Antibacterial AZT derivative regulates metastasis of breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2020.112233

Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential.

Full text of DOI:10.1016/j.ejmech.2020.112233

European Journal of Medicinal Chemistry 193 (2020) 112233
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Antibacterial AZT derivative regulates metastasis of breast cancer cells
,
,
Sridhar Chirumarry a 1, Nak-Kyun Soung b 1, Junyeol Han b, Eun Young Kim c,
Eun Kyoung Ryu a d, Young-Ho Lee a d, Song Yub Shin c, Pethaiah Gunasekaran a
,
,
,
, **
Jeong Kyu Bang a
, d, *
a Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), Ochang, Chung Buk, 28119, Republic of Korea
b Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongju, 28116, Republic of Korea
c Department of Biomedical Science, Graduate School, Cellular and Molecular Medicine, Chosun University, Gwangju, 61452, Republic of Korea
d Department of Bio-analytical Science, University of Science & Technology, Daejeon, 34113, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern
treatments. However, long peptide length and protease instability are the most addressing factors, which
hampers their further development as therapeutic agents. In view of this, herein, we designed and
synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups
and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of
AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-
negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e
as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant
bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic
ability by investigating the cancer cell migration and invasiveness through scratch wound-healing
assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also per-
formed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer
cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation,
indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an
appealing class of antibacterial agents with anti-metastasis potential.
Received 4 February 2020
Received in revised form
10 March 2020
Accepted 10 March 2020
Available online xxx
Keywords:
Antibacterials
Amphipathic small molecule
Anti-Metastasis
Cell motility
Lamellipodia
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
numerous cancer drugs due to the poor life span antibiotics as they
develop the resistance over a period, which eventually diminishes
The most concerning and challenging threats faced by global
health care is the emergence of multidrug resistance [1,2]. A
continuous misuse of antibiotics and a sharp decline in the inven-
tion of new therapeutics are the key causes of exacerbating the
drug resistance. As a result of antibiotic resistance, 700,000 people
die worldwide every year, and the number may increase to 10
million by 2050 at the absence of preventive measures taken
against drug resistance. The number of new antibiotics in the
clinical development is also sharply decreased compared to that of
the profits and interest of developing new antibiotics [3]. To combat
the drug resistance, cationic peptide antibiotics (CPAs), known as
host defense peptides, play crucial roles as a new class of novel
antibiotics [1,4]. CPAs are amphipathic peptides that constitute
significant portions of cationic and hydrophobic residues. In gen-
eral, structural components of mammalian and bacterial mem-
brane determines the bacterial selectivity [5]. Zwitterionic lipids,
including cholesterol, phosphatidylcholine, and sphingomyelin are
the prime components of the outer part of the mammalian cell
membrane, while the inner part sequenced with the phospholipids
with negative charges. In contrast, the bacterial outer membranes
constitute negatively charged phospholipids and lipopolysaccha-
rides [6,7]. The cationic and hydrophobic groups of CPAs interact
with anionic phosphate and lipid portions of the bacterial mem-
brane, respectively, which promotes the creation of stable pores
and consequently removes the bacterial membrane. Thus, CPAs
* Corresponding author. Division of Magnetic Resonance, Korea Basic Science
Institute (KBSI), Ochang, Chung Buk, 28119, Republic of Korea.
** Corresponding author.
E-mail addresses: gunaharaks@kbsi.re.kr (P. Gunasekaran), bangjk@kbsi.re.kr
(J.K. Bang).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2020.112233
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

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S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
bacterial killing is very rapid and declines the propensity for the
development of bacterial resistance. Despite there are remarkable
features of CPAs, their advancements into pharmaceutical drugs are
impeded by few factors, including long chain length that relates to
their expensive manufacturing cost and poor SAR study, suscepti-
bility to protease degradation and hemolytic activity. To overcome
these disadvantages, small molecules antibacterials were found as
an alternative to peptide based antibacterials, and also they are
scalable to a large quantity [4]. Thus, designing small molecules
with structural features of CPAs are considered as a key strategy for
the discovery of novel antibiotics [8,9]. For instance, recently, small
molecules derived from pyridopyrimidine [10], hydrazone de-
rivatives [11], pyrazoles [12] core residues were reported as anti-
bacterial agents. In view of this, we synthesized amphiphilic 30-
azido-30-deoxythymidine (AZT) based antibacterials with cationic
residues, including amine and guanidine resembling the CPAs with
lysine and arginine respectively, and various hydrophobic residues.
