Stereochemical aspects in the synthesis of novel N-(purin-6-yl)dipeptides as potential antimycobacterial agents

Article abstract of DOI:10.1007/s00726-021-02958-0

The synthesis of purine conjugates with natural amino acids is one of the promising directions in search for novel therapeutic agents, including antimycobacterial agents. The purpose of this study was to synthesize N-(purin-6-yl)dipeptides containing the

Full text of DOI:10.1007/s00726-021-02958-0

Amino Acids (2021) 53:407–415
https://doi.org/10.1007/s00726-021-02958-0
ORIGINAL ARTICLE
Stereochemical aspects in the synthesis of novel N‑(purin‑6‑yl)
dipeptides as potential antimycobacterial agents
Vera V. Musiyak1 · Irina A. Nizova1 · Evgeny N. Chulakov1 · Liliya Sh. Sadretdinova1 · Andrey A. Tumashov1,2
Galina L. Levit1 · Victor P. Krasnov1,2
·
Received: 29 July 2020 / Accepted: 6 February 2021 / Published online: 18 February 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
The synthesis of purine conjugates with natural amino acids is one of the promising directions in search for novel therapeutic
agents, including antimycobacterial agents. The purpose of this study was to synthesize N-(purin-6-yl)dipeptides contain-
ing the terminal fragment of (S)-glutamic acid. To obtain the target compounds, two synthetic routes were tested. The frst
of them is based on coupling of N-(purin-6-yl)-(S)-amino acids to dimethyl (S)-glutamate in the presence of carbodiimide
coupling agent followed by the removal of ester groups. However, it turned out that this coupling process was accompanied
by racemization of the chiral center of N-(purin-6-yl)-α-amino acids and in all cases led to mixtures of (S,S)- and (R,S)-
diastereomers (6:4). Individual (S,S)-diastereomers were obtained using an alternative approach based on the nucleophilic
substitution of chlorine in 6-chloropurine or 2-amino-6-chloropurine with corresponding dipeptides as nucleophiles. The
enantiomeric purity of the target compounds was confrmed by chiral HPLC. To test the assumption that racemization of
the chiral center of N-(purin-6-yl)-α-amino acids occurs with the participation of nitrogen atoms of the imidazole ring via
the stage of formation of a chirally labile intermediate, we obtained such structural analogs of N-(purin-6-yl)-(S)-alanine as
N-(9-benzylpurin-6-yl)-(S)-alanine and N-(7-deazapurin-6-yl)-(S)-alanine. It was found that coupling of these compounds
to dimethyl (S)-glutamate was also accompanied by racemization. This indicates that the imidazole fragment does not play a
crucial role in this process. When testing the antimycobacterial activity of some of the obtained compounds, conjugates with
moderate activity against the laboratory Mycobacterium tuberculosis H37Rv strain (MIC 3.1–6.25 μg/mL) were identifed.
Keywords Dipeptides · Racemization · Purine · Coupling · Nucleophilic substitution · Antimycobacterial activity
Introduction
Al-Harbi and Abdel-Rahman 2012), and infuenza viruses
(Pautus et al. 2013). It was also reported about purinyl amino
acids exhibiting antitumor (Ward et al. 1961), antimycobac-
terial (Voynikov et al. 2014; Stavrakov et. al. 2016; Gru-
zdev et al. 2017, 2018; Krasnov et al. 2016, 2020), and other
efects.
The synthesis of purine conjugates with natural amino acids
is one of the promising directions in search for novel ef-
cient therapeutic agents. These compounds are known to
be highly active against herpes viruses (Beauchamp et al.
1992; Krasnov et al. 2019), HIV (McGuigan et al. 2005,
2006), hepatitis C (McGuigan et al. 2010; Chang et al. 2011;
The modifcation of purine core at position N9 leading
to the analogs of natural nucleosides has been widely used
(Wang et al. 2015; Mehellou 2016). At the same time, the
synthesis of C6-substituted derivatives is also an important
area of the purine chemistry (Legraverend and Grierson
2006; Legraverend 2008). The most common synthetic route
to purine conjugates bearing an amino acid fragment at posi-
tion C6 is the nucleophilic substitution of the chlorine atom
in 6-chloropurine (Ward et al. 1961; Niu et al. 2012; Boer-
ema et al. 2016). Depending on the chosen synthetic strat-
egy, reaction conditions and the amino acid structure, the
Handling editor: P. Mefre.
* Victor P. Krasnov
ca@ios.uran.ru
1
Postovsky Institute of Organic Synthesis of RAS (Ural
Branch), 22/20, S. Kovalevskoy/Akademicheskaya St.,
Ekaterinburg 620108, Russia
2
Ural Federal University, 19, Mira St., Ekaterinburg 620002,
Russia
Vol.:(0123456789)
1 3

408
V. V. Musiyak et al.
target compounds may be obtained in enantiomerically pure
form or as a mixture of enantiomers (Vigorov et al. 2014).
The search for novel antimycobacterial agents is an urgent
task. According to the World Health Organization, tubercu-
losis (TB) was diagnosed in 10 million people worldwide
and 1.5 million people died of TB in 2018 (WHO 2019).
Moreover, at present during the COVID-19 pandemic, there
is an increased risk for unfavorable outcome in TB patients
(Motta et al. 2020; WHO 2020). TB treatment is often com-
plicated because of the emergence of mycobacterium strains
resistant to clinically used anti-TB drugs such as isoniazid
and rifampicin (Tripathi et al. 2005; de Wet et al. 2019).
