1,3,5-Triazino Peptide Derivatives: Synthesis, Characterization, and Preliminary Antileishmanial Activity

Article abstract of DOI:10.1002/cmdc.201700770

A library of short di-, tri-, and tetra-peptides with an s-triazine moiety at the N terminus and either an amide or ethyl ester C terminus was prepared in solution and on the solid phase. The two remaining positions of the s-triazine moiety were substitut

Full text of DOI:10.1002/cmdc.201700770

Accepted Article
Title: 1,3,5-Triazino-Peptide Derivatives: Synthesis, Characterization
and Preliminary Antileishmanial Activity
Authors: Sherine N Khattab, Hosam H Khalil, Adnan A Bekhit,
Mohamed M Abd El-Rahman, Beatriz G de la Torre, Ayman
El-Faham, and Fernando Albericio
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To be cited as: ChemMedChem 10.1002/cmdc.201700770
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10.1002/cmdc.201700770
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1,3,5-Triazino-Peptide Derivatives: Synthesis, Characterization
and Preliminary Antileishmanial Activity
Sherine N. Khattab,* [a,b] Hosam H. Khalil,[a] Adnan A. Bekhit,[b,c] Mohamed M. Abd El-Rahman,[a] Beatriz G. de la
Torre,[d] Ayman El-Faham,*[a,e] and Fernando Albericio,[e,f,g,h]
Abstract: A library of short di- to tetra-peptides with s-triazine moiety
at the N-terminal and the C-terminal in the form of either ethyl ester
or amide were prepared in solution and in solid-phase. The two
remaining positions of the s-triazine moiety were substituted by
dimethoxy, dimorpholino, or dipiperidino groups. All the synthesized
peptide derivatives were analyzed by HPLC and fully characterized
by IR, NMR (1H-NMR and 13C-NMR), elemental analysis, and mass
spectra analysis (MALDI TOF/TOF). A preliminary study of the
antileishmanial activity of the 1,3,5-triazinyl-peptide derivatives
revealed that four dipeptide amide derivatives showed better
antipromastigote or antiamastigote activity than that of the reference
standard drug miltefosine with no significance acute toxicity.
disorders and they are also used as antimicrobial, antiviral and
analgesic agents, among others.[4] To date, radiolabeled small
peptides have mainly been utilized in oncology for diagnostic
imaging and targeting radiotherapy.[5] Furthermore, given that
peptides can be taken up by cells, these compounds are
particularly useful for drug delivery purposes.[6]
Antimicrobial peptides (AMPs) deserve special mention as they
are found endogenously in many species of animals and plants.
Most AMPs have a low molecular mass and serve as defense
and protection against predators and microorganisms.[7] Amino
acid and dipeptide esters that contain at least one hydrophobic
amino acid show leishmanicidal activities.[8] The intralesional
administration of several compounds also restricts the growth of
mouse lesions.[8] However, the esters are known to be toxic in
vitro for monocytes and certain lymphoid cells.[9] Furthermore,
triazines, which act as dihydrofolate reductase inhibitors
(DHFRs),[10] also show potential as antileishmanial agents.[11]
The World Health Organisation considers leishmaniasis to be
among the seventeen most negelected dangerous tropical
protozoal diseases.[12] Indeed, the prevalence of this disease
continues to increase, causing morbidity worldwide.
Leishmaniasisis is caused by insect vectorborne protozoan
parasites and it affects an estimated 12 million people a year,
with a further 350 million people at risk of infection.[13] Despite
attempts to control this disease over the last few decades, its
prevalence has increased in developing countries.[14]
Introduction
Peptides are characterized by broad chemical diversity, which is
translated into a range of biological activities of interest.[1] In this
regard, even small peptides have marked physiological effects
on living organisms; for example, the tripeptide glutathione is
found in most living cells; the pentapeptide enkephalin is a
naturally occurring analgesic; and the nonapeptide oxytocin
induces the contraction of the uterine muscle in women during
labor.[2] Small peptides (3-9 amino acid residues) find
applications in diverse therapeutic areas including cancer,[3]
asthma, allergy, Ca2+ metabolism, and central nervous system
The various clinical forms of leishmaniasis constitute a serious
public health concern. Visceral leishmaniasis (VL) is usually
fatal when untreated, muco-cutaneous leishmaniasis (MCL) is a
mutilating disease, diffuse cutaneous leishmaniasis (DCL) is a
disabling disease, and cutaneous leishmaniasis (CL) is also
disabling when lesions are multiple.[15] Cutaneous leishmaniasis
(CL) is endemic in over 80 countries. In the Americas, CL is
widely distributed, from southern Texas to northern Argentina.
Other endemic areas include the Middle East, India, Pakistan,
Iran, and North and East Africa.[15a, 16] It is worth mentioning that
Cutaneous leishmaniasis is caused mainly by the following
species L. major, L. tropica, and L. aethiopica in the Old World
while by L. mexicana, L. amazonensis, L. braziliensis, L.
panamanesis, and L. guyanensis in the New World.[15a, 16]
[a] Prof. Sh. N. Khattab; Dr. H. H. Khalil; Prof. M. M. Abd El-Rahman;
Prof. A. El-Faham
Department of Chemistry, Faculty of Science, Alexandria University,
P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt. E-mail:
sh.n.khattab@gmail.com
[b] Prof. Sh. N. Khattab; Prof. Adnan A. Bekhit
Cancer Nanotechnology Research Laboratory (CNRL), Faculty of
Pharmacy, Alexandria University, Alexandria 21521, Egypt.
[c] Prof. Adnan A. Bekhit
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Alexandria University, Alexandria 21521, Egypt.
[d] Prof. Beatriz G. de la Torre
KRISP, School of Health Sciences, University of KwaZulu-Natal,
Durban 4001, South Africa.
[e] Prof. A. El-Faham; Prof. Fernando Albericio
Department of Chemistry, College of Science, King Saud University,
P.O. Box 2455, Riyadh11451, Saudi Arabia. E-mail:
aymanel_faham@hotmail.com
Almost all the drugs currently used to treat the disease show
resistance and side effects.[14]
A
vaccine to prevent
leishmaniasis is not available despite considerable research
efforts in this field.[17] Consequently, the treatment of this
condition relies entirely on a considerably limited arsenal of
chemotherapeutics. Both VL and CL are treated mostly with
pentavalent antimonials, such as sodium stibogluconate
(Pentostam) and meglumine antimoniate (Glucantime),[18] which
are highly toxic. However, an increasing non-susceptibility of the
parasites to antimonials injection in the area with the world’s
highest prevalence of disease (North Bihar, India), led to
widespread treatment failure and a shift to amphotericin B and
Miltefosine.[19] Different formulations of amphotericin B are
[f] Prof. Fernando Albericio
School of Chemistry and Physics, University of KwaZulu-Natal, Durban
4001, South Africa.
[g] Prof. Fernando Albericio
CIBER-BBN, Networking Centre on Bioengineering Biomaterials and
Nanomedicine, Barcelona Science Park, Barcelona 08028, Spain.
[h] Prof. Fernando Albericio
Department of Organic Chemistry, University of Barcelona, Barcelona
08028, Spain
Supporting information for this article is given via a link at the end of the
document.
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10.1002/cmdc.201700770
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currently available, and amphotericin
B
deoxycholate
as confirmed by elemental analysis, IR, and NMR spectroscopy
(supplementary data).
(Fungizone) is the most highly effective.[19d, 19f] Amphotericin B
deoxycholate has some unfortunate side effects and some
infusion-related side effects such as fever, chills, and
thrombophlebitis.[20] Miltefosine was the first oral antileishmanial
drug that reached the market and has been used for treatment
of VL and CL.[19d, 19f] However, side effects include disturbance
of the gastrointestinal tract[21] and an elevation of hepatic
enzymes.[22] Furthermore, miltefosine has a teratogenic effect
and is contra indicated for use during pregnancy.[19f, 21] Similarly,
mandatory contraception is also suggested for women of child-
bearing age. [21] In addition, Paromomycin,[19f, 23] pentamidine, [19d,
19f, 20] and Sitamaquine[19f, 24] were also investigated.
The 13C-NMR spectrum for the dimethoxy-s-triazine
derivatives 4a, 4b, 4c, 4e, 4f and 7g showed three peaks
corresponding to the triazine ring. For example, compound
4f showed peaks at δ 168.13, 170.26, 172.21, 172.38 and
173.28 ppm, corresponding to the two carbonyl carbons (amide
CO and ester CO) and the triazine ring carbons. The
observation of three peaks for the triazine ring carbons confirms
that the two methoxy groups on the triazine ring are non-
equivalent, thereby allowing the formation of two conformers.