In general, AZTs are nucleosides, where nucleobase linked to sugar
that play pivotal roles in controlling the heredity in all living or-
ganisms. Over the years great attention has been drawn to AZT
based therapeutics due to their remarkable medicinal properties,
including an antiviral drug in HIV treatment [13], antitumor agents
[14], antioxidant [15], antimalarial agents [16] and AZT prodrugs for
anticancer and antiviral effects [17,18]. Similarly, several natural
nucleosides and synthetic analogs were also reported to have
moderate to good antibacterial activity against different bacterial
strains [19,20].
Usually, membrane phenotypes of both bacteria and cancerous
cells are enriched with negatively charged phosphatidylserine that
is susceptible to electrostatic interactions with cationic groups of
CPAs [21]. Thus numerous naturally occurring and synthetic CPAs
are reported as anticancer agents [22]. Metastasis is a cancer cell
spreading process from tumor to surrounding tissues and organs,
and remains a key cause of mortality in more than 90% of cancer
patients. Cell motility plays a key role in the metastatic cascade that
involves detachment of tumor cells, mobilizing into the blood-
stream, seeding at different sites, invade into the normal cells and
proliferates as tumors [21,22]. As a result, significant attention has
been paid to the molecular mechanics of cell motility and their
impact on cancer metastasis. Cell motility directly depends on the
cancer cells plasma membrane tension that determines various
biological processes [23]. Thus, variation in the cell membrane
tension affects the cell motility and morphology of the cell mem-
brane. One of the key strategies for the development of cell motility
inhibitors is the insertion of amphipathic compounds into the
membrane, which reduces the membrane tension, eventually,
obstruct the movement of the cancer cell [24]. For instance,
recently, Futaki et al. reported an amphipathic peptide derived
from influenza that showed a remarkable ability in cell membrane
insertion, which decreased the cell movement [25].
of AZT-based small molecular cationic antibacterial agents (Fig. 1)
and screened them against both Gram-negative and Gram-positive
strains. Also, potent compounds were evaluated for the cell selec-
tivity against sheep red blood cells. Finally, in view of identifying
the antibacterial agent with anticancer potential, the most potent
compound ADG-2e was examined for metastasis inhibitory po-
tential in breast cancer cells (MDA-MB-231) using wound healing
assay, transwell invasive assay, cell tracking analysis and lamelli-
podium formation.
2. Results and discussions
2.1. Design and synthesis
In the design of an antibacterial agent, we used 30-azidothymi-
dine (AZT) as a core skeleton due to the above mentioned merits
and functionalized 30 and 50 positions of the sugar through an
amide bond to anchor hydrophobic residues, including alkyl and
alkylaryl functionalities. For achieving the cationicity, norspermi-
dine and its functionalized guanidine were incorporated at the C4
position of a pyrimidine to mimic the properties of CPAs possessing
lysine and arginine, respectively [4]. Thus, we synthesized three
kinds of amphipathic derivatives using mono-, di-guanidines and
di-amine analogs as shown in Fig. 1.
