Asymmetric synthesis, biological activity and molecular docking studies of some unsaturated α-amino acids, derivatives of glycine, allylglycine and propargylglycine

Article abstract of DOI:10.1016/j.molstruc.2020.127850

New enantiomerically enriched unsaturated tailor-made amino acids have been obtained. As a starting amino acid synthon for the asymmetric synthesis of tailor-made unsaturated amino acids, Ni(II) square-planar complexes of Schiff's bases of propargylglycine, allylglycine and glycine with chiral auxiliary (S)-2-N-(N’-benzylprolyl)-aminobenzophenone ((S)-BPB) were used. The C_α-alkylation of propargylglycine, allylglycine and glycine moieties resulted in the asymmetric synthesis of novel (S)-α-propargylglycine, (S)-α-allylglycine and glycine derivatives containing an aromatic group in the side chain (de 80–95,5%). After purification and cleavage of the metal complexes, the amino acids were isolated in high enantiomeric purity (ee >99%). Of the obtained seven tailor-made amino acids four showed inhibitory activity to collagenase G. The amino acid with an acetylene bond in the side chain (IC₅₀ = 1.29 ± 0.02 mM) had the best result. Molecular docking showed that the amino acids with activity to collagenase G contained hydrogen and π-π bonds with the enzyme.

Full text of DOI:10.1016/j.molstruc.2020.127850

Journal Pre-proof
Asymmetric synthesis, biological activity and molecular docking studies of some
unsaturated α-amino acids, derivatives of glycine, allylglycine and propargylglycine
Anna F. Mkrtchyan, Ashot S. Saghyan, Liana A. Hayriyan, Armen S. Sargsyan, Ani J.
Karapetyan, Anna S. Tovmasyan, Avetis H. Tsaturyan, Ella V. Minasyan, Artavazd S.
Poghosyan, Ani M. Paloyan, Henrik A. Panosyan, Lusine Yu. Sahakyan
PII:
S0022-2860(20)30174-5
DOI:
https://doi.org/10.1016/j.molstruc.2020.127850
MOLSTR 127850
Reference:
To appear in: Journal of Molecular Structure
Received Date: 30 November 2019
Revised Date: 31 January 2020
Accepted Date: 4 February 2020
Please cite this article as: A.F. Mkrtchyan, A.S. Saghyan, L.A. Hayriyan, A.S. Sargsyan, A.J.
Karapetyan, A.S. Tovmasyan, A.H. Tsaturyan, E.V. Minasyan, A.S. Poghosyan, A.M. Paloyan, H.A.
Panosyan, L.Y. Sahakyan, Asymmetric synthesis, biological activity and molecular docking studies of
some unsaturated α-amino acids, derivatives of glycine, allylglycine and propargylglycine, Journal of
Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.127850.
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Ashot S. Saghyan Supervision, Writing - Review & Editing
Anna F. Mkrtchyan, Supervision, Project administration, Writing - Original Draft
Liana A. Hayriyan, Investigation, validation, methodology
Armen S. Sargsyan Investigation, Data Curation, Formal analysis
Ani J. Karapetyan, Investigation, methodology
Anna S. Tovmasyan Investigation
Avetis H. Tsaturyan, Data Curation, Formal analysis
Ella V. Minasyan, Data Curation, Formal analysis
Artavazd S. Poghosyan, Investigation
Ani M. Paloyan, Investigation
Henrik A. Panosyan Data Curation
Lusine Yu. Sahakyan , Investigation

Graphical Abstract
Asymmetric synthesis, biological activity and
molecular docking studies of some unsaturated
α-amino acids, derivatives of glycine, allylglycine and propargylglycine
a,b
Ashot S.Saghyan a,b, Anna F. Mkrtchyan
, Liana A. Hayriyan a,b, Armen S. Sargsyan a,b, Ani J. Karapetyan a,b,
Anna S. Tovmasyan a,b, Avetis H. Tsaturyan a,b, Ella V. Minasyan a, Artavazd S. Poghosyan a,b, Ani M. Paloyanb,
Henrik A. Panosyanc
a Scientific and Production Center “Armbiotechnology” of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia
b Institute of Pharmacy, Yerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia
c Scientific and Technological Center of Organic and Pharmaceutical Chemistry of NAS RA, 26 Azatutyan Ave.,0014 Yerevan, Armenia

Asymmetric synthesis, biological activity and molecular docking studies of some unsaturated α-amino acids, derivatives
of glycine, allylglycine and propargylglycine
a,b
Anna F. Mkrtchyan
, Ashot S.Saghyan a,b, Liana A. Hayriyan a, Armen S. Sargsyan a,b, Ani J. Karapetyan a,b, Anna S.
Tovmasyan a,b, Avetis H. Tsaturyan a,b, Ella V. Minasyan a, Artavazd S. Poghosyan a, Ani M. Paloyana, Henrik A. Panosyanc,
Lusine Yu. Sahakyanb
anna_mkrtchyan@ysu.am
a Scientific and Production Center “Armbiotechnology” of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia
b Institute of Pharmacy, Yerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia
c Scientific and Technological Center of Organic and Pharmaceutical Chemistry of NAS RA, 26 Azatutyan Ave.,0014 Yerevan,
Armenia
ABSTRACT: New enantiomerically enriched unsaturated tailor-made amino acids have been obtained. As a starting amino acid
synthon for the asymmetric synthesis of tailor-made unsaturated amino acids, Ni(II) square-planar complexes of Schiff’s bases
of propargylglycine, allylglycine and glycine with chiral auxiliary (S)-2-N-(N`-benzylprolyl)- aminobenzophenone ((S)-BPB)
were used. The Cα-alkylation of propargylglycine, allylglycine and glycine moieties resulted in the asymmetric synthesis of
novel (S)-α-propargylglycine, (S)-α-allylglycine and glycine derivatives containing an aromatic group in the side chain (de 80–
95,5%). After purification and cleavage of the metal complexes, the amino acids were isolated in high enantiomeric purity (ee
>99%). Of the obtained 7 tailor-made amino acids 4 showed inhibitory activity to collagenase G. The amino acid with an
acetylene bond in the side chain (IC50=1.29±0.02mM) had the best result. Molecular docking showed that the amino acids with
activity to collagenase G contained hydrogen and π-π bonds with the enzyme.
KEYWORDS: Tailor-made amino acid, Enantiomerically enriched, Collagenase, Inhibitor, Molecular docking
Introduction
Amino acids contained in natural products such as the antibiotics bacitracin and vancomycin, and in peptides such as insulin
have played a significant role in drugs from the earliest days of modern drug discovery [1]. For this reason, as well as due to the
growing awareness of the relevance of these molecules in several technological contexts, tailor-made amino acids have been the
subject of a relatively large number of scientific investigations in recent decades[2].
In this domain, unsaturated amino acids represent very interesting building blocks. Alkyne and allyl group containing amino
acids are versatile structures readily available by a number of methods and are accessible using very few transformations from
economical starting materials [3]
.
Matrix metalloproteases (ММРs) are a major group of enzymes that regulates cell-matrix composition. Matrix metalloproteases
play an important role in degradation of extracellular matrix in both norm and various pathologies [4,5,6]. Metalloproteases are
targets for a wide range of medications, including antitumor and anti-inflammatory drugs [7,8,9]. However, the majority of
inhibitors such as calprotectin are zinc-chelating compounds of a wide spectrum of action that did not have a specific effect. This
could be one of the reasons that clinical trials of MMP inhibitors on oncological patients have not revealed a therapeutic effect so
far [10]
.
In this work, the search for new highly specific inhibitors of metalloproteases has been carried out. Closridium (Hathewaya)
histolyticum collagenase was used in this work as a target for identification of metalloproteases inhibitors by using structure-
based drug design approach. This enzyme has a high level of homology with the mammal’s collagenases[11]. Previously the
inhibition of collagenase by several heterocycle-substituted non-protein amino acids was described [12]
.
Here, we present stereoselective approaches to synthesize unsaturated α-amino acids in an optically active form and investigate
their biological activity.
Over the past decades, numerous enantiomerically enriched non-protein amino acids have been synthesized by our scientific
group [13]
.
Result and discussion
Chemistry synthesis
Here, we present a stereoselective approach to synthesize unsaturated α-amino acids in an optically active form. As a starting
amino acid synthon for the asymmetric synthesis of amino acids, Ni (II) square-planar complexes of Schiff’s bases of glycine,
allylglycine and propargylglycine with chiral auxiliary (S)-2-N-(N`-benzyl-prolyl)aminobenzophenone (BPB) were taken
1

