Design, synthesis and docking studies of novel macrocyclic pentapeptides as anticancer multi-targeted kinase inhibitors

Article abstract of DOI:10.3390/molecules23102416

A series of macrocyclic pyrido-pentapeptide candidates 2-6 were synthesized by using N,N-bis-[1-carboxy-2-(benzyl)]-2,6-(diaminocarbonyl)pyridine 1a,b as starting material. Structures of the newly synthesized compounds were established by IR, 1H and 13C-NMR, and MS spectral data and elemental analysis. The in-vitro cytotoxicity activity was investigated for all compounds against MCF-7 and HepG-2 cell lines and the majority of the compounds showed potent anticancer activity against the tested cell lines in comparison with the reference drugs. Out of the macrocyclic pyrido-pentapeptide based compounds, 5c showed encouraging inhibitory activity on MCF-7 and HepG-2 cell lines with IC50 values 9.41 1.25 and 7.53 1.33 M, respectively. Interestingly, 5c also demonstrated multitarget profile and excellent inhibitory activity towards VEGFR-2, CDK-2 and PDGFR? kinases. Furthermore, molecular modeling studies of the compound 5c revealed its possible binding modes into the active sites of those kinases.

Full text of DOI:10.3390/molecules23102416

molecules
Article
Design, Synthesis and Docking Studies of Novel
Macrocyclic Pentapeptides as Anticancer
Multi-Targeted Kinase Inhibitors
Abd El-Galil E. Amr 1,2,
* ,
, Mohamed H. Abo-Ghalia 3 , Gaber O. Moustafa 3
Mohamed A. Al-Omar 1 , Eman S. Nossier 4 and Elsayed A. Elsayed 5,6
1
Pharmaceutical Chemistry Department, Drug Exploration & Development Chair (DEDC),
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; malomar1@ksu.edu.sa
Applied Organic Chemistry Department, National Research Centre, 12622 Dokki, Giza, Egypt
Department of Peptide Chemistry, National Research Centre, 12622 Dokki, Giza, Egypt;
mhaboghalia@yahoo.com (M.H.A.-G.); gosman79@gmail.com (G.O.M.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, 11754 Cairo,
Egypt; dr.emannossier@gmail.com
Bioproducts Research Department, Zoology Department, Faculty of Science, King Saud University,
Riyadh 11451, Saudi Arabia; eaelsayed@ksu.edu.sa
Chemistry of Natural and Microbial Products Department, National Research Centre, 12622 Dokki,
Cairo, Egypt
2
3
4
5
6
*
Correspondence: aeamr1963@yahoo.com; Tel.: +966-565-148-750
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 September 2018; Accepted: 18 September 2018; Published: 20 September 2018
Abstract: A series of macrocyclic pyrido-pentapeptide candidates
2–
6
were synthesized by using
N,N-bis-[1-carboxy-2-(benzyl)]-2,6-(diaminocarbonyl)pyridine 1a
,b as starting material. Structures
of the newly synthesized compounds were established by IR, 1H and 13C-NMR, and MS spectral
data and elemental analysis. The in-vitro cytotoxicity activity was investigated for all compounds
against MCF-7 and HepG-2 cell lines and the majority of the compounds showed potent anticancer
activity against the tested cell lines in comparison with the reference drugs. Out of the macrocyclic
pyrido-pentapeptide based compounds, 5c showed encouraging inhibitory activity on MCF-7 and
HepG-2 cell lines with IC50 values 9.41
demonstrated multitarget profile and excellent inhibitory activity towards VEGFR-2, CDK-2 and
PDGFR kinases. Furthermore, molecular modeling studies of the compound 5c revealed its possible
±
1.25 and 7.53
±
1.33 µM, respectively. Interestingly, 5c also
β
binding modes into the active sites of those kinases.
Keywords: macrocyclic pentapeptides; in vitro anticancer activity; multitarget; molecular
modeling studies
1. Introduction
Cancer is still considered one of the most serious diseases threatening human life. In the past three
decades, there enormous efforts have been undertaken to confront cancer diseases. Within such efforts,
many therapeutic agents have been developed to treat cancer patients in their early, as well as late
cancer-developing stages [1]. These agents vary from antibiotics, chemically synthesized compounds,
and natural products-based drugs.
Macrocyclic motifs are commonly defined as a ring system containing 12 or more atoms [2]. They
are privileged scaffolds in the fields of chemistry, biology, and medicine [3–5]. There are different
classes of macrocycles like peptidic and nonpeptidic natural products, non-natural (synthetic) peptides
and non-natural (synthetic) macrocycles [6].
Molecules 2018, 23, 2416; doi:10.3390/molecules23102416
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2416
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Furthermore, peptides constitute a major class of important anticancer therapeutic agents [
7].
Chemically synthesized peptides have been reported to exhibit antimicrobial, anti-inflammatory [
8–
16],
as well as anticancer properties [17
characteristics of some bis-amino acid and peptide conjugates of dipicolinic acid [22]. Our studies of
these compounds exemplified by compound [ ] (Figure 1) revealed an interesting anticancer activity,
–21]. We have previously explored the analytical and biological
A
probably via DNA intercalation, as well as an outstanding metal sensor property, particularly, for
pollutant lead (Pb2+) cations [23].
O
O
N
NH
HN
HN
O
O
O
O
NH
HN
NH
O
OMe
A
Figure 1. Structure of the anticancer agent.
The advances in molecular biology and genetics help in identification of molecular targets that
are related to cancer cells or overexpressed on them. The design of compounds affecting these
targets improves the development of more selective anticancer drugs with less toxic side effects [24].
Macrocycles were reported to display antitumor properties, which may be attributed to inhibition
of different enzymes involved in carcinogenesis cases. The prominent examples of macrocycles,
illustrated in Figure 2, exhibited a potent inhibitory activity against various kinases, e.g., CDK-2,
VEGFR-2, JAK-2, FLT-3, PDK-1 and EGFR [6,25–28].
Br
O
H
N
NCH3
N
N
HN
S
N
HN
O
O
N
N
H
B (CDK-2 &VEGFR-2 inhibitor)
SG1317/TG02 (CDK-2, JAK-2 & FLT-3 inhibitor)
O
NH
O
N
Cl
Cl
N
Zn
N
N
Cl
O
N
N
N
N
N
N
H
H
H
H
D (EGFR inhibitor)
C (PDK-1, CDK-2, Flt-3 & JAK-2 inhibitor)
Figure 2. Reported and proposed macrocyclic conjugates with anticancer and different kinases
inhibitory activity.

Molecules 2018, 23, 2416
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In view of these observations and as continuation of our previous works in peptide heterocyclic
chemistry, we have herein synthesized some new linear and macrocyclic peptidopyridine derivatives,
and tested their anticancer activity in comparison to Tamoxifen and 5-Fluorouracil® as positive
controls. Furthermore, they were screened for their inhibitory activity against VEGFR-2, EGFR,
PDGFRβ and CDK-2 enzymes. Additionally, molecular modeling study was performed to explore the
most appropriate binding modes of the most potent target compounds.
2. Results and Discussion
2.1. Chemistry
In the previous work [9,29], L-amino acid methyl esters was initially coupled with dipicolinic
acid via the conventional acid chloride method to give the corresponding 2,6-bis-Nα-L-diamino
acid pyridine methyl ester derivatives. In the present work, a series of linear and macrocyclic
pyridopentapeptide derivatives
(diamino-carbonyl)pyridine (1a
2
–
6
were synthesized based on N,N-bis-[1-carboxy-2-(benzyl)]-2,6-
,
b) and they are screened as anticancer agents. Treatment of with
1
L-amino acid methyl ester hydrochloride in the presence of ethyl chloroformate in dichloromethane
afforded the corresponding tetrapeptide pyridine methyl ester derivatives 2a
–
c, respectively
(Scheme 1).
O
O
N
R1
O
O
OH
OH
O
N
ClH H2N
CH3
NH
HN
O
R1
R1
O
TEA
O
O
O
CH3
CH3
O
O
N
Cl
Cl
NaOH/MeOH
R2
O
O
O
ClH H2N
CH3
O
O
N
N
O
NH
HN
O
R1
R1
NH
HN
R1
R1
O
O
H
O
HN
O
O
O
O
NH
1a,bH
R2
R2
OMe
OMe
a, R1 = CH(CH3)CH2CH3;
b, R1 = CH2Ph
2a-c
a, R1 = R2 = CH(CH3)CH2CH3;
b, R1 = CH(CH3)CH2CH3;R2 = CH2Ph
c, R1 = CH2Ph; R2 = CH(CH3)CH2CH3
Scheme 1. Synthetic pathway for compounds 2a–c.