To synthesis the mono-guanidine derivatives, AMG-1(a-e) as
delineated in Scheme 1, initially, the hydroxyl group of 4 was
converted to azide using tosylation and followed by azidation re-
action. Thus, 4 was treated with p-tosyl chloride in presence of
triethylamine and catalytic amount of 4-(dimethylamino)pyridine
(DMAP) to yield the tosylated product, which was treated with
sodium azide in N,N-dimethylformamide (DMF) to result in the
formation of 5 in 88% yield. In order to functionalize the C4 carbonyl
group of pyrimidine ring, initially, triazole was introduced using
phosphorus oxychloride, 1,2,4-triazole and triethylamine. Then the
nucleophilic substitution reaction of aminoguanidine, 7 resulted in
the formation of di-Boc guanidine adduct 8 in 96% yield. To incor-
porate various alkyl/aryl substituents to enhance the hydropho-
bicity, the azide groups were reduced to amines using Pd/C and
hydrogen. Subsequent coupling with various alkyl/aryl carboxylic
acids (9) using EDC, HOBt, in presence of DIEA derived the desired
products 10(a-e). Finally, Boc-deprotection of 10(a-e) using TFA in
DCM (1:1) resulted in the formation of desired AZT-based mono-
guanidine, AMG-1(a-e).
To synthesize of ADL and ADG as shown in Scheme 2, firstly, di-
Boc-norspermidine, 11 was treated with 6 in the presence of K2CO3
in acetonitrile resulted in the formation of 12 in 87% yield. To
incorporate various alkyl derivatives, the azide groups were
reduced to amine using Pd/C and H2, which upon subsequent
coupling with various alkyl and aryl carboxylic acids in presence of
EDC, HOBt and DIEA afforded the diamide compound 13. Removal
of Boc groups in 13 using 1.25M HCl solution in methanol resulted
in the desired AZT-based lysine mimetics ADL-3(a-e). Further, bis-
The present study stems as part of our research program on the
discovery of antibacterial agents [26e29]. We synthesized a series
Fig. 1. Structures of AMG, ADG, and ADL

S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
3
Scheme 1. Synthesis of AMG-1(a-e). Reagents and conditions; a) (i) TsCl, TEA, DMAP, CH2Cl2, 0 ꢀC - rt, 15 h; (ii) NaN3, DMF, 60 ꢀC, 12 h, 88% (for two steps). b) 1,2,4-Triazole, POCl3,
TEA, CH3CN, 0 ꢀC - rt, 20 h, 98%. c) K2CO3, CH3CN, rt, 24 h, 96%. d) (i) Pd/C, H2, MeOH, rt, 6 h; (ii) ReCOOH (9), EDC, HOBt, DIEA, DMF, rt, 10 h; e) TFA:CH2Cl2 (1:1), rt, 4 h.
Scheme 2. Synthesis of ADL and ADG. Reagents and conditions; a) K2CO3, CH3CN, 40 ꢀC, 24 h, 87%. b) (i) Pd/C, H2, MeOH, rt, 6 h; (ii) ReCOOH(9), EDC, HOBt, DIEA, DMF, rt, 10 h. c)
1.25M HCl in MeOH, 50 ꢀC, 1 h. d) (i). DIEA, DMF, rt, 12 h; (ii). 1.25M HCl in MeOH.
guanidinylation on the amine ADL-3(a-e) was performed using 1H-
pyrazole-1-carboxamidine hydrochloride solution and DIEA, and
subsequent treatment of 1.25M HCl solution in methanol afforded
the desired arginine mimetics ADG-2(a-e) in good yields.
by introducing 4-phenylbutyl residue (AMG-1b), which didn’t
show appreciable antibacterial effects all the strains, except for
P. aeruginosa. Further reducing the hydrophobicity by introducing
cyclohexyl (AMG-1c) and n-hexyl (AMG-1d) didn’t result in an
appreciable activity. Lastly, we choose 1-adamantanemethyl as a
lipophilic group to synthesize AMG-1e because adamantane
anchored thiosemicarbazones and thiocarbonohydrazones were
found to be anticancer and antibacterial agents, and ada-
mantylthiozoles was also reported to possess remarkable anti-
inflammatory effects [30,31]. Interestingly, introducing the 1-
adamantanemethyl group (AMG-1e) displayed increased antibac-
terial effects against Gram-positive bacterial strains, which dis-
played two folds better antibacterial effects than the melittin.