Design of novel antimycobacterial agents difering in the
mechanism of action from known drugs may be the solution
to the problem of multidrug resistance (MDR).
active and exhibited high antimycobacterial activity against
both the standard laboratory strain (M. tuberculosis H37Rv)
and the MDR-TB strain (Krasnov et al. 2016).
It has been found that the analogs of compounds (S)-
1a,b in which the (S)-glutamic acid fragment is replaced
by residues of other α-amino acids, as well as the analogs
in which the glycine fragment is replaced by residues of
various ω-amino acids, do not exhibit substantial antimy-
cobacterial activity (Musiyak et al. 2019). The purpose of
this work was to obtain the analogs of compounds (S)-1a,b
containing fragments of natural α-amino acids instead of
glycine moiety. We used the following α-amino acids as
simple structures without any additional functional groups:
(S)-alanine, (S)-phenylalanine (common aromatic α-amino
acid), and (S)-valine (common branched aliphatic α-amino
acid). Moreover, special attention was paid to monitoring the
enantiomeric purity of the target compounds, since stereo
confguration is known to signifcantly afect the biological
activity of substances (Islam et al. 1997; Melchert and List
2007; Lin et al. 2011).
To identify highly efective anti-TB agents, we carried out
a screening of a large group of novel purine conjugates with
amino acids and short peptides. Among them, N-(purin-6-yl)
glycyl-(S)-glutamic acid ((S)-1a, Fig. 1) and N-(2-aminopu-
rin-6-yl)glycyl-(S)-glutamic acid ((S)-1b) were the most
Results and discussion
Initially, synthesis of purine conjugates containing (S)-
alanine (4a), (S)-phenylalanine (4b) or (S)-valine (4c) frag-
ments instead of glycine residue was carried out by cou-
pling of N-(purin-6-yl)-(S)-amino acids (S)-2a–c to dimethyl
(S)-glutamate followed by alkaline hydrolysis (Scheme 1).
The starting compounds, N-(purin-6-yl)-(S)-amino acids
(S)-2a–c, were synthesized by the nucleophilic substitu-
tion of chlorine in 6-chloropurine with the corresponding
Fig. 1 Structures of the parent compounds (S)-1a,b (Krasnov et al.
2016)
Scheme 1 Synthesis of compounds 4a–c, (R)-5 and (S)-5
1 3

Stereochemical aspects in the synthesis of novel N-(purin-6-yl)dipeptides as potential…
409
(S)-amino acids in aqueous Na2CO3 under refux accord-
ing to the described procedure (Ward et al. 1961). It should
be noted that in the original work (Ward et al. 1961) there
was no information about enantiomeric purity of the purinyl
amino acids obtained under rather drastic conditions. So, we
decided to check enantiomeric purity of compounds 2a–c by
the example of phenylalanine derivatives 2b, thereby con-
frming the absence of racemization at the stage of nucleo-
philic substitution. To do this, we converted compounds (R)-
2b and (S)-2b to methyl esters (R)-5 and (S)-5, respectively
(Scheme 1), and then performed analysis of their enantio-
meric purity (>99% ee) by chiral HPLC.
with dipeptides (S,S)-7a–c (Scheme 2). The protected dipep-
tides (S,S)-6a–c obtained according to the known procedure
(Deng et al. 2008) were used as the starting compounds. The
subsequent deprotection of ester protecting groups by alka-
line hydrolysis followed by Boc-group cleavage via treat-
ment with trifuoroacetic acid led to dipeptides (S,S)-7a–c,
their characteristics were identical to those described in the
literature (McGregor and Carpenter 1961; Gray and Khoujah
1969; Deng et al. 2008). The interaction of dipeptides (S,S)-
7a–c with 6-chloropurine in aqueous Na2CO3 under refux
led to the products (S,S)-4a–c (Scheme 2). Diastereomeric
1
purity of these compounds was confrmed by H and 13C
The coupling of N-(purin-6-yl)-(S)-amino acids (S)-2a–c
to dimethyl (S)-glutamate was carried out in the presence of
N,N′-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotria-
zole (HOBt), and N,N-diisopropylethylamine (DIEA). We
NMR spectral data.
This approach was also applied for the synthesis of 2-ami-
nopurine derivatives, the structural analogs of compound
(S)-1b. Interaction of dipeptides (S,S)-7a,b with 2-amino-
6-chloropurine (ACP) in aqueous Na2CO3 under reflux
aforded the target diastereomerically pure products (S,S)-
8a,b (Scheme 2). It has been found that part of the starting
ACP does not react under these conditions. In the case of the
synthesis of compound (S,S)-8a, 6% of unreacted ACP was
isolated; taking this into account, the yield of product (S,S)-
8a was 34%. In the case of compound (S,S)-8b, signifcantly
more ACP (39%) was isolated from the reaction mixture, and
the product yield was 28%, relative to reacted ACP.
During the synthesis of conjugates (S,S)-4b,c and (S,S)-
8b, we observed unexpected intramolecular cyclization of
dipeptides (S,S)-7b,c with the formation of diketopipera-
zines (S,S)-9b,c (Scheme 2). Because of this side process,
the yield of target products was decreased, and their puri-
fcation was rather laborious. The structure of compounds
(S,S)-9b,c was confrmed by 1H and 13C NMR spectroscopy
and CHN elemental microanalysis; the characteristics of
(S)-phenylalanine derivative (S,S)-9b were identical to those
described in the literature (Kleinsmann and Nachtsheim
2013).