Molecules 2018, 23, 2416
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Hydrolysis of 2a
–c
with methanolic sodium hydroxide to afford the corresponding tetrapeptide
pyridine derivatives 3a
afford the corresponding cyclic pentapeptide esters 4a
4a was hydrolyzed with methanolic sodium hydroxide to give the corresponding cyclic pentapeptide
acids 5a , or by hydrazonolysis with hydrazine hydrate in methanol to give the corresponding cyclic
pentapeptide acid hydrazides 6a–c, respectively (Scheme 2).
–
c, which were cyclized with L-lysine methyl ester by different methods to
–
c, respectively. The cyclized pentapeptide esters
–
c
–c
O
O
O
O
N
N
NH
HN
R1
HN
R1
NH
NH
HN
R1
HN
R1
NH
NaOH/MeOH
O
O
O
O
O
O
O
O
R2
R2
R2
R2
OMe
OMe
OH
OH
2a-c
3a-c
O
O
ClH.H2N
NH2.HCl
OMe
N
( )3
NH
HN
R1
R1
NH
O
HN
O
O
O
O
R2
R2
HN
NH
O
OMe
4a-c
NH2NH2.H2O
NaOH/MeOH
O
O
O
O
N
N
NH
HN
NH
HN
R1
R1
NH
R1
R1
NH
HN
O
O
O
O
HN
O
O
O
O
R2
HN
R2
R2
R2
NH
OH
HN
NH
5a-c
O
O
NHNH2
6a-c
2-6
a, R1 = R2 = CH(CH3)CH2CH3;
b, R1 = CH(CH3)CH2CH3;R2 = CH2Ph
c, R1 = CH2Ph; R2 = CH(CH3)CH2CH3
Scheme 2. Synthetic pathway for compounds 3–6.

Molecules 2018, 23, 2416
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2.2. Anticancer Activity
The cytotoxic effects of all newly synthesized compounds were evaluated against MCF-7 and
HepG-2 cell lines. Results obtained (Figure 3) showed that all prepared compounds affected both cell
lines in a dose-dependent manner, where increasing the applied concentration gradually decreased
cell viability. All tested compounds exhibited moderate to excellent cytotoxic activities on both tested
cell lines in comparison with control drugs. Compounds 4a
at lower range of tested concentrations. However, compounds 2c
inactive against MCF-7 cells, since they showed no practical IC50 (>100
(Table 1), it was noticed that only compounds 6a and 6b affected MCF-7 cells more than HepG-2 cells
1.10 M for MCF-7, and 12.44 1.3 and 11.53 1.70 M for HepG-2,
,
4b
,
5a,
5b
,
6a and 6b were more potent
4a and 6c were considered
M). Concerning IC50 data
,
3a
,
µ
(
IC50 = 11.83 ± 1.62 and 10.87
±
µ
±
±
µ
respectively). While, the rest of the prepared compounds showed more noticeable anticancer activities
in case of HepG-2 cells rather than MCF-7 cells. Comparing the obtained IC50 values for HepG-2
cells with those of the control drugs explored that compound 5c was the most potent in comparison
with tamoxifen and 5-fluorouracil (IC50 = 7.53 ± 1.33, 29.38
Compounds 2a 2b 3b 4a 5a and 6a afforded higher cytotoxic activity, while compounds 3a
and 3c gave approximately equipotent cytotoxic activity (IC50 = 26.01
2.35 and 26.64 ± 1.85 µM
respectively). For MCF-7 cell lines, 5c was also the most active derivative in comparison with tamoxifen
(IC50 = 9.41 1.25 and 22.40 2.42 M, respectively), followed by compounds 4b 6b 5b 6a and 6a
(IC50 = 10.45 ± 1.33, 10.87 ± 1.10, 11.32 ± 1.15, 11.83 ± 1.62 and 12.67 ± 2.40 µM, respectively).
±
1.15 and 43.84
±
1.84 µM, respectively).
,
,
,
–
c
,
–
c
–c
±
,
±
±
µ
,
,
,
110
100
90
80
70
60
50
40
30
20
10
110
100
90
Cpd2a
Cpd3a
Cpd4c
Cpd2b
Cpd3b
Cpd5c
Cpd2c
Cpd3c
Cpd6c
Cpd4a
Cpd5b
Cpd4b
Cpd6a
Cpd5a
Cpd6b
80
70
60
50
40
30
0
10
20
30
40
50
0
2
4
6
8
10
Conc. ( M)
μ
Conc. ( M)
μ
HepG2
HepG2
110
100
90
80
70
60
50
40
30
20
10
110
100
90
Cpd2a
Cpd3a
Cpd4c
Cpd2b
Cpd3b
Cpd5c
Cpd2c
Cpd3c
Cpd6c
Cpd4a
Cpd5b
Cpd4b
Cpd6a
Cpd5a
Cpd6b
80
70
60
50
40
30
0
10
20
30
40
50
0
2
4
6
8
10
Conc. ( M)
μ
Conc. ( M)
μ
Figure 3. Effect of different concentrations of the prepared compounds on the viability of HepG-2 and
MCF-7 cell lines. Data are expressed as means ± SEM (standard error mean).

Molecules 2018, 23, 2416
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Table 1. IC50 of the tested compounds 2–6 against MCF-7 and HepG-2 cell lines.
IC50 (Mean ± SEM) (µM)
Compound
MCF-7
HepG-2
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
31.64 ± 1.30
32.58 ± 1.50
-
20.37 ± 1.36
15.80 ± 1.66
35.52 ± 1.83
26.01 ± 2.35
13.54 ± 1.45
26.64 ± 1.85
11.59 ± 2.70
10.25 ± 2.20
18.84 ± 1.47
11.19 ± 1.95
10.09 ± 2.05
7.53 ± 1.33
12.44 ± 1.3
11.53 ± 1.70
12.07 ± 1.68
29.38 ± 1.15
43.84 ± 1.84
-
25.33 ± 1.18
29.55 ± 2.06
-
10.45 ± 1.33
29.15 ± 1.39
12.67 ± 2.40
11.32 ± 1.15
9.41 ± 1.25
11.83 ± 1.62
10.87 ± 1.10
-
Tamoxifen
22.40 ± 2.42
5-Fluorouracil®
-
IC50: Compound concentration required to inhibit the cell viability by 50%, SEM = standard error mean; each value
is the mean of three values.
By analysis of the previous results, the cytotoxic activities of the tested compounds, with the
exception of compound 4c, were in the following order compound
structure-activity relationships of the synthesized compounds, it was observed that the open chain
derivatives and have lower activity than the cyclized derivatives . Furthermore, insertion
of benzyl moiety at R1 adjacent to the pyridine scaffold in 2c and 3c gave a marked decrease in
the cytotoxic activity compared to their series 2a and 3a , respectively. This decrease may be
due to formation of steric hindrance. The enhanced anticancer activities of compounds may be
5 > 6 > 4 > 3 > 2. Concerning the
2
3
4–6
,
b
,b
4–6
contributed to the cyclization of these compounds. Contrary to what was found in the open chain
derivatives 2c and 3c, the increased aromaticity of the cyclopeptide by the location of the phenyl
group of phenylalanine neighboring the pyridine nucleus may be the cause of improved activity of
compound 5c > 5b > 5a.
2.3. In Vitro Enzymatic Assays
Upon cellular screening on MCF-7 and HepG-2, compound 5c exhibited higher anticancer
activity in comparison with tamoxifen and 5-fluorouracil. So, it was subjected for in vitro inhibition
assessment against a panel of four different kinases; VEGFR-2, EGFR, PDGFR
using staurosporine as multitarget inhibitor. As shown in Table 2 and Figure 4, compound 5c
exhibited three folds increase in the inhibitory effect against VEGFR-2 (IC50 = 0.01 1.25 M),
two folds increase against CDK-2 (IC50 = 0.06 1.27 M) and approximately equipotent against
PDGFR 1.10, 0.11 1.13
IC50 = 0.08 ± 1.45 µM) in comparison with staurosporine (IC50 = 0.03
and 0.07 ± 1.65 µM, respectively). Furthermore, the inhibitory activity of compound 5c was moderate
against EGFR in comparison with the reference (IC50 = 0.14 1.00 and 0.02 1.32 M, respectively).
These data suggested that compound 5c is a promising multitarget kinase inhibitor.
β and CDK-2 kinases
±
µ
±
µ
β
(
±
±
±
±
µ