(Figure 1). Recently, these complexes have been applied to Michael additions, Mannich reactions, aldol reactions, SN2 reactions,
[14-
151617 19,20].
18
Sonogashira and Heck couplings and etc.
-
O
O
O
H
H
O
O
O
N
N
Ni
N
N
N
Ni
N
N
N
Ni
N
O
O
O
(S)-BPB-(S)-AllylGly (3)
(S)-BPB-(S)-PGly (2)
(S)-BPB-Gly (1)
Figure 1. The structures of initial complexes: Ni(II)-(S)-BPB-Gly, Ni(II)-(S)-BPB-(S)-PGly, Ni(II)-(S)-BPB-(S)-AllylGly.
Initial complexes 1-3 were synthesized according to the literature [21,22]
.
Scheme 1 presents the reaction of Cα-alkylation of amino acid fragment of 1-3 complexes with formation of α-substituted
glycine, propargylglycine and allylglycine analogs. The asymmetric addition of various electrophilic agents 4-bromobenzyl-,
4-(trifluoromethyl)benzyl-, 3-(trifluoromethyl)benzyl bromides, α, α'-p-xylene and others to the α-carbon of amino acids
fragments of 1-3 complexes afforded a variety of products with very good diastereoselectivity (Scheme 1).
Scheme 1. The asymmetric addition of various electrophilic agents to the α-carbon of amino acid fragments of 1-3
complexes. Conditions. a) 1 (1.0 equiv.), propargyl bromide (1.2 equiv.), NaOH (2.5 equiv.), CH3CN, r.t.; a') 1 (1.0 equiv.), allyl
bromide, NaOH (2.5 equiv.), DMF, r.t.; b) and b') 2 or 3 (1.0 equiv.), alkyl halide (1.5 equiv.), NaOH (3.0 equiv.), DMF.; c) 1
(1.0 equiv.), alkyl halide (1.2 equiv.), NaOH (3.5 equiv.), DMF.
On the example of addition of g to 3 complex optimal reaction conditions were selected (Table 1).
Thus, the optimal conditions for this reaction are DMF as a solvent, room temperature, the ratio of complex/NaOH;
alkylating agent: 1eq./3eq./1.5eq.
Table 1. Selection of optimal reaction conditions.
(S)-BPB-(S)-AllylGly (3): NaOH: 3-(Trifluoromethyl)benzyl bromide (g)
Time
(minute)
120
120
300
480
160
240
50
Yield
(%)
30
30
35
35
40
40
75
T oC
(eq.)
1:1:1.5
1:1:1.5
1:2:3
1:3:3
1:4:2
1:4:3
1:3:1.5
25
65
65
25
70
70
25
* The conditions of reaction: DMF as a solvent, room temperature.
The reaction was monitored by TLC (silica gel, CHCl3/EtOAc = 1:3). However, the formation of a significant amount of
undesired by-products (up to 10%) was also observed. The diastereomeric excess (de) of complexes 4a, 4g, 4c, 5b-h, and 6i, 6j
1
was determined by H NMR analysis of the chemical shifts of the doublet signals assigned to the methylene protons of the N-
benzyl group of the BPB fragment for both diastereomers. The de of the obtained complexes was also confirmed by HPLC
analysis of the isolated amino acids mixture (before chromatography). The results are presented in Table 2. The major
2

diastereomers of complexes 4a,4g, 4c, 5b-h, and 6i, 6j were isolated by crystallization from methanol. The absolute
configurations of the amino acid fragments present in complexes 4a,4g, 4c, 5b-h, and 6e-g were determined by measuring the
specific rotation at 589 nm as was previously done for related complexes of other amino acids [23,24] -
Table 2. Asymmetric addition of electrophile agents to complexes 1-3
Major diastereomers of
Time (min)
de %
Yield %
complex
240
90
86.6
90.2
88.6
85.6
80.4
95.4
88.6
86.4
87.5
90.5
91.4
30
78
33
59
55.5
55
65
51
63
61
4a
4g
4c
5b
5c
5d
5e
5f
260
120
240
120
300
120
240
180
60
5g
5h
6i
56.8
a Diastereomeric excess based on 1H NMR data
The next step was isolation of the obtained amino acids by hydrolysis of complexes 4g, 5b-h, and 6i, 6j (Scheme 2).
Hydrolysis of the diastereomerically pure complexes with aqueous HCl, followed by recovery of the hydrochlorides of the (S)-
BPB and purification of the amino acids by an ion-exchange method and by crystallization from a mixture of water and ethanol
(1:1) afforded the desired amino acids 7 (a-e), 8f and 9g in Scheme 2.
The enantiomeric excess of the isolated amino acids was determined by the chiral HPLC. The enantiomeric purity of
obtained amino acids was over 97%.
Scheme 2. Isolation of the obtained amino acids by hydrolysis of complexes 4g, 5b-h and 6i, 6j. i) 2M HCl, 500C; ii) Dowex 50;
iii) crystallization C2H5OH: H2O 1:1.
3

Molecular docking
As described above, 7(b-e) and 8f were identified as the most promising inhibitors of collagenase G. To better understand
structure-activity relationship, we carried out a molecular docking study of 7(b-e) and 8f on collagenase G using AutoDock Vina
software. As shown in Figure 2, compounds 7(b-e) and 8f have a different docking mode. The 7(b-e) compounds have similar
contracture, in addition, 7c and 7e are isomers. Considering the molecular structure of 7b and 8f compounds, the main
differences are in the presence of a CF3 group in the aromatic ring in the case of 8f, and a triple bond instead of a double one.
When the detailed investigation of these compounds was carried out, the most noticeable interactions were hydrogen bonding
and π-π interactions. The binding free energy of 8f (-7.1 kcal/mol) was much higher than ꢀG of 7b (-6.3 kcal/mol), 7c and 7e
(-6.4 kcal/mol).
a)
b)
c)
d)
Figure 2. a) Hydrogen-bond and π-π interactions of compound 7b and amino acid residues near the ligand; b) Hydrogen-bond
interactions of compound 7c and amino acid residues near the ligand; c) Hydrogen-bond interactions of compound 7e and
amino acid residues near the ligand; d)Hydrogen-bond and π-π interactions of compound 8f and amino acid residues near the
ligand.
As shown in Figure 2, the amino group of 8f was found to form a hydrogen-bond with the carbonyl oxygen atom of carboxyl
group of Gly250 (2.234 Å); and the phenyl ring of 8f could form a π-π interaction with Tyr201 (3.9125 Å). These interactions of 8f
might strengthen its binding, which is beneficial for enhancing the activity of 8f. In the case of 7b, interactions with the enzyme
were also detected, in particular, the H-bond length between the carbonyl oxygen atom of carboxyl group of 7b and amino group
of Gln215 (2.206 Å). The π-π interactions were also detected and the length was calculated. The phenyl ring of 7b compound
could form a π-π interaction with Tyr201 (4.203 Å).
Despite the fact that 7c and 7e are isomers, have the same free energy and form both hydrogen and π-π bonds with the same
sites of the enzyme, the distance of the bonds is different. For example, the H-bond length between the carbonyl oxygen atom of
carboxyl group of 7c and amino group of Gln215 is 2.172 Å, but in the case of 7e it is 2.236. The phenyl ring of 7c compound
could form a π-π interaction with Tyr201 is 3.373 Å, but in the case of 7e is 4.262 Å. In addition, the amino group of 7c was found
to form a hydrogen bond with the oxygen of amide group of Asn251 (2.249 Å).
In vitro performed inhibition assays on the collagenase
All investigated compounds were checked on the ability to influence the collagenase activity. The activity of collagenase has
been determined in the presence of investigated tailor-made amino acids. Investigated amino acids were added in the
concentration range 0.5-5 mM. The obtained results are presented in Table 3.
According to the obtained results (Table 3), collagenase has been inhibited by the following compounds: 7a
(IC50=3.25±0.03mM), 7b (IC50=2.65±0.01mM), 7c (IC50=4.98±0.03mM), 8f (IC50=1.29±0.02mM). The rest of the investigated
amino acids had no influence on the enzyme activity.
Based on the structure of amino acids, it is obvious that the presence of an acetylene bond affects the activity of the
compound. Thus, in the case of 8f amino acid, which is a derivative of propargylglycine, the highest inhibitory activity is
4