This observation is in agreement with the data reported for
methoxy-s-triazine amino acid derivatives by Loeser and
Babiak.[26]
In this regard, the search for new drugs with strong
leishmanicidal activity and a safer profile is critical if we are to
tackle this devastating disease. Here we studied the potential
The HPLC chromatogram for N-(4,6-dipiperidino-1,3,5-triazin-2-
yl)-Ile-Gly-OC2H5 5g revealed two conformers at retention times
tR = 5.14 and 11.19 min in a ratio of 15:85, respectively. The two
conformers were confirmed by the 1H-NMR spectrum. The
chemical shift of the two α-CHs and of the two NHs of these
conformers differed. Two peaks were observed to be equivalent
to one proton at δ 5.55 and 4.25 ppm, in a ratio of 85:15,
corresponding to the α-CH. In addition, four peaks were
observed to be equivalent to two protons, corresponding to the
two NHs. NMR analysis indicated that the conversion between
the two conformations was slow enough to be observed by NMR
spectrum at room temperature (rt).[26]
Furthermore, the 1H-NMR spectrum of 6e also revealed two
conformers in a ratio of 56:44. The chemical shift of the two α-
CH and of the two NH’s of these conformers differed.
The purity of N-(4,6-dimethoxy-1,3,5-triazin-2-yl)-Gly-Phe-Val-
NH2 8 was 100%, as detected by reverse- phase HPLC at
retention time tR = 9.05 min. Product 8 was identified from its
mass spectral analysis, and it showed an exact mass [M+Na]+ =
antileishmanial activity of
a new family of compounds,
considered hybrid molecules of 1,3,5-triazine moiety and short
hydrophobic peptides, against L. aethiopica, the causative agent
of CL.
Results and Discussion
Chemistry
Although the solid-phase methodology is the method of choice
for the synthesis of most peptides, very short peptides are best
synthesized in solution, because this approach involves only one
or two reactions.[25] Furthermore, C-terminal esters are difficult to
synthesize in solid-phase due to the inherent characteristic of
the solid-phase, which is based on anchoring the first amino acid
to the solid support through the carboxylic function.
Synthesis of N-(4,6-Disubstituted-1,3,5-triazin-2-yl)dipeptide
/tripeptide ethyl esters/amides (4-10). 1,3,5-Triazine-based
amino acid derivatives [N-(4,6-disubstituted-1,3,5-triazin-2-
yl)amino acid], which were prepared from 4,6-disubstituted-
1,3,5-triazine and the corresponding L-amino acid, were coupled
to glycine ethyl ester hydrochloride, L-valine amide
hydrochloride, and L-phenylalanyl-L-valine amide p-toluene
sulfonic acid salt to give di- and tri-peptide-1,3,5-triazine
derivatives, respectively (Scheme 1). Reactions were performed
482.201
(Supplementary data).
Reverse-phase HPLC
chromatogram for N-(4,6-dipiperidino-1,3,5-triazin-2-yl)-Gly-Phe-
Val-NH2 9 revealed two conformers at retention times tR = 5.11
and tR = 6.49 min in a ratio of 91:9, respectively, while only one
mass signal at m/z = [M+H]+ = 566.613 was observed. In
addition, reverse-phase HPLC chromatogram for N-(4,6-
dimorpholino-1,3,5-triazin-2-yl)-Gly-Phe-Val-NH2 10 showed the
presence of two conformers with retention times tR = 8.54 min
and tR = 9.25 min in a ratio of 85 :15, with an exact mass [M+H]+
= 570.568.
using
N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-
Synthesis
of
N-(4,6-Disubstituted-1,3,5-triazin-2-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-
oxide (HATU) as a coupling reagent in the presence of DIEA as
base, followed by reaction with a solution of amino acid ethyl
ester/amide hydrochloride/p-toluensulphonate and DIEA in DMF
at 0°C to afford the desired derivatives in high yields and purities,
yl)tetrapeptide amides (11-13). The peptide derivatives 11-13
were manually assembled stepwise on Fmoc-Rinkamide-AM-PS
resin, Scheme 2. Preactivation was carried out using Fmoc-L-
amino acid (4-fold excess), HATU (4-fold excess), and DIEA (8-
fold excess) in DMF, and then the solution was added to the
resin, and the resulting mixture was periodically stirred for 2 h.
The loaded resin was washed with DMF, and the Fmoc group
was removed with 20% piperidine in DMF. Washing of the
deblocked resin with DMF, CH2Cl2, and DMF was followed by an
analogous coupling step with the second Fmoc-L-amino acid.

.
For compounds 8-10, H-Phe-Val-NH2 p-Tos was first prepared starting by the
coupling of Boc-L-Phe-OH and HCl.L-Val-NH2 with HATU in presence of DIEA
as a base and DMF solvent at 0°C for 1 h and overnight at room temperature
to afford the dipeptide Boc-Phe-Val-NH2. The crude Boc-Phe-Val-NH2 was
treated with p-toluene sulfonic acid in acetonitrile, and stirred for 2 h at room
temperature to remove the Boc-group forming the H-Phe-Val-NH2.p-TsOH salt,
which was used without further purification.
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Scheme 1. Synthesis of N-(4,6-disubstituted-1,3,5-triazin-2-yl)dipeptide/tripeptide ethyl esters/amides 4-10.
The assembly of the peptide derivatives was achived via one of
the following two routes (Scheme 2):
give the corresponding N-(4,6-disubstituted-1,3,5-triazin-2yl)
tetrapeptide 11/12/13, respectively (Scheme 2).
Route (i): The tripeptide H-Gly-Phe-Val-NH2 was cleaved from
the resin with TFA-DCM (9:1) and then coupled to N-(4,6-
Route (ii): The tetrapeptide H-Gly-Gly-Phe-Val-NH2 was allowed
to react with 2-chloro-4,6-dimethoxy-1,3,5-triazine in the
presence of Et3N at rt overnight to give N-(4,6-dimethoxy-1,3,5-
triazin-2yl)-peptide 11 (Scheme 2).
disubstituted-1,3,5-triazin-2yl)-Gly-OH
1b/2b/3b
using
HATU/DIEA in DMF solvent at 0°C for 1 h and overnight at rt to
Scheme 2. Synthesis of N-(4,6-disubstituted-1,3,5-triazin-2-yl)tetrapeptide amides 11-13.
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within this series was 7c, which contains alanine adjacent to the
Biological Activity
s-triazine.
In vitro antipromastigote activity. A quantitative colorimetric
assay using the oxidation-reduction indicator Alamar blue® was
developed to measure cytotoxicity of the synthesized
compounds against the protozoan parasite Leishmania.[27]
Alamar blue assay was used to determine the viability of
promastigotes and evaluate the antileishmanial activity of the
synthesized compounds, using as references the drugs
miltefosine and amphotericin B.[7b, 28]
The dimethoxy triazine derivatives
4
showed greater
antileishmanial activity than the corresponding dipepridino 5 and
dimorpholino 6 derivatives.
Compounds 7a, 7d, 7e, and 7g showed greater antileishmanial
activity than miltefosine (Table 1). The most active compound,
7a (IC50 = 1.4 µM), showed about 5-fold the activity of
miltefosine (IC50 = 7.8 µM). On the other hand, all the
compounds tested showed lower antipromastigote activity than
amphotericin B.
Table 1. Antipromastigote activity (IC50) of the compounds synthesized and
reference standards in μM.
Compound
number
Compound
number
IC50*
IC50*
Figure 1. Structure of compounds 7a, 7d, 7e, and 7g, which showed greater
activity than miltefosine.
4a
4b
13.6±0.18
15.9±0.16
6g
7a
28.4±0.22
1.4±0.04
4c
4e
4f
15.0±0.22
12.7±0.18
16.8±0.26
19.7±0.18
16.8±0.14
17.9±0.36
19.5±0.28
17.0±0.32
28.7±0.24
31.4±0.22
21.7±0.26
7c
17.2±0.28
4.7±0.11
2.3±0.06
5.0±0.08
13.1±0.16
23.3±0.22
27.5±0.36
9.4±0.26
24.0±0.32
25.6±0.34
7.8±0.34
In vitro antiamastigote activity. Axenic amastigote were
produced appling the method described by Teixeira et al.[30] The
most active compounds, namely 7a, 7d, 7e, 7g, were further
tested for their antiamastigote activity.[31] They showed 3.4-, 1.6-,
2- and 1.4-fold activity of that of miltefosine, respectively, while
they showed lower activity than the standard reference drug
amphotericin B (Table 2). The most active compounds were 7a
and 7e, which have iminodiacetic acid and phenylalanine
adjacent to the s-triazine. Compounds 7a, 7d, 7e, 7g showed
slightly lower activity than amphotericin B. However, 7a showed
an activity very close to amphotericin B (Table 2).
7d
7e
5a
5b
5d
5e
5g
6a
6b
6d
7g
8
9
10
11
12
13
Miltefosine
Table 2. Antiamastigote activity of the compounds synthesized and the
reference standards in μM.
Amphotericin B
deoxycholate
6e
29.2±0.38
0.04±0.01
Compound number
IC50*
* IC50: values indicate the effective concentration of a compound required to
achieve 50% growth inhibition in μM. IC50 is expressed as mean ± SD of
triplicate experiments, P < 0.05.