Molecules 2018, 23, 2416
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Table 2. Inhibitory evaluation of compound 5c against VEGFR-2, EGFR, PDGFRβ and CDK-2 kinases.
IC50 (Mean±SEM) (µM)
Kinase
5c
Staurosporine
VEGFR-2
EGFR
PDGFRβ
CDK-2
0.01 ± 1.25
0.14 ± 1.00
0.08 ± 1.45
0.06 ± 1.27
0.03 ± 1.10
0.02 ± 1.32
0.07 ± 1.65
0.11 ± 1.13
IC50: Compound concentration required to inhibit the enzyme activity by 50%, SEM = Standard error mean; each
value is the mean of three values.
Figure 4. IC50 of compound 5c against VEGFR-2, EGFR, PDGFRβ and CDK-2 kinases in comparison
with staurosporine.
2.4. Molecular Modeling Studies
The kinase inhibitory assay revealed that compound 5c showed promising inhibitory activity
against three kinases, namely VEGFR-2, CDK-2 and PDGFR . So, docking simulation was performed
using Molecular Operating Environment (MOE®) 2008.10 [30
31] to predict the binding modes,
affinities, and orientations of compound 5a at the active sites of them. The X-ray crystallographic
structure of PDGFR was not fully resolved [32]. On the other hand, the X-ray crystallographic
β
,
β
structures were reported for VEGFR-2 (pdb code: 4ASD) [33] with sorafenib and for CDK-2 (PDB ID:
2J9M) [34] with PY8. So the docking study was achieved for both VEGFR-2 and CDK-2 kinases
The binding model, shown in Figure 5, was exemplified by the interaction of compound 5c with
VEGFR-2. The carboxylic moiety of 5c shared in the binding pattern with three hydrogen bond donors;
one H-bond was between CO group and the backbone of His1026 (distance: 2.99 Å), and the others were
between OH group and the sidechain of Asp1046 (distance: 1.92 and 2.65 Å). Moreover, the residues
Ser884, Arg1027 and Leu1049 were inserted nicely inside the centre of cyclic pentapeptide scaffold.

Molecules 2018, 23, 2416
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(A)
(B)
Figure 5. Docking solution for 5c in the active site of VEGFR-2; (A,B) showing 2D and 3D
ligand-receptor interactions (hydrogen bonds are illustrated as arrows; C atoms are colored gray,
N blue and O red).
Interaction of compound 5c with the binding site of CDK-2 kinase was illustrated in Figure 6.
There was H-bond acceptor between CO of the carboxylic group and the backbone of Asp86 (distance:
2.89 Å). Another H-bond acceptor was established between the amide nitrogen and the backbone of
Ile10 (distance: 1.80 Å). Furthermore, carbonyl group placed at p-2 of pyridine ring was linked to
backbone of Glu12 (distance: 2.66 Å). In addition, Lys89 located in the centre of cyclic pentapeptide
scaffold formed arene-cation interaction with centroid of benzyl ring.

Molecules 2018, 23, 2416
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(A)
(B)
Figure 6. Docking solution for 5c in the active site of CDK-2; (A,B) showing 2D and 3D ligand-receptor
interactions (hydrogen bonds are illustrated as arrows; C atoms are colored gray, N blue and O red).
Finally, it is deduced from molecular docking studies that the good fitting of compound 5c in the
active sites of VEGFR-2 and CDK-2 enzymes via different types of interactions could be attributed to
the presence of carboxylic group and cyclic pentapeptide scaffold.
3. Materials and Methods
3.1. Chemistry
Melting points were determined in an “Electro Thermal” Digital melting point apparatus
(Shimadzu, Tokyo, Japan), (model: IA9100). Elemental analysis was found within the acceptable
limits of the calculated values (Microanalytical Unit, NRC). Infrared spectra (KBr) were recorded

Molecules 2018, 23, 2416
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on a Nexus 670 FTIR Nicolet, Fourier Transform infrared spectrometer (Perkin Elmer, Hopkinton,
MA, USA). Proton nuclear magnetic resonance (1H-NMR) spectra were run in [d6] DMSO on Jeol 270
MHz or 500 MHz instruments ((Tokyo, Japan). Chemical shifts d are given in ppm. Mass spectra
were run on a MAT Finnigan SSQ 7000 spectrometer (Shimadzu, Kyoto, Japan; Model: QP2010
ultra), using the electron impact technique (EI). Analytical thin layer chromatography (TLC) was
performed on silica gel aluminum sheets, 60 F254 (E. Merck). Specific optical rotations were measured
with a A. Krawss, Optronic, P8000a polarimeter (A. Krüss, Hamburg, Germany), in a 1 dm length
observation tube, at the indicated conditions, and according to the equation: [a]T D =100. a = (c l)
where a = observed rotation angle; D = sodium line (l = 589 nm); c =concentration (g = 100 mL)
,
;
l = path length in dm; and T = temperature (◦C). The following solvent systems (by volume) were
used as eluents for the development of the plates: S: chloroform-methanol-acetic acid (85:10:5); S1:
S-petroleum ether (b.p. 40–60 ◦C) (1:1); S2: S-petroleum ether (b.p. 40–60 ◦C) (3:2); S3: S-petroleum
ether (b.p. 40–60 ◦C) (1:2) and S4: butanol-water-acetic acid-pyridine (120:48:12:40). It is generally
known that basic reaction media enhance racemization. However, under the reaction conditions
employed in this work, especially short reaction times and temperatures below (0 ◦C), only negligible
racemization was observed.
3.1.1. Synthesis of Nα-dipicolinoyl-bis[dipeptide methyl ester] Derivatives (2a–c)
◦
Ethyl chloroformate (0:2 mL, 2 mmol) was added to a stirred and cold (
−
15 C) dichloromethane
b
solution (20 mL) of the corresponding Nα-dipicolinoyl-bis[amino acid] (1a
,
) (1 mmol), containing
N-methylmorpholine (0:2 mL, 2 mmol). The reaction mixture was stirred for additional 10 min, then a
cold dichloromethane solution (20 mL) of the free amino acid methyl esters, namely, L-isolucine-OMe
or L-phenyalanine-OMe (2 mmol), was added. Stirring was maintained for 3 h at (
15 ◦C), then for
−
12 h at room temperature. The reaction mixture was washed with water, 1 N sodium bicarbonate, 1
N potassium hydrogen sulfate and water, and dried over anhydrous calcium chloride. The solvent
was evaporated under reduced pressure to dryness, and the obtained oily residue was solidified
by trituration with a dry ether-n-hexane mixture. The obtained solid was collected by filtration
and crystallized from ethanol-n-hexane to give the corresponding 2,6-pyridine-bis-dipeptide ester
derivatives (2a–c), respectively.
Nα-Dipicolinoyl-bis[L-ILe-L-ILe-methyl ester] (2a). Yield: 80%; m.p. 106–108 C, [α]2D5
:
−
71 (C, 0.02,
◦
MeOH). IR (KBr, cm−1):
(C=O, ester), 1654 and 1440 (C=O amide I and II, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
δ = 8.54–8.51 (m, 3H, Pyr-H), 8.40–8.20 (s, 4H, 4NH, D2O exchangeable), 4.56 (d, 2H, J = 8.8 Hz
ν = 3304 (NH stretching), 3070 (CH, aromatic), 2966 (CH, aliphatic), 1746
1
,
2NHCH, L-Ile), 4.51 (d, 2H, J = 8.8 Hz, 2NHCH, L-Ile), 3.61 (s, 6H, 2OCH3), 3.32–3.30 (m, 4H,
4NHCHCH, L-Ile), 1.23–1.16 (m, 8H, 4CH2, L-Ile), 0.96–0.81 (m, 24H, 8CH3, L-Ile). 13C-NMR (125 MHz,
ppm, DMSO-d6): δ = 171.6, 170.9 (2C, 2
COOCH3), 162.8 (2C, 2CO), 148.7 (2CO, Pyridine dicarbonyl),
139.9 (2 C, pyridine, C2, C6), 130.3 (1C, pyridine, C4), 124.7 (2 C, pyridine, C3, C5), 56.7–54.5 (4 C,
NHCH), 50.2 (2 C, 2OCH3), 40.3, 39.3 (4 C, 4NHCHCH, L-Ile), 39.0, 36.0 (4C, 2 CH2), 24.8, 11.0 (8 C,
8CH3, L-Ile). MS (EI, 70 eV): m/z (%) = 648 (M+ + 1, 3.62), 647 (M+, 3.74), 591 (18.87), 475 (100), 330
(16.79), 302 (14.78), 57 (1.16), 55 (0.53). Analysis for C33H53N5O8 (647.80): Calcd. C, 61.18; H, 8.25; N,
10.81. Found: C 61.15, H 8.22, N 10.77.
Nα-Dipicolinoyl-bis[L-ILe-L-Phe-methyl ester] (2b). Yield: 70%; m.p. 122–124 C, [α]2D5
:
−
57 (C, 0.02,
◦
MeOH). IR (KBr, cm−1):
ν = 3330 (NH stretching), 3087 (CH, aromatic), 2967 (CH, aliphatic), 1724
1
(C=O, ester), 1653 and 1532 (C=O amide I and II, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
δ = 8.57–8.53 (m, 3H, Pyr-H), 8.54 (s, 4H, 4NH, D2O exchangeable), 8.25–8.20 (m, 10H, Ar-H, L-Phe-ala),
4.56 (t, 2H, J = 5.02 Hz, 2NHCH, L-Phe-ala), 4.21 (d, 2H, J = 8.8 Hz, 2NHCH, L-Ile), 4.16 (d, 4H,
J = 8.1 Hz, 2CH2, L-Phe-ala), 3.31 (s, 6H, 2 OCH3), 2.98–2.92 (m, 2H, 2NHCHCH, L-Ile), 1.43–1.36 (m,
4H, 2CH2, L-Ile), 0.96–0.81 (m, 12H, 4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
(2 C, 2 OOCH3), 170.7, 162.8 (2 O, L-Ile), 148.7 (2 O, Pyridine dicarbonyl), 130.0 (2C, pyridine, C2,
δ = 172.6
C
C
C