1
were surprised to observe double sets of signals in the H
1
and 13C NMR spectra of products 3a–c (according to H
NMR, in a 6:4 ratio). It should be noted that when conduct-
ing coupling of achiral carboxy component (N-(purin-6-yl)
glycine and N-(purin-6-yl)-beta-alanine) to chiral amino
component (methyl (S)-tyrosinate, methyl (S)-serinate, or
dimethyl (S)-glutamate) in the presence of a DCC–HOBt
mixture, we did not observe any racemization at the chiral
centers of the target pyrinyl dipeptides (Musiyak et al. 2019).
So, we can conclude that in the present case racemization
occurs at the chiral center of the carboxy component, thus
leading to the formation of a mixture of (S,S)- and (R,S)-
diastereomers. Varying the reaction conditions (changing
the order of adding the reagents, loading reagents at 0 °C,
using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
tetrafuoroborate (TBTU) instead of DCC/HOBt mixture)
did not afect the diastereomeric composition of the products
3a–c. Alkaline hydrolysis of diesters 3a–c led to compounds
4a–c, which were also mixtures of diastereomers in a 6:4
ratio (according to 1H NMR spectroscopy).
To obtain the target conjugates 4a–c in diastereomerically
pure form, we used an alternative approach that was based
on the nucleophilic substitution of chlorine in 6-chloropurine
Compounds (S,S)-4a–c were used for identifcation of
the diastereomers formed via the coupling of N-(purin-
6-yl)-α-amino acids (S)-2a–c to dimethyl (S)-glutamate
Scheme 2 Synthesis of compounds (S,S)-4a–c, (S,S)-8a,b and (S,S)-3a–c
1 3

410
V. V. Musiyak et al.
(Scheme 1). The diastereomerically pure dimethyl esters
(S,S)-3a–c were obtained from conjugates (S,S)-4a–c
(Scheme 2), which made it possible to assign correspond-
ing peaks in HPLC chromatograms of diastereomeric
7-deaza-6-chloropurine (10b) with (S)-alanine in aqueous
Na2CO3 under refux (Scheme 4).
It has been found that the coupling of N-(9-benzylpu-
rin-6-yl)-(S)-alanine (S)-11a to dimethyl (S)-glutamate in
the presence of DCC, HOBt, and DIEA is accompanied by
racemization and leads to the product 12a that is a mix-
ture of diastereomers in a 6:4 ratio (Scheme 4), as in the
case of N9-unsubstituted analogs (Scheme 1). In the case
of N-(7-deazapurin-6-yl)-(S)-alanine (S)-11b, racemization
also occurred, despite the absence of the N7 atom (analog
of Nπ atom in histidine) in the purine fragment, resulting in
product 12b as a 6:4 mixture of diastereomers. The alkaline
hydrolysis of compound 12b led to the product 13b as a 7:3
1
mixtures 3a–c. When comparing the HPLC and H, 13C
NMR spectral data of individual diastereomers (S,S)-3a–c
(> 99% de) with the data obtained for mixtures 3a–c, we
found that in all cases the coupling of compounds (S)-
2a–c to dimethyl (S)-glutamate (Scheme 1) resulted in
predominant formation of (S,S)-diastereomers; that is,
products 3a–c were mixtures of (S,S)-and (R,S)-diastere-
omers in a 6:4 ratio. Moreover, it has been shown that
when N-(purin-6-yl)-(R)-phenylalanine [(R)-2b] was used
as a carboxy component, exactly the same mixture of dias-
tereomers [(S,S)-3b/(R,S)-3b 6:4] was formed as in the
case of N-(purin-6-yl)-(S)-phenylalanine (S)-2b. Thus, the
confguration of chiral center of the starting N-(purin-6-
yl)-α-amino acid does not afect the diastereomeric com-
position of products. So, we suppose that (S,S)- and (R,S)-
diastereomers are formed from the same chirally labile
intermediate.
1
mixture of diastereomers, according to the H NMR data
(Scheme 4). The results obtained indicate that the imidazole
nitrogen atoms of N-(purin-6-yl)-α-amino acids either are
not involved in the racemization process during the coupling
reaction in the presence of DCC at all, or do not play a key
role in this process. In general, the question of the mecha-
nism of N-(purin-6-yl)-α-amino acid racemization requires
further study.
It is known that histidine (imidazole-substituted amino
acid), which can be considered as a structural analog of
N-(purin-6-yl)-α-amino acids, is racemized while DCC
treatment (Scheme 3) (Jones et al. 1980). Histidine is
supposed to form the corresponding O-acylisourea while
interaction with DCC. Racemization of the chiral center
occurs with the participation of the imidazole Nπ atom that
acts as a proton acceptor (mechanism A) or as a nucleo-
phile interacting with a carbonyl fragment (mechanism
B). It is noted that the presence of Nτ-protecting group is
not a hindrance for this process; at the same time, in the
case of Nπ-protected histidine racemization does not occur.
We suppose that the racemization of the chiral center
of N-(purin-6-yl)-α-amino acids occurs with the partici-
pation of the imidazole fragment, as in the case of histi-
dine. Therefore, we obtained N-(9-benzylpurin-6-yl)-(S)-
alanine ((S)-11a) and N-(7-deazapurin-6-yl)-(S)-alanine
((S)-11b) by the interaction of 9-benzyl-6-chloropurine
(10a) (Kelley et al. 1988) or the commercially available
To identify diastereomers in mixtures 12a and 12b, dias-
tereomerically pure conjugates (S,S)-13a,b were synthesized
by the nucleophilic substitution of chlorine in 9-benzyl-
6-chloropurine (10a) or in 7-deaza-6-chloropurine (10b)
with dipeptide (S,S)-7a (Scheme 5). It should be noted
that the conversion of compound 10b was only about 50%;
so, the yield of the product (S,S)-13b was 42% relative to
reacted 10b. To fnd more efcient synthetic approach to
(S,S)-13b, we varied the reaction conditions. Unfortunately,
we did not observe the product formation neither in aque-
ous HCl (Liu and Robins 2007), nor while using DMF as a
solvent (Terrier 2013), nor without any solvent (Novosjolova
et al. 2015).