observed: IC50=1.29±0.02mM. For the manifestation of activity, the presence of the phenyl ring in the side chain of amino acids
is also very important.
Thus, for example, in the case of 9g, where there is a double bond, but there is no phenyl fragment in the side chain, no
inhibitory effect is observed. The derivatives of аllylglycine 7a-7c, containing the benzyl fragment in the side chain, exhibit
inhibitory activity, and the presence of fluorine in the phenyl ring in the meta position enhances, and in para - reduces the
inhibitory activity compared to 7a, which does not contain fluorine in the phenyl ring. The presence of the bulky group OCH3
(7d) or CF3 (7e) in the phenyl ring sharply reduces the inhibitory properties of the compound.
Table 3. Biological studies of synthesized amino acids
Compounds
Structure
IC50 (mM)
3.25±0.03
7a
2.65±0.01
4.98±0.03
7b
7c
No
influence
7d
No
influence
7e
1.29±0.02
8f
No
influence
9g
Conclusion
In conclusion, in the frame of the scientific work, we managed to obtain 7 previously not described in the literature novel tailor-
made amino acids, derivatives of glycine, with more than 98% enantiomeric purity, as well as their intermediate complex-
products, respectively. The obtained amino acids and intermediate complexes open a new way to the synthesis of
enantiomerically pure amino acids containing practically unlimited number of radicals of different nature attached to the triple
and double C-C bond. Inclusion of trifluoromethyl group into the aglycon composition is widely used in many pharmacological
drugs and agrochemicals due to enhancement of chemical and metabolic stability, improvement of lipophilicity and
bioaccessibility of the substance. The presence of fluorine in the composition of the amino acid allows to use this compound as
an intermediate for realization of a great many reactions. Intermediate complexes can also serve as potential initial compounds
4
for the study of the reactions of Sonogashira, Heck, Glaser, etc. Tailor-made amino acids of the obtained 7 had inhibitory
activity to collagenase G. The best result showed the amino acid with an acetylene bond in the side chain (IC50=1.29±0.02mM).
Molecular docking showed that the amino acids that exhibited activity to collagenase G contained hydrogen and π-π bonds with
the enzyme.
Experimental
Chemisrty synthesis
Materials
All reagents were obtained from commercial sources and used without further purification. Thin-layer chromatography (TLC)
was carried out on Merck aluminium foil backed sheets precoated with 0.2 mm Kielselgel 60 F254. Column chromatography
was performed on Fluka silica gel 60 (0.063-0.200 mm, 70-320 mesh) on a glass column. Melting points (mp) were determined
1
by “Elektrothermal”. H and 13C NMR spectra were recorded on ”Varian Mercury300” 300 MHz spectrometer using TMS as
internal standard. The NMR spectra were calibrated by solvent at 7.27 (CDCl3), 3.31 (CD3OD), 4.79 (D2O), 2.50 ((CD3)2SO) for
1H and 77.1 (CDCl3), 49.15 (CD3OD), 39.5 ((CD3)2SO) for 13C. Elemental analysis was done by Euro EA3000. For the cation
exchange column Dowex-50 (H+ form) was used. The enantiomeric purity of the amino acids was determined by HPLC (“Waters
Alliance 2695 HPLC System”) on a chiral phase of the Diaspher 110-Chirasel-E-1 6µ, 4.0x250mm, mobile phase consisted of
methanol: sodium phosphate monobasic buffer (20:80). Enantiomeric yield was proved by chiral HPLC analysis of the isolated
amino acids.
General Procedures for the reaction of C-alkylation of α-H amino acid fragment of 1-3 complexes.
5