Molecules 2018, 23, 2416
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C6), 128.3 (1C, pyridine, C4), 125.7 (2 C, pyridine, C3, C5), 138.2, 127.5, 126.1, 125.4 (12C, Ph-C), 56.8,
56.3 (4C, NH H), 52.9 (2C, 2O H3), 40.4, 40.1 (2C, 2 H2, L-Phe-ala), 39.8–38.7 (2C, 2NHCH H, L-Ile),
37.2–36.1 (2C, CH2), 24.7, 11.0 (4C, 4
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 717 (M+ + 1, 0.85), 620
C
C
C
C
C
(1.95), 563 (7.83), 461 (100), 404 (3.59), 330 (30.42), 302 (43.45), 69 (8.42), 57 (3.89), 51 (0.96). Analysis for
C39H49N5O8 (715.80): Calcd. C, 65.44; H, 6.90; N, 9.78. Found: C 65.42, H 6.88, N 9.76.
Nα-Dipicolinoyl-bis[L-Phe-L-ILe- methyl ester] (2c). Yield: 65%; m.p. 150–152 C, [α]2D5
:
−
30 (C, 0.02,
◦
MeOH). IR (KBr, cm−1):
(C=O, ester), 1695 and 1600 (C=O amide I and II, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
= 8.90–8.85 (m, 3H, Pyr-H), 8.50–8.41 (s, 4H, 4NH, D2O exchangeable), 7.60–7.25 (m, 10H, Ar-H,
ν = 3338 (NH stretching), 3100 (CH, aromatic), 3000 (CH, aliphatic), 1780
1
δ
L-Phe-ala), 4.55 (t, 2H, J = 5.02 Hz, 2NHCH, L-Phe-ala), 4.40 (d, 2H, J = 8.8 Hz, NHCH, L-Ile), 4.25 (d, 4H,
J = 8.1 Hz, 2CH2, L-Phe-ala), 3.30–3.20 (s, 6H, 2OCH3), 2.90–2.70 (m, 2H, 2NHCHCH, L-Ile), 1.40–1.25
(m, 4H, 2CH2, L-Ile), 1.05–0.80 (m, 12H, 4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6): δ = 174.0
(2CO, 2
C6), 138.0 (1C, pyridine, C4), 126.1 (2 C, pyridine, C3, C5), 139.2, 127.7, 126.4, 125.3 (12C, Ph-C), 61.0,
55.0 (4C, NH H), 45.1 (2C, 2 H2, L-Phe-ala), 40.8, 40.5 (2C, 2NHCH H, L-Ile), 39.0, 37.0 (2C, 2CH2),
24.0 (2C, 2O H3), 18.0, 11.0 (4C,
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 716 (M+, 18.00), 600 (22.12), 484
COOCH3), 171.9 (2CO, L-Phe-ala), 160.0 (2CO, Pyridine dicarbonyl), 150.5 (2C, pyridine, C2,
C
C
C
C
C
(34.00), 350 (29.39), 290 (32.67), 205 (75.81), 125 (100), 91 (82.32), 70 (33), 57 (45.87), 50 (18.85). Analysis
for C39H49N5O8 (715.80): Calcd. C, 65.44; H, 6.90; N, 9.78. Found: C 65.40, H 6.83, N 9.77.
3.1.2. Synthesis of Nα-Dipicolinoyl-bis[dipeptide]derivatives (3a–c)
To a cold ( –c) (1 mmol) in methanol
15 ◦C) solution of the corresponding tetrapeptide ester (2a
−
(20 mL) with stirring, sodium hydroxide (1 N, 25 mL) was gradually added. The reaction mixture was
stirred for 2 h at the same temperature then for 3 h at room temperature. The solvent was distilled
off under reduced pressure, and the remaining aqueous solution was cooled and acidified with 1
N hydrochloric acid to pH = 3. The obtained solid was filtered off, washed with water, dried and
crystallized from ethanol/water to give the corresponding tetrapeptide acids (3a–c), respectively.
Nα-Dipicolinoyl-bis[L-ILe-L-ILe]acid (3a). Yield: 88%; m.p. 178–180 ◦C, [α]2D5
(KBr, cm−1):
= 3280 (NH stretching), 3060 (CH, aromatic), 2964 (CH, aliphatic), 1748 (C=O, acid),
1651 and 1537 (C=O amide I and II, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
:
−
101 (C, 0.02, MeOH). IR
ν
1
δ
= 12.48 (s,
2 H, 2 OH, D2O exchangeable), 8.56–8.53 (m, 3H, Pyr-H), 8.39–8.22 (s, 4H, 4NH, D2O exchangeable),
4.52 (d, 4H, J = 8.8 Hz, 4NHCH, L-Ile), 3.61–3.58 (m, 4H, 4NHCHCH, L-Ile), 1.90–1.15 (m, 8H, 4CH2,
L-Ile), 0.91–0.82 (m, 24H, 8CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
162.6 (2CO, L-Ile), 160.5 (2C
δ
= 171.5 (2C, 2
O, Pyridine dicarbonyl), 139.7 (2C, pyridine, C2, C6), 137.0 (1C, pyridine,
C4), 124.3 (2 C, pyridine, C3, C5), 56.9, 51.6 (4C, 4NH H), 40.3, 39.2 (4C, 4NHCH H, L-Ile), 38.9, 36.4
(4C, 4CH2, L-Ile), 23.8, 10.9 (8C, 8
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 621 (M+ + 1, 9.35), 537 (20.12),
COOH),
C
C
C
509 (100), 330 (25.60), 302 (49.23), 69 (17.34), 59 (52.77), 57 (18.36), 50 (0.92). Analysis for C31H49N5O8
(619.70): Calcd. C, 60.08; H, 7.97; N, 11.30. Found: C 60.08, H 7.92, N 11.29.
Nα-Dipicolinoyl-bis[L-ILe-L-Phe]acid (3b). Yield: 82%; m.p. 184–186 ◦C, [α]2D5
(KBr, cm−1):
= 3329 (NH stretching), 3072 (CH, aromatic), 2965 (CH, aliphatic), 1726 (C=O, acid),
1652 and 1532 (C=O amide I and II, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
:
−
106 (C, 0.02, MeOH). IR
ν
1
δ
= 12.65 (s,
2H, 2OH, D2O exchangeable), 8.52–8.43 (m, 3H, Pyr-H), 8.40–8.20 (s, 4H, 4NH, D2O exchangeable),
7.23–7.08 (m, 10H, Ar-H, L-Phe-ala), 4.45 (t, 2H, J = 5.02 Hz, 2NHCH, L-Phe-ala), 4.40 (d, 2H, J = 8.8 Hz,
2NHCH, L-Ile), 4.31 (d, 4H, J = 8.1 Hz, 2CH2, L-Phe-ala), 3.00–2.91 (m, 2H, 2NHCHCH, L-Ile), 1.9–1.5
(m, 4H, 2CH2, L-Ile), 1.15–0.8 (m, 12H, 4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
(2C, 2COOH), 169.1 (2C, 2CO, L-Ile), 162.6 (2C, 2C
δ
= 172.5
O, Pyridine dicarbonyl), 148.5 (2C, pyridine, C2, C6),
139.7 (1C, pyridine, C4), 126.00 (2 C, pyridine, C3, C5), 139.0, 127.2, 126.4, 125.6 (12C, Ph-C), 56.9, 53.2
(4C, 4NH H), 40.3, 40.1 (2C, 2 H2, L-Phe-ala), 39.8, 38.7 (2C, 2NHCH H, L-Ile), 37.0, 36.5 (2C, 2CH2,
L-Ile), 23.8, 10.9 (4C, 4
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 689 (M+ + 1, 1.78), 688 (M+, 4.41), 687
C
C
C
C

