty acids of 14 carbon atoms were considerably
more active than their neighbors of C12 and C16. Contrary to our results, in the same article the
respective esters were completely inactive, but the activity on C18 fatty acids increased along with
the degree of unsaturation.
Table 1. Antimycobacterial activity of the compounds prepared.
Comp.
n
-
-
R1
R2
Yielda
72
M. tuberculosis MIC (µ
M)b
1
2
CH2OH C8H17
CH2OH C10H21 66
CH2OH C13H27 85
473
209
3
-
44
4
-
-
COOH C8H17
COOH C10H21 91
73
86
14
52
5
20
23
6
- COOMe C8H17
7
- COOMe C10H21 83
8
- COOMe C13H27 78
323
c
9
1
1
1
1
1
1
2
2
3
3
3
4
4
4
5
5
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
C3H7
C5H11
C8H17
C10H21 86
C13H27 91
C15H31 85
505
c
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Lengthd
111
1.9
1.7
296
17
C8H17
C10H21 60
72
22
20
C5H11
C8H17
C10H21 90
80
90
12
21
309
309
C5H11
C8H17
C10H21 92
82
74
23
40
37
C3H7
C5H11
C8H17
78
72
77
394
22
C12 C14 C16 C17 C18 C19 C20 C21 C22 C24
a %, after purification
bM. tuberculosis H37rv
c Previously reported[37]
d Equivalency to linear fatty acids
It seems possible that the activity displayed by our collection is caused by a synergetic effect
between the ester and the triazole, which not only gives rigidity to the chain. A possible additional
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role may involve another specific effect, different from the double bond replacement. Besides the
general use of 1,2,3-triazoles as a linker or structural mimetic of aromatic rings, there are
examples where there behavior exceeds that role. It has been observed on crystal structures of
1,2,3-triazole inhibitor-protein complexes that nitrogen´s triazole forms hydrogen bond with
specific residues of the amino acids on the active site of the protein contributing to their
stabilization.[47, 48]
The antimicrobial activity of long chain fatty acids have also been reported, being markedly
influenced by the number of carbon atoms present in the alkanol chain and the presence of
unsaturations.[49] In the case of M. tuberculosis the activity of primary linear alcohols from C6 to
C11 have been recently reported.[50] The alcohols are considerably less active than the
corresponding acids, being 1-decanol the most active with MIC of 800 µM. A different report
explored longer-chain alcohols that displayed an antimycobacterial activity with MIC of 5 μg/mL
for C12 (27 μM), C14 (23 μM) and C16 (21 μM).[51] The activity of the alcohol 1 to 3 range from
473 to 44 μM are considerably less active than the corresponding fatty alcohols, indicating that
the triazoles have a negative effect on the activity.
The reported activity of alkynoic acids on M. smegmatis[39] has some interesting coincidence with
some of our results. In that work it was demonstrated that among the analogs with the alkyne on
C2, the 2-octadecynoic acid was the most active, that matches the molecular area what is the
molecular area of our most potent analogs. From a series of octadecynoic acids with the triple
bond between C2 to C5, 2-octadecynoic acid was the most active. In our case, the series members
with the triazole moiety on C2 were considerably less active (compounds 6-8), probably suggesting
a different target than for 2-alkynoic acids. The activity of long fatty acids amide has also been
reported.[52] The activity of those compounds was not as good as the corresponding ester, with
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no members of the collection showing MIC below 12.5
µM. Those results are another indication
that low polarity and lipophilicity do not provide active compounds without specific interactions.
Having completed the analysis of the influence on the chain length, the position of the triazole and
the functional group requirements on C1, it was necessary to determine if the substitution pattern
on the triazole may affect the activity. With that idea in mind, the 1,5-disubstituted analogs (26
and 27) of compounds 11 and 12 were prepared using the same precursors. Azides and alkynes
were refluxed in benzene under CP*Ru(PPh3)2Cl catalysis following Jia´s procedure.[53] The
reaction was not completely selective in our hands[54] and produced, in both cases, a mixture the
1,5- and 1,4-1,2,3-triazoles in a 4:1 ratio, respectively.(Scheme 2) Fortunately, both isomers were
separable, characterizing the 1,5 product by comparison with previously prepared 1,4-analog and
by nOe experiments. The new analogs 26 and 27 were evaluated against M. tuberculosis H37rv
being manifestly less active than their 1,4 regioisomer. Triazole 26 has an MIC of 93
µM,
meanwhile that of 27 was 84 M, being both 1,5-isomers 49 times less active than the
µ
corresponding 1,4. Those results clearly define another important structural feature required for
the pharmacophore.
Scheme 2. Synthesis and antitubercular activity of 1,5-derivatives of the 1,2,3-triazoles most active
analogs.
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2.2.2 In vitro activity against Mt InhA.