Diastereomerically pure compounds (S,S)-13a,b were
converted to dimethyl esters (S,S)-12a,b (Scheme 5). This
made it possible to assign corresponding peaks in HPLC
chromatograms of diastereomeric mixtures 12a,b. When
comparing the HPLC data (Fig. 2) and 1H, 13C NMR spec-
tra of individual diastereomers (S,S)-12a,b (>99% de) with
Scheme 3 Proposed mechanisms of the histidine racemization (Jones et al. 1980)
1 3

Stereochemical aspects in the synthesis of novel N-(purin-6-yl)dipeptides as potential…
411
Scheme 4 Synthesis of compounds (S)-11a,b, 12a,b and 13b
Scheme 5 Synthesis of diastereomerically pure compounds (S,S)-13a,b and (S,S)-12a,b
the data of mixtures 12a,b, we found that the coupling of
N-(9-benzylpurin-6-yl)-(S)-alanine and N-(7-deazapurin-
6-yl)-(S)-alanine (compounds (S)-11a and (S)-11b) to
dimethyl (S)-glutamate (Scheme 5) also led to predominant
formation of (S,S)-diastereomers as it was observed for the
coupling reaction of N-(purin-6-yl)-(S)-amino acids ((S)-
2a–c) (Scheme 1).
derivative 4a was found to exhibit the highest inhibitory
activity (MIC 3.1 μg/mL), while phenylalanine (4b) and
valine (4c) derivatives were not signifcantly active (MIC
6.2–12.5 μg/mL). Moreover, individual diastereomer
(S,S)-4a exhibits the same antimycobacterial activity as
the mixture 4a of (S,S)- and (R,S)-diastereomers in 6:4
ratio (MIC 3.1 μg/mL). It has been also demonstrated that
the modifcation of structure 4a by introducing an amino
group at position C2 of the purine core ((S,S)-8a) or by
replacement of the imidazole moiety with pyrrole frag-
ment (13b) leads to the decreased inhibitory activity (MIC
6.2–12.5 μg/mL). In general, these results show that the
replacement of the glycine fragment in structures (S)-1a,b
with other α-amino acid residues leads to a decrease in
antimycobacterial activity.
The in vitro antimycobacterial activity of conjugates
4a–c, (S,S)-4a, (S,S)-8a, and 13b against Mycobacte-
rium tuberculosis H37Rv strain was studied in the Ural
Research Institute of Phthisiopulmonology (National
Medical Research Center, Ministry of Healthcare of the
Russian Federation, Ekaterinburg, Russia). The minimum
inhibitory concentrations (MIC) of these compounds
were determined by the previously described procedure
(Krasnov et al. 2016). Among tested compounds, alanine
1 3

412
V. V. Musiyak et al.
Fig. 2 HPLC chromatograms of compounds: a 3a,b (S,S)-3a (S,S-Whelk-O1, 0.025 N aqueous AcONa–MeOH, 55:45, 0.75 mL/min, detection
at 280 nm); c 12b,d (S,S)-12b (S,S-Whelk-O1, H2O–MeOH, 55:45, 0.8 mL/min, detection at 280 nm)
Conclusion
Materials and methods
In summary, we developed the synthetic approaches to
novel purine and 2-aminopurine conjugates with dipep-
tides bearing the (S)-glutamic acid terminal fragment,
and methods for monitoring their enantiomeric and dias-
tereomeric composition. It has been shown that approach
based on the nucleophilic substitution of chlorine in
6-chloropurine and 2-amino-6-chloropurine with dipep-
tides is racemization-free and leads to the diastereomeri-
cally pure target products. It has been found that another
approach based on the coupling of N-(purin-6-yl)-(S)-
amino acids to dimethyl (S)-glutamate is accompanied
by racemization of the carboxy component chiral center
and leads to the mixtures of (S,S)- и (R,S)-diastereomers
in a 6:4 ratio.
General
N-(Purin-6-yl)-(S)-alanine (S)-2a (Ward et al. 1961), dime-
thyl N-Boc-(S)-phenylalaninyl-(S)-glutamate (S,S)-6b (Deng
et al. 2008) and 9-benzyl-6-chloropurine 10a (Kelley et al.
1988) were obtained as previously described. N-(Purin-
6-yl)-α-amino acids (S)-2b,c, (R)-2b (Ward et al. 1961),
dimethyl N-Boc-(S)-alaninyl-(S)-glutamate (S,S)-6a, and
dimethyl N-Boc-(S)-valyl-(S)-glutamate (S,S)-6c (Deng
et al. 2008) were synthesized by analogy with the known
methods. Other reagents are commercially available and
were purchased from Alfa Aesar (UK) and Sigma-Aldrich
(Germany).
The solvents were purified according to traditional
methods and used freshly distilled. Melting points were
obtained on a SMP3 apparatus (Barloworld Scientifc, UK).