Molecules 2018, 23, 2416
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(8.50), 625 (71.56), 524 (100), 495 (92.50), 477 (51.54), 330 (56.17), 86 (36.66), 57 (12.05), 50 (2.34). Analysis
for C37H45N5O8 (687.80): C, 64.61; H, 6.59; N, 10.18. Found: C 64.58, H 6.57, N 10.17.
Nα-Dipicolinoyl-bis[L-Phe-L-ILe]acid (3c). Yield: 77%; m.p. 202–204 ◦C, [α]2D5
(KBr, cm−1):
= 3321 (NH stretching), 3031 (CH, aromatic), 2967 (CH, aliphatic), 1725 (C=O, acid),
1655 and 1530 (C=O amide I and II, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
2 H, 2OH, D2O exchangeable), 9.60–8.50 (m, 3H, Pyr-H), 8.23–8.09 (s, 4H, 4NH, D2O exchangeable),
7.44–7.12 (m, 10H, Ar-H, L-Phe-ala), 4.90 (t, J = 5.02 Hz, 2H, 2NHCH, L-Phe-ala), 4.29 (d, 2H, J = 8.8 Hz
:
−
70 (C, 0.02, MeOH). IR
ν
1
δ
= 12.70 (s,
,
2NHCH, L-Ile), 4.00 (d, 4H, J = 8.1 Hz, 2CH2, L-Phe-ala), 3.34–3.13 (m, 2H, 2NHCHCH, L-Ile), 1.23–1.16
(m, 4H, 2CH2, L-Ile), 0.92–0.80 (m, 12H, 4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6): δ = 173.2
(2C, 2
C2, C6), 139.6 (1C, pyridine, C4), 125.6 (2 C, pyridine, C3, C5), 138.15 126.8, 126.3, 125.8 (12C, Ph-C),
59.8, 56.4 (4C, 4NH H), 43.1, 40.4 (2C, 2 H2, L-Phe-ala), 39.8, 39.2 (2C, 2NHCH H, L-Ile), 39.0, 36.0
(2C, H2), 24.7, 11.0 (4C,
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 689 (M+ + 1, 2.05), 688 (M+, 4.22), 669
COOH), 172.6, 170.8 (2CO, L-Phe-ala), 164.9, 163.4 (2CO, Pyridine dicarbonyl), 148.6 (2C, pyridine,
C
C
C
C
C
(8.33), 625 (22.60), 558 (67.30), 529 (49.57), 461 (18.79), 416 (26.29), 370 (100), 302 (11.74), 86 (29.02), 57
(15.14), 51 (5.33). Analysis for C37H45N5O8 (687.80): Calcd. C, 64.61; H, 6.59; N, 10.18. Found: C 64.60,
H 6.60, N 10.18.
3.1.3. Synthesis of Cyclo-(Nα-Dipicolinoyl)-bis-[dipeptide]-L-Lys-OMe (cyclic pentapeptide methyl
esters) (4a–c)
To a stirred and cold (
Nα-dipicolinoyl-bis[dipeptide] (3a
ethyl chloroformate (0.2 mL, 2 mmol) was added and stirred for 20 min. To the reaction mixture,
free L-lysine methyl ester (1 mmol) in dichloromethane (20 mL) was added at (
15 ◦C) with stirring.
Stirring was maintained for 3 h, at (
15 ◦C), then for 12 h at room temperature. The reaction mixture
−
15 ◦C) dichloromethane solution (20 mL) of the corresponding
) (1 mmol), containing N-methylmorpholine (0:2 mL, 2 mmol),
–c
−
−
was washed with water, 1 N sodium bicarbonate, 1 N potassium hydrogen sulfate and water, and
then dried over anhydrous calcium chloride. The solvent was evaporated under reduced pressure to
dryness, and the obtained oily residue was solidified by trituration with dry ether-n-hexane mixture.
The crude product was purified by preparative thin layer chromatography using S3 as eluent to give
the corresponding cyclic pentapeptide methyl esters (4a–c), respectively.
Cyclo-(Nα-dipicolinoyl)-bis-[L-ILe-L-ILe]-L-Lys-OMe (4a). Yield: 78%; m.p. 77–79 ◦C, [α]2D5
MeOH). IR (KBr, cm−1):
= 3398 (NH stretching), 2945 (CH, aromatic), 2867, 2863 (CH, aliphatic),
1642 (C=O, ester), 1530, 1454 and 1396 (C=O amide I, II and III, respectively). H-NMR (500 MHz,
ppm, DMSO-d6): = 8.90–8.76 (m, 3H, Pyr-H), 8.32, 8.21 (s, 6H, 6NH, D2O exchangeable), 4.66 (d, 4H,
:
−
13 (C, 0.02,
ν
1
δ
J = 8.8 Hz, 4NHCH, L-Ile), 4.45 (t, 1H, J = 9.7 Hz, CH, L-Lys), 3.84 (s, 3H, OCH3), 3.57–3.55 (m, 2H, CH2,
NHCH2), 2.70–2.55 (m, 4H, 4NHCHCH, L-Ile), 2.00–1.45 (m, 6H, 3CH2, NHCH2CH2CH2CH2CHNH),
1.30–1.15 (m, 8H, 4CH2, L-Ile), 1.05–0.80 (m, 24H, 8CH3). MS (EI, 70 eV): m/z (%) = 743 (M+, 13.34), 568
(10.65), 460 (11.38), 129 (23.58), 96 (28.34), 85 (34.33), 69 (69.08), 60 (74.04), 57 (77.97), 55 (100), 50 (14.79).
Analysis for C38H61N7O8 (743.90): Calcd. C, 61.35; H, 8.26; N, 13.18. Found: C 61.30, H 8.22, N 13.14.
Cyclo-(Nα-Dipicolinoyl)-bis-[L-ILe-L-Phe]-L-Lys-OMe (4b). Yield: 81%; m.p. 104–106 ◦C, [α]2D5
0.02, MeOH). IR (KBr, cm−1):
= 3368 (NH stretching), 2945 (CH, aromatic), 2835 (CH, aliphatic),
1657 (C=O, ester), 1528, 1425 and 1222 (C=O amide I, II and III, respectively). H-NMR (500 MHz,
ppm, DMSO-d6): = 8.60–8.58 (m, 3H, Pyr-H), 8.43, 8.21 (2s, 6H, 6NH, D2O, exchangeable), 7.22–7.10
:
−
28 (C,
ν
1
δ
(m, 10H, Ar-H, L-Phe-ala), 4.46 (t, 2H, J = 5.02 Hz, 2NHCH, L-Phe-ala), 4.44 (d, 2H, J = 8.8 Hz,
2NHCH, L-Ile), 4.43 (t, 1H, CH, L-Lys), 4.41 (d, 4H, J = 8.1 Hz, 2CH2, L-Phe-ala), 3.61–3.57 (m, 2H,
CH2, NHCH2, L-Lys), 3.32 (s, 3H, OCH3), 3,00-2.95 (m, 2H, 2NHCHCH, L-Ile), 1.9–1.5 (m, 6H, 3CH2,
NHCH2C
(125 MHz, ppm, DMSO-d6):
139.8 (1C, pyridine, C4), 127.1 (2 C, pyridine, C3, C5), 138.8, 127.6, 126.8, 125.2 (12C, Ph-C), 56.9–51.7
(5C, 5NH H), 40.34 (1C, NH H2, L-Lys), 40.1 (2C, 2 H2, L-Phe-ala), 39.8, 39.5 (2C, 2NHCH H, L-Ile),
H
2CH
2CH
2CHNH), 1.4–1.35 (m, 4H, 2CH2, L-Ile), 0.91–0.81 (m, 12H, 4CH3, L-Ile). 13C-NMR
= 171.6 ( OOCH3), 170.6, 162.7 (6C, 6 O), 148.5 (2C, pyridine, C2, C6),
δ
C
C
C
C
C
C