Having in mind that the library has been inspired on the InhA inhibitor 8PP reported by Sullivan et
al,[38] analogs 9, 10, 11 and 12 were assayed on the mycobacterial enzyme InhA. The assay was
performed at a fixed enzyme concentration of 20 µM determining the reaction velocity in absence
(v)0 and presence of the inhibitor (v)I The results showed that the inhibition is very poor for the
most potent mycobacterial analog 12 with 99.3 % of enzyme activity and the analog 9 with 69% of
enzyme activity remaining, which is the least active of the four collection members assayed.(Table
2)
The reported enoyl reductases inhibitors are divided based on whether they form a covalent
adduct with the NAD+ cofactor.[55] Based on their structure the prepared analogs should not
require activation by the mycobacterial KatG enzyme. A comparison between the assayed analogs
and FabI/InhA experimentally validated inhibitors revealed that functional groups that should
interact with critical amino acids on the active pocket are not present. Triclosan and their diphenyl
ether inspired InhA analogs contain a phenolic hydroxyl on their structure. The interaction
between this hydroxyl group and the Tyr158 is critical for the inhibition as can be seen on their
crystal structure (2B37.pdb,[38]). The 1,2,3-triazole will probably display similar interactions and
the long fatty acid tail will probably fill the hydrophobic pocket but those interactions do not
provide the required stabilization energy to inhibit the enzyme. Many structurally different
compounds have shown to inhibit FabI,[55] including unsaturated fatty acids. Palmitoleic, oleic,
linoleic, linolenic and arachidonic acids inhibited FabI with an average IC50 of 40 µM being at least
50 time more active that their saturated counterpart and their esters.[56] The binding of those
compounds is facilitated by their carboxylate being more suitable to inhibit the enzyme than 1,2,3-
triazolyl analogs.
Therefore, it is clear that InhA is not the main molecular target of the prepared compounds as they
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are, having at best, a minor contribution on the activity profile of the triazolyl fatty esters activity.
In spite of that, although unlikely, mycobacterial metabolism of the compounds may be required
for activity display, thus activity on inhA over-expressing M. tuberculosis strains would finally
clarify this point. Thus, the action mechanism of the compounds is a matter of future studies. We
believe that based on the scaffold of the compounds it may be connected to the mycobacterial
lipid metabolism.
Table 2. Enzyme inhibition values of the selected analogs.
MIC
% Remaining
activityb
69.0
Comp. a
µM
9
505
111
1.9
10
11
12
73.2
90.1
99.3
1.7
a [Inh] = 20
µM
bInhA remaining activity =vi / v0
2.2.3 In vitro cytotoxicity assay on Vero cells.
The prepared compounds were assayed against Vero cells (kidney Green monkey) to determine
the selectivity against the mycobacteria. All the compounds were non-cytotoxic at the maximum
concentration tested of 4.75
µg/mL. Taking in consideration those values, the selected hits of the
series compounds 11 (cytotoxicity IC50>17.8
µ
M) and 12 (cytotoxicity IC50> 16.1 M) have a
µ
therapeutic index that higher than 10 which is very promising[57] being good candidate to move
to further in vivo studies.
2.2.4 ADME-Tox calculations
One third of new chemical entities failed before reaching clinical trial phase due to poor
pharmacokinetics. The required good oral availability can be achieved by balancing their
partitioning and solubility properties. To rationalize the profile of our analogs computational
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studies of all the synthesized compounds were performed to predict their adsorption, distribution,
metabolism and excretion (ADME) properties, Lipinski’s rule of five, toxicity liabilities and drug
likeness. The calculation were performed using the web-based software Molinspiration,[58]and
Osiris.[59] The Molinspiration platform uses a sum of fragment-based contributions and correction
factors being able to calculate parameters of most of the organic and even organometallic
molecules. According to Lipinski’s rule of five a potential drug candidate molecule should be orally
active if their molecular weight is
≤
500 Da, logP
≤ 5, the number of hydrogen bond acceptors ≤ 10
and the number of hydrogen bond donors
≤
5. The analysis of the collection revealed that all the
compound have a MW<500 (187 to 365 Da), there are 8 that violated the rule with logP > 5, and all
fulfill the restrictions on the number of hydrogen donors (4 or 5) and acceptors (0 or 1). Since an
antimycobacterial drug has to cross the waxy mycobacterial cell wall, it is usual to find a
correlation between the compound activity and its partition coefficient, with the more lipophilic
being more active. A chart correlating the log (MIC µM) vs logP did not have a connection for all
the compounds, but it was clear that two set of compounds followed a linear correlation as was
expected.(Figure 5, red and blue, respectively) An interesting outcome of this chart is the location
of compounds 11 and 12. Those are the most active compounds and did not follow the same
behavior than the rest of the collection. The fact that those compounds are considerable more
active due to their lipophilicity could be the result of a specific interaction with an unknown
molecular target than enhance their activity beyond their partition coefficient.
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Figure 5.Correlation between logP and antitubercular activity.
The analysis by the Osiris platform provided the toxicity risk, drug likeness and a drug score. The
results revealed that all the compounds did not have any toxicity risk, undesired effects;
tumorigenic features or effect on the reproductive system. The solubility is a critical property
which aids in the circulation of a drug after the administration and into the bloodstream. More
than 80% of marketed drugs have logS > -4. The Osiris estimation, measured in mol/liter, provides
a promising range of -4.32 to -1.62 for the studied 1,2,3-triazoles. In terms of drug likeness the
prepared collection displayed a poor profile on (-9.70 to -39.79) compared to commercial drugs
like fluconazole or rifampicin (1.99 and 10.5 respectively). That poor behavior is balanced with the
rest of the parameters used to calculate the drug score (solubility, logP, MW and toxicity risk)
delivering moderate to good values compared with a standard drug (0.25 to 0.49).