Optical rotations were measured on a Perkin Elmer 341
To verify the hypothesis that the racemization of the
chiral center of N-(purin-6-yl)-α-amino acids proceeds
with the participation of the imidazole nitrogen atoms via
the stage of formation of a chirally labile intermediate, we
synthesized N-(purin-6-yl)-(S)-alanine analogs differing
in the structure of the imidazole fragment: N-(9-benzyl-
purin-6-yl)-(S)-alanine and N-(7-deazapurin-6-yl)-(S)-
alanine. Since the coupling of these compounds to dime-
thyl (S)-glutamate was also accompanied by racemization;
it can be assumed that the imidazole fragment does not
play a crucial role in the course of this process.
1
polarimeter (Perkin Elmer, USA). The H NMR spectra
were recorded on Bruker DRX-400 (400 MHz) or Bruker
Avance 500 (500 MHz) (Bruker, Germany); the 13C NMR
spectra were recorded on Bruker Avance 500 (125 MHz);
TMS was used as internal reference. CHN elemental micro-
analyses were performed using Perkin Elmer 2400 II ana-
lyser (Perkin Elmer, USA). Analytical TLC was performed
using Sorbfl plates (Imid, Russia). Flash-column chroma-
tography procedures were performed using Silica gel 40
(230–400 mesh) (Alfa Aesar, UK). For ion exchange resin
Some of the obtained compounds were found to exhibit
moderate activity against M. tuberculosis H37Rv.
1 3

Stereochemical aspects in the synthesis of novel N-(purin-6-yl)dipeptides as potential…
413
purifcation, we used Amberlite IR-120(H) resin (Alfa Aesar,
UK). Analytical chiral HPLC of compounds 3a, (S,S)-3a,
12b, (S,S)-12b was performed on a Agilent 1100 instru-
ment (Agilent Technologies, USA) using a S,S-Whelk-O1
column (250 × 4.6 mm, 5 μm) (Phenomenex, USA); ana-
lytical chiral HPLC of compounds 3b, (S,S)-3b, 3c, (S,S)-
3c, (R)-5, (S)-5, 12a, (S,S)-12a was performed on Knauer
Smartline-1100 instrument (Knauer, Germany) using the
following columns: Chiralcel OD-H (250×4.6 mm, 5 μm)
(Daicel, Japan) for compounds 3b, (S,S)-3b, 12, (S,S)-12;
Chiralpak AD (250 × 4.6 mm, 5 μm) (Daicel, Japan) for
compounds 3c, (S,S)-3c, or S,S-Whelk-O1 (250×4.6 mm,
5 μm) (Phenomenex, USA) for compounds (R)-5, (S)-5. Pre-
parative HPLC of compounds (S,S)-4b, (S,S)-4c, (S,S)-8b,
(S,S)-9b was performed on a Shimadzu Prominence LC-20
instrument (Shimadzu, Japan) using a Phenomenex Luna
C18(2) column (250×21.2 mm, 5 μm) (Phenomenex, USA).
The high-resolution mass spectra were obtained on a Bruker
maXis Impact HD mass spectrometer (Bruker, Germany),
electrospray ionization with direct sample inlet (4 dm3/min
fow rate).
(3 × 0.5 mL). In the case of 3c, the resulting solution was
treated with ion-exchange resin Amberlite IR-120(H); the
resin was washed with water; then the target compound was
eluted with a water–pyridine 9:1 mixture; the eluate was
evaporated to dryness under reduced pressure.
General procedure for the synthesis of methyl
esters (R)‑5, (S)‑5 and dimethyl esters (S,S)‑3a–c,
(S,S)‑12a,b
SOCl2 (38 μL, 0.330 mmol (for (R)-2b, (S)-2b), 76
μL, 0.660 mmol (for (S,S)-4b and (S,S)-13a), 101 μL,
0.880 mmol (for (S,S)-4a,c) or 114 μL, 0.990 mmol (for
(S,S)-13b)) was added to a suspension of compound (R)-2b,
(S)-2b, (S,S)-4a–c or (S,S)-13a,b (0.220 mmol) in absolute
MeOH (3 mL) under stirring at 0 °C. The reaction mixture
was stirred at 0 °C for 30 min and then at room tempera-
ture for 24 h. The solvent was evaporated to dryness under
reduced pressure; the residue was treated with cold Et2O,
and the resulting precipitate was fltered of.
General procedure for the synthesis of dipeptides
(S,S)‑7a–c
General procedure for the coupling reactions
(synthesis of compounds 3a–c, 12a,b)
0.5 M aqueous LiOH (40 mL, 25.1 mmol) was added to
a solution of compound (S,S)-6a–c (8.37 mmol) in EtOH
(10 mL) at to 0 °C. The reaction mixture was stirred at 0 °C
for 30 min, then at room temperature for 4 h. The reaction
mixture was washed with EtOAc (2×10 mL), acidifed with
4 M HCl to pH 3 and extracted with EtOAc (3 × 15 mL).
The combined organic layers were dried over MgSO4 and
evaporated to dryness under reduced pressure. The residue
was dissolved in trifuoroacetic acid (20 mL); then the reac-
tion mixture was stirred at room temperature for 1 h and
evaporated under reduced pressure. The residue was dis-
solved in water (20 mL); the solution was alkalized with
5% aqueous NaHCO3 to pH 5. The resulting solution was
treated with ion-exchange resin Amberlite IR-120(H); the
resin was washed with water; then the target compound was
eluted with a water–pyridine 9:1 mixture. The eluate was
evaporated to dryness under reduced pressure.