Next, we tried to increase the cationic charge because the bal-
ance between cationic charge and hydrophobicity is one of the key
factors in achieving antibacterial effects [32]. To increase the
cationic charge, bis-guanidyl norspermidine residue was appended
fixedly and SAR was performed by varying the alkyl groups at 30 and
50 positions of the sugar moiety (Fig. 1). Initially, 1-naphthylmethyl
2.2. Antibacterial activity
Initially, the AMG set of compounds (Fig. 1) were investigated
for their antibacterial effects against both Gram-negative and
Gram-positive bacterial strains (Table 1). In the design of AMG
compounds, variable alkyl/aryl groups were appended at 30 and 50
positions of the sugar for achieving hydrophobicity and an ami-
noethylguanidine was positioned at the C-4 position of pyrimidine
for deriving hydrophilicity in the AZT core moiety. Initially, we
synthesized AMG-1a with 1-naphthylmethyl group as a hydro-
phobic component, which displayed 2 and 4 folds of reduced ac-
tivity against B. subtilis and S. aureus, respectively, compared to that
of reference, melittin, and lost its activity entirely against Gram-
negative bacterial strains. Further, we reduced the hydrophobicity

4
S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
Table 1
MIC values of the synthesized compounds.
Compounds
MICa
Gram-negative
(mg/mL)
Gram-positive
E. coli [KCTC 1682]
P. aeruginosa [KCTC 1637]
B.subtilis [KCTC 3068]
S. aureus [KCTC 1621]
AMG-1a
AMG-1b
AMG-1c
AMG-1d
AMG-1e
ADG-2a
ADG-2b
ADG-2c
ADG-2d
ADG-2e
ADL-3a
ADL-3b
ADL-3c
ADL-3d
ADL-3e
melittin
>64
>64
>64
>64
64
>64
32
>64
64
>64
16
16
16
16
4
8
4
64
8
4
16
64
>64
>64
4
4
8
32
32
2
32
64
>64
>64
4
64
8
8
>64
>64
>64
16
32
32
2
16
32
>64
>64
4
64
4
>64
>64
>64
32
16
64
64
2
16
16
8
8
a
MICs were determined as the lowest concentration of compounds that causes 100% inhibition of microbial growth.
was appended (ADG-2a) that displayed significant antibacterial
effect, except for E.coli. It displayed similar potencies in the case of
P. aeruginosa and S. aureus compared to that of reference, mellitin.
In the case of B. subtilis, it displayed two folds of superior potency
than melittin. Further, reducing the hydrophobicity by introducing
4-phenylbutyl residue resulted in ADG-2b. Except for E.coli, it dis-
played a similar antibacterial effect as melittin against all the
strains. ADG-2c and ADG-2d were synthesized by appending
cyclohexyl and n-hexyl residues, both of them showed similar
antibacterial effects. However, the antibacterial effects were
slightly reduced compared to that of ADG-2b and ADG-2a. Further,
inspired by the proficient activity of 1-adamantanemethyl residue
(AMG-1e), we synthesized ADG-2e by introducing ada-
mantanemethyl residue, which displayed the most potent anti-
bacterial effects compared to the rest of the substituents. For
instance, compared to melittin, it showed four folds of a better
activity against P. aeruginosa, B. subtilis, and S. aureus, while dis-
playing similar potency against E.coli (Table 1).