o
To a solution of complex 1-3 in DMF NaOH and alkyl halogenide were added at room temperature or 65 C with stirring. The
reaction was monitored by TLC (SiO2, CHCl3/ethylacetate = 1:3) following the disappearance of the spot of the initial complexes
1-3. Upon completion of the reaction, the mixture was precipitated by H2O. The products were crystallized from methanol.
Complex 4a
To a solution of complex 1 eq. (1g, 1.87mmol) of complex 2 in DMF were added 3eq. (0.22g, 5.61mmol) of NaOH and 3eq
(0.69mL, 5.61mmol) of α, α'-p-xylene bromide, at 250C with stirring. The reaction was monitored by TLC (SiO2,
CHCl3/ethylacetate = 1:3) following the disappearance of the spot of the initial complex 1. Upon completion of the reaction, the
mixture was precipitated by H2O. The products were crystallized from methanol. Complex 4a was isolated in 30% (0.4g,
0.556mmol).
20
Anal. calc. for C38H34BrN3NiO3(719.26): C 63.45; H 4.76; N 5.84; Found: C 63,71; H 4.85; N 5.78. Mp. 165-167 OC. [α]D
= + 1’765. 0° (с 0.2, CH3OH).
1H NMR: (300 MHz, CDCl3): δ =1.46-1.57 (m, 1H); 1.74-1.90 (m, 2H); 2.06 (dd, 1H, J=17.3, 2.6, CH2 C≡CH); 2.04-2.18
(m, 1H); 2.20-2.36 (m, 1H), 2.40 (t, 1H, J=2.6, ≡CH); 2.75 (d, 1H, J=13.7, CH2 C6H4); 2.89 (dd, 1H, J= 17.3, 2.6, CH2 C≡CH);
3.00-3.10 (m, 1H), 3.05 (d, 1H, J= 13.7, CH2 C6H4); 3.21 (dd, 1H, J=10.1, 6.9, α-CH Pro); 3.28 (d, 1H, J=12.4, CH2 Ph); 4.25 (d,
1H, J=12.4, CH2 Ph); 4.76 (s, 2H, CH2 Br); 6.55-6.63 (m, 2H, 3.4-CH C6H4); 7.06 (ddd, 1H, J=8.7, 5.7, 2.8, 5-CH C6H4); 7.10 (tt,
1H, J= 7.5, 1.1, 4-CH PH); 7.21-7.32 (m, 3H, Ar), 7.40-7.59 (m, 7H, Ar), 7.93-8.00 (m, 2H, Ar), 8.27-8.32 (m, 2H, Ar). 13C
NMR (75.46, CDCl3): δ = 22.6 (γ-CH2 Pro); 30.7 (β-CH2 Pro); 31.7 (CH2 C≡CH); 43.3 (CH2 C6H4); 58.3 (δ-CH2 Pro); 64.6 (CH2
Ph); 64.9 (CH2 Br); 70.7 (α-CH Pro); 72.9 (C CH2 C≡CH); 80.2 (C≡CH); 81.0 (≡CH); 120.5 (4-CH, C6H4); 123.7 (6-CH C6H4);
127.5 (2-CH); 127.7 (CH);, 127.8 (CH); 127.8, 128.0 (CH); 128.8 (CH); 128.9 (2CH); 129.6 (CH); 130.0 (CH); 131.1 (2CH);
131.6 (2CH); 131.8 (CH), 133.8 (CH); 134.5, 135.4, 136.8, 140.9, 142.5, 172.8, 179.4, 180.5.
Complex 4c
Starting with 1 eq. (1g, 1.87mmol) of complex 2 1.5 eq. (0.39mL, 2.805mmol) of 3-(methoxy)benzyl bromide, 3 eq. (0.22g,
5.61mmol) of NaOH and of DMF at 650C with stirring. Complex 4c was isolated in 33% yield (0.404g, 0.615mmol).
o
20
Anal. calc. for C38H35N3NiO4 (656.4), C 69.53; H 5.37; N 6.40; Found: C 69.83; H 5.71; N 6.11. Mp. 259-260 C. [α]D
=+2’082.4. 0° (с 0.15, CH3OH).
1H NMR (300 MHz, CDCl3) δ =1.45–1.66 (m, 2H); 1.90 (t, 2H, J = 8.7 Hz); 1.98 – 2.11 (m, 1H); 2.19 (dd, 2H, J = 16.7, 12.5
Hz, CH2C≡CH); 2.39 (t, 1H, J = 2.3 Hz, ≡CH); 2.72 (d, 1H, J = 13.6 Hz, CH2C6H4OCH3); 2.90 (d, 1H, J=17.3 Hz,
CH2C6H4OCH3); 3.04 (d, 2H, J = 13.7 Hz, α-H Pro); 3.17 – 3.26 (m, 1H); 3.32 (d, 1H, J = 12.4 Hz, CH2Ph); 3.72 (s, 3H, CH3);
4.29 (d, 1H, J = 12.4 Hz, CH2Ph); 6.58 (d, 2H, J = 5.7 Hz); 6.94 – 7.18 (m, 5H); 7.31 (d, 3H, J = 7.6 Hz); 7.42 (d, 2H, J=16.8
Hz); 7.52 – 7.60 (m, 2H); 7.97 (d, 1H, J = 8.5 Hz); 8.03 (d, 1H, J=8.6 Hz, H6 C6H4); 8.30 (d, 2H, J = 7.3 Hz, H2,2’ Ph). 13C NMR
(75.46, CDCl3): δ = 22.8 δ =22.82 (CH3); 30.74 (β-CH2 Pro); 31.8 (CH2); 43.74 (CH2); 55.09 (δ-CH2 Pro); 58.43; 64.65 (CH2Ph);
70.75 (α-CH Pro); 72.9 (≡CH); 80.37 (CCH2C≡C); 80.97 (C≡); 96.33; 114.28; 115.70; 120.39 (CH4 C6H4); 123.43 (CH6 C6H4);
123.86; 127.79 (CH); 127.86; 128.02 128.79; 128.88 (CH); 129.66 (CH); 129.97 (CH); 131.70 (2CH); 131.89 (CH); 133.75
(CH); 134.46 (CH); 136.97; 137.75 (CH); 142.72; 160.16; 172.59; 179.15; 180.46.
Complex 4g
Starting with 1 eq. (1g, 1.87mmol) of complex 2 1.5 eq. (0.343mL, 2.805mmol) of 3-(trifluoromethyl)benzyl bromide, 3 eq
(0.22g, 5.61mmol) of NaOH and of DMF at 250C with stirring. Complex 4g was isolated in 78% yield (1.01g, 1,15mmol).
20
Anal. calc. for C38H32F3N3NiO3 (694.37), C, 65.73; H, 4.65; N, 6.05; Found: C 65.83; H 4.81; N 6.31. Mp. 259-260 oC. [α]D
=+2’082.4. 0° (с 0.15, CH3OH).
1H NMR - (300 MHz, CDCl3) δ = 1.50-1.63 (m, 1H); 1.72-1.90 (m, 2H); 1.98-2.09 (m, 1H); 2.05 (dd, 1H, J 17.3, 2.5,
CH2C≡CH); 2.16-2.30 (m, 1H); 2.42 (t, 1H, J 2.5, ≡CH); 2.82 (d, 1H, J 13.7, CH2C6H4CF3); 2.90 (dd, 1H, J 17.3, 2.5,
CH2C≡CH); 3.09 (d, 1H, J=13.7, CH2C6H4CF3); 3.05-3.14 (m, 1H); 3.22.96 (dd, 1H, J 10.4, 6.3, α-H Pro); 3.28 (d, 1H, J 12.4) &
4.27 (d, 1H, J 12.4, CH2Ph); 6.54-6.62 (m, 2H, H3,H4 C6H4); 7.08 (ddd, 1H, J 8.6, 6.2, 2.4, H5 C6H4); 7.12 (tt, 1H, J 7.5, 1.2, H4
Ph); 7.21-7.25 (m, 1H); 7.26-7.33 (m, 1H); 7.42-7.50 (m, 1H); 7.54-7.66 (m, 5H); 7.71-7.76 (m, 1H); 7.93-7.98 (m, 1H); 8.03
(dd, 2H, J 8.7, 1.2, H6 C6H4); 8. 28-8.32 (m, 2H, H2,2’ Ph). 13C NMR (75.46, CDCl3): δ = 22.8 (γ-CH2 Pro); 30.6 (β-CH2 Pro);
31.7 (CH2); 43.4 (CH2); 58.4 (δ-CH2 Pro); 64.5 (CH2Ph); 70.4 (α-CH Pro); 73.1 (≡CH); 80.1 (CCH2C≡C); 80.6 (≡C); 120.5 (CH4
C6H4); 123.8 (CH6 C6H4); 124.1 (q, JC,F 272.3, CF3); 124.7 (q, JC,F 3.7, CH); 127.5; 127.6 (CH); 127.9 (q, JC,F 3.7, CH); 128.0
(CH); 128.1 (CH); 128.8 (CH); 128.9 (2CH); 129.3 (CH); 129.6 (CH); 130.1 (CH); 131.7 (2CH); 131.7 (q, JC,F 32.6, CH); 132.1
(CH); 133.8 (CH); 134.2 (CH); 134.4; 136.8; 137.4; (CH); 142.8; 173.1; 178.9; 180.6.
Complex 5b
Starting with 1 eq. (1g, 1.85mmol) of complex 3, of 1.5eq. (0.33mL, 2.78mmol) benzyl bromide, 5eq. (0.3g, 9.2mmol) of
NaOH and DMF at 250C with stirring. Complex 5b was isolated in 59% yield (0.64g, 1.09mmol).
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20
Anal. Calc. for C37H35N3NiO3 (628.39): C 70.72; H 5.61; N 6.69; Found: C 70.52; H 5.83; N 6.53. Mp. 245 C. [α]D
=+1755.3° (с 0.15, CH3OH).
1H NMR - (300 MHz, CDCl3): δ =1.44 – 1.59 (m, 1H), 1.75 – 1.95 (m, 2H), 2.12 – 2.33 (m, 3H), 2.58 (dd, 1H, J = 16.0, 6.8
Hz), 2.81 (d, 1H, J = 14.1 Hz), 3.01– 3.13 (m, 2H), 3.21 (dd, 1H, J = 9.6, 7.5 Hz), 3.46 (d, 1H, J = 12.5 Hz), 4.17 (d, 1H, J =
12.5 Hz), 5.29 (dd, 2H, J = 20.8, 13.9 Hz), 5.68 – 5.92 (m, 1H), 6.50 – 6. 65 (m, 2H), 7.10 (ddd, 1H, J = 8.5, 6.5, 2.0 Hz), 7.17 –
7.28 (m, 2H), 7.34 (t, 2H, J = 7.6 Hz), 7.40 – 7.67 (m, 9H), 8.07 (t, 3H, J = 8.1 Hz). 13C NMR (75.46, CDCl3): δ =22.69 (γ-CH2
Pro); 30.48 (β-CH2 Pro); 43.78 (CH2); 43.86(CH2); 57.67 (δ-CH2 Pro); 64.19 (CH2Ph); 70.69 (α-CH Pro); 82.39 (CH2 C CH2);
114.4 (d, J=20.7, C6H4); 119.12 (d, J=21.2, C6H4);120.46 (CH, =CH2); 120.45 (C6H4); 123.85 (6-CH C6H4); 127.23 (d, J=2.8,
C6H4); 127.40 (CH); 127.96(CH); 128.03; 128.82 (CH); 129.03 (3,3'-CHPh); 129.79(CH); 130.96(CH); 131.71(CH); 132,37
(CH, g, J=8.1, C6H4); 133.60 (δ-CH C6H4); 134.06; 136.81; 137.00 (g, J=7.2, C6H4); 142.46; 172.18; 179.73; 180.24.
Complex 5c
Starting with 1 eq. (1g, 1.85mmol) of complex 3, of 1.5eq. (0.39mL, 2.78mmol) 3-(methoxy)benzyl bromide, 5eq. (0.3g,
9.2mmol) of NaOH and DMF at 250C with stirring. Complex 5c was isolated in 55,5% yield (0.679g, 1.02mmol).
Anal. Calc. for C38H37N3NiO4 (658.41): C 69.32; H 5.66; N 6.38; Found: C 69.73; H 5.81; N 6.19. Mp. 220 oC. [α]D20 =+736°
(с 0.15, CH3OH).
6