DIEA (345 μL, 1.98 mmol), HOBt (0.27 g, 1.98 mmol),
and DCC (0.41 g, 1.98 mmol) were successively added to
a suspension of compound (S)-2a–c, (R)-2b, or (S)-11a,b
(1.98 mmol) in DMSO or DMF (10 mL) under stirring at
room temperature. After 15 min, dimethyl (S)-glutamate
hydrochloride (0.42 g, 1.98 mmol) was added to the reac-
tion mixture. The reaction mixture was stirred at room
temperature for 48 h. The resulting precipitate was fltered
of and washed with DMSO or DMF (3 ×0.5 mL), the fl-
trate and washings were poured in cold water (150 mL) and
extracted with n-BuOH (4×25 mL). The combined organic
layers were washed with 5% aqueous NaHCO3 (3×20 mL),
saturated aqueous NaCl (3×20 mL), then evaporated to dry-
ness under reduced pressure. In the case of (S)-11a, EtOAc
(4×25 mL) was used for extraction; combined organic lay-
ers were dried over MgSO4 before evaporation. In all cases
the residue was purifed by fash column chromatography on
silica gel (CHCl3–EtOH as an eluent).
General procedure for the nucleophilic substitution
reactions (synthesis of compounds (S,S)‑4a–c,
(S,S)‑8a,b, (S)‑11a,b, and (S,S)‑13a,b)
General procedure for alkaline hydrolysis (synthesis
of compounds 4a–c, 13b)
Compound 3a–c or 12b (0.500 mmol) was dissolved in
0.5 M aqueous LiOH (3 mL, 1.50 mmol). The reaction
mixture was stirred at room temperature for 24 h, and then
cooled to 0 °C (ice bath); the pH was adjusted to 3 by con-
centrated HCl. In the cases of 3a,b and 12b, the resulting
precipitate was filtered off and washed with cold water
Na2CO3 (0.16 g, 1.52 mmol) and 6-chloropurine, 2-amino-
6-chloropurine or compounds 10a,b (1.02 mmol) were
successively added to a solution of compound (S,S)-7a–c
or (S)-alanine (2.03 mmol) in water (5 mL). The reaction
mixture was refuxed for 4–6 h; the resulting solution was
cooled to 0 °C and acidifed with concentrated HCl to pH
1 3

414
V. V. Musiyak et al.
3. Further operations are described for each compound in
Electronic supplementary material.
References
Al-Harbi RAK, Abdel-Rahman AAH (2012) Synthesis and antiviral
evaluation of α-amino acid esters bearing N6-benzyladenine
side chain. Acta Pol Pharm 69:917–925
Analytical data for all compounds can be found in Elec-
tronic supplementary material.
Beauchamp LM, Orr GF, de Miranda P, Burnette T, Krenitsky TA
(1992) Amino acid ester prodrugs of acyclovir. Antiviral Chem
Chemother 3:157–164. https://doi.org/10.1177/0956320292
00300305
Assessment of antimycobacterial activity
Boerema DJ, Tereshko VA, Zhangc J, Kent SBH (2016) Chemi-
cal synthesis and enzymatic properties of RNase A analogues
designed to enhance second-step catalytic activity. Org Biomol
Chem 14:8804–8814. https://doi.org/10.1039/C6OB01163B
Chang W, Bao D, Chun BK, Naduthambi D, Nagarathnam D, Racha-
konda S, Reddy PG, Ross BS, Zhang HR, Bansal S, Espiritu
CL, Keilman M, Lam AM, Niu C, Steuer HM, Furman PA, Otto
MJ, Sofa MJ (2011) Discovery of PSI-353661, a novel purine
nucleotide prodrug for the treatment of HCV infection. ACS
Med Chem Lett 2:130–135. https://doi.org/10.1021/ml100209f
de Wet TJ, Warner DF, Mizrahi V (2019) Harnessing biological
insight to accelerate tuberculosis drug discovery. Acc Chem Res
52:2340–2348. https://doi.org/10.1021/acs.accounts.9b00275
Deng G, Liu Z, Ye F, Luo X, Zhu W, Shen X, Liu H, Jiang H (2008)
Tryptophan-containing dipeptide derivatives as potent PPARγ
antagonists: design, synthesis, biological evaluation, and
molecular modeling. Eur J Med Chem 43:2699–2716. https://
doi.org/10.1016/j.ejmech.2008.01.032
The inhibitory efect of compounds 4a–c, (S,S)-4a, (S,S)-
8a and 13b was studied against M. tuberculosis H37Rv
strain. The minimal inhibitory concentration (MIC) for
each compound was determined by the micro broth dilution
method (Krasnov et al. 2016). All compounds tested were
dissolved in DMSO and their 1/2 dilutions were added in
tubes with 5 ml of the Löwenstein–Jensen medium. Each
compound was tested at six concentrations 12.5, 6.25, 3.1,
1.5, 0.7, and 0.35 μg/mL. Tubes with Löwenstein–Jensen
medium (5 mL) containing tested compounds and those
without them (controls) were inoculated with a suspension
of M. tuberculosis H37Rv strain and incubated at 37 °C
for 10 days. The mycobacterial growth was assessed by the
standart procedure: the appearance of zones of mycobacte-
rial growth delay (more than 10 mm) indicated the pres-
ence of antimycobacterial properties of tested compounds
at the studied concentrations. The value of growth retarda-
tion zone (in mm) was proportional to the antimycobacte-
rial activity of the compounds. Growth delay (100 mm or
more) was considered as a complete mycobacteria growth
inhibition. Isoniazid (Alfa Aesar, UK) was used as a refer-
ence drug (MIC 0.1 μg/mL).