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A detailed analysis of compounds 11 and 12 shown that 11 has a better drug score (0.45 vs 0.41),
is more soluble (-1.73 vs -2.97), has lower MW (267.37 vs 295.43), logP (3.97 vs 4.98) and equal
drug likeness. Considering that both compounds have almost the same antimycobacterial potency,
it promotes 11 as the selected candidate for future studies.
2.2.7 Activity of 11 on M. tuberculosis drug resistant strains
As was mentioned before, the appearance of resistant M. tuberculosis strains is a major problem
for TB treatment. For that reason, we evaluated the activity of the most promising analog, triazole
11, on different antibiotic resistant strains. The compound was assayed on a panel of 19 clinical
isolates that were resistant to rifampicin, streptomycin, isoniazid, pyrazinamide or their
combinations.(Table 3) As can be seen, the compound maintained its potency (MIC 1.9 µM)on
pansusceptible strains (164, 304, 598; except strain 51 that was more sensitive),the rifampicin
resistant strains (110, 277, 300, 925 and 958)and the isoniazid resistant strains (277b and 620).
The compound was equally potent toward the MDR strains (396, 681, 780, 914, 675), showing
uneven behavior on the two triple-resistant strains (122 is more sensitive and 501 less active).
However, in MIC for both strains are in a narrow concentration range and could be explain by
strain differences unrelated to the susceptibility to compound 11.
In summary, the compound maintained the activity on most of the 15 clinically isolated strains
indicating that the resistance mechanisms present in the clinical isolates do not affect 11.
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Table 3. Activity of 11 on different resistant and multiresistant
M. tuberculosis strains.
Strain
H37Rv
51
164
304
598
110
277
300
Resistance
Strain
277b
620
396
681
780
914
675
122
Resistance
Inh
MIC µM
1.9
0.45
1.9
MIC µM
1.9
-
-
-
-
-
Rif
Rif
Rif
Rif
Rif
Inh
1.9
1.9
1.9
1.9
1.9
1.9
0.9
3.7
Inh-Sm
Inh-Sm
Inh-Sm
Inh-Sm
Rif -Sm
Rif-Inh-Pza
Rif-Inh-Pza
1.9
1.9
1.9
1.9
1.9
1.9
1.9
925
958
501
a) Rif= rifampicin, Sm=streptomycin, Inh= isoniazid, Pza=pyrazinamide
3. Conclusions
In the present study a set of twenty five 1,5-subtituted-1,2,3-triazolyl fatty esters were
prepared with good yields. The collection was assayed against M. tuberculosis H37rv and thirteen
of the newly prepared compounds have MIC of below 60 µM. The activity profile of the series
allowed to reach a well-defined structural-activity relationship that in turn allowed setting the
structural requirements that define a new antimycobacterial scaffold. The pharmacophore
requirements are described on the Figure 6. To maximize their activity the compounds have to
contain an ester on C1, the triazole has to have a 1,4-alkyl-substitution that has to be between C17
to C19 long. The studies conducted to validate the target of 11 revealed that are not linked to InhA
inhibition. Also, 11 activity was not affected by the resistant mechanisms to rifampicin,
streptomycin, isoniazid and pyrazinamide present in clinical isolates. In general, the prepared
compounds showed suitable drug score and good ADME-tox properties and are expected to
present good bioavailability profile. In summary, 11 has emerged as a valuable candidate
considering its simple preparation and its high potency. The possibility that their activity could
target pathway(s) different than the ones targeted by standard therapeutic antimycobacterial
drugs encourages further mechanistic studies and additional exploration of structural
modifications with enhanced activity.
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Figure 6.Structural requirements for activity.
4. Material and methods
4.1. General
Chemical reagents were purchased from commercial suppliers and were used without further
purification unless otherwise noted. All the solvents (hexane, ethyl acetate, CH2Cl2, Et2O) were
distilled prior to use. Yields were calculated for material judged homogenous by thin layer
chromatography and nuclear magnetic resonance (NMR). Reaction progress was monitored by
thin layer chromatography (TLC) performed on silica gel 60 F254 pre-coated aluminum sheets.
Spots were visualized under 254 nm UV lamp and/or by dipping the TLC plate into a solution of p-
anisaldehyde or phosphomolibdic acid followed by heating with a heat gun. Flash column
chromatography was performed using silica gel 60 (230 – 400 mesh). 1H and 13C NMR spectra were
recorded in a Bruker Avance II 300 MHz (75.13 MHz) using CDCl3 as solvent unless otherwise
noted. Chemical shifts for proton nuclear magnetic resonance (1H NMR) spectra are reported in
parts per million relative to the signal of tetramethylsilane at 0 ppm (internal standard) and
coupling constants (J) are reported in hertz (Hz). Chemical shifts for carbon nuclear magnetic
resonance (13C NMR) spectra are reported in parts per million relative to the center line of the
CDCl3triplet at 76.9 ppm. Assignments of proton resonances were confirmed by correlated
spectroscopy. ESI-HRMS were recorded at the University of Mississippi, Department of Medicinal
Chemistry on a Waters LC-QTof micro.