Next, we wanted to investigate the effect of amine derivatives in
the antibacterial activity, thus, we fixed the norspermidine as a
Fig. 2. Concentration-response curves of percent hemolysis of the designed antimi-
mimic of lysine derivatives and varied the alkyl residues for the
identification of the most potent compounds (Fig. 1). Initially, we
introduced the 1-naphthylmethyl residue (ADL-3a) that resulted in
a moderate activity by displaying a two-fold better activity than
that of the reference against P. aeruginosa, B. subtilis. However,
compared to mellitin, it displayed two and four folds of weak ac-
tivity against S. aureus and E.coli, respectively. Further reducing the
hydrophobicity by introducing the 4-phenylbutyl residues resulted
in a decreased activity compared to ADL-3a. In contrast, against
P. aeruginosa, an increase in activity was observed. Further, we
derivatized ADL-3c and -3d using cyclohexyl and n-hexyl residues,
respectively, but they almost lost their antibacterial activity against
all the strains compared to that of ADL-3b. Still, ADL-3d displayed
crobial small molecules against sheep red blood cells (s-RBCs).
displayed excellent potency against all tested bacteria. From the
SAR study, it was observed that (i) even though 1-naphthylmethyl
and 4-phenylbutyl residues showed better antibacterial effect, 1-
adamantanemethyl residue was required for enhanced antibacte-
rial effects, (ii) two charge residues are essential for balancing the
alkyl residues, (iii) structurally, guanidine derivatives showed bet-
ter antibacterial effect compared to the amine derivatives, which
may be attributed to the fact that the guanidines are capable of
spreading the charge at physiological conditions.
the considerable activity of MIC 8
mg/mL against P. aeruginosa.
2.3. Hemolytic activity
Finally, the 1-adamantanemethyl residue was introduced to obtain
ADL-3e that displayed a notable antibacterial effect against all the
strains. In the case of P. aeruginosa and B. subtilis, ADL-3e displayed
4 folds of superior activity, and in the case of S. aureus, two folds
better activity compared to that of melittin. And two folds of
diminished activity were observed against E.coli compared to the
reference.
In view of identifying the bacterial selectivity, the compounds
that possess significant antibacterial effects in each series namely,
AMG-1e, ADG-2e, and ADL-3e were evaluated for their hemolytic
activity against sheep red blood cells (sRBCs) (Fig. 2).
Interestingly, our compounds didn’t show any hemolysis until
16
mg/mL. In contrast, the reference mellitin showed toxicity even
As a whole, among the ADL, ADG and AMG series, ADG-2e
at 4
m
g/mL (12%), and reached the maximum at 32 g/mL. However,
m

S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
5
Table 2
In the present work, we hypothesized that the amphipathic
structure of our AZT compounds may interact with cancer cells
surface via electrostatic interaction, consequently, which may
reduce the membrane tension and affect cell migration. To verify
this hypothesis, the most potent antibacterial compound ADG-2e
was investigated for their potential on cancer cell migration
through scratch wound-healing assay using metastatic breast
cancer cell line MDA-MB-231. Initially, ADG-2e was examined for
its inhibitory effect on the proliferation of MDA-MB-231 cells by the
antiproliferative assay. There were no significant changes observed
Therapeutic index (TI) of AMG-1e, ADG-2e, ADL-3e, and melittin.
compound
GM(
m
g/mL)a
MHC(
m
g/mL)b
TI(MHC/GM)c
AMG-1e
ADG-2e
ADL-3e
Melittin
50
6
10.5
12
43
25
52
4
0.9
4.2
5
0.3
a
GM denotes the geometric mean of MIC values from selected four bacterial
strains.
b
MHC is the lowest peptide concentration that produces 10% hemolysis of sheep
red blood cells.
on the proliferation of MDA-MB-231 cells, until the 50
centration of ADG-2e, indicating the low or negligible cytotoxicity
towards this cell line (Fig. 3). Thus, we chose 20 and 40 M doses of
mM con-
c
Therapeutic index (TI) is the ratio of the MHC value (
mL).
mg/mL) over the GM (mg/
m
ADG-2e as a non-lethal concentration for performing the wound
healing assay that represents a method for analyzing cell mobility
(Fig. 4a). In this assay, a confluent monolayer of MDA-MB-231 cells
was grown and scratched with a pipette tip to result in the wound
area marked in redlines. The extent of wound closure represents
wound healing efficacy. ADG-2e was treated and kept for the in-
cubation periods of 18 h and 36 h.