Gray SG, Khoujah AM (1969) Selective cleavage of the t-butoxycar-
bonyl protecting group. Tetrahedron Lett 10:2647–2650. https
://doi.org/10.1016/S0040-4039(01)88233-2
Gruzdev DA, Chulakov EN, Levit GL, Kravchenko MA, Krasnov
VP, Charushin VN (2017) Synthesis and antimycobacterial
activity of novel purin-6-yl and 2-aminopurin-6-yl conjugates
with (S)-aspartic and (S)-glutamic acids. Mendeleev Commun
27:547–549. https://doi.org/10.1016/j.mencom.2017.11.002
Gruzdev DA, Musiyak VV, Levit GL, Krasnov VP, Charushin VN
(2018) Purine derivatives with antituberculosis activity. Russ
Chem Rev 87:604–618. https://doi.org/10.1070/RCR4772
Islam MR, Mahdi JG, Bowen ID (1997) Pharmacological impor-
tance of stereochemical resolution of enantiomeric drugs. Drug
Saf 17:149–165. https://doi.org/10.2165/00002018-19971
7030-00002
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00726-021-02958-0.
Acknowledgements We are grateful to Dr. Marionella A. Kravchenko
[Ural Research Institute of Phthisiopulmonology (National Medical
Research Center, Ministry of Healthcare of the Russian Federation,
Ekaterinburg, Russia)] for conducting biological trials. Analytical
studies were carried out using equipment of the Centre for Joint Use
“Spectroscopy and Analysis of Organic Compounds” at the Postovsky
Institute of Organic Synthesis, Ural Branch of the Russian Academy
of Sciences.
Jones JH, Ramage WI, Witty MJ (1980) Mechanism of racemisation of
histidine derivatives in peptide synthesis. Int J Peptide Protein Res
15:301–303. https://doi.org/10.1111/j.1399-3011.1980.tb02581.x
Kelley JL, Krochmal MP, Linn JA, McLean EW, Soroko FE (1988)
6-(Alkylamino)-9-benzyl-9H-purines. A new class of anti-
convulsant agents. J Med Chem 31:606–612. https://doi.
org/10.1021/jm00398a019
Kleinsmann AJ, Nachtsheim BJ (2013) Phenylalanine-containing
cyclic dipeptides—the lowest molecular weight hydrogelators
based on unmodifed proteinogenic amino acids. Chem Com-
mun 49:7818–7820. https://doi.org/10.1039/C3CC44110E
Krasnov VP, Vigorov AYu, Musiyak VV, Nizova IA, Gruzdev DA,
Matveeva TV, Levit GL, Kravchenko MA, Skornyakov SN,
Bekker OB, Danilenko VN, Charushin VN (2016) Synthesis
and antimycobacterial activity of N-(2-aminopurin-6-yl) and
N-(purin-6-yl) amino acids and dipeptides. Bioorg Med Chem
Lett 26:2645–2648. https://doi.org/10.1016/j.bmcl.2016.04.017
Krasnov VP, Musiyak VV, Vozdvizhenskaya OA, Galegov GA,
Andronova VL, Gruzdev DA, Chulakov EN, Vigorov AYu,
Ezhikova MA, Kodess MI, Levit GL, Charushin VN (2019)
N-[ω-(Purin-6-yl)aminoalkanoyl] derivatives of chiral heterocy-
clic amines as promising anti-herpesvirus agents. Eur J Org Chem
2019:4811–4821. https://doi.org/10.1002/ejoc.201900727
Funding The work was fnancially supported by the Russian Science
Foundation (Grant 19-13-00231).
Data availability The authors declare that the data supporting the fnd-
ings of this study are available within the article and its supplementary
information fles.
Compliance with ethical standards
Conflict of interest The authors declare no conficts of interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
1 3

Stereochemical aspects in the synthesis of novel N-(purin-6-yl)dipeptides as potential…
415
Krasnov VP, Levit GL, Musiyak VV, Gruzdev DA, Charushin VN
(2020) Fragment-based approach to novel bioactive purine deriva-
tives. Pure Appl Chem. https://doi.org/10.1515/pac-2019-1214
Legraverend M (2008) Recent advances in the synthesis of purine
derivatives and their precursors. Tetrahedron 64:8585–8603. https
://doi.org/10.1016/j.tet.2008.05.115
Niu HY, Bai SX, Wu S, Qu GR, Guo HM (2012) Synthesis of chiral
N-(Purin-6-yl)amino acid derivatives by using natural amino acids
as starting materials. Asian J Org Chem 1:238–244. https://doi.
org/10.1002/ajoc.201200081
Novosjolova I, Bizdēna E, Turks M (2015) Synthesis and applications
of azolylpurine and azolylpurine nucleoside derivatives. Eur J Org
Chem 2015:3629–3649. https://doi.org/10.1002/ejoc.201403527
Pautus S, Sehr P, Lewis J, Fortune A, Wolkerstorfer A, Szolar O, Guil-
ligay D, Lunardi T, Decout JL, Cusack S (2013) New 7-methyl-
guanosine derivatives targeting the infuenza polymerase PB2
cap-binding domain. J Med Chem 56:8915–8930. https://doi.
org/10.1021/jm401369y
Legraverend M, Grierson DS (2006) The purines: Potent and versatile
small molecule inhibitors and modulators of key biological tar-
gets. Bioorg Med Chem 14:3987–4006. https://doi.org/10.1016/j.