7a
7d
7e
7g
0.22±0.02
0.47±0.08
0.37±0.06
0.55±0.08
0.74±0.04
From a structure-activity point of view and taking into account
that compounds 7a, 7d, 7e, 7g (Figure 1) showed greater or
comparable activity to that of miltefosine, we can conclude that
hydrophobic amino acids with an amide at the C-terminal show
the best results. The presence of a methoxy group on the
triazine moiety enhanced the activity of the s-traizine derivatives,
as reported in the literature.[29] Compound 7a showed the
greatest activity. This observation may be explained by the
combination of two valine amides and the dimethoxy groups on
the triazine moiety. Furthermore, the least active compound
Miltefosine
Amphotericin B deoxycholate
0.15±0.02
*IC50: effective concentration required to achieve 50% growth inhibition in µM.
IC50 is expressed as mean ± SD of triplicate experiments, P < 0.05.
In vitro cytotoxicity assay. The cytotoxicity of the most active
compounds, 7a, 7d, 7e and 7g, was tested in a VERO cell line
using the Mosmann method with modifications, as described in
the literature.[32] Briefly, the cells were incubated for 72 h with a
range of dilutions of the selected compounds, using MTT as
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reagent for the detection of cytotoxicity. 50% cytotoxic
concentration (CC50) values represent the concentration of
compound required to kill 50% of the cells (Table 3).
Combining the cytotoxic and antiparasitic activities of 7a, 7d, 7e
and 7g by means of the selectivity index (Table 3), these
compounds showed greater selectivity toward L. aethiopica than
mammalian cells, thereby indicating a safe toxicity profile.
Conclusions
New hybrid molecules that hold a 1,3,5-triazine moiety along
with di-tri-and tetra-peptides were synthesized and tested for
leishmanicidal activity. The piperidino and moroholino
derivatives showed low activities while the combination of
dimethoxy and valine amide on the s-triazine ring demonstrated
the highest antipromastigote and antiamastigote activity. The in
vitro antipromastigote activity revealed 7a, 7d, 7e, and 7g as
having a better IC50 for promastigotes and amastigotes than that
of the standard drug miltefosine. Refering to amphotericin B,
these compounds showed an IC50 in the same range for
amastigotes and less active for promastigotes. It is worth
mentioning that amphotericin B uses to show higher activity
toward L. Mexicana promastigote than amastigote, as occurs
with other active compounds.[37]
The compounds presenting greatest activity, namely 7a, 7d, 7e,
and 7g, showed low toxicity against mammalian cells and
therefore a promising SI. In addition, in vivo acute toxicity
studies for these compounds indicated their safety when
administered orally and parenterally up to 250 and 100 mg/kg of
body weight, respectively.
Table 3. CC50 and therapeutic values of the most active compounds on
VERO cells.
a
a
Compound number
CC50
IC50
SIb
7a
7d
7e
7g
533.61
705.42
310.6
0.22±0.02
0.47±0.08
0.37±0.06
0.55±0.08
2457.88
1503.45
841.27
678.55
1240.96
a
CC50 is the concentration at which 50% cells survive and IC50 is the
concentration causing 50% cell death.
b SI is the selectivity index regarding antiamastigote activity; SI = CC50 / IC50
.
In vivo acute toxicity testing. The most active antileishmanial
compounds, 7a, 7d, 7e and 7g were tested for their toxicity in
mice, following the reported method.[33] The mice did not show
any sign of toxicity after treatment. There was no significant
difference in the weight of the mice and no deaths were
recorded during three days of observation after administration of
the compounds (data not shown). Oral administration of the
compounds up to 250 mg/kg of body weight were well tolerated
by the animals. Moreover, the toxicity of the compounds when
administered via the parenteral route was tested. All of them
were found to be non-toxic up to 100 mg/kg of body weight.
The kidney, spleen and liver of mice receiving 250 mg/kg of
body weight orally or 100 mg/kg parenterally showed normal
textures. Histopathological studies of samples of these organs
did not show any abnormalities.
Therefore, compounds 7a, 7d, 7e, and 7g emerge as promising
derivatives for the development of a new class of antileishmanial
agents and deserve further attention. In special, 7a is more
active than miltefosin towards both proamastigote and
amastigote and of similar activity than amphotericin B for
amastigote. Taking into account its great SI makes it an
excellent HIT for a medicinal chemistry program.
Experimental Section
Chemistry
Solvents and all reagents were purchased from Sigma-Aldrich.
All 1,3,5-triazine L-amino acid derivatives 1a-g, 2a,b,d,e,g and
3a,b,d,e,g were prepared earlier in our lab.[38] Unless otherwise
stated, normal workup from organic solvent involved drying over
Na2SO4 and rotary evaporation. TLC was performed using
aluminum-backed Merck Silica Gel 60 F-254 plates using
suitable solvent systems, with spots being visualized by a
Spectroline UV Lamp (254 or 365 nm) or I2 vapor. Melting points
were obtained in open capillary tubes by using a MEL-Temp II
melting point apparatus and they are uncorrected. Infrared (IR)
spectra were recorded on a Perkin-Elmer 1600 series Fourier
transform instrument as KBr pellets. The absorption bands
( max) are given in wave numbers (cm-1). Nuclear magnetic
resonance (NMR) spectra (1H-NMR and 13C-NMR) were
recorded on JEOL 400 MHz and JEOL 500 MHz spectrometers
at ambient temperature. Chemical shifts are reported in parts
per million (ppm) and are referenced relative to residual solvent
(e.g. CHCl3 at δ 7.26 ppm for CDCl3, DMSO at δ 2.50 ppm for
[D6]DMSO). Elemental analyses were performed on a Perkin-
Elmer 2400 elemental analyzer, and the values found were
within ±0.3% of the theoretical ones. HPLC analysis was
performed using a reverse-phase Agilent 1200 HPLC separation
module, coupled to an Agilent 1200 PDA UV detector. The
The results of this study compare favourable with others in the
literature for both triazine derivatives and peptides. For instance,
Kgokong, et al.[34] studied the antileishmanial effect of 1,3,5-
triazine derivatives, where the most active compound
(supporting info.) had an IC50 of 4.01 μM and toxicity (CC50
=
227.04 μM) with an SI of 56.57. Furthermore, the antileishmanial
effect of much more structurally complex peptides has also been
reported. For example, viridamide A (supporting info.) obtained
from Oscillatoria nigrowiridis showed antileishmanial activity
toward L. mexicana with an IC50 of 1.5 μM;[35] symplocamide A
(supporting info.) exhibited antileishmanial activity toward L.
donovani with an IC50 of 9.5 μM;[36] and the most active cecropin
A-melittin hybrid peptide KWKLFKKIGAVLKVL-amide had an
IC50 of 1.8 μM (SI = 26.8) against L. donovani.[7d] In comparison
to these observations, some of the 1,3,5-triazine-peptide hybrids
synthesized herein showed an improvement in both anti-
leishmanial activity and SI regarding antiamastigote activity, as
exemplified by 7a (IC50 = 1.4±0.04 μM and SI = 2457.88), while
other derivatives, such as 7d, 7e, and 7g, showed an
improvement in the SI.
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chromatograms were processed with Empower software.
Separation was accomplished using an Eclipse plus C18 column
(3.5 µm 4.6x100 mm) or Eclipse plus C8 column (3.5 µm
4.6x250 mm), and linear gradients of solvent A [0.045%
trifluoroacetic acid (TFA) in H2O] in solvent B (0.036% TFA in
CH3CN) with a flow = 1.0 mL min–1. Exact mass measurements
were carried out using a Bruker Daltonics Ultraflex MALDI
TOF/TOF Mass Spectrometer in a solvent mixture of acetonitrile:
0.1% aqueous TFA (30/70).
amine), 3293 (NH, amide), 1759 (CO, ester), 1658 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 1.10-1.15 (m, 3H,
CH3CH2), 1.26-1.30 (m, 3H, CH3CH), 3.75-3.82 (m, 2H, α-CH2),
3.75-3.82 (m, 6H, 2 × OCH3), 4.01-4.03 (m, 2H, CH2CH3), 4.37-
4.42 (m, 1H, α-CH), 7.97 (s, 1H, NH), 8.26 (s, 1H, NH amide);
13C-NMR (125 MHz, [D6]DMSO): 14.54, 18.26, 41.52, 50.52,
54.67, 60.89, 167.74, 170.28, 172.22, 172.35, 173.54. Anal.
calcd for C12H19N5O5: C, 46.00; H, 6.11; N, 22.35. Found: C,
46.27; H, 6.19; N, 22.06.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Phe-Gly-OEt (4e).
The product was obtained as a white solid, 0.37 g (95.3%; mp)
yield: 166-168oC; tR = 11.37 min. (100%). IR (KBr): 3446 (NH,
amine), 3283 (NH, amide), 1748 (CO, ester), 1652 (CO, amide)
General procedure for the synthesis of N-(4,6-dimethoxy-
1,3,5-triazin-2-yl) dipeptide ethyl ester (4a,b,c,e,f)
HATU (0.38 g, 1 mmol) was added to an ice-cold solution of Nα-
(4,6-dimethoxy-1,3,5-triazin-2-yl) amino acid (1 mmol) and DIEA
(0.34 mL, 2 mmol) in DMF with stirring. A solution of glycine
ethyl ester hydrochloride (0.14 g, 1 mmol) and DIEA (0.17 mL, 1
mmol) in DMF was then added to this mixture. The mixture was
left overnight with stirring at rt (in the case of IDA derivative, (2
mmol) HATU and amino acid ester were used with equiv. DIEA).