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4.2. Synthesis
4.2.1. General procedure for Azides and Alkynes reaction under click chemistry conditions:
Alkyne (1 eq) and the azide (1 eq) were suspended in mL/eq of t-BuOH:H2O (1:1) and then
aqueous 1M CuSO4 solution and finally aqueous 1M sodium ascorbate solution were added and
the mixture stirred overnight at room temperature. Brine was added and the solution was
extracted with dichloromethane. Combined organic extracts were dried with sodium sulphate and
evaporated. The resulting residue was purified by column chromatography over silica gel using an
increasing AcOEt/hexane gradient to afford desired pure products.
4.2.1.1. (1-octyl-1H-1,2,3-triazol-4-yl)methanol (1)
White solid. M.p. 49.0-49.8 °C. 1H NMR (300 MHz, CDCl3):
δ7.50 (s, 1H), 4.79 (s, 2H), 4.33 (t, J = 7.3
Hz, 2H), 1.91-1.85 (m, 2H), 1.65 (m, 2H), 1.31-1.25 (m, 10H), 0.87 (t, J = 6.3 Hz, 3H).13C NMR (75
MHz, CDCl3): δ147.5 (C), 121.4 (CH), 56.7 (CH2), 50.4 (CH2), 31.7 (CH2), 30.3 (CH2), 29.0 (CH2), 28.9
(CH2), 26.5 (CH2), 22.6 (CH2), 14.0 (CH3), IR (KBr) υ: 3400, 2953, 2922, 2852, 1651, 1465, 1220, 1145
cm-1. ESI-HRMS Calcd for (M+H+) C11H21N3O 212.1763, found 212.1768.
4.2.1.2. (1-decyl-1H-1,2,3-triazol-4-yl)methanol (2)
White solid. Mp: 61.3-61.0 °C. 1H NMR (300 MHz, CDCl3):
δ 7.51 (s, 1H), 4.79 (s, 2H), 4.34 (t, J = 7.3
Hz, 2H), 1.89 (q, J = 5.8 Hz, 2H), 1.67-1.50 (m, 2H), 1.31-1.25 (m, 18H), 0.87 (t, J = 6.3 Hz, 3H).13C
NMR (75 MHz, CDCl3): δ130.9 (C), 121.4 (CH), 56.6 (CH2), 50.4 (CH2), 31.7 (CH2), 30.3 (CH2), 29.0
(CH2), 27.7 (CH2), 19.2 (CH2), 22.6 (CH2), 14.0 (CH3). IR (KBr) υ: 3385, 2954, 2920, 2846, 1730, 1462,
1274, 1122, 1072, 746 cm-1. ESI-HRMS Calcd for (M+H+) C13H26N3 O 240.2076, found 240.2083.
4.2.1.3. (1-tridecyl-1H-1,2,3-triazol-4-yl)methanol (3)
White solid. M.p. 78.8-79.6 °C. 1H NMR (300 MHz, CDCl3):
δ 7.51 (s, 1H), 4.79 (s, 2H), 4.34 (t, J = 7.2
Hz, 2H), 1.92-1.87 (m, 2H), 1.68 (m, 2H), 1.31-1.25 (m, 20H), 0.87 (t, J = 6.3 Hz, 3H).13C NMR (75
MHz, CDCl3):
δ
121.4 (C), 56.6 (CH2), 50.4 (CH2), 31.9 (CH2), 30.3 (CH2), 9.6 (CH2), 29.5 (CH2), 29.4
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(CH2), 29.3 (CH2), 29.0 (CH2), 26.5 (CH2), 22.7 (CH2), 14.1 (CH3). IR (KBr) υ: 3415, 2918, 2847, 1643,
1465, 1274, 860, 750 cm-1. ESI-HRMS Calcd for (M+H+) C16H32N3 O 282.2545, found 282.2539.
4.2.1.4. 1-octyl-1H-1,2,3-triazole-4-carboxylic acid (4)
White solid. Mp: 158.8-159.5 °C.1H NMR (300 MHz, DMSO-d6):
δ 8.68 (s, 1H), 4.37 (t, J = 7.0 Hz,
2H), 3.35 (t, J = 4.5 Hz, 2H), 1,83 (q, J = 10.8 Hz, 2H), 1.23 (m,12H), 0.83 (t, J = 6.9 Hz, 3H).13C NMR
(75 MHz, DMSO):
δ 161.2 (C), 139.0 (C), 128.2 (CH), 49.1 (CH2), 30.6 (CH2), 28.9 (CH2), 27.9 (CH2),
27.7 (CH2), 25.1 (CH2), 21.5 (CH2), 13.4 (CH3). IR (KBr) 3117, 2957,2920, 2848, 2355, 1681, 1467,
1234, 1047, 602 cm-1. ESI-HRMS Calcd for (M+H+) C11H20N3O2 226.1556, found 226.1546.