Molecules 2018, 23, 2416
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39.22, 36.39 (5C, 5CH2), 23.2 (1C, O
C
H3), 15.3, 11.0 (4C, 4
C
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 796
(M+, 1.05), 715 (9.49), 659 (5.53), 509 (71.40), 449 (9.59), 347 (26.17), 302 (100), 180 (62.98), 86 (48.45), 69
(30.62), 52 (0.14). Analysis for C44H57N7O8 (811.96): Calcd. C, 65.09; H, 7.08; N, 12.08. Found: C, 65.00;
H, 7.00; N, 12.00.
Cyclo-(Nα-Dipicolinoyl)-bis-[L-Phe-L-ILe]-L-Lys-OMe (4c). Yield: 75%; m.p. 140–142 ◦C, [α]2D5
0.02, MeOH). IR (KBr, cm−1):
= 3320 (NH stretching), 3065 (CH, aromatic), 2965 (CH, aliphatic), 1783
(C=O, ester), 1663, 1527 and 1447 (C=O amide I, II and III, respectively). H-NMR (500 MHz, ppm,
DMSO-d6): = 8.80–8.78 (m, 3H, Pyr-H), 8.63, 8.51 (s, 6H, 6NH, D2O exchangeable), 7.52–7.10 (m,
10H, Ar-H, L-Phe-ala), 4.65 (t, 2H, J = 5.02 Hz, 2NHCH, L-Phe-ala), 4.50 (d, 2H, J = 8.8 Hz, 2NHCH
:
−
55 (C,
ν
1
δ
,
L-Ile), 4.40 (t, 1H, J = 9.7 Hz, CH, L-Lys), 4.36 (d, 4H, J = 8.1 Hz, 2CH2, L-Phe-ala), 3.82–3.74 (m,
2H, CH2, NHCH2, L-Lys), 3.52 (s, 3H, OCH3), 3,20–3.05 (m, 2H, 2NHCHCH, L-Ile), 1.85–1.45 (m,
6H, 3CH2, NHCH2CH2
L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
pyridine, C2, C6), 140.2 (1C, pyridine, C4), 125.6 (2 C, pyridine, C3, C5), 138.2 127.0, 126.2, 125.4 (12C,
Ph-C), 60.3–55.9 (5C, 5NH H), 48.40 (1C, NH H2, L-Lys), 45.0 (2C, 2 H2, L-Phe-ala), 42.0, 41.5 (2C,
2NHCH H, L-Ile), 40.20, 38.44 (5C, 5CH2), 25.5 (1C, O H3), 13.3, 10.5 (4C, 4CH3, L-Ile). MS (EI, 70 eV):
C
H2
C
H2CHNH), 1.38–1.36 (m, 4H, 2CH2, L-Ile), 0.90–0.75 (m, 12H, 4CH3,
δ
= 174.2 ( OOCH3), 172.8, 168.0 (6C, 6 O), 150.6 (2C,
C
C
C
C
C
C
C
m/z (%) = 813 (M+ + 1, 25.06), 812 (M+, 42.13), 755 (33.32), 720 (37.79), 698 (22.27), 596 (21.32), 571
(30.36), 543 (32.52), 495 (20.68), 458 (25.09), 430 (56.12), 370 (100), 91 (14.24), 69 (10.40), 57 (3.84), 50
(3.80). Analysis for C44H57N7O8 (811.96). Calcd. C, 65.09; H, 7.08; N, 12.08. Found: C, 65.00; H, 7.00; N,
12.00.
3.1.4. Synthesis of Cyclo-(Nα-dipicolinoyl)-bis[dipeptide]-L-Lys-OH (cyclic pentapeptides) (5a–c)
◦
To a stirred and cold methanolic solution (
methyl ester (4a
−
5 C, 20 mL) of the corresponding cyclic pentapeptide
–
c
) (1 mmol), sodium hydroxide (1 N, 25 mL) was gradually added. The reaction
mixture was stirred for 2 h at the same temperature, then for 3 h at room temperature. The solvent was
distilled off under reduced pressure, and the remaining aqueous solution was cooled and acidified
with 1 N hydrochloric acid to pH = 3. The obtained solid was filtered off, washed with water, dried and
crystallized from ethanol-water to give the corresponding cyclic pentapeptides (5a–c), respectively.
Cyclo-(Nα-dipicolinoyl)-bis [L-ILe-L-ILe]-L-Lys-OH (5a). Yield: 69%; m.p. 108–110 C, [α]2D5
:
−
21 (C,
◦
0.02, MeOH). IR (KBr, cm−1):
1724 (C=O, acid), 1655, 1533 and 1456 (C=O amide I, II and III, respectively). H-NMR (500 MHz,
ppm, DMSO-d6): = 12.60 (s, 1H, OH, D2O exchangeable), 8.51–8.49 (m, 3H, Pyr-H), 8.46, 8.20 (2s,
ν = 3317 (NH stretching), 3081 (CH, aromatic), 2967 (CH, aliphatic),
1
δ
6H, 6NH, D2O exchangeable), 4.70- (d, 4H, J = 8.8 Hz, 4 NHCH, L-Ile), 4.18 (t, 1H, J = 9.7 Hz, CH,
L-Lys), 3.31–3.28 (m, 2H, CH2, NHCH2, L-Lys), 1.94–1.92 (m, 4H, 4NHCHCH), 1.90–1.88 (m, 6H, 3CH2,
NHCH2CH2CH2CH2), 1.50–1.16 (m, 8H, 4CH2, L-Ile), 0.96–0.81 (m, 24H, 8CH3, L-Ile). MS (EI, 70 eV):
m/z (%) = 730 (M+, 22.28), 682 (35.08), 621 (48.97), 565 (35.49), 395 (44.98), 285 (31.09), 244 (25.03), 69
(68.36), 57 (100), 51 (14.03). Analysis for C37H59N7O8 (729.90): Calcd. C, 60.88; H, 8.15; N, 13.43. Found:
C 60.84, H 8.11, N 13.40.
Cyclo-(Nα-dipicolinoyl)-bis [L-ILe-L-Phe]-L-Lys-OH (5b). Yield: 73%; m.p. 128–130 C, [α]2D5
:
−
35 (C,
◦
0.02, MeOH). IR (KBr, cm−1):
1725 (C=O, acid), 1655, 1531 and 1448 (C=O amide I, II and III, respectively). H-NMR (500 MHz,
ppm, DMSO-d6): = 12.70 (s, 1H, OH, D2O exchangeable), 8.43–8.38 (3H, Pyr-H), 8.22, 8.20 (2s, 6H,
ν = 3312 (NH stretching), 3067 (CH, aromatic), 2965 (CH, aliphatic),
1
δ
6NH, D2O exchangeable), 7.23–7.08 (m, 10H, Ar-H, L-Phe-ala), 4.45–4.42 (m, 5H, 5CH), 4.40 (t, 4H, J
= 8.1 Hz, 2CH2, L-Phe-ala), 3.41–3.38 (m, 2H, NHCH2, L-Lys), 3,05–2.91 (m, 2H, 2NHCHCH, L-Ile),
1.95–1.50 (m, 6H, 3CH2, NHCH2CH2CH2CH2CHNH), 1.18 (m, 4H, 2CH2, L-Ile), 0.90–0.80 (m, 12H,
4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
(2
C3, C5), 139.0, 127.8, 126.7, 125.5 (12C, Ph-C), 57.0, 53.3 (5C, 5 NH
39.8 (2C, 2 H2, L-Phe-ala), 39.5, 39.3 (2C, 2NHCH H, L-Ile), 39.0, 36.5 (5C, 5CH2), 23.9, 11.0 (4C, 4CH3,
δ
= 172.6 (
O, Pyridine dicarbonyl), 148.6 (2C, pyridine, C2, C6), 139.7 (1C, pyridine, C4), 128.1 (2 C, pyridine,
H), 40.4 (1C, NH H2, L-Lys), 40.1,
COOH), 170.4, 170.3 (4C, 4CO), 162.7
C
C
C
C
C