1H NMR - (300 MHz, CDCl3): δ =1.95 (ddd, 2H, J = 20.5, 19.4, 9.3 Hz, γ,δ-Ha Pro); 2.19 – 2.34 (m, 3H); 2.58 (dd, 1H, J =
16.0, 6.8 Hz, β-Ha Pro); 2.81 (d, 1H, J = 14.1 Hz, CH2-All); 3.05 (d, 1H, J = 14.0 Hz, γ-Hb Pro); 3.14 (dd, 1H, J = 8.9, 5.2 Hz,
CH2Ar); 3.19 – 3.29 (m, 1H, α-CH Pro); 3.45 – 3.53 (m, 1H, CH2Ph); 3.73 (s, 3H, CH3); 4.19 (d, 1H, J = 12.5 Hz, CH2Ph); 5.30
(dd, 2H, J = 23.4, 13.3 Hz, =CH2); 5.81 (tt, 1H, J = 12.4, 6.3 Hz, =CH2); 6.52 – 6.66 (m, 2H, =CH, All); 6.97 (d, 1H, J = 8.3 Hz);
7.03 – 7.16 (m, 3H, Ar); 7.20 (d, 1H, J = 7.5 Hz, Ar) 7.38 (dd, 4H, J = 18.1, 12.2 Hz, Ar); 7.51 (dd, 3H, J = 12.6, 4.4 Hz H-6
C6H4, Ar); 8.09 (t, 3H, J = 7.4 Hz, 2,2’-H Ph, Ar): 13C NMR (75.46, CDCl3): δ =22.82 (γ-CH2, Pro); 30.53 (β-CH2 Pro); 43.92
(CH2); 44.01 (CH2); 55.07 (δ- CH2 Pro); 57.87 (CH3); 64.31 (CH2Ph); 70.85 (α-CH Pro); 82.44 (C-CH2); 96.28; 113.73; 115.89;
119.23; 120.53; 123.29; 124.00 (q, JC,F=274.4, CF3); 127.33; 128.06(q, JC,F=6.1); 128.13; 128.90; 129.11; 129.80; 129.87;
131.63; 131.83; 132.42; 133.66; 134.16; 137.10; 138.29; 142.56; 160.03; 172.23; 179.85; 180.36:
Complex 5d
Starting with 1 eq. (1g, 1.85mmol)of complex 3, of 1.5eq. (0.34mL, 2.78mmol) 3-(fluoro)benzyl bromide, 5eq. (0.3g,
9.2mmol) of NaOH and DMF at 550C with stirring. Complex 5d was isolated in 55% yield (0.66g, 1.02mmol).
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Anal. Calc. for C37H34FN3NiO3 (646.38): C 68.75; H 5.30; N 6.50; Found: C 68.55; H 5.21; N 6.28. Mp. 226 C. [α]D
=+2137.30 (c 0.15, CH3OH).
1H NMR - (300 MHz, CDCl3): δ =1.61-1.72 (m, 1H, γ-Hα Pro); 1.87 (ddd, 1H, J=10.7, 10.0, 6.3, δ-Hα Pro); 2.05-2.20(m,
1H); 2.24-2.34 (m, 1H); 2.34 (ddt, 1H, J=15.8, 5.9, 1.4, CH2-All); 2.58 (ddt, 1H, J=15.8, 6.9, 1.4, CH2-All); 2.84 (d, 1H, J=14.3,
CH2 C6H4F); 2.99 (d, 1H, J=14.3, CH2 C6H4F); 3.22 (ddd, 1H, J=10.7, 6.3, 2.8, δ-Hb Pro); 3.26 (dd, 1H, J=9.5, 7.4, α-H Pro);
3.50 (d, 1H, J=12.5, CH2Ph); 4.22 (d, 1H, J=12.5, CH2Ph); 5.27 (ddt, 1H, J=17.2, 1.6, 1.4, =CH2); 5.36 (dq, 1H, J=10.5, 1.6, 1.4,
=CH2); 5.84 (dddd, 1H, J=17.2, 10.5, 6.9, 5.9, =CH); 6.55 (dd, 1H, J=8.4, 1.8, 3-H C6H4); 6.61 (ddd, 1H, J=8.4, 6.7, 1.2, 4-H
C6H4); 7.09-7.16 (m, 2H); 7.17-7.28 (m, 3H); 7.29-7.47 (m, 5H); 7.51-7.56 (m, 3H); 8.07 (dd, 1H, J=8.7, 1.2, H-6 C6H4); 8.09-
8.13 (m, 2H, 2,2'-H Ph). C6H4CF3); 8.05-8.10 (m, 3H, Ar): 13C NMR (75.46, CDCl3): δ =22.9 (γ-CH2 Pro); 30.7 (β-CH2 Pro);
43.4 (CH2); 44.4(CH2); 57.9 (δ-CH2 Pro); 64.4 (CH2Ph); 70.8 (α-CH Pro); 82.0 (CH2 C CH2); 114.4 (d, JC,F+20.7, C6H4F); 118.0
(d, JC,F=21.2, C6H4F);119.5 (CH, =CH2); 120.6 (4-CH C6H4); 124.1 (6-CH C6H4); 126.5 (CH, d, JC,F=2.8, C6H4F); 127.4 (CH);
128.1(CH); 128.1; 128.2 (CH); 129.0 (3,3'-CHPh); 129.0(CH); 129.1(CH); 130.0(CH); 130,2 (CH, g, JC,F=8.1, C6H4F); 133.7 (δ-
CH C6H4); 134.1; 137.1; 139.4 (g, JC,F=7.2, C6H4F); 142.6; 163.3 (g, JC,F=246.5, C6H4F); 172.6; 179.8; 180.4.
Complex 5e
Starting with 1 eq. (1g, 1.85mmol) of complex 3, of 1 eq. (0.22mL, 1.85mmol) 4-(fluoro)benzyl bromide, 3eq. (0.22g,
5.55mmol) of NaOH and DMF at 550C with stirring. Complex 5e was isolated in 65% yield (0.78g, 1.21mmol).
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Anal. Calc. for C37H34FN3NiO3 (646.38): C 68.75; H 5.30; N 6.50; Found: C 68.35; H 5.45; N 6.14. Mp. 262 C. [α]D
=+1880.00 (c 0.1, CH3OH).
1H NMR - (300 MHz, CDCl3): δ =1.57-1.68 (m, 1H); 1.81-1.91 (m, 1H); 1.92-2.05 (m, 1H); 2.15-2.38 (m, 3H); 2.56 (ddt,
1H, J=15.9, 6.9, 1.4, CH2 All); 2.79 (d, 1H, J=14.1, CH2 C6H4F); 3.05 (d, 1H, J=14.1, CH2 C6H4F); 3.08-3.16 (m, 1H); 3.25 (dd,
1H, J=10.0, 7.0, α-CH Pro); 3.47 (d, 1H, J=12.4, CH2Ph); 4.17 (d, 1H, J=12.4, CH2Ph); 5.25 (ddt, 1H, J=17.2, 1.6, 1.4, =CH2);
5.33 (ddt, 1H, J=10.5, 1.6, 1.4, =CH2); 5.79 (dddd, 1H, J=17.2, 10.5, 6.9, 5.7, =CH); 6.54 (dd, 1H, J=8.5, 1.8, H-3 C6H4); 6.60
(ddd, 1H, J=8.5, 6.7, 1.2, H-4 C6H4); 7.11 (ddd, 1H, J=8.6, 6.7, 1.8, H-5 C6H4); 7.14-7.25 (m, 4H, Ar); 7.32-7.39 (m, 2H, Ar);
7.41-7.48 (m, 3H, Ar); 7.50-7.58 (m, 3H, Ar); 8.06 (dd, 1H, J=8.6, 1.2, H-6 C6H4); 8.07-8.11 (m, 2H, Ar); 13C NMR (75.46,
CDCl3): δ =22.7 (γ-CH2 Pro); 30.6 (β-CH2 Pro); 43.0 (CH2); 43.8 (CH2); 57.8 (δ-CH2 Pro); 64.4 (CH2 Ph); 70.8 (α-CH Pro);
82.5; 115.7 (d, JC,F 21.0, 2,2ʹ-CH C6H4F); 119.3 (=CH2); 120.6 (4-CH C6H4); 124.0 (6-CH C6H4); 127.4 (CH); 127.9 (CH);
128.0; 128.1 (CH); 128.9 (3.3ʹ -CH Ph); 129.0 (CH); 129.1 (CH); 129.9 (CH); 131.6 (2.2ʹ-CH Ph); 131.9 (CH); 132.3 (5-CH
C6H4); 132.5 (d, J=7.6, 3.3ʹ-CH C6H4F); 132.6 (d, JC,F =3.5, 4-C C6H4F); 133.7 (5-CH, C6H4); 134.1, 137.0; 142.6; 162.7 (d,
J
C,F=246.4, C-1 C6H4F); 172.4; 179.7; 180.4.
Complex 5f
Starting with 1 eq. (1g, 1.85mmol) of complex 3, of 1.5eq. (0.66g, 2.78mmol) 2-(trifluoromethyl)benzyl bromide, 3eq.
(0.22g, 5.55mmol) of NaOH and DMF at 250C with stirring. Complex 5f was isolated in 51% yield (0.66g, 0.95mmol).
Anal. Calc. for C38H34F3N3NiO3 (696.38): C 65.54; H 4.92; N 6.03; Found: C 65.82; H 5.15; N 5.86. Mp. 196-197 oC. [α]D
=+1656,67 (C=0,15, CH3OH)
20
1H NMR - (300 MHz, CDCl3): δ =2.04-2.15 (m, 2H, γ,δ-Ha Pro); 2.49-2.63 (m, 3H); 2.67-2.7 (m, 1H, β-Ha Pro); 2.78 (dd,
1H, J=14.7, 6.9, CH2-All); 3.12-3.29 (m, 1H, γ,-Hb Pro); 3.43 (d, 1H, J=18.2, CH2Ar); 3.48 (dd, 1H, J=10.6, 6.0, α-CH Pro);
3.64 (d, 1H, J=12.6, CH2Ph); 3.80 (dd, 1H, br. J=9.2, 6.6, δ-Ha Pro); 4.52 (d, 1H, J=12.6, CH2Ph); 5.30 (ddt, 1H, J=17.1, 1.5,
1.5, =CH2); 5.47 (ddt, 1H, J=10.3, 1.5, 1.5, =CH2); 6.18 (ddt, 1H, J=17.1, 10.3, 7.0, =CH, All); 6.40 (d, 1H, br. J=7.8, 2-H C6H5);
6.58 (ddd, 1H, J=8.4, 6.8, 1.2, H-4, C6H4); 7.06-7.14 (m, 2H, Ar; 7.29 (tt, 1H, J=7.4, 1.2, H-4 Ph); 7.36-7.53 (m, 6H, Ar); 7.66
(d, 1H, br. J=7.8, Ar); 7.76 (t, 1H, br. J=7.7, Ar); 7.95 (dd, 1H, J=8.6, 1.2 H-6 C6H4); 8.21-8.25 (m, 2H, 2,2’-H Ph); 9.47 (d, 1H,
J=7.8, Ar) 13C NMR (75.46, CDCl3): δ =23.4 (γ-CH2, Pro); 31.0 (β-CH2 Pro); 39.9 (CH2); 47.7 (CH2); 58.0 (δ- CH2 Pro); 64.3
(CH2Ph); 70.5 (α-CH Pro); 79.7 (C-CH2); 119.6; 120.9; 124.0 (q, JC,F=274.4, CF3); 124.5; 126,6; 126,9; 127,0 (q, JC,F=6.1);
127.4; 127.7; 128.4; 128.5; 136.3 (br.); 136.5; 141.8; 173.2; 180.3; 180.6
Complex 5g
Starting with 1 eq. (1g, 1.85mmol) of complex 3, of 1.5eq. (0.42mL, 2.78mmol) 3-(trifluoromethyl)benzyl bromide, 5eq.
(0.3g, 9.2mmol) of NaOH and DMF at 250C with stirring. Complex 5g was isolated in 63% yield (0.816g, 1.17mmol).
Anal. Calc. for C38H34F3N3NiO3 (696.38): C 65.54; H 4.92; N 6.03; Found: C 65.32; H 5.19; N 5.76. Mp. 197 oC. [α]D20 =+1470
(C=0,15, CH3OH)
1H NMR - (300 MHz, CDCl3): δ =1.53 – 1.68 (m, 1H, Pro); 1.78 -2.05 (m, 3H, Pro); 2.07 -2.34 (m, 3H, CH2, All); 2.57 (dd,
1H, J = 15.8, 7.0 Hz, CH2, All); 2.98 (dd, 2H, J = 36.3, 14.2 Hz, CH2C6H4CF3); 3.13-3.28 (m, 2H, CH2Ph, Pro); 3.45 (1H, d, J =
12.5 Hz, α-CH Pro); 4.17 (d, 1H, J = 12.5 Hz, CH2Ph); 5.30 (ddd, 2H, J = 18.8, 13.8, 1.6 Hz, =CH2); 5.75-5.90 (m, 1H, =CH,
All); 6.57 (dtd, 2H, J = 10.2, 8.5, 1.5 Hz, C6H4); 7.12 (ddd, 1H, J = 8.6, 6.7, 1.8 Hz, Ar); 7.20 (dd, 2H, J = 13.1, 7.6 Hz, Ar);
7.50-7.59 (m, 3H, Ar); 7.58-7.64 (m, 2H, C6H4CF3); 7.71 (dd, 2H, J = 13.6, 7.7 Hz, C6H4CF3); 8.04-8.14 (m, 3H, Ar); 13C NMR
7