The reaction mixture was diluted with 70 mL ethyl acetate and
then washed with 5% citric acid (2 × 10 mL), a saturated
NaHCO3 solution (2 × 10 mL), and a saturated NaCl solution (2
× 10 mL). The organic layer was dried over anhydrous Na2SO4
and filtered. The solvent was removed in vacuo with the aid of a
water aspirator. The purity of compounds 4a,b,c,e,f was
detected by reverse-phase HPLC using the following conditions:
detection at 220 nm (Agilent 1200 PDA detector); Eclipse plus
C18 column (3.5 µm 4.6 x 100 mm); linear gradient over 16 min
(25 to 50% CH3CN in H2O/ 0.1% TFA); and flow rate 1.0 mL/min.
1
cm-1; H-NMR (500 MHz, [D6]DMSO): δ 1.13 (t, 3H, J = 6.9 Hz,
2
3
CH3CH2), 2.88 (dd, 1H, J = 13.8 Hz, J = 10.7, CH2-Ph), 3.07
(dd, 1H, 2J = 13.8 Hz, 3J = 3.9 Hz, CH2-Ph), 3.747 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 3.77-3.87 (m, 2H, α-CH2), 4.04 (q, 2H, J =
6.9 Hz, CH2CH3), 4.64-4.69 (m, 1H, α-CH), 7.13-7.31 (m, 5H,
Ph-H), 8.02 (d, 1H, J = 8.4 Hz, NH), 8.53 (t, 1H, J = 6.1 Hz, NH
amide); 13C-NMR (125 MHz, [D6]DMSO): 14.54, 37.59, 41.35,
54.65, 56.58, 60.95, 126.80, 128.62, 129.69, 138.81, 168.15,
170.27, 172.13, 172.23, 172.58. Elemental Analysis Calcd for
C18H23N5O5: C, 55.52; H, 5.95; N, 17.98. Found: C, 55.31; H,
5.76; N, 17.69.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Leu-Gly-OEt (4f).
The product was obtained as a white solid, 0.30 g (85.3%) yield;
mp: 126-128oC; tR = 9.34 min. (100%). IR (KBr): 3441 (NH,
amine), 3282 (NH, amide), 1747 (CO, ester), 1656 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 0.82 (d, 3H, J = 6.9 Hz,
CH3CH), 0.86 (d, 3H, J = 6.1 Hz, CH3CH), 1.13 (t, 3H, J = 6.9 Hz,
CH3CH2), 1.43-1.49 (m, 1H, CH), 1.56-1.65 (m, 2H, CH2), 3.78
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.69-3.83 (m, 2H, α-CH2),
4.03 (q, 2H, J = 6.9 Hz, CH2CH3), 4.39-4.44 (m, 1H, α-CH), 7.95
(d, 1H, J = 7.6 Hz, NH), 8.28 (t, 1H, J = 6.1 Hz, NH amide); 13C-
NMR (125 MHz, [D6]DMSO): 14.52, 21.90, 23.58, 24.75, 40.20,
53.38, 54.66, 60.87, 168.13, 170.26, 172.21, 172.38, 173.28.
Elemental Analysis Calcd for C15H25N5O5: C, 50.69; H, 7.09; N,
19.71. Found: C, 50.91; H, 6.93; N, 19.51.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-IDA-(Gly-OEt)2 (4a).
The product was obtained as a white solid, (0.41 g, 91.7%); mp:
164-166oC; tR = 4.46 min, purity 100% as detected by HPLC. IR
(KBr): 3316 (NH, amide), 1740 (CO, ester) 1657 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 1.13 (t, 3H, J = 6.9 Hz,
CH3CH2), 3.74 (s, 3H, OCH3), 3.83 (d, 2H, J = 4.6 Hz, CH2NH),
4.04 (q, 2H, J = 6.9 Hz, OCH2CH3), 4.24 (s, 2H, CH2N), 8.82 (s,
1H, NH); 13C-NMR (125 MHz, [D6]DMSO): 14.54, 40.35, 52.42,
54.88, 60.99, 167.74, 170.08, 172.20. Anal. calcd for
C17H26N6O8: C, 46.15; H, 5.92; N, 19.00. Found: C, 46.01; H,
6.12; N, 19.17.
General procedure for the synthesis of N-(4,6-dipiperidino-
1,3, 5-triazin-2-yl) dipeptide ethyl ester (5a,b,d,e,g)
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Gly-Gly-OEt (4b).
The product was obtained as a white solid, 0.29 g (96.1%) yield;
mp: 163-165oC; tR = 10.99 min. (93.60%). IR (KBr): 3459 (NH,
amine), 3275 (NH, amide), 1733 (CO, ester) 1632 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 1.14 (t, 3H, J = 6.9 Hz,
CH3CH2), 3.78 (s, 3H, OCH3), 3.78 (d, 2H, J = 6.1 Hz, α-
CH2COO), 3.79 (s, 3H, OCH3), 3.86 (d, 2H, J = 6.1 Hz, α-
CH2CONH), 4.03 (q, 2H, J = 6.9 Hz, CH2CH3), 8.03 (t, 1H, J =
6.1 Hz, NH), 8.29 (t, 1H, J = 6.1 Hz, N-H amide); 13C-NMR (125
MHz, [D6]DMSO): 14.57, 41.19, 44.00, 54.66, 60.92, 168.55,
169.94, 170.29, 172.21, 172.39. Anal. calcd for C11H17N5O5: C,
44.14; H, 5.73; N, 23.40. Found: C, 43.91; H, 5.99; N, 23.47.
HATU (0.19 g, 0.5 mmol) was added to an ice-cold solution of
Nα-(4,6-dipiperidino-1,3,5-triazin-2-yl) amino acid (0.5 mmol)
and DIEA (0.17 mL, 1 mmol) in DMF with stirring. A solution of
glycine ethyl ester hydrochloride (0.07 g, 0.5 mmol) and DIEA
(0.09 mL, 0.5 mmol) in DMF was added to this mixture. The
mixture was left overnight at rt with stirring (in the case of IDA
derivative, 2 mmol of HATU and amino acid ester were used
with equiv. DIEA). The reaction mixture was diluted with 70 mL
ethyl acetate and then washed with 5% citric acid (2 × 10 mL), a
saturated NaHCO3 solution (2 × 10 mL), and a saturated NaCl
solution (2 × 10 mL). The organic layer was dried over
anhydrous Na2SO4 and filtered. The solvent was removed in
vacuo with the aid of a water aspirator.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Ala-Gly-OEt (4c).
The product was obtained as a white solid, 0.26 g (85.8%) yield;
mp: 108-110oC; tR = 2.73 min. (100%). IR (KBr): 3444 (NH,
N-(4,6-Dipiperidino-1,3,5-triazin-2-yl)-IDA-(Gly-OEt)2 (5a).
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10.1002/cmdc.201700770
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The product was obtained as a white solid, 0.18 g (80.6%) yield;
mp:138-140oC; IR (KBr): 3306 (NH, amide), 1744 (CO, ester),
0.79-0.85 (m, 3H, CH3), 0.88-0.94 (m, 3H, CH3), 1.15 (t, 3H, J =
6.6 Hz, CH3CH2O), 1.35-1.61 (m, 14H, 6 × CH2, CH2CH3), 1.89-
1.93 (m, 1H, CHCH3), 3.54-3.71 (m, 8H, 4 × CH2N), 4.05 (q, 2H,
J = 6.6 Hz, OCH2CH3), 4.25 (t, 1H, J = 8.8 Hz, α-CH), 4.70 (d,
2H, J = 9.5 Hz, α-CH2), 6.27 (d, 1H, J = 8.1 Hz, NH), 8.21-8.27
(m, 1H, NH). 13C-NMR (100 MHz, [D6]DMSO): 11.41, 11.77,
14.56, 16.10, 18.30, 24.71, 25.02, 25.83, 25.98, 44.45, 60.99,
164.53, 167.90, 170.13, 170.61, 175.00. Anal. calcd for
C23H39N7O3: C, 59.85; H, 8.52; N, 21.24. Found: C, 59.99; H,
8.37; N, 21.04.
1
1662 (CO, amide) cm-1; H-NMR (500 MHz, [D6]DMSO): δ 1.13
(t, 6H, J = 7.7 Hz, 2×CH3), 1.37-1.39 (m, 8H, 4×b-CH2), 1.54
(quint, 4H, J = 6.2 Hz, 2×c-CH2), 3.57 (t, 8H, J = 6.2 Hz, 4×a-
CH2), 3.80 (d, 2H, J = 6.1 Hz, 2×α-CH2NH), 4.03 (q, 4H, J = 7.7
Hz, 2× OCH2CH3), 4.10 (s, 2H, 2×α-CH2N), 8.87 (t, 2H, J = 6.1
Hz, 2×NH); Anal. calcd for C25H40N8O6: C, 54.73; H, 7.35; N,
20.42. Found: C, 54.59; H, 7.47; N, 20.33.
N-(4,6-Dipiperidino-1,3,5-triazin-2-yl)-Gly-Gly-OEt (5b).