4.2.1.5. 1-decyl-1H-1,2,3-triazole-4-carboxylic acid (5)
White solid. M.p. 150.4-151.1 °C. 1H NMR (300 MHz, DMSO):
δ 8.67 (s, 1H), 4.37 (t, J = 6.4 Hz, 2H),
3.35 (t, J = 4.5 Hz, 2H), 1,80 (t, J = 12.8 Hz, 2H), 1.21 (m, 14H), 0.85 (t, J = 6.3 Hz, 3H).13C NMR (75
MHz, DMSO): δ161.7 (C), 139.5 (C), 128.7 (CH), 60.7 (CH2), 49.6 (CH2), 31.2 (CH2), 29.4 (CH2), 28.8
(CH2), 28.8 (CH2), 28.6 (CH2), 28.2 (CH2), 25.6 (CH2), 22.0 (CH2), 13.9 (CH3). IR (KBr) 3117, 2359,
1541, 1465, 1339, 1234, 1045, 779 cm-1. ESI-HRMS Calcd for (M+Na+) C13H23N3O2Na 276.1688,
found 276.1680.
4.2.1.6. Methyl 1-octyl-1H-1,2,3-triazole-4-carboxylate (6)
1
White solid. M.p. 75.7-76.5 °C. H NMR (300 MHz, CDCl3):
δ 8.07 (s, 1H), 4.40 (t, J = 7.2 Hz, 2H),
3.95 (s, 3H), 1.97-1.90 (m, 2H), 1.62 (s, 2H), 1.31-1.26 (m, 10H), 0.87 (t, J = 6.3 Hz, 3H).13C NMR (75
MHz, CDCl3): 161.3 (C), 139.9 (C), 127.2 (CH), 52.2 (CH2), 50.7 (CH2), 31.6 (CH2), 30.1 (CH2), 29.0
δ
(CH2), 28.9 (CH2), 26.3 (CH2), 22.6 (CH2), 14.0 (CH3). IR (KBr) υ: 3120, 2953, 2914, 2846, 1730, 1539,
1469, 1384, 1348, 1234, 1047, 881, 777 cm-1. ESI-HRMS Calcd for (M+H+) C12H21N3 O2 240.1712,
found 240.1724.
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4.2.1.7. Methyl 1-decyl-1H-1,2,3-triazole-4-carboxylate (7)
1
White solid. M.p. 95.4-96.0 °C. H NMR (300 MHz, CDCl3):
δ 8.07 (s, 1H), 4.40 (t, J = 7.2 Hz, 2H),
3.95 (s, 3H), 1.92 (q, J = 6.4 Hz, 2H), 1.31-1.24 (m, 18H), 0.87 (t, J = 6.3 Hz, 3H).13C NMR (75 MHz,
CDCl3):
δ 161.3 (C), 139.9 (C), 127.2 (CH), 52.2 (CH3), 50.7 (CH2), 31.8 (CH2), 30.1 (CH2), 29.4 (CH2),
29.3 (CH2), 29.2 (CH2), 28.9 (CH2), 26.3 (CH2), 22.6 (CH2), 14.1 (CH3). IR (KBr) υ: 3130, 2999, 2958,
2358, 1753, 1726, 1543, 1377, 1022, 871, 779 cm-1. ESI-HRMS Calcd for (M+H+) C14H26N3O2
268.2025, found 268.2015.
4.2.1.8. Methyl 1-tridecyl-1H-1,2,3-triazole-4-carboxylate (8)
White solid. Mp: 96.2-96.9 °C. 1H NMR (300 MHz, CDCl3):
δ 8.07 (s, 1H), 4.39 (t, J = 7.2 Hz, 2H), 3.94
(s, 3H), 1.92 (q, J = 6.9 Hz, 2H), 1.31-1.24 (m, 20H), 0.87 (t, J = 6.3 Hz, 3H).13C NMR (75 MHz, CDCl3):
δ
161.3 (C), 139.9 (C), 127.2 (CH), 52.2 (CH3), 50.7 (CH2), 31.9 (CH2), 31.3 (CH2), 29.6 (CH2), 29.6
(CH2), 29.5 (CH2), 29.3 (CH2), 28.9 (CH2), 26.4 (CH2), 22.7 (CH2), 14.1 (CH3). IR (KBr) υ: 3120, 2915,
2846, 2355, 1732, 1543, 1468, 1242, 1051, 777, 719 cm-1. ESI-HRMS Calcd for (M+H+) C17H32N3O2
310.2495, found 310.2491.
4.2.1.9. Ethyl 2-(4-decyl-1H-1,2,3-triazol-1-yl)acetate (12)
White solid. Mp: 61.1-62.0 °C. 1H NMR (300 MHz, CDCl3):
Hz, 2H), 2.73 (t, J = 7.7 Hz, 3H), 1.70-1.63 (m, 3H), 1.29 (t, J = 7.1 Hz, 3H), 1.26 (m, 16H), 0.88 (t, J =
6.7 Hz, 3H).13C NMR (75 MHz, CDCl3):
166.7 (C), 149.2 (C), 122.1 (CH), 62.5 (CH2), 51.0 (CH2), 32.1
δ 7.40 (s, 1H), 5.11 (s, 2H), 4.26 (q, J = 7.1
δ
(CH2), 29.9 (CH2), 29.8 (CH2), 29.6 (CH2), 29.4 (CH2), 25.9 (CH2), 22.9 (CH2), 14.3 (CH3), 14.3 (CH3). IR
(KBr) υ: 3122, 3064, 2954, 2918, 2848, 1745, 1467, 1377, 1220, 1055, 1033, 721 cm-1. ESI-HRMS
Calcd for (M+H+) C16H30N3O2 296.2338, found 296.2341.