(75.46, CDCl3): δ =22.82 (γ-CH2); 30.48 (β-CH2); 43.60 (CH2); 44.11 (CH2); 57.88 (δ- CH2); 64.23 (CH2Ph); 70.59 (α-CH Pro);
82.06; 96.27; 119.49; 120.61; 124.00 (q, JC,F=272.0); 124.34 (q, JC,F=3.7); 127.54; 127.91; 128.16; 128.93; 129.02; 129.11;
130.03 (q, JC,F=32.5); 131.60; 132.09; 133.70; 133.88; 134.12; 137.04; 137.88; 142.63; 172.76; 179.60; 180.51:
Complex 5h
Starting with 1 eq. (1g, 1.85mmol) of complex 3, of 1.5eq. (0.43mL, 2.78mmol) 4-(trifluoromethyl)benzyl bromide, 5eq.
(0.3g, 9.2mmol) of NaOH and DMF at 250C with stirring. Complex 5h was isolated in 61% yield (0.789g, 1.13mmol).
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Anal. Calc. for C38H34F3N3NiO3 (696.38): C 65.54; H 4.92; N 6.03; Found: C 65.22; H 4.55; N 6.14. Mp. 247 C. [α]D
=+1813 (C=0,15, CH3OH).
1H NMR - (300 MHz, CDCl3): δ =1.50-1.64 (m, H); 1.69-1.85 (m, 2H); 2.05-2.16 (m, 1H); 2.20-2.31 (m, 1H); 2.32 (ddt, 1H,
J=15.9, 5.7, 1.4, CH2, All); 2.58 (ddt, 1H, J=15.9, 6.9, 1.4, CH2, All); 2.84 (d, 1H, J=14.0, CH2C6H4CF3); 3.01-3.10 (m, 1H);
3.13 (d, 1H, J=14.0, CH2C6H4CF3); 3.21 (dd, 1H, J=10.3, 6.6, α-CH Pro); 3.45 (d, 1H, J=12.5, CH2Ph); 4.16 (d, 1H, J=12.5,
CH2Ph); 5.27 (ddt, 1H, J=17.2, 1.5, 1.4, =CH2); 5.36 (ddt, 1H, J=10.5, 1.5, 1.4, =CH2); 5.81 (dddd, 1H, J=17.2, 10.5, 6.9, 5.7,
=CH, All); 6.53 (dd, 1H, J=8.5, 1.6, C6H4); 6.60 (ddd, 1H, J=8.5, 6.8, 1.2, C6H4); 7.12 (ddd, 1H, J=8.6, 6.8, 1.6, C6H4); 7.16-7.26
(m, 2H, Ar); 7.31-7.38 (m, 2H, Ar); 7.41-7.48 (m, 1H, Ar); 7.50-7.58 (m, 4H, Ar); 7.58-7.63 (m, 2H, C6H4CF3); 7.74-7.79 (m,
2H, C6H4CF3); 8.05-8.10 (m, 3H, Ar): 13C NMR (75.46, CDCl3): δ =22.6 (γ-CH2); 30.6 (β-CH2); 43.4 (CH2); 44.0 (CH2); 57.7 (δ-
CH2); 6.3 (CH2Ph); 70.7 (α-CH Pro); 82.2; 119.5; 120.6; 124.0; 124.3 (q, JC,F=272.0); 125.7 (q, JC,F=3.7); 127.5; 127.9; 127.9;
128.2; 128.9; 129.0; 129.2; 130.0; 130.1 (q, JC,F=32.5); 131.4; 131.5; 132.0; 133.7; 134.1; 137.0; 141.0; 142.7; 172.6; 179.4;
180.4:
Complex 6i
Starting with 1 eq. (1g, 2mmol) of complex 1, of 1,2 eq. (0.24, 2.4mmol) 4-bromobut-1-ene, 3,5eq. (0.28g, 7mmol) of NaOH
and DMF at 250C with stirring. Complex 6i was isolated in 56.8% yield (0.63g, 1.14mmol).
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Anal. Calc. for C31H31N3NiO3 (552.29): C 67.42; H 5.66; N 7.61; Found: C 67.71; H 5.93; N 7.59. Mp. 196 C. [α]D
=+2230.60 (c 0.15, CH3OH).
1H NMR - (300 MHz, CDCl3): δ =1.64-1.75 (m, 1H, γ-H Pro); 2.03-2.33 (m, 5H); 2.46-2.60 (m, 1H, β-H Pro); 2.66-2.83 (m,
2H); 3.46 (dd, 1H, J 10.9, 5.8, α-H Pro); 3.51-3.61 (m, 2H); 3.60 (d, 1H, J 12.7, CH2Ph); 3.89 (dd, 1H, J 8.6, 3.6, CH); 4.46 (d,
1H, J 12.7, CH2Ph); 4.87 (ddt, 1H, J 10.2, 1.7, 1.2, =CH2); 4.96 (ddt, 1H, J 17.1, 1.7, 1.5, =CH2); 5.53 (ddt, 1H, J 17.1, 10.2, 6.4,
=CH); 6.62 (dd, 1H, J 8.2, 2.2, 3-H, C6H4); 6.65 (dd, 1H, J 8.2, 6.4, 1.1, 4-H, C6H4); 6.91-6.95 (m, 1H, Ar.); 7.14 (ddd, 1H, J 8.6,
6.4, 2.2, 5-H, C6H4); 7.20 (tt, 1H, J=7.5, 1.2, 4-H, Ar); 7.25-7.29 (m, 1H, Ar.); 7.32-7.39 (m, 2H, Ar.); 7.43-7.56 (m, 3H, Ar.);
8.02-8.06 (m, 2H, 2,2’-H, NCH2Ph); 8.17 (dd, 1H, J 8.6, 1.1, 6-H, C6H4).13C NMR (75.46, CDCl3): δ = 23.9 (γ-CH2 Pro); 29.6
(CH2); 30.9 (β-CH2 Pro); 35.3 (CH2); 57.1 (δ-CH2 Pro); 63.2 (CH2Ph); 69.9 (CH); 70.3 (α-CH Pro); 115.9 (=CH2); 120.8 (4-CH,
C6H4); 123.9 (6-CH, C6H4); 126,5; 127.4 (CH); 127.6 (CH); 129.0 (CH); 129.0 (3,3’-CH, CH2Ph); 129.1 (CH); 129.8 (CH);
131.7 (2,2’-H, CH2Ph); 132.2 (CH); 133.3 (CH); 134.0; 136.7; 142.5; 170.4; 179.0; 180.4.
General procedure. Isolation of amino acids
Isolation of amino acids from the complexes was carried out according to the standard procedure12. Complexes were dissolved in
50 mL of MeOH and then slowly added to 50 mL of 2M HCl solution and the mixture was heated at 50 oC. After disappearance
of the red color of the metal complex, the solution was concentrated in vacuum, then 50 mL of water was added and the
precipitated (S)-BPBxHCl was filtered. The optically active amino acid was isolated from the water layer by ion-exchange
sorption and desorption using Ku-2x8H+ for cation exchange and an aq. solution of NH4OH (5%) as eluent. The eluate was
concentrated in vacuum and the amino acid was recrystallized from a mixture of water and EtOH (1:2). All compounds were
isolated in analytically pure form.
In the case of complexes 4a, 4c, and 5f, the amino acid failed to be isolated.
(S)-2-amino-2-benzylpent-4-enoic acid-7a
Yield 65,28% (0.21g, 1.02mmol). Anal. calc. for C12H15NO2 (205,25) C 70.22; H 7.37; N 6.82. Found: C 70.14; H 7.18; N
6.60. M.p. 255 OC, (decomposed and became black), [α]D20 = + 6 (c 0,5; 6N HCl).
1H NMR (300 MHz, D2O): δ =2.60 (dd, 1H, J=14.5, 8.7, CH2, All); 2.91 (dd, 1H, J=15.4, 6.2, CH2 All); 3.11 (d, 1H, J=14.3,
CH2Ph); 3.44 (d, 1H, J=14.3, CH2,Ph); 5.35-5.42 (m, 2H, = CH2); 5.80-5.94 (m, 1H, =CH); 7.35-7.39 (m, 2H); 7.43-7.53 (m,
3H):
(S)-2-amino-2-(3-fluorobenzyl)pent-4-enoic acid-7b
Yield 62% (0.214g, 0.95mmol). Anal. calc. for C12H14FNO2 (223,24) C 64.56; H 6.32; N 6.27. Found: C 64.71; H 6.18; N
6.06. M.p. 226 OC (decomposed and became black), [α]D20 = + 14.55 (c 1,5; 6N HCl).
1H NMR (300 MHz, D2O): δ =20.60 (ddt, 1H, J=14.5, 8.6, 0.8, CH2, All); 2.90 (ddt, 1H, J=14.5, 6.3, 1.3, CH2 All); 3.12 (d,
1H, J=14.3, CH2); 3.43 (d, 1H, J=14.3, CH2); 5.35-5.42 (m, 2H, =CH2); 5.80-5.94 (m, 1H, =CH2); 7.09-7.23 (m, 3H, C6H4); 7.44-
7.52 (m, 1H, C6H4):
(S)-2-amino-2-(4-fluorobenzyl)pent-4-enoic acid-7c
Yield 60% (0.207g, 0.927mmol). Anal. calc. for C12H14FNO2 (223,24) C 64.56; H 6.32; N 6.27. Found: C 64.35; H 6.62; N
6.16. M.p. 231 OC (decomposed and became black), [α]D20 = + 14.55 (c 0,2; 6N HCl).
1H NMR (300 MHz, D2O): δ =2.59 (ddt, 1H, J=14.6, 8.7, 0.7, CH2 All); 2.89 (ddt, 1H, J=14.6, 6.3, 1.1, CH2 All); 3.09 (d,
1H, J=14.5, CH2Ar); 3.40 (d, 1H, J=14.5, CH2Ar); 5.35-5.41 (m, 2H, =CH2); 5.87 (dddd, 1H, J=17.8,11.5, 8.7, 6.3, =CH); 7.17-
7.25(m, 2H, C6H4F); 7.32-7.39 (m, 2H, C6H4F):
(S)-2-amino-2-(4-(trifluoromethyl)benzyl)pent-4-enoic acid-7d
Yield 61% (0.239g, 0.875mmol). Anal. calc. for C13H14F3NO2 (273,25) C 57.14; H 5.16; N 5.13. Found: C 57.05; H 5.32; N
4.87. M.p. 221 OC (decomposed and became black), [α]D20 = + 15.5 (c 0,2, 6N HCl).
1H NMR (300 MHz, D2O): δ =20.60 (ddt, 1H, J=14.5, 8.6, 0.8, CH2, All); 2.90 (ddt, 1H, J=14.5, 6.3, 1.3, CH2 All); 3.12 (d,
1H, J=14.3, CH2); 3.43 (d, 1H, J=14.3, CH2); 5.35-5.42 (m, 2H, =CH2); 5.80-5.94 (m, 1H, =CH2); 7.09-7.23 (m, 3H, C6H4); 7.44-
7.52 (m, 1H, C6H4).
8