The product was obtained as a white solid, 0.14 g (70.8%) yield;
mp: 152-154oC; IR (KBr): 3421 (NH, amine), 3287 (NH, amide),
General procedure for the synthesis of N-(4,6-dimorpholino-
1,3,5-triazin-2-yl) dipeptide ethyl ester (6a,b,d,e,g)
1
1749 (CO, ester), 1665 (CO, amide) cm-1; H-NMR (500 MHz,
CDCl3): δ 1.21-1.26 (m, 3H, CH3), 1.51-1.66 (m, 12H, 6×CH2),
3.61-3.74 (m, 8H, 4×CH2N), 3.93 (d, 1H, J = 5.4 Hz, α-CH2),
4.00 (d, 1H, J = 5.4 Hz, α-CH2), 4.10-4.16 (m, 2H, CH2), 4.66-
4.68 (m, 1H, α-CH2), 4.83 (d, 2H, J = 5.4 Hz, α-CH2), 6.73 (t, 1H,
J = 5.4 Hz, NH), 7.10 (t, 1H, J = 5.4 Hz, NH); Anal. calcd for
C19H31N7O3: C, 56.28; H, 7.71; N, 24.18. Found: C, 56.07; H,
7.61; N, 24.49.
HATU (0.19 g, 0.5 mmol) was added to an ice-cold solution of
Nα-(4,6-dimorpholino-1,3,5-triazin-2-yl) amino acid (0.5 mmol)
and DIEA (0.17 mL, 1 mmol) in DMF with stirring. A solution of
glycine ethyl ester hydrochloride (0.07 g, 0.5 mmol) and DIEA
(0.09 mL, 0.5 mmol) in DMF was added to this mixture. The
mixture was left stirring at rt overnight (in the case of IDA
derivative, 2 mmol of HATU and amino acid ester were used
with equiv. DIEA). The reaction mixture was diluted with ethyl
acetate and then washed with 5% citric acid (2 × 10 mL), a
saturated NaHCO3 solution (2 × 10 mL), and a saturated NaCl
solution (2 × 10 mL). The organic layer was dried over
anhydrous Na2SO4 and filtered. The solvent was removed in
vacuo with the aid of a water aspirator.
N-(4,6-dipiperidino-1,3,5-triazin-2-yl)-Val-Gly-OEt (5d).
The product was obtained as a white solid, 0.18 g (76.8%) yield;
mp: 88-90oC; IR (KBr): 3433 (NH, amine), 1692 (CO, amide),
1
1749 (CO, ester) cm-1; H-NMR (500 MHz, CDCl3): δ 0.94-1.08
(m, 6H, 2×CH3), 1.22-1.26 (m, 3H, CH2CH3), 1.41-1.67 (m, 12H,
6×CH2-pip), 2.18-2.24 (m, 1H, CH), 3.63-4.01 (m, 10H, 4×CH2N,
α-CH2), 4.11-4.4.16 (m, 2H, CH2), 4.64-4.72 (m, 1H, α-CH),
7.41-7.52 (m, 1H, NH), 8.69 (br. s, 1H, NH). Anal. calcd for
C22H37N7O3: C, 59.04; H, 8.33; N, 21.91. Found: C, 59.31; H,
8.07; N, 22.05.
N-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-IDA-(Gly-OEt)2 (6a).
The product was obtained as a white solid, 0.24 g (86.9%) yield;
mp: 211-214oC; IR (KBr): 3299 (NH, amide), 1748 (CO, ester),
1
1657 (CO, amide) cm-1; H-NMR (500 MHz, [D6]DMSO): δ 1.13
N-(4,6-Dipiperidino-1,3,5-triazin-2-yl)-Phe-Gly-OEt (5e).
The product was obtained as a white solid, 0.21 g (83.9%) yield;
mp: 112-115oC; IR (KBr): 3424 (NH, amine), 3296 (NH, amide),
(t, 6H, J = 6.9 Hz, 2×CH3), 3.50-3.53 (m, 8H, 4×CH2N), 3.55-
3.58 (m, 8H, 4×CH2O), 3.80 (d, 4H, J = 5.4 Hz, 2×α-CH2COO),
4.02 (q, 4H, J = 6.9 Hz, 2×OCH2CH3), 4.10 (s, 4H, 2×α-
CH2CONH), 8.88 (t, 2H, J = 5.4 Hz, 2×NH amide); 13C-NMR
(125 MHz, [D6]DMSO): 14.54, 43.73, 52.74, 56.57, 60.93, 66.54,
164.97, 165.35, 170.14, 171.41. Anal. calcd for C23H36N8O8: C,
49.99; H, 6.57; N, 20.28. Found: C, 49.84; H, 6.71; N, 20.35.
1
1749 (CO, ester), 1687 (CO, amide) cm-1; H-NMR (125 MHz,
CDCl3): δ 1.22-1.26 (m, 3H, CH3), 1.41-1.76 (m, 12H, 6×CH2-
pip), 2.97-3.36 (m, 2H, CH2-Ph), 3.51-3.70 (m, 8H, 4×CH2N),
3.75-4.01 (m, 2H, α-CH2), 4.09-4.14 (m, 2H, CH2), 5.39-5.48 (m,
1H, α-CH), 6.98-7.35 (m, 7H, 5Ph-H, 2NH); Anal. calcd for
C26H37N7O3: C, 63.01; H, 7.52; N, 19.78. Found: C, 63.21; H,
7.27; N, 19.91.
N-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-Gly-Gly-OEt (6b).
The product was obtained as a white solid, 0.20 g (94.2%) yield;
mp: 204-206oC; IR (KBr): 3419 (NH, amine), 3287 (NH, amide),
1
1753 (CO, ester), 1663 (CO, amide) cm-1; H-NMR (500 MHz,
N-(4,6-Dipiperidino-1,3,5-triazin-2-yl)-Ile-Gly-OEt (5g).
The product was obtained as a white solid, 0.19 g (83.3%) yield;
mp: 127-130oC; the purity of 32 was detected by reverse-phase
HPLC with conditions: detection at 240 nm, using Agilent 1200
PDA detector; Eclipse plus C8 column (3.5 µm 4.6 x 250 mm);
linear gradient over 16 min (40 to 90 % CH3CN in H2O/ 0.1%
[D6]DMSO): δ 1.13 (t, 3H, J = 6.9 Hz, CH3), 3.53-3.65 (m, 8H,
4×CH2N), 3.53-3.65 (m, 8H, 4×CH2O), 3.76-3.79 (m, 2H, α-CH2),
4.03 (q, 2H, J = 6.9 Hz, CH2), 4.52-4.63 (m, 2H, α-CH2), 6.93-
6.97 (m, 1H, NH), 8.05-8.10 (m, 1H, NH). Anal. calcd for
C17H27N7O5: C, 49.87; H, 6.65; N, 23.95. Found: C, 50.06; H,
6.43; N, 23.87.
TFA); flow rate 1.0 mL/min., tR = 5.14 min. (14.86 %) and tR
=
11.19 min. (85.14 %). IR (KBr): 3429 (NH, amine), 3381 (NH,
amide), 1749 (CO, ester), 1690 (CO, amide) cm-1; 1H-NMR (400
MHz, [D6]DMSO) Isomer A (85.14%): δ 0.79-0.85 (m, 3H, CH3),
0.88-0.94 (m, 3H, CH3), 1.15 (t, 3H, J = 6.6 Hz, CH3CH2O), 1.35-
1.61 (m, 14H, 6 × CH2, CH2CH3), 1.89-1.93 (m, 1H, CHCH3),
3.54-3.71 (m, 8H, 4 × CH2N), 3.75-3.90 (m, 2H, α-CH2), 4.05 (q,
2H, J = 6.6 Hz, OCH2CH3), 5.55 (t, 1H, J = 8.8 Hz, α-CH), 6.18-
6.23 (m, 1H, NH), 7.71-7.82 (m, 1H, NH); Isomer B (14.86 %): δ
N-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-Val-Gly-OEt (6d).
The product was obtained as a white solid, 0.20 g (87.1%) yield;
mp: 161-164oC; IR (KBr): 3445 (NH, amine), 3289 (NH, amide),
1
1729 (CO, ester), 1655 (CO, amide) cm-1; H-NMR (500 MHz,
[D6]DMSO): δ 0.86 (d, 3H, J = 6.9 Hz, CH3CH), 0.88 (d, 3H, J =
6.9 Hz, CH3CH), 1.12 (t, 3H, J = 6.9 Hz, CH3CH2O), 1.95 (octet,
1H, J = 6.9 Hz, CH), 3.52 (br. s, 8H, 4×CH2N), 3.58 (br. s, 8H,
4×CH2O), 3.70 (dd, 1H, J = 16.8 Hz, J = 4.6 Hz, α-CH2), 3.81 (dd,
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10.1002/cmdc.201700770
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1H, J = 16.8 Hz, J = 5.4 Hz, α-CH2), 4.02 (q, 2H, J = 6.9 Hz,
OCH2CH3), 4.10-4.15 (m, 1H, α-CH), 6.47 (d, 1H, J = 8.4 Hz,
NH), 8.23 (t, 1H, J = 5.4 Hz, NH); 13C-NMR (125 MHz,
[D6]DMSO): 14.55, 19.25, 19.82, 30.76, 41.15, 43.71, 60.33,
60.88, 66.59, 165.07, 165.18, 166.21, 170.31, 173.02. Anal.
calcd for C20H33N7O5: C, 53.20; H, 7.37; N, 21.71. Found: C,
53.55; H, 7.18; N, 21.54.