4.2.1.10 Ethyl 2-(4-tridecyl-1H-1,2,3-triazol-1-yl)acetate (13)
White solid. M.p. 70.2-71.1 °C.1H NMR (300 MHz, CDCl3):
δ 7.40 (s, 1H), 5.11 (s, 2H), 4.26 (q, J = 7.1
Hz, 2H), 2.73 (t, J = 7.8 Hz, 3H), 1.70-1.62 (m, 2H), 1.29-1.25 (m, 26H), 0.87 (t, J = 6.6 Hz, 3H).13C
24

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NMR (75 MHz, CDCl3):
δ
166.7 (C), 149.1 (C), 122.1 (CH), 62.5 (CH2), 50.9 (CH2), 32.1 (CH2), 29.8
(CH2), 29.7 (CH2), 29.5 (CH2), 29.4 (CH2), 25.8 (CH2), 22.9 (CH2), 14.3 (CH3), 14.2 (CH3). IR (KBr)
υ: 3064, 2953, 2916, 2848, 1745, 1469, 1456, 1375, 1220, 1055, 1035, 719 cm-1. ESI-HRMS Calcd
for (M+H+) C19H36N3O2 338.2808, found 338.2808.
4.2.1.11. Ethyl 2-(4-pentadecyl-1H-1,2,3-triazol-1-yl)acetate (14)
White solid. Mp: 76.2-77.1 °C.1H NMR (300 MHz, CDCl3):
Hz, 2H), 2.73 (t, J = 7.8 Hz, 3H), 1.69-1.65 (m, 3H), 1.29 (t, J = 14.3 Hz, 3H), 1.25 (m, 23H), 0.87 (t, J
= 6.3 Hz, 3H).13C NMR (75 MHz, CDCl3):
166.7 (C), 149.1 (C), 122.1 (CH), 62.5 (CH2), 51.0 (CH2),
δ 7.39 (s, 1H), 5.11 (s, 2H), 4,25 (q, J = 7.1
δ
32.1 (CH2), 29.9 (CH2), 29.7 (CH2), 29.5 (CH2), 29.4 (CH2), 25.8 (CH2), 22.9 (CH2), 14.3 (CH3), 14.23
(CH3). IR (KBr) υ: 3064, 2954, 2916, 2848, 1747, 1469, 1222, 1197, 1056 cm-1. ESI-HRMS Calcd for
(M+H+) C21H40N3O2 336.3121, found 366.3136.
4.2.1.12. Ethyl 3-(4-octyl-1H-1,2,3-triazol-1-yl)propanoate (15)
1
Colourless oil. H NMR (300 MHz, CDCl3):
δ
7.34 (s, 1H), 4.60 (t, J = 6.4 Hz, 2H), 4.15 (q, J = 7.1 Hz,
2H), 2.94 (t, J = 6.6 Hz, 2H), 2.68 (t, J = 8.1 Hz, 2H), 1.67-1.59 (m, 7H), 1.28 (m, 14H), 1.24 (t, J = 7.1
Hz, 3H), 0.87 (t, J = 6.6 Hz, 3H).13C NMR (75 MHz, CDCl3):
170.6 (C), 148.3 (C), 121.4 (CH), 61.1
δ
(CH2), 45.3 (CH2), 34.8 (CH2), 31.8 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 25.6 (CH2),
22.6 (CH2), 14.0 (CH3). IR (film) υ: 2956, 2852, 1732, 1552, 1446, 1195, 1018, 945, 858, 723 cm-1.
ESI-HRMS Calcd for (M+H+) C15H28N3O2 282.2182, found 282.2192.
4.2.1.13. Ethyl 3-(4-decyl-1H-1,2,3-triazol-1-yl)propanoate (16)
White solid. Mp:43.1-44.0 °C. 1H NMR (300 MHz, CDCl3):
(q, J = 7.0 Hz, 2H), 2.93 (t, J = 6.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 1.67-1.59 (m, 7H), 1.26 (t, J = 7.1
Hz, 3H), 1.25 (m, 14H), 0.87 (t, J = 6.9 Hz, 3H).13C NMR (75 MHz, CDCl3):
170.6 (C), 148.3 (C), 121.4
δ 7.34 (s, 1H), 4.60 (t, J = 6.4 Hz, 2H), 4,15
δ
(CH), 61.1 (CH2), 45.3 (CH2), 34.8 (CH2), 31.8 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2),
25

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25.6 (CH2), 22.6 (CH2), 14.0 (CH3). IR (KBr) υ: 2926, 2854, 1753, 1556, 1456, 1375, 1211, 1026, 987,
819 cm-1. ESI-HRMS Calcd for (M+H+) C17H32N3O2 310.2495, found 310.2502.