(S)-2-(3-methoxybenzyl)-2-aminopent-4-enoic acid-7e
Yield 58% (0.207g, 0.879mmol). Anal. calc. for C13H17NO3 (235,28) C 66.36; H 7.28; N 5.95. Found: C 66.75; H 7.54; N
5.87. M.p. 238OC (decomposed and became black), [α]D20 = +6 (c 0,2, 6N HCl).1H NMR (300 MHz, DMSO) δ= 2. 29–2.41 (m,
1H, CH2, All); 2.52 – 2.63 (m, 1H, CH2 All); 2.81 (d, 1H, J = 13.5 Hz, CH2); 3.09 (d, 1H, J = 13.4 Hz CH2); 3.75 (s, 3H, OCH3);
5.11 (t, 2H, J = 14.6 Hz, =CH2); 5.87 (dd, 1H, J = 24.8, 11.9 Hz, CH=); 6.64 – 6.73 (m, 1H, C6H4); 6.81 – 6.92 (m, 2H, C6H4);
7.10 (t, 1H, J = 7.8 Hz, C6H4),: 13C NMR (75.46 MHz, DMSO): 38.65(CH2), 38.93(CH2), 40.04, 40.32, 41.46, 42.12,
54.31(CH3), 62.24(C-NH2), 95.47, 111.85, 115.56, 118.32, 122.39, 128.26, 132.86, 137.17, 158.70(C-O), 172.68(C=O):
(S)-2-amino-2-(3-(trifluoromethyl)benzyl)pent-4-ynoic acid 8f
Yield 85,89 % (0.336g, 0.12mmol). Anal. calc. for C13H12F3NO2 (271,24) C 57.57; H 4.46; F 21.01; N 5.16. Found: C 57.86;
H 4.72; N 5.32. M.p. 220 OC (decomposed and became black), [α]D20 = + 11,5 (c 0,2, 6N HCl). 1H NMR (300 MHz, DMSO –
d6/CCl4 + CF3COOD): δ =2.74-2.92 (m, 3H, CH2C≡CH), 3.31 (s, 2H, CH2-Ar), 7.44-7.61 (m, 4H, C6H4). 13C NMR (75.46,
CDCl3): δ =24.8 (CH2); 39.5 (CH2); 62.3; 74.8 (CH Acet.); 75.5 (C Acet.); 115.5 (q, J~285, CF3); 125.4 (q, J 3.5, 2CH); 130.2
(2CH); 129.2 (q, J 30.0); 136.1; 170.6 (CO).
(S)-2-aminohex-5-enoic acid 9g
Yield 68% (0.159g, 0.123mmol). Anal. calc. for C6H11NO2 (129,16) C 55.80; H 8.58; N 10.84. Found: C 55.96; H 8.67; N
10.93. M.p. 231 OC (decomposed and became black), [α]D20 = +7.74 (c 0.15; 6N HCl).
1H NMR (300 MHz, D2O): δ =1.94-2.14 (m, 2H, CH2); 2.21-2.30 (m, 2H, CH2); 3.83 (dd, 1H, J=6.8, 5.4, CH); 5.14-5.26 (m,
2H, =CH2); 5.97 (ddt, 1H, J=17.2, 10.4, 6.5, =CH):
Molecular docking
Amino acids structures were built by ChemBioOffice 2010 (ChemBio3D Ultra12.0). Ligand free energy was minimized using
MM2 force field and truncated Newton–Raphson method. Crystallographic structures of collagenase G were taken from
http://www.rcsb.org website (PDB-ID: 2Y50). Docking of ligand to enzyme has been done by AutoGrid 4 and AutoDock Vina
software [25]. AutoDock uses the Lamarckian genetic algorithm by alternating local search with selection and crossover [26]. The
ligands were ranked using an energy-based scoring function and a grid-based protein-ligand interaction was used to speedup the
score calculation.
Determination of collagenase activity
Collagenase G from Closridium (Hathewaya) histolyticum (EC 3.4.24.3) and miscellaneous reagents were purchased from
Sigma-Aldrich (now Merck).
Collagenase activity was measured by the known method [27]. The reaction mixture contained 0.05 M HEPES (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 7.2, 10 mg/mL gelatin and 0.025 mg/mL collagenase (activated by
o
0.21 M CaCl2). The aliquot (50 ꢁl) was taken and the remaining mixture was incubated at 37 C. Aliquot was taken every 30
min. The reaction was stopped by addition of 6 ꢁl of 30% trichloroacetic acid. The concentration of free amino groups in the
reaction mixture was determined by ortho-phthalaldehyde (OPA) reagent containing 0.2 M borate buffer, pH 9.7, 0.1667 mg/ml
OPA and 1.18 mM mercaptoethanol. The reaction mixture (50 ꢁl) was added to OPA reagent (1.5 mL) and H2O (1.5 mL). A340
was recorded after 5 min incubation at room temperature.
Acknowledgment
This work was supported by the RA MES State Committee of Science and Russian Foundation for Basic Research (RF) in
the frames of the joint research projects SCS 18RF-073, 18T-1D317 and in the frames of the research project ISTC А-2289 and
ANSEF microbio-5133.
Disclosure statement
The authors announce no competing interest.
Supporting Information
Full experimental detail, 1H and 13C NMR spectra, HPLC traces. This material can be found via the “Supplementary Content”
section of this article’s webpage.
References
[1] Blaskovich MA (2016) Unusual Amino Acids in Medicinal Chemistry. J. Med. Chem 5924:10807-10836.
[2] Stevenazzi A, Marchini M, Sandrone G, Vergani B, Lattanzio M. (2014) Amino acidic scaffolds bearing unnatural side
chains: An old idea generates new and versatile tools for the life sciences. Bioorg. Med. Chem. Lett. 24: 5349–5356.
[3] Kaul R, Surprenant S, Lubell WD (2005) J Org Chem 70: 3838–3844.
[4] Close DR (2001) Matrix metalloproteinase inhibitors in rheumatic diseases. Ann Rheum Dis., 60: iii62-67.
[5] Baker AH, Edwards DR, Murphy G (2002) Metalloproteinase inhibitors: biological actions and therapeutic opportunities.
J. Cell Science 115: 3719–3727.
[6] Mittal R, Patel A.P, Debs L.H, Nguyen D, Patel K, Grati M, Mittal J, Yan D, Chapagain P, Liu XZ (2016) Intricate
Functions of Matrix Metalloproteinases in Physiological and Pathological Conditions. Journal of cellular physiology
231(12):2599–2621.
9