bmc.2005.12.060
Lin GQ, You QD, Cheng JF (2011) Chiral drugs. Wiley, Hoboken
Liu J, Robins MJ (2007) SNAr Displacements with 6-(fuoro, chloro,
bromo, iodo, and alkylsulfonyl) purine nucleosides: synthesis,
kinetics, and mechanism. J Am Chem Soc 129:5962–5968. https
://doi.org/10.1021/ja070021u
Stavrakov G, Valcheva V, Voynikov Y, Philipova I, Atanasova M, Kon-
stantinov S, Peikov P, Doytchinova I (2016) Design, synthesis,
and antimycobacterial activity of novel theophylline-7-acetic
acid derivatives with amino acid moieties. Chem Biol Drug Des
87:335–341. https://doi.org/10.1111/cbdd.12676
McGregor WH, Carpenter FH (1961) Synthesis of l-leucyl-l-alanyl-
l-valyl-l-glutamic acid and intermediate peptides. J Org Chem
26:1849–1854. https://doi.org/10.1021/jo01065a039
Terrier F (2013) The S
NAr reactions: mechanistic aspects. In: Terrier
McGuigan C, Harris SA, Daluge SM, Gudmundsson KS, McLean EW,
Burnette TC, Marr H, Hazen R, Condreay LD, Johnson L, De
Clercq E, Balzarini J (2005) Application of phosphoramidate pro-
nucleotide technology to Abacavir leads to a signifcant enhance-
ment of antiviral potency. J Med Chem 48:3504–3515. https://doi.
org/10.1021/jm0491400
F (ed) Modern nucleophilic aromatic substitution. Wiley-VCH
Verlag GmbH & Co, Weinheim, pp 1–84
Tripathi RP, Tewari N, Dwivedi N, Tiwari VK (2005) Fighting tuber-
culosis: an old disease with new challenges. Med Res Rev 25:93–
131. https://doi.org/10.1002/med.20017
Vigorov AYu, Krasnov VP, Gruzdev DA, Men’shikova AA, Demin
AM, Levit GL, Charushin VN (2014) Novel synthetic routes
to N-(2-amino-9H-purin-6-yl)-substituted amino acids. Men-
deleev Commun 24:35–36. https://doi.org/10.1016/j.menco
m.2013.12.011
McGuigan C, Hassan-Abdallah A, Srinivasan S, Wang Y, Siddiqui
A, Daluge SM, Gudmundsson KS, Zhou H, McLean EW, Peck-
ham JP, Burnette TC, Marr H, Hazen R, Condreay LD, Johnson
L, Balzarini J (2006) Application of phosphoramidate ProTide
technology signifcantly improves antiviral potency of carbocyclic
adenosine derivatives. J Med Chem 49:7215–7226. https://doi.
org/10.1021/jm060776w
Voynikov Y, Valcheva V, Momekov G, Peikov P, Stavrakov G (2014)
Theophylline-7-acetic acid derivatives with amino acids as anti-
tuberculosis agents. Bioorg Med Chem Lett 24:3043–3045. https
://doi.org/10.1016/j.bmcl.2014.05.026
McGuigan C, Madela K, Aljarah M, Gilles A, Brancale A, Zonta N,
Chamberlain S, Vernachio J, Hutchins J, Hall A, Ames B, Goro-
vits E, Ganguly B, Kolykhalov A, Wang J, Muhammad J, Patti
JM, Henson G (2010) Design, synthesis and evaluation of a novel
double pro-drug: INX-08189. A new clinical candidate for hepa-
titis C virus. Bioorg Med Chem Lett 20:4850–4854. https://doi.
org/10.1016/j.bmcl.2010.06.094
Wang C, Song Z, Yu H, Liu K, Ma X (2015) Adenine: an important
drug scafold for the design of antiviral agents. Acta Pharm Sin B
5:431–441. https://doi.org/10.1016/j.apsb.2015.07.002
Ward DN, Wade J, Walborg EF Jr, Osdexe TS (1961) The Synthesis
of N-(6-Purinyl)amino acids. Amino acids with a single reactive
amino group. J Org Chem 26:5000–5005. https://doi.org/10.1021/
jo01070a054
Mehellou Y (2016) The ProTides Boom. ChemMedChem 11:1114–
1116. https://doi.org/10.1002/cmdc.201600156
WHO (2019) Global Tuberculosis Report 2019. https://www.who.int/
tb/global-report-2019. Accessed 30 June 2020
Melchert M, List A (2007) The thalidomide saga. Int J Biochem Cell
Biol 39:1489–1499. https://doi.org/10.1016/j.biocel.2007.01.022
Motta I, Centis R, D’Ambrosio L, García-García JM, Goletti D,
Gualano G, Lipani F, Palmieri F, Sánchez-Montalvá A, Pontali
E, Sotgiu G, Spanevello A, Stochino C, Tabernero E, Tadolini M,
van den Boom M, Villa S, Visca D, Migliori GB (2020) Tuber-
culosis, COVID-19 and migrants: Preliminary analysis of deaths
occurring in 69 patients from two cohorts. Pulmonology 26:233–
240. https://doi.org/10.1016/j.pulmoe.2020.05.002
WHO (2020) COVID-19: Considerations for tuberculosis (TB) care.
https://www.who.int/docs/default-source/documents/tuberculos
is/infonote-tb-covid-19.pdf?sfvrsn=b5985459_18. Accessed 20
July 2020
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Musiyak VV, Nizova IA, Matveeva TV, Levit GL, Krasnov VP,
Charushin VN (2019) Synthesis of new purine derivatives con-
taining α- and ω-amino acid fragments. Russ J Org Chem 55:762–
770. https://doi.org/10.1134/S1070428019060046
1 3