4.2.1.14. Ethyl 4-(4-pentyl-1H-1,2,3-triazol-1-yl)butanoate (17)
1
Colourless oil. H NMR (300 MHz, CDCl3):
δ 7.26 (s, 1H), 4.38 (t, J = 6.8 Hz, 2H), 4,13 (q, J = 7.1 Hz,
2H), 2.69 (t, J = 7.5 Hz, 2H), 2.32 (t, J = 6.6 Hz, 2H), 2.25-2.15 (m, 2H), 1.68-1.63 (m, 3H), 1.35-1.30
(m, 4H), 1.28 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3):
172.3 (C), 148.4
δ
(C), 120.7 (CH), 60.5 (CH2), 48.9 (CH2), 31.3 (CH2), 30.7 (CH2), 29.0 (CH2), 25.5 (CH2), 25.4 (CH2), 22.3
(CH2), 14.1 (CH3), 13.9 (CH3). ESI-HRMS Calcd for (M+H+) C13H24N3O2 254.1869, found 254.1861.
4.2.1.15. Ethyl 4-(4-octyl-1H-1,2,3-triazol-1-yl)butanoate (18)
White solid. M.p. 42.7-43-3 °C.1H NMR (300 MHz, CDCl3):
(q, J = 7.1 Hz, 2H), 2.69 (t, J = 8.0 Hz, 2H), 2.32 (t, J = 6.6 Hz, 2H), 2.25-2.15 (m, 2H), 1.68-1.60 (m,
3H), 1.31-1.26 (m, 10H), 1.25 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3):
δ 7.26 (s, 1H), 4.38 (t, J = 6.8 Hz, 2H), 4,13
δ
172.3 (C), 148.5 (C), 120.7 (CH), 60.6 (CH2), 49.0 (CH2), 31.8 (CH2), 30.7 (CH2), 29.4 (CH2), 29.3 (CH2),
29.2 (CH2), 25.6 (CH2), 25.5 (CH2), 25.6 (CH2), 22.6 (CH2), 14.1 (CH3), 14.0 (CH3). IR (KBr) υ: 3109,
3057, 2918, 2850, 1732, 1467, 1215, 1184, 1053, 871, 806, 721 cm-1. ESI-HRMS Calcd for (M+H+)
C16H30N3O2 296.2338, found 296.2331.
4.2.1.16. Ethyl 4-(4-decyl-1H-1,2,3-triazol-1-yl)butanoate (19)
1
White solid. M.p. 49.1-49.8 °C. H NMR (300 MHz, CDCl3):
δ
7.26 (s, 1H), 4.38 (t, J = 6.8 Hz, 2H),
4.13 (q, J = 7.1 Hz, 2H), 2.69 (t, J = 7.5 Hz, 2H), 2.32 (t, J = 6.6 Hz, 2H), 2.25-1.17 (m, 2H), 1.67-1.62
(m, 2H), 1.31-1.24 (m, 14H), 1.25 (t, J = 14.3 Hz, 3H), 0.87 (t, J = 6.3 Hz, 3H). 13C NMR (75 MHz,
CDCl3): δ172.3 (C), 148.4 (C), 120.6 (CH), 60.5 (CH2), 48.9 (CH2), 31.8 (CH2), 30.7 (CH2), 29.5 (CH2),
29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.2 (CH2), 25.6 (CH2), 25.4 (CH2), 22.6 (CH2), 14.1
(CH3), 14.0 (CH3). IR (KBr) υ: 2927, 2856, 1759, 1554, 1462, 1375, 1209, 1026, 819 cm-1.
4.2.1.17. Ethyl 5-(4-pentyl-1H-1,2,3-triazol-1-yl)pentanoate (20)
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1
Colourless oil. H NMR (300 MHz, CDCl3):
2H), 2.70 (t, J = 7.5 Hz, 2H), 2.33 (t, J = 6.6 Hz, 2H), 1.99-1.89 (m, 2H), 1.67-1.61 (m, 4H), 1.33 (m,
4H), 1.24 (t, J = 7.1 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3):
172.9 (C), 148.4 (C),
δ7.25 (s, 1H), 4.33 (t, J = 6.8 Hz, 2H), 4.69 (q, J = 7.1 Hz,
δ
120.5 (CH), 60.4 (CH2), 49.6 (CH2), 33.4 (CH2), 31.4 (CH2), 29.6 (CH2), 29.1 (CH2), 25.6 (CH2), 22.3
(CH2), 21.8 (CH2), 14.1 (CH3), 13.9 (CH3). IR (film) υ: 2954, 2931, 2358, 1732, 1458, 1375, 1182,
1047, 1029 cm-1. ESI-HRMS Calcd for (M+H+) C14H26N3O2 268.2025, found 268.2038.
4.2.1.18. Ethyl 5-(4-octyl-1H-1,2,3-triazol-1-yl)pentanoate (21)
1
Colourless oil. H NMR (300 MHz, CDCl3):
δ 7.24 (s, 1H), 4.31 (t, J = 7.1 Hz, 2H), 4,11 (q, J = 7.1 Hz,
2H), 2.69 (t, J = 7.5 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.98-1.88 (m, 2H), 1.67-1.61 (m, 4H), 1.25 (m,
10H), 1.24 (t, J = 7.0 Hz, 3H), 0.87 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3):
173.0 (C), 148.5
δ
(C), 120.5 (CH), 60.4 (CH2), 49.7 (CH2), 33.4 (CH2), 31.9 (CH2), 29.7 (CH2), 29.5 (CH2), 29.3 (CH2), 29.3
(CH2), 29.2 (CH2), 25.7 (CH2), 22.7 (CH2), 21.8 (CH2), 14.2 (CH3), 14.1 (CH3). IR: 3134, 2957, 2936,
2870, 2359, 1730, 1551, 1462, 1373, 1213, 1182, 1034 cm-1. ESI-HRMS Calcd for (M+H+) C17H32N3O2
310.2495, found 310.2501.