The microscopic images of migration suggested that in the
control DMSO, a significant amount of cancer cell population
prevalent in the wounded area after 18 and 36 h. In contrast, the
treatment of ADG-2e didn’t show any considerable population at
our compounds showed negligible toxicity at 32 mg/mL. Further,
therapeutic index (TI) of AMG-1e, ADG-2e, ADL-3e was investi-
gated. In general, the therapeutic potential of antibacterial agents,
referred as therapeutic index (TI) values, denotes the relative safety
of a drug to selectively kill the bacteria. Investigation of the ther-
apeutic index (Table 2) of ADG-2e and ADL-3e displayed 14 and 17
folds better TI values compared to that of melittin, which suggests
that these compounds may represent a therapeutic model for
designing the AZT based antibacterial.
2.4. Antibacterial activity of ADG-2e against drug-resistant bacteria
the wounded area at the maximum concentration (40 mM), sug-
gesting its inhibitory potential on the migration of cancer cells. To
verify this, further, cell migration of ADG-2e was confirmed by
transwell invasive assay that measures the capacity of cell motility
and invasiveness. Thus, ADG-2e was examined using transwell
assay for 16 h to quantitatively determine their anti-motility ac-
tivity. As shown in Fig. 4b, in the control, the population of invaded
cells was relatively high; in contrast, ADG-2e treated chamber
didn’t contain a considerable amount of invaded cells, proving the
inhibitory potential of ADG-2e on cancer cell migration.
Even though wound healing assay and the invasion assay are
widely using for cell migration, these methods represent an indi-
rect method for determining cell migration. Thus, to evaluate the
effect of ADG-2e on cell migration directly, we performed time-
lapse cell tracking analysis using the holoMonitor m4 time-lapse
cytometer (Phase Holographic Imaging) that is an analyzer for
adherent cells to monitor movement patterns and motility by
tracking of individual cells using time-lapse videos. The ADG-2e
treated cells were tracked over a period of 24 h at 30 min in-
tervals, and the acquired data were computationally processed to
create holographic images over a range of focal distances. As shown
in Fig. 5a and b, the migration distance of ADG-2e treated cells was
Drug resistance becomes an escalating threat to worldwide
health care society. In particular, in the hospital- and community-
acquired infections, methicillin-resistant S. aureus (MRSA) and
multidrug-resistant P. aeruginosa (MDRPA) are the key contributors
[33]. A combination of scarcity in the discovery of new antibiotics
and evolving drug resistance are the alarming call for the invention
of new and alternative antibiotics for treating the drug-resistant
bacteria. In this view, we have evaluated the most potent com-
pound, ADG-2e against MRSA and MDRPA bacterial strains
(Table 3). Interestingly, ADG-2e showed 2 folds better antibacterial
activity against all the strains compared to that of melittin. In
contrast, conventional antibiotics, including chloramphenicol, cip-
rofloxacin and oxacillin failed to display any considerable antibac-
terial effects. Even though conventional antibiotics lost its
antibacterial effect, our compound showed good antibacterial ef-
fects, demonstrating the therapeutic potential against the drug-
resistant pathogens.
2.5. Cancer metastasis inhibition
Similarities in the phenotypes of both cancer and bacterial
membrane surface offer an advantage for a single promising
molecule that can act as both antibacterials as well as an anti-
cancer agent [34,35]. As tumor cell migration remains a sole
cause for the tumor metastasis, inhibition of which is considered a
potential approach for antitumor treatment. Thus, studies per-
taining to cell migration have drawn a key focus in the field of
discovery of anti-cancer agents. In general, an increase in mem-
brane tension results in unidirectional cell movement, eventually,
increases cell migration. Recently, Futaki et al. reported an
amphipathic peptide derived from influenza that showed a notable
ability in cell membrane insertion, which affected the cell migra-
tion and membrane morphology. Even though a couple of reports
demonstrated that the insertion of amphipathic compounds and an
inorganic complex on the membrane surface reduced the mem-
brane tension that affected the cell movement [23,24,36,37], to the
best of our knowledge, AZT based amphipathic small molecules
having antibacterial activity have not investigated for the inhibition
of metastasis yet.
remarkably decreased compared to that of control, at 40 mM con-
centration. The reduced distance can be attributed to the fact that
the possible insertion of ADG-2e on the cancer cell surface,
consequently affected the cell movement.
Membrane tension and the cytoskeletal protein, actin are the
key factors, which regulate cell movement and morphogenesis
[38]. An increase in membrane tension forbids the lateral poly-
merization of actin and organize them in a protrusion toward the
direction of migration, which triggers the cell migration. However,
a decrease in cell membrane tension enhances the formation of
lateral polymerization of actin, which results in the formation of
multiple actin-rich veils called, lamellipodia, hindering the cell
migration [39e41]. A recent report demonstrated that arginine-
rich amphipathic peptides showed increased lamellipodium for-
mation, thereby affecting cell mobility [25]. Arginine has the ability
to form bidentate hydrogen bonds with phospholipids [42] and also
can disperse the cationic charge on the cancer cell surface through
electrostatic interactions. Thus, most of the cell penetrating pep-
tides are rich in arginine [43].

6
S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
Table 3
Antimicrobial activities of ADG-2e, melittin and conventional antibiotics against antibiotic-resistant bacterial strains.
MIC ( g/mL)
m
Microorganisms
ADG-2e
Melittin
Chloramphenicol
Ciprofloxacin
Oxacillin
MRSAa
CCARM 3089
CCARM 3090
CCARM 3095
MDRPAb
8
8
8
16
16
16
512
256
512
512
512
512
256
512
256
CCARM 2095
CCARM 2109
8
8
16
16
>512
>512
128
16
>512
>512
a
MRSA: methicillin-resistant Staphylococcus aureus.
MDRPA: multidrug-resistant Pseudomonas aeruginosa.
b
Fig. 3. Effect of ADG-2e on MDA-MB-231 cell viability. The cells were treated with
different concentrations of the compound for 24 h. Cell viability was measured with
the EZ-Cytox cell viability assay kit. Values are the mean SD of three independent
experiments.
Fig. 4. Inhibitory effect of ADG-2e on the invasion of MDA-MB-231 cells. (a) Repre-
sentative images of MDA-MB-231 cells treated with ADG-2e or DMSO in the Matrigel-
coated upper chamber. After 18 h or 36 h incubation, the invaded cells were captured
by a microscope at a 100x magnification. (b) The cells were cultured in the modified
Boyden chamber with DMSO or ADG-2e (40 mM) contained media, after incubation for
Inspired by this, we investigated the lamellipodium formation
ability of our most potent antibacterial agent ADG-2e that pos-
sesses a couple of guanidines. Interestingly, our results suggested
that ADG-2e treated cells showed an increase in lamellipodium
generation by distributing the actin in a multi-directional pattern
(Fig. 6). In contrast, control didn’t show lamellipodium formation.
This could be likely due to fact that cationic charges of guanidine
residues in ADG-2e might have interacted with anionic phosphate
in the membrane of cancer cells through electrostatic interactions,
eventually reduced the cell membrane tension, thus forming mul-
16 h, cells were stained with the crystal violet. And then the invaded cells were
observed by a microscope. (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
(Fig. 2 and Table 2). The MDA-MB-231 antiproliferative assay sug-
gests that until 100
toxicity (Fig. 3). In this regard, ADG-4e displayed anti-bacterial ef-
fect ranging 2e16 g/mL and maximum anti-metastasis effects
with 40 M (31.58 g/mL) concentration. Thus, these results sug-
mM, ADG-2e didn’t show any considerable
m
m
m
tiple lamellipodia. These results suggest that ADG-2e has
a
gest that the administration of ADG-2e is safe during the treatment
of both bacterial infections and metastasis. Collectively, our study
represents a model for developing a promising class of cationic
small molecule antibacterial agents with good anti-metastasis
activity.
noticeable potential to become a model for designing anti-
metastasis agents by reducing cell migration through actin reor-
ganization and multiple lamellipodium formation.
3. Conclusion
4. Experimental section
In summary, the detailed SAR studies describe that dicationic
species with a larger hydrophobic group, especially, the presence of
1-adamantanemethyl group was a key factor in achieving the
antibacterial effect. While addressing the emergence of multidrug
resistance, the most potent antimicrobial compound ADG-2e dis-
played a good antibacterial effect against drug resistance patho-
gens. Considering the role of cell motility in exacerbating cancer
metastasis and the influence of amphipathic compounds in cell
motility [36,37], ADG-2e was investigated for their anti-metastasis
potential. Results of wound healing, transwell, Locomotion studies
suggested that ADG-2e has a notable potential to inhibit the cancer
cell migration. Further, ADG-2e displayed an ability to form mul-
tiple lamellipodia formation by reorganizing the actin, which drops
the cell migration. In hemolysis, ADG-2e didn’t show any hemolysis
4.1. Chemistry
Greater than 99.9% of purity of organic solvents were purchased
from Aldrich and used directly. All reagents and starting materials
were supplied by the commercial chemical suppliers including
Sigma-Aldrich, TCI and across organics and used as received. Merck
aluminum sheets with silica gel 60 F254 was used for thin-layer
chromatography (TLC) and visualized by ultraviolet light and
staining with KMnO4 and ninhydrin. Column chromatography was
performed on Merck silica gel 60 (70e230 mesh or 230e400 mesh)
for the purification of compounds. 1H and 13C NMR spectra were
recorded on Bruker DRX-400, DRX-500, Bruker 800 and 900
spectrometer. Chemical shifts (
d) are reported in parts per million
until 16 mg/mL, and showed a negligible hemolysis at 32 mg/mL
(ppm) measured relative to an internal standard and coupling

S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
7
Fig. 5. (a) The holographic records of cell movement are presented as individual colored lines. MDA-MB-231 cells were treated with the 40
mM of ADG-2e and captured every
30 min for 24 h. (b) The average migration distance (mm) was analyzed using the HoloStudio software and represented in the bar graph.
Fig. 6. Immunofluorescence staining of microtubules in MDA-MB-231 cells. Cells were treated with DMSO or indicated concentrations of ADG-2e for 6 h. Cells were then fixed and
stained with Texa Red-conjugated phalloidin to visualize F-actin and Hoechst 33342 to visualize -tubulin and DNA, respectively. Scale bar, 10 m. The arrows indicated lamel-
lipodia. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
a
m
constants (J) are expressed in hertz (Hz). Mass spectra were
recorded using Shimadzu (MALDI-TOF) mass spectrometer.
Reverse-phase HPLC analysis (RP-HPLC) was carried out at 230 nm
on an Agilent HPLC system equipped with a C18 analytical column
colorless sticky liquid. 1H NMR (500 MHz, CDCl3)
d 8.73 (brs, 1H),
7.30e7.27 (m, 1H), 6.14 (t, J ¼ 6.0 Hz, 1H), 4.25 (dt, J ¼ 7.7, 5.5 Hz,
1H), 3.94 (dt, J ¼ 5.5, 3.4 Hz, 1H), 3.77 (dd, J ¼ 13.4, 3.3 Hz, 1H), 3.57
(dd, J ¼ 13.4, 3.5 Hz, 1H), 2.49e2.36 (m, 2H), 1.94 (d, J ¼ 1.1 Hz, 3H).
(250 ꢁ 22 mm, 10
m
m). Two different linear gradients of 0.05% aq.
13C NMR (100 MHz, CDCl3)
d