The product was obtained as a white solid, 0.40 g (85.0%) yield;
mp: 260-262oC; IR (KBr): 3397 (NH, amide), 1654 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 0.74 (d, 6H, J = 6.9 Hz,
2×CH3), 0.77 (d, 6H, J = 6.1 Hz, 2×CH3), 1.96 (m, 2H, 2×CH),
3.75 (s, 6H, 2×OCH3), 4.05-4.09 (m, 2H, 2×α-CH), 4.17-4.30 (m,
4H, 2×α-CH2), 6.99 (s, 2H, 2×NH), 7.23 (s, 2H, 2×NH), 8.53 (d,
2H, J = 9.2 Hz, 2×NH); 13C-NMR (125 MHz, [D6]DMSO): 18.12,
19.74, 30.62, 53.31, 54.81, 58.18, 167.61, 169.72, 172.12,
173.39. Anal. calcd for C19H32N8O6: C, 48.71; H, 6.88; N, 23.92.
Found: C, 48.55; H, 7.17; N, 23.69.
N-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-Phe-Gly-OEt (6e).
The product was obtained as a white solid, 0.22 g (89.7%) yield;
mp: 135-138oC; IR (KBr): 3428 (NH, amine), 1745 (CO, ester),
1681 (CO, amide) cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ
isomer A (55.6 %): δ 1.12-1.16 (m, 3H, CH3), 2.94-3.12 (m, 2H,
CH2-Ph), 3.45-3.56 (m, 8H, 4×CH2N), 3.45-3.56 (m, 8H,
4×CH2O), 3.78-3.83 (m, 2H, α-CH2), 4.01-4.06 (m, 2H, CH2),
4.54-4.58 (m, 1H, α-CH), 6.76 (d, 1H, J = 7.7 Hz, NH), 7.02-7.18
(m, 5H, Ph-H), 7.22 (t, 1H, J = 6.9 Hz, NH); isomer B (44.4 %): δ
1.12-1.16 (m, 3H, CH3), 2.94-3.12 (m, 2H, CH2-Ph), 3.45-3.56
(m, 8H, 4×CH2N), 3.45-3.56 (m, 8H, 4×CH2O), 4.01-4.06 (m, 2H,
CH2), 5.44-5.42 (m, 2H, α-CH2), 5.93-5.96 (m, 1H, α-CH), 7.02-
7.18 (m, 5H, Ph-H), 7.29 (d, 1H, J = 7.7 Hz, NH), 7.43 (t, 1H, J =
6.9 Hz, NH); Anal. calcd for C24H33N7O5: C, 57.70; H, 6.66; N,
19.63. Found: C, 57.89; H, 6.51; N, 19.49.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Ala-Val-NH2 (7c).
The product was obtained as a white solid, 0.29 g (88.2%) yield;
mp: 244-246oC; IR (KBr): 3463 (NH, amide), 1648 (CO, amide)
1
cm-1; H-NMR (500 MHz, [D6]DMSO): δ 0.73-0.82 (m, 6H, 2×a-
CH3), 1.22-1.26 (m, 3H, b-CH3), 1.60-1.94 (m, 1H, CH), 3.77 (s,
3H, OCH3), 3.78 (s, 3H, OCH3), 4.03-4.09 (m, 1H, α-CHCH),
4.40-4.49 (m, 1H, α-CHCH3), 7.00-7.02 (m, 1H, NH2), 7.37-7.46
(m, 1H, NH2), 7.62-7.76 (m, 1H, NH), 7.88-8.01 (m, 1H, N-H);
13C-NMR (125 MHz, [D6]DMSO): 11.34, 14.42, 18.41, 19.77,
31.25, 50.61, 54.70, 57.90, 167.67, 172.20, 172.40, 172.79,
173.41. Anal. calcd for C13H22N6O4: C, 47.84; H, 6.79; N, 25.75.
Found: C, 47.61; H, 6.99; N, 25.86.
N-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-Ile-Gly-OEt (6g).
The product was obtained as a white solid, 0.21 g (86.8%) yield;
mp: 170-172oC; IR (KBr): 3438 (NH, amine), 3301 (NH, amide),
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Val-Val-NH2 (7d).
The product was obtained as a white solid, 0.29 g (83.1%) yield;
mp: 203-206oC; IR (KBr): 3441 (NH, amide), 1662 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 0.77 (d, 3H, J = 7.7 Hz,
a-CH3), 0.78 (d, 3H, J = 7.7 Hz, b-CH3), 0.86 (d, 6H, J = 6.9 Hz,
2×c-CH3), 1.85-1.89 (m, 1H, CH), 2.03 (octet, 1H, J = 6.9 Hz,
CH), 3.80 (s, 6H, 2 × OCH3), 4.07-4.12 (m, 1H, α-CH), 4.29 (t,
1H, J = 7.7 Hz, α-CH), 7.00 (s, 1H, NH2), 7.38 (s, 1H, NH2), 7.69
(d, 1H, J = 8.4 Hz, NH), 7.89 (d, 1H, J = 9.1 Hz, NH); 13C-NMR
(125 MHz, [D6]DMSO): 18.24, 18.52, 19.17, 19.75, 19.80, 30.63,
31.23, 54.72, 57.79, 60.85, 168.34, 171.31, 172.23, 172.38,
173.24. Anal. calcd for C15H26N6O4: C, 50.83; H, 7.39; N, 23.71.
Found: C, 51.07; H, 7.11; N, 23.61.
1
1727 (CO, ester), 1651 (CO, amide) cm-1; H-NMR (500 MHz,
[D6]DMSO): δ 0.79 (t, 3H, J = 6.9 Hz, CH3CH2), 0.85 (d, 3H, J =
6.1 Hz, CH3CH), 1.12 (t, 3H, J = 6.9 Hz, CH3CH2O), 1.10-1.14
(m, 1H, CH2CH3), 1.43-1.50 (m, 1H, CH2CH3), 1.70-1.75 (m, 1H,
CHCH3), 3.52 (br. s, 8H, 4×CH2N), 3.58 (br. s, 8H, 4×CH2O),
3.68-3.84 (m, 2H, α-CH2), 4.02 (q, 2H, J = 6.9 Hz, OCH2CH3),
4.19 (t, 1H, J = 7.7 Hz, α-CH), 6.48 (d, 1H, J = 7.7 Hz, NH), 8.23
(t, 1H, J = 5.4 Hz, NH); 13C-NMR (125 MHz, [D6]DMSO): 11.47,
14.54, 15.88, 25.27, 36.93, 41.16, 43.70, 59.05, 60.87, 66.59,
165.09, 165.19, 166.09, 170.29, 173.00. Anal. calcd for
C21H35N7O5: C, 54.18; H, 7.58; N, 21.06. Found: C, 54.41; H,
7.43; N, 21.11.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Phe-Val-NH2 (7e).
The product was obtained as a white solid, 0.34 g (83.5%) yield;
mp: 224-226oC; IR (KBr): 3282 (NH, amide), 1664 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 0.75-0.86 (m, 6H,
2×CH3), 1.92 (octet, 1H, J = 6.9 Hz, CH), 2.84-2.94 (m, 1H, CH2-
Ph), 2.99-3.05 (m, 1H, CH2-Ph), 3.75 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3), 4.08-4.15 (m, 1H, α-CHCH), 4.70-4.75 (m, 1H, α-
CHCH2), 7.03 (s, 1H, NH), 7.10-7.31 (m, 5H, Ph-H), 7.41 (s, 1H,
NH), 7.90 (d, 1H, J = 9.2 Hz, NH), 8.02 (d, 1H, J = 8.4 Hz, NH
amide); 13C-NMR (125 MHz, [D6]DMSO): 18.38, 18.61, 19.74,
31.28, 37.51, 54.66, 56.49, 57.85, 58.18, 126.77, 128.58, 129.71,
138.70, 168.07, 171.73, 172.12, 172.26, 173.26. Anal. calcd for
C19H26N6O4: C, 56.70; H, 6.51; N, 20.88. Found: C, 56.96; H,
6.23; N, 20.76.