Molecules 2018, 23, 2416
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L-Ile). MS (EI, 70 eV): m/z (%) = 797 (M+, 16.93), 708 (16.34), 621 (28.74), 562 (22.24), 504 (24.21), 321
(18.01), 281 (17.72), 170 (18.21), 85 (19.59), 71 (40.85), 61 (20.37), 59 (52.07), 57 (100), 51 (17.22). Analysis
for C43H55N7O8 (797.90): Calcd. C, 64.72; H, 6.95; N, 12.29. Found: C 64.69, H 6.94, N 12.27.
Cyclo-(Nα-dipicolinoyl)-bis [L-Phe-L-ILe]-L-Lys-OH (5c). Yield: 81%; m.p. 178–180 C, [α]2D5
:
−
30 (C, 0.02,
◦
MeOH). IR (KBr, cm−1):
(C=O, acid), 1659, 1527 and 1448 (C=O amide I, II and III, respectively). H-NMR (500 MHz, ppm,
DMSO-d6): = 12.85 (s, 1H, OH, D2O exchangeable), 8.60–8.45 (m, 3H, Pyr-H), 8.33, 8.25 (2s, 6H,
6NH, D2O exchangeable), 7.55–7.10 (m, 10H, Ar-H, L-Phe-ala), 4.60–4.45 (m, 5H, 5CH), 4.35 (t, 4H,
J = 8.1 Hz, 2CH2, L-Phe-ala), 3.50–3.45 (m, 2H, 1CH2, NHCH2, L-Lys), 3,25–3.10 (m, 2H, 2NHCHCH
L-Ile), 2.00–1.70 (m, 6H, 3CH2, NHCH2CH2CH2CH2), 1.30 (m, 4H, 2CH2, L-Ile), 1.10–0.70 (m, 12H,
4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
= 176.0 ( OOH), 174.5, 172.8 (4 O, L-Phe-ala,
L-Ile), 169.0 (2 O, Pyridine dicarbonyl), 150.3 (2C, pyridine, C2,C6), 140.9 (1C, pyridine, C4), 125.9 (2C,
pyridine, C3, C5), 138.4, 127.0, 126.5, 125.7 (12C, Ph-C), 61.0, 56.3 (5C, 5NH H), 45.5 (1C, NH H2), 46.1,
40.8 (2C, 2 H2, L-Phe-ala), 39.5, 39.3 (2C, 2NHCH H), 38.0, 36.5 (5C, 5 CH2), 24.5, 11.0 (4C, 4 CH3,
ν = 3327 (NH stretching), 3065 (CH, aromatic), 2965 (CH, aliphatic), 1721
1
δ
,
δ
C
C
C
C
C
C
C
L-Ile). MS (EI, 70 eV): m/z (%) = 797 (M+, 6.08), 681 (6.44), 636 (37.92), 621 (100), 588 (8.55), 565 (44.24),
304 (7.27), 89 (7.27), 67 (4.61), 57 (11.72), 55 (11.41). Analysis for C43H55N7O8 (797.90): Calcd. C, 64.72;
H, 6.95; N, 12.29. Found: C 64.70, H 6.6.90, N 12.22.
3.1.5. Synthesis of Cyclo-(Nα-dipicolinoyl)-bis[dipeptide]-L-Lys-NHNH2 (cyclic pentapeptide
hydrazides) (6a–c)
A mixture of cyclic pentapeptide methyl esters (4a–c) (1 mmol) and anhydrous hydrazine hydrate
(0.35 mL, 10 mmol) in methanol (20 mL) was stirred at room temperature for 30 min., and then it was
for 3 h. The reaction mixture was evaporated under reduced pressure; the obtained residue oil was
solidified with ether, filtered off and crystallized from methanol/ether to afford the corresponding
cyclic pentapeptide hydrazides (6a–c), respectively.
Cyclo-(Nα-dipicolinoyl)-bis[L-ILe-L-ILe]-L-Lys-NHNH2 (6a). Yield: 60%; m.p. 180–182 ◦C, [α]D25
0.02, MeOH). IR (KBr, cm−1):
= 3280 (NH stretching), 30,867 (CH, aromatic), 2964 (CH, aliphatic),
1644, 1532, 1450 and 1383 (C=O amide I, II, III and IV, respectively). 1H-NMR (500 MHz, ppm,
DMSO-d6): = 9.02 (s, 1H, CONH, D2O exchangeable), 8.75–8.70 (3H, Pyr-H), 8.20, 8.10 (2s, 6H, 6NH,
:
−
12 (C,
ν
δ
D2O exchangeable), 4.55 (d, 4H, J = 8.8 Hz, 4NHCH, L-Ile), 4.35–4.33 (m, 1H, CH, L-Lys), 4.20 (s, 2H,
NH2), 3.70–3.65 (m, 2H, NHCH2, L-Lys), 2.55–2.40 (m, 4H, 4NHCHCH, L-Ile), 2.10–1.65 (m, 6H, 3CH2,
NHCH2CH2CH2CH2CHNH), 1.40–1.25 (m, 8H, 4CH2, L-Ile), 1.15–0.75 (m, 24H, 8CH3, L-Ile). MS (EI,
70 eV): m/z (%) = 743 (M, 1.39), 709 (2), 656 (60.81), 616 (94.4), 584 (70.53), 543 (27.33), 515 (20.75),
471 (100), 443 (67.27), 358 (55.32), 302 (80.56), 86 (80.07), 69 (42.70), 57 (15.46), 50 (1.24). Analysis for
C37H61N9O7 (743.90): Calcd. C, 59.74; H, 8.26; N, 16.95. Found: C 59.71, H 8.17, N 16.92.
Cyclo-(Nα-dipicolinoyl)-bis[L-ILe-L-Phe]-L-Lys-NHNH2 (6b). Yield: 65%; m.p. 161–163 ◦C, [α]D25
0.02, MeOH). IR (KBr, cm−1):
= 3289 (NH stretching), 3063 (CH, aromatic), 2964 (CH, aliphatic), 1650,
1529, 1448 and 1382 (C=O amide I, II, III and IV, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
:
−
40 (C,
ν
1
δ
= 9.12 (s, 1H, CONH, D2O exchangeable, Hydrazide), 8.53–8.51 (m, 3H, Pyr-H), 8.24, 8.19 (2s, 6H, 6NH,
D2O exchangeable), 7.23–7.05 (m, 10H, Ar-H, L-Phe-ala), 4.50–4.48 (m, 6H, 2 NHCH, 2CH2, L-Phe-ala),
4.45–4.38 (m, 3H, 3CH), 4.35 (s, 2H, NH2), 3.32 (t, 2H, 2NHCH2, L-Lys), 2.79–2.75 (m, 2H, 2NHCHCH
-Ile), 1.35–1.34 (m, 6H, 3CH2, NHCH2CH2CH2CH2CHNH), 1.23–1.20 (m, 4H, 2CH2, L-Ile), 0.90–0.60
(m, 12H, 4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
= 173.4, 173.0 (4C, 4 O, L-Phe-ala,
L-Ile), 170.0 (1C, O, Hydrazide), 164.5, 163.8 (2 O, Pyridine dicarbonyl), 150.7 (2C, pyridine, C2, C6),
142.9 (1C, pyridine, C4), 128.1 (2 C, pyridine, C3, C5), 139.0, 127.8, 126.7, 125.5 (12C, Ph-C), 60.6, 54.0
(5C, 5NH H), 48.0 (1C, NH H2, L-Lys), 44.4, 42.6 (2C, 2 H2, L-Phe-ala), 37.5, 37.0 (2C, 2NHCH H,
L-Ile), 34.0, 30.9 (5C, 5CH2), 24.2, 10.5 (4C, 4
H3, L-Ile). MS (EI, 70 eV): m/z (%) = 813 (M++1, 2.42),
,
L
δ
C
C
C
C
C
C
C
C
724 (13.09), 684 (40.82), 669 (30.09), 549 (37.88), 477 (55.84), 302 (100), 84 (24.44), 55 (22.15), 50 (3.04).
Analysis for C43H57N9O7 (812.00): Calcd. C, 63.61; H, 7.08; N, 15.53. Found: C 63.59, H 7.02, N 15.47.