[7] Migliaccio A, Castoria G, Giovannelli P, Auricchio F. (2010) Cross talk between epidermal growth factor (EGF) receptor
and extra nuclear steroid receptors in cell lines. Mol Cell Endocrinol 327(1-2):19-24.
[8] McCawley LJ, Matrisian LM (2001) Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol
13: 534-540.
[9] Partridge JJ, Madsen MA, Ardi VC, Papagiannakopoulos T, Kupriyanova TA, Quigley JP, Deryugina EI (2007)
Functional analysis of matrix metalloproteinases and tissue inhibitors of metalloproteinases differentially expressed by variants
of human HT-1080 fibrosarcoma exhibiting high and low levels of intravasation and metastasis. J Biol Chem. 282(49):35964-
35977.
[10] Isaksen B, Fagerhol MK (2001) Calprotectin inhibits matrix metalloproteinases by sequestration of zinc. J.Clin
pathol:Mol pathol., 54:289-282.
[11] Edkins TJ, Koller-Eichhorn R, Alhadeff JA, Mayer U, Faust H, Del Tito BJ (2012) Assessment of potential cross-
reactivity of human endogenous matrix metalloproteinases with collagenase Clostridium histolyticum antibodies in human sera
obtained from patients with Dupuytren's contracture. Clin Vaccine Immunol. 19(4): 562-569.
[12] Sargsyan AS, Babayan BG, Avetisyan NS, Mkrtchyan AG,
Hovhannisyan AM, Hovhannisyan NA (2018)
Heterocyclic substituted non-protein amino acids as inhibitors of Clostridium histolyticum collagenase. Biolog. Journal of
Armenia 1 (70): 12-15.
[13] Saghyan AS, Langer P (2016) Asymmetric synthesis of non-proteinogenic amino acids, Wiley-VCH, 376 page.
[14] Sorochinsky AE, Acen˜a JL, Moriwaki H, Sato T, Soloshonok V(2013) Asymmetric synthesis of α-amino acids via
homologation of Ni(II) complexes of glycine Schiff bases; Part 1: alkyl halide alkylations, Amino Acids, 45 (4), 691–718.
[15] Sorochinsky AE, Acen˜a JL, Moriwaki H, Sato T, Soloshonok V(2013) Asymmetric synthesis of a-amino acids via
homologation of Ni(II) complexes of glycine Schiff bases. Part 2: Aldol, Mannich addition reactions, deracemization and (S) to
(R) interconversion of a-amino acids Amino Acids, 45, 1017–1033
[16] Luis AJ., Sorochinsky AE., Soloshonok V (2014) Asymmetric synthesis of a-amino acids via homologation of Ni(II)
complexes of glycine Schiff bases. Part 3: Michael addition reactions and miscellaneous transformations. Amino Acids 46
(9), 2047-2073.
[17] Romoff TT, Ignacio BE, Mansour N, Palmer AB, Creighton CJ, Abe H, Moriwaki H, Han J, Konno H, Soloshonok VA
(2019) Large-Scale Synthesis of the Glycine Schiff Base Ni(II) Complex Derived from (S)- and (R)-N-(2-Benzoyl-4-
chlorophenyl)-1-[(3,4-dichlorophenyl)methyl]-2-pyrrolidinecarboxamide, Org. Process Res. Dev., 23, 629-634.
[18] Yin Z, Moriwaki H, Abe H, Miwa T, Han J, Soloshonok VA (2019) Large-Scale Asymmetric Synthesis of Fmoc-(S)-2-
Amino-6,6,6-Trifluorohexanoic Acid ChemistryOpen, 8, 701-704
[19]Mei H, Han J, Takeda R, Sakamoto T, Miwa T, Minamitsuji Yu, Moriwaki H, Abe H, Soloshonok V.A. (2019) Practical
Method for Preparation of (S) 2-Amino-5,5,5-trifluoropentanoic Acid via Dynamic Kinetic Resolution, ACS Omega 2019, 4,
11844−11851.
[20] Mei H, Hiramatsu T, Takeda R, Moriwaki H, Abe H, Han J, Soloshonok V.A. (2019) Expedient Asymmetric Synthesis
of (S)-2-Amino-4,4,4-trifluorobutanoic Acid via Alkylation of Chiral Nucleophilic Glycine Equivalent Organic Process Research
& Development, 23 (4), 629-634.
[21] Collet S, Bauchat P, Danion-Bougot R, Danion D (1998) Stereoselective, nonracemic synthesis of ω-borono-α-amino
acids. Tetrahedron: Asymmetry 9: 2121–2131.
[22] Saghyan AS, Petrosyan SGh, Mkrtchyan AF, Mkrtchyan GM, Langer P. (2013) Asymmetric synthesis of
enantiomerically enriched aino acids containing an acetylene bond in the side radical. Chem. Journal of Armenia 66: 588-596.
[23] Belokon' YuN, Saghiyan AS, Djamgaryan SM, Bakhmutov VI, Struchkov YuT, Belikov VM (1990) General method of
the Asymmetric Synthesis of antidiadiastereoisomers of β-Substituted L-2-Aminobutanoic Acids via Chiral Ni(II) Schiff's Base
Complexes of Dehydroaminobutanoic Acid J. Chem Soc. Pekin Trans. I. : 2301-2310.
[24] Belokon' YuN, Saghiyan AS, Djamgaryan SM, Bakhmutov BI, Belikov VM (1988) Asymmetric Synthesis of β-
Substituted α-Amino Acids via a Chiral Ni(II) Complexes of Dehydroalanine. Тetrahedron 44: 5507-5514.
[25] Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function,
efficient optimization, and multithreading. J.Comput. Chem, 31:455–46125.
[26] Morris G, Goodsell D, Halliday R, Huey R, Hart W, Belew R, Olson A (1998) Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry, 19:1639-1662.
[27] Gade W, Brown J (1981) Purification, characterization and possible function of α-N-acylamino acid hydrolase from
bovine liver. Biochemica and Biophysica Acta 13: 86-93
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Highlights
•
•
•
Seven previously not described in the literature novel enantiomerically enriched
unsaturated tailor-made amino acids have been obtainedwith more than 98%
enantiomeric purity, as well as their intermediate complex-products, respectively.
Of the obtained seven tailor-made amino acids four showed inhibitory activity to
collagenase G. The amino acid with an acetylene bond in the side chain
(IC50=1.29±0.02mM) had the best result.
Molecular docking showed that the amino acids with activity to collagenase G
contained hydrogen and π-π bonds with the enzyme.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.