General procedure for the synthesis of N-(4,6-dimethoxy-
1,3,5-triazin-2-yl) dipeptide amide derivatives (7a,c,d,e,g)
HATU (0.38 g, 1 mmol) was added to an ice-cold solution of Nα-
(4,6-dimethoxy-1,3,5-triazin-2-yl) amino acid (1 mmol) and DIEA
(0.34 mL, 2 mmol) in DMF with stirring. A solution of L-valine
amide hydrochloride (0.15 g, 1 mmol) and DIEA (0.17 mL, 1
mmol) in DMF was added to this mixture. The mixture was left
overnight at rt with stirring (in the case of IDA derivative, 2 mmol
of HATU and amino acid ester were used with equiv. DIEA). The
reaction mixture was diluted with 70 mL ethyl acetate and then
washed with 5% citric acid (2 × 10 mL), a saturated NaHCO3
solution (2 × 10 mL), and a saturated NaCl solution (2 × 10 mL).
The organic layer was dried over anhydrous Na2SO4 and filtered.
The solvent was removed in vacuo with the aid of a water
aspirator.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Ile-Val-NH2 (7g).
The product was obtained as a white solid, 0.35 g (94.0%) yield;
mp: 205-208oC; IR (KBr): 3439 (NH, amide), 1656 (CO, amide)
cm-1; 1H-NMR (500 MHz, [D6]DMSO): δ 0.76-0.83 (m, 12H,
4×CH3), 1.10-1.24 (m, 1H, CH), 1.41-1.44 (m, 1H, CH), 1.81-
1.90 (m, 2H, CH2), 3.79 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.10
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-IDA-(Val-NH2)2 (7a).
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700770
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(t, 1H, J = 8.4 Hz, α-CH), 4.33 (t, 1H, J = 8.4 Hz, α-CH), 7.01 (s,
1H, NH), 7.41 (s, 1H, NH), 7.77 (d, 1H, J = 8.4 Hz, NH), 7.93 (d,
1H, J = 8.4 Hz, NH); 13C-NMR (125 MHz, [D6]DMSO): 11.27,
15.91, 18.54, 19.75, 25.25, 31.20, 36.57, 54.69, 54.76, 57.85,
59.61, 168.19, 171.36, 172.22, 172.39, 173.28. Anal. calcd for
C16H28N6O4: C, 52.16; H, 7.66; N, 22.81. Found: C, 51.00; H,
7.81; N, 22.67.
(0.041 mL, 0.23 mmol) in DMF was added to this mixture. The
reaction mixture was stirred at 0°C for 1 h and overnight at rt.
The mixture was diluted with 50 ml of ethyl acetate and then
washed with 5% citric acid (2 × 10 mL), saturated NaHCO3 (2 ×
10 mL), and saturated NaCl solution (2 × 10 mL). The organic
layer was dried over anhydrous Na2SO4 and filtered. The solvent
was removed in vacuo with the aid of a water aspirator. The
crude product was dried under reduced pressure. Yield: 0.073 g
(56.2 %), mp: 242-246°C. The purity of 9 was measured by
reverse-phase HPLC with the following conditions: detection at
220 nm using an Agilent 1200 PDA detector; Eclipse plus C18
column (3.5 µm 4.6 x 100 mm); linear gradient over 18 min (0 to
50 % CH3CN in H2O/ 0.1% TFA); and flow rate 1.0 mL/min., tR =
5.11 min. (90.84 %), tR = 6.49 min. (9.16%). For exact mass
determination, a sample of 9 was prepared, by dissolution in
H2O/CH3CN and dilution in H2O/CH3CN/1%TFA: m/z = 566.613
[M+H]+.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Gly-Phe-Val-NH2 (8).
HATU (0.76 g, 2 mmol) was added to an ice-cold solution of
Boc-L-phenyl alanine (0.53 g, 2 mmol) and DIEA (0.7 mL, 4
mmol) in DMF with stirring. A solution of L-valine amide
hydrochloride (0.30 g, 2 mmol) and DIEA (0.35 mL, 2 mmol) in
DMF was added to this mixture. The reaction mixture was stirred
at 0C for 1 h and overnight at rt. The mixture was diluted with
70 ml of ethyl acetate and then washed with 5% citric acid (2 ×
10 mL), saturated NaHCO3 (2 × 10 mL), and saturated NaCl
solution (2 × 10 mL). The organic layer was dried over
anhydrous Na2SO4 and filtered. The solvent was removed in
vacuo with the aid of a water aspirator. The crude product was
dried under reduced pressure, to afford a white solid of Boc-
Phe-Val-NH2 in an overall yield 0.65 g (89.5%), m.p. 193-194°C.
The crude product (0.64 g, 1.76 mmol) and p-toluene sulfonic
acid (1.01 g, 5.29 mmol) were dissolved in acetonitrile (10 mL),
and stirred for 2 h to remove the Boc-group, thereby forming the
H-Phe-Val-NH2.p-TsOH. The product was filtered and washed
with acetonitrile to give an overall yield of 0.651 g (85%), m.p.
234-235°C. The crude H-Phe-Val-NH2.p-TsOH was allowed to
undergo a further coupling as follows: HATU (0.062 g, 0.069
mmol) was added to a stirred solution of N-(4,6-Dimethoxy-
1,3,5-triazin-2-yl)-Gly-OH 1b (0.015 g, 0.069 mmol), the crude
H-Phe-Val-NH2.p-TsOH (0.03 g, 0.069 mmol) and DIEA (0.015
mL, 0.207 mmol) in DMF (3 mL) at 0C. The reaction mixture
was stirred at 0C for 1 h and overnight at rt. The mixture was
diluted with 70 ml of ethyl acetate and then washed with 5 %
citric acid (2 x 10 ml), saturated NaHCO3 (2 x 10 ml), and
saturated NaCl (2 x 10 ml). The organic layer was dried over
anhydrous Na2SO4 and filtered. The solvent was removed in
vacuo with the aid of a water aspirator. The crude N-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-Gly-Phe-Val-NH2 was dried under
reduced pressure. The purity of 8 was measured by reverse-
phase HPLC with the following conditions: detection at 220 nm,
using an Agilent 1200 PDA detector; Eclipse plus C18 column
(3.5 µm 4.6 x 100 mm); linear gradient over 14 min (0 to 50 %
CH3CN in H2O/ 0.1% TFA); and flow rate 1.0 mL/min., tR = 9.05
min. (100 %). Yield: 0.02 g (66.7 %), mp: 264-266oC. For exact
mass determination, a sample of 8 was prepared by dissolution
in H2O/CH3CN and dilution in H2O/CH3CN/1%TFA: m/z =
482.201 [M+Na]+.
N-(4,6-dimorpholino-1,3,5-triazin-2-yl)-Gly-Phe-Val-NH2 (10).
HATU (0.087 g, 0.23 mmol) was added to an ice-cold solution of
N-(4,6-dimorpholino-1,3,5-triazin-2-yl)-Gly-OH 3b (0.075 g, 0.23
mmol) and DIEA (0.081 mL, 0.46 mmol) in DMF with stirring. A
solution of H-Phe-Val-NH2.p-TsOH (0.1 g, 0.23 mmol) and DIEA
(0.041 mL, 0.23 mmol) in DMF was added to this mixture. The
reaction mixture was stirred at 0C for 1 h and overnight at rt. It
was then diluted with 50 ml of ethyl acetate, and then washed
with 5% citric acid (2 × 10 mL), saturated NaHCO3 (2 × 10 mL),
and then saturated NaCl (2 × 10 mL). The organic layer was
dried over anhydrous Na2SO4 and filtered. The solvent was
removed in vacuo with the aid of a rotary evaporator. The crude
product was dried under reduced pressure. The purity of 10 was
measured by reverse-phase HPLC with the following conditions:
detection at 220 nm (Agilent 1200 PDA detector); Eclipse plus
C8 column (3.5 µm 4.6 x 250 mm); linear gradient over 15 min
(30 to 55 % CH3CN in H2O/ 0.1% TFA); and flow rate 1.0
mL/min., tR = 8.54 min. (84.50 %), tR = 9.25 min. (15.50 %).
Yield: 0.05 g (38.5 %), mp: 259-262°C. For exact mass
determination, a sample of 10 was prepared by dissolution in
H2O/CH3CN and dilution in H2O/CH3CN/1%TFA: m/z = 570.568
[M+H]+.
General Procedure for Solid-Phase Assembly of Model
Peptides (11-13)
The general synthesis was carried out manually using a
disposable plastic syringe attached to a water aspirator as a
reaction vessel. The synthesis was carried out as follows: 100
mg of Fmoc-Rinkamide-AM-PS resin (0.059 mmol/g) in a 10-mL
disposable syringe fitted with a teflon filter was washed with
CH2Cl2 (3 x 10 mL) and DMF (3 x 10 mL) and deprotected with
10 mL of 20% piperidine in DMF for 7 min. The deprotected
resin was washed with DMF (3 x 10 mL), CH2Cl2 (3 x 10 mL)
and again with DMF (3 x 10 mL). Preactivation was carried out
for 5 min using Fmoc-L-Val-OH or Fmoc-L-Leu-OH (0.236 mmol,
4 equiv.), HATU (0.09 g, 0.236 mmol, 4 equiv.), and DIEA (0.09
mL, 0.472 mmol, 8 equiv.) in 0.2 ml of DMF. The solution of the
activated amino acid was added to the resin, and the resulting
mixture was periodically stirred with a teflon stick every 5 min
over a period of 2 h. The loaded resin was washed with DMF (3
N-(4,6-dipiperidino-1,3,5-triazin-2-yl)-Gly-Phe-Val-NH2 (9)
HATU (0.087 g, 0.23 mmol) was added to an ice-cold solution of
N-(4,6-dipiperidino-1,3,5-triazin-2-yl)-Gly-OH 2b (0.074 g, 0.23
mmol) and DIEA (0.081 mL, 0.46 mmol) in DMF with stirring. A
solution of H-Phe-Val-NH2.p-TsOH (0.1 g, 0.23 mmol) and DIEA
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700770
ChemMedChem
FULL PAPER
x 10 mL), and the Fmoc group was removed with 10 mL of 20 %
piperidine in DMF for 7 min. Washing of the deblocked resin with
DMF, CH2Cl2, and DMF was followed by an analogous coupling
step with the second Fmoc-L-amino acid. Subsequent L-amino
acids were added in the same manner. To complete the
synthesis of the desired peptides, the previously synthesized
peptide-resin underwent one of the following three routes (route i
or ii).
Route i: The peptide-resin was treated with 10 mL of 50% TFA
in CH2Cl2 with shaking for 2.5 h. The peptide formed was filtered
and concentrated in vacuo and then precipitated by the addition
of cold ether (40 mL). The crude peptide was filtered. The free
peptide underwent an additional coupling step with N-(4,6-
disubstituted-1,3,5-triazin-2yl)-Gly-OH (1b, 2b or 3b) to afford
the desired N-(4,6-disubstituted-1,3,5-triazin-2yl)-peptides.
H2O/CH3CN and dilution in H2O/CH3CN/1%TFA: m/z (MALDI-
TOF/TOF) = [M+H]+ = 627.350.
Biology
The protozoan used in this study was L. aethiopica, which is the
main causal agent of cutaneous leishmaniasis in Ethiopia.
Culture medium for anti-leishmanial activity
RPMI-1640 (Gibco, Invitrogen Co., UK), 10% heat-inactivated
fetal calf serum (HIFCS), penicillin-streptomycin and 1% L-
glutamine, all from Sigma Chem. Co., St. Louis, USA, were used
to make complete culture media.
In vitro antipromastigote activity
All the compounds, dissolved in DMSO to a final concentration
of 1 mg/mL, were evaluated for antileishmanial activity. The final
concentration of DMSO did not exceed 0.1% and thus had no
effect on the parasite. Both test and standard solutions were
serially diluted to appropriate concentrations using fresh
complete media.[7b, 28a,b] The compounds were prepared by serial
dilutions (starting from 10 to 0.04 μg/mL). Amphotericin B
deoxycholate and miltefosine were used as positive controls for
comparison of the antileishmanial activity of the selected
compounds and were used in serial dilutions. Promastigote
forms of L. aethiopica were used for the assay. 100 μL of culture
media containing 3 x 106 promastigotes of L. aethiopica was
seeded in each well of a 96-well flat-bottomed plate. Various
dilutions of the test compounds (10, 3.33, 1.11, 0.37, 0.12, 0.04
μg/mL) were added to the parasites. The assay was done in
triplicate. Wells containing only the parasites, media and DMSO
were used as negative controls. The plates were then kept at rt
(21 ± 1°C). After 24 h, 10 μL of Alamar blue (12.5 mg resazurin
dissolved in 100 ml of distilled water)[28c] was added to each well.
Absorbance of the resulting mixture was measured after 48 h at
540 and 630 nm using a plate reader. Alamar blue works
through the conversion of resazurin (7-hydroxy-3H-phenoxazine-
3-one-10-oxide), the active ingredient of Alamar blue® (blue and
non-fluorescent), to resorufin (pink and highly fluorescent)
through reduction reactions of metabolically active cells. The
amount of fluorescence produced is proportional to the number
of living cells.[28d,e] It is worth mentioning that the assay method
used in this article was validated according to the reported
procedures.[27] It was found that, there is a direct and linear
correlation between the fluorescence intensity and the cell
concentration (promastigote or amastigote).
Route ii: The peptide-resin was treated with 10 mL of 50 % TFA
in CH2Cl2 with shaking for 2.5 h. The peptide formed was filtered
and concentrated in vacuo and then precipitated by the addition
of cold ether (40 mL). The crude peptide was filtered. The free
peptide reacts with 2-chloro-4,6-dimethoxy-1,3,5-triazine in the
presence of triethyl amine at rt overnight to give N-(4,6-
dimethoxy-1,3,5-triazin-2yl)-peptide after neutralization with 1N
HCl.
N-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-Gly-Gly-Phe-Val-NH2 (11).
Route i or Route ii, the sticky product was obtained, yield 0.03 g
(58.8%). Purity was detected by reverse-phase HPLC with the
following conditions: detection at 220 nm (Agilent 1200 PDA
detector); Eclipse plus C18 column (3.5 µm 4.6 x 100 mm); linear
gradient over 14 min (0 to 50 % CH3CN in H2O/ 0.1% TFA); and
flow rate 1.0 mL/min., tR = 3.42 min. (100%). For exact mass
determination, a sample of 11 was prepared by dissolution in
H2O/CH3CN and dilution in H2O/CH3CN/1%TFA: m/z (MALDI-
TOF/TOF) = [M+H] + = 517.644.
N-(4,6-Dipiperidino-1,3,5-triazin-2-yl)-Gly-Gly-Phe-Val-NH2
(12).
Route i, the product was obtained as a white solid, 0.042 g
(68.0 %) yield; mp: 234-237oC. Purity was detected by reverse-
phase HPLC with the following conditions: detection at 220 nm
(Agilent 1200 PDA detector); Eclipse plus C18 column (3.5 µm
4.6 x 100 mm); linear gradient over 20 min (0 to 50 % CH3CN in
H2O/ 0.1% TFA); and flow rate 1.0 mL/min., tR = 10.71 min.
(98.28 %). For exact mass determination, a sample of 12 was
prepared by dissolution in H2O/CH3CN and dilution in
H2O/CH3CN/1%TFA: m/z (MALDI-TOF/TOF)= [M+H] + = 623.548.
In vitro anti-amastigote activity
N-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-Gly-Gly-Phe-Val-NH2
(13).
Axenic amastigote were produced appling the method described
by Teixeira et al.[30] The compounds were serially diluted in a 96-
well microtitre plate to a final test concentration of 0.04-10 μg/mL
in 50 μL culture medium, and 50-μL suspensions containing 2 x
107 cells/ml axenic amastigotes were added to each well. The
plate contents were then incubated in a humidified atmosphere
at 31oC under a 5% CO2 for 72 h. After 68 h of incubation, a 10
μL of fluorochrome resazurin solution (12.5 μg dissolved in 100
mL of PBS (pH = 7.2)) was added to each well and the
fluorescence intensity was measured after a total incubation
time of 72 h using 37 Victor3 Multilabel Counter at excitation
Route i, the product was obtained as a white solid, 0.045 g
(71%) yield; mp: 242-245°C. Purity was determined by reverse-
phase HPLC with the following conditions: detection at 220 nm
(Agilent 1200 PDA detector); Eclipse plus C18 column (3.5 µm
4.6 x 100 mm); linear gradient over 20 min (0 to 50 % CH3CN in
H2O/ 0.1% TFA); and flow rate 1.0 mL/min., tR = 16.54 min.
(70.79 %), tR = 17.41 min. (29.21 %). For exact mass
determination, a sample of 13 was prepared by dissolution in
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700770
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FULL PAPER
wavelength of 530 nm and emission wavelength of 590 nm. The Ethical conduct of research
IC50 value for each compound was evaluated from sigmoidal
dose-response curves using Graph pad prism 3.0 software. The
results were expressed as mean ± SD of triplicate experiments
with each test concentration measured in duplicate. Assays with
standard anti-leishmanial drugs and negative controls (medium
alone and 1% DMSO) were also performed to have reference
values. The background fluorescence intensity of each
compound and reference drug was measured.[31]
The protocols used in this study followed the guidelines set in
‘The Guide for the Care and Use of Laboratory Animals,’ and got
approval by ACUC, Faculty of Pharmacy, Alexandria University,
Project No. ACUC17/18, at 29/4/2017 ACUC17/18.
Conflicts of Interest
The authors declare no conflict of interest.
In vitro cytotoxicity assay
The cytotoxicity of the compounds was tested in the Vero cell
line using the Mosmann method, with certain modifications, as
described in the literature.[32] Briefly, the cells were incubated for
72 h with a range of dilutions of the selected compounds, using
MTT as reagent for the detection of cytotoxicity. 50% cytotoxic
concentration (CC50) values represent the concentration of
compound required to kill 50% of the cells. The SI (Table 3) was
calculated using the formula, SI = CC50/IC50
Keywords: 1,3,5-Triazine derivatives • Peptide • Morpholine •
Piperidine • Antileishmanial
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The authors thank the Alexandria University-Research
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Entry for the Table of Contents
The antileishmanial activity of the 1,3,5-triazino-peptide derivatives revealed that four
dipeptide amide derivatives showed better antipromastigote or antiamastigote activity than
that of the reference standard drug miltefosine with no significance acute toxicity.
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