Molecules 2018, 23, 2416
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Cyclo-(Nα-Dipicolinoyl)-bis[L-Phe-L-ILe]-L-Lys-NHNH2 (6c). Yield: 78%; m.p. 208–210 ◦C, [α]D25
:
−
66 (C,
0.02, MeOH). IR (KBr, cm−1):
ν = 3282 (NH stretching), 3065 (CH, aromatic), 2963 (CH, aliphatic), 1645,
1
1524, 1447 and 1382 (C=O amide I, II, III and IV, respectively). H-NMR (500 MHz, ppm, DMSO-d6):
= 9.35 (s, 1H, CONH, D2O exchangeable, Hydrazide), 9.10–8.90 (m, 3H, Pyr-H), 8.11, 8.09 (2s, 6H,
δ
6NH, D2O exchangeable), 7.41–7.13 (m, 10H, Ar-H, L-Phe-ala), 4.82–4.80 (m, 6H, 2NHCH, 2CH2,
L-Phe-ala), 4.25–4.00 (m, 3H, 3CH), 3.98 (s, 2H, NH2), 3.30–3.19 (m, 2H, NHCH2, L-Lys), 2.75–2.70
(m, 2H, 2NHCHCH, L-Ile), 1.4–1.32 (m, 6H, NHCH2CH2CH2CH2CHNH), 1.14–1.12 (m, 4H, 2CH2,
L-Ile), 0.80–0.79 (m, 12H, 4CH3, L-Ile). 13C-NMR (125 MHz, ppm, DMSO-d6):
L-Phe-ala, L-Ile), 172.0 (1C, O, Hydrazide), 168.5, 166.9 (2C, 2
pyridine, C2, C6), 144.0 (1C, pyridine, C4), 126.1 (2C, pyridine, C3, C5), 138.5, 127.0, 126.7, 125.8 (12C,
Ph-C), 61.0, 57.5 (5C, 5NH H), 50.0 (1C, NH H2, L-Lys), 47.4, 46.9 (2C, 2 H2, L-Phe-ala), 39.0, 38.8
(2C, 2
H, L-Ile), 36.0, 34.4 (5C, 5CH2), 27.7, 11.0 (4C, 4CH3, L-Ile). MS (EI, 70 eV): m/z (%) = 812 (M+,
δ
= 174.6, 174.0 (4C, 4
CO,
C
CO, Pyridine dicarbonyl), 148.5 (2C,
C
C
C
C
0.8), 808 (2.6), 621 (1.00), 423 (1.2), 391 (1.2), 373 (1.8), 295 (3.6), 268 (11.9), 213 (11.3), 149 (17.2), 121
(26.9), 104 (36.2), 91 (100), 86 (43.4), 77 (35.8), 50 (24.2). Analysis for C43H57N9O7 (812.00): Calcd. C,
63.61; H, 7.08; N, 15.53. Found: C 63.58, H 7.07, N 15.49.
3.2. Anticancer Screening
In vitro bioassay on human cancer cell lines was adopted against two cancer cell lines (MCF-7 and
HepG-2). HepG2, human hepatocellular carcinoma and MCF-7, human breast cancer cell lines were
obtained from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. Tamoxifen and 5-fluorouracil
were used as reference standards according to a previously reported method [35–39].
3.3. In Vitro Enzymatic Assays
The in vitro enzyme inhibition determination for compound 5c was carried out in confirmatory
diagnostic unit, Vacsera, Egypt. The evaluation performed profiling of the compound 5c against a
range of four protein kinases [VEGFR-2, EGFR, PDGFRβ and CDK-2] by ELISA assay method using
staurosporine as a reference according to the previously reported methods [40–42].
3.4. Molecular Modeling Studies
To evaluate the inhibitory activity and confirm best interactions between compound 5c and
target proteins, molecular docking studies were performed with the help of Molecular Operating
Environment (MOE®) 2008.10 [30
,31]. The three-dimensional X-ray structures of VEGFR-2 (PDB code:
4ASD) [33] and CDK-2 (PDB code: 2J9M) [34] were obtained from the Protein Data Bank through
the internet.
4. Conclusions
In summary, a series of macrocyclic derivatives bearing pyrido-pentapeptide moiety were
designed and synthesized from N,N-bis-[1-carboxy-2-(benzyl)]-2,6-(diaminocarbonyl)pyridine. Two
human cancer cell lines (MCF-7 and HepG-2) were used to evaluate the anticancer potency of all
synthesized compounds. Compared with tamoxifen, compounds 4a
excellent potency against one or both cell lines. Furthermore, compound 5c showed the strongest
cytotoxic activities against both cell lines (IC50 = 9.41 1.25 and 7.53 1.33 M, respectively) in
comparison with tamoxifen and 5-fluorouracil. On the other hand, enzyme inhibition assay of
compound 5c against VEGFR-2, CDK-2 and PDGFR demonstrated promising inhibitory activity with
IC50 0.01 1.25, 0.06 1.27 and 0.08 1.45 M, respectively compared to staurosporine. Molecular
, 4b, 5a, 5b, 5c, 6a, 6b, 6c exhibited
±
±
µ
β
±
±
±
µ
docking study showed the ability of the compound to fit into the active sites of these enzymes with the
best binding modes. It is worthy to mention macrocyclic pyrido-pentapeptide based compounds serve
as a useful template for the further development of the anticancer multi-targeted agents.

Molecules 2018, 23, 2416
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Author Contributions: The listed authors contributed to this work as described in the following. A.E.-G.E.A.,
M.H.A.-G., G.O.M. gave the concepts of the work, interpreted the results, the experimental part and prepared the
manuscript, M.A.A.-O. cooperated in the preparation of the manuscript and E.S.N. and E.A.E. performed the
anticancer and biochemical, as well as docking studies of the new compounds. All authors read and approved the
final manuscript.
Funding: This research was funded by research group project “RGP-1435-047” of the Deanship of Scientific
Research, King Saud University.
Acknowledgments: The authors are grateful to the Deanship of Scientific Research, King Saud University for
funding this work through research group project “RGP-1435-047”.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).