4.2.1.19. Ethyl 5-(4-decyl-1H-1,2,3-triazol-1-yl)pentanoate (22).
White solid. M.p. 46.9-47.9 °C.1H NMR (300 MHz, CDCl3):
(q, J = 7.1 Hz, 2H), 2.69 (t, J = 7.5 Hz, 2H), 2.33 (t, J = 7.3 Hz, 2H), 1.99-1.89 (m, 2H), 1.69-1.62 (m,
4H), 1.26 (m, 14H), 1.25 (t, J = 7.1 Hz, 3H), 0.88 (t, J = 6.6 Hz, 3H).13C NMR (75 MHz, CDCl3):
172.9
δ7.25 (s, 1H), 4.32 (t, J = 7.1 Hz, 2H), 4.12
δ
(C), 148.4 (C), 120.5 (CH), 60.3 (CH2), 49.6 (CH2), 33.4 (CH2), 31.8 (CH2), 29.6 (CH2), 29.5 (CH2), 29.5
(CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 25.66 (CH2), 22.6 (CH2), 21.8 (CH2), 14.2 (CH3), 14.04 (CH3).
IR (KBr) υ: 3055, 2918, 2848, 1730, 1552, 1469, 1267, 1029, 736 cm-1. ESI-HRMS Calcd for (M+H+)
C19H36N3O2338.2808, found 338.2800.
4.2.1.20. Ethyl 6-(4-propyl-1H-1,2,3-triazol-1-yl)hexanoate (23)
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Colourless oil. H NMR (300 MHz, CDCl3):
δ 7.24 (s, 1H), 4.30 (t, J = 7.1 Hz, 2H), 4.10 (q, J = 7.1 Hz,
2H), 2.68 (t, J = 7.5 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.95-1.85 (m, 2H), 1.73-1.62 (m, 4H), 1.73-1.62
(m, 4H), 1.37 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H), 0.95 (t, J = 7.3 Hz, 3H).13C NMR (75 MHz, CDCl3):
δ
173.3 (C), 148.1 (C), 120.6 (CH), 60.2 (CH2), 49.8 (CH2), 33.9 (CH2), 30.0 (CH2), 27.6 (CH2), 25.9
(CH2), 24.2 (CH2), 22.6 (CH2), 14.2 (CH3), 13.7 (CH3). IR (film) υ: 2956, 2935, 2870, 2358, 1730, 1550,
1462, 1373, 1213, 1182, 1033 cm-1. ESI-HRMS Calcd for (M+H+) C13H24N3O2 254.1869, found
254.1874
4.2.1.21. Ethyl 6-(4-pentyl-1H-1,2,3-triazol-1-yl)hexanoate (24)
Colourless oil. 1H NMR (300 MHz, CDCl3):
δ7.24 (s, 1H), 4.30 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz,
2H), 2.70 (t, J = 7.5 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.95-1.85 (m, 2H), 1.73-1.62 (m, 4H), 1.71-1.61
(m, 4H), 1.40-1.29 (m, 6H), 1.24 (t, J = 7.1 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H).13C NMR (75 MHz, CDCl3):
δ
173.3 (C), 148.4 (C), 120.5 (CH), 60.3 (CH2), 49.8 (CH2), 33.9 (CH2), 31.4 (CH2), 30.0 (CH2), 29.1
(CH2), 25.9 (CH2), 25.6 (CH2), 24.2 (CH2), 22.4 (CH2), 14.2 (CH3), 14.0 (CH3). IR (film) υ: 3134, 2931,
2858, 1730, 1550, 1462, 1373, 1180, 1047 cm-1. ESI-HRMS Calcd for (M+H+) C15H28N3O2 282.2182,
found 282.2194.
4.2.1.22. Ethyl 6-(4-octyl-1H-1,2,3-triazol-1-yl)hexanoate (25)
Colourless oil. M.p. 34.2-35.0 °C.1H NMR (300 MHz, CDCl3):
δ 7.24 (s, 1H), 4.30 (t, J = 7.2 Hz, 2H),
4,11 (q, J = 7.1 Hz, 2H), 2.69 (t, J = 7.8 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.95-1.85 (m, 2H), 1.71-1.63
(m, 4H), 1.34-1.26 (m, 12H), 1.25(t, J = 7.1 Hz, 3H), 0.87 (t, J = 6.3 Hz, 3H).13C NMR (75 MHz, CDCl3):
δ
173.4 (C), 148.5 (C), 120.5 (CH), 60.3 (CH2), 49.9 (CH2), 34.0 (CH2), 31.9 (CH2), 30.1 (CH2), 29.5
(CH2), 29.4 (CH2), 29.3 (CH2), 29.3 (CH2), 26.0 (CH2), 25.7 (CH2), 24.3 (CH2), 22.7 (CH2), 14.3 (CH3),
14.1 (CH3). IR (film) υ: 2926, 2854, 2358, 1735, 1463, 1375, 1180, 1047 cm-1. ESI-HRMS Calcd for
(M+H+) C18H34N3O2 324.2651, found 324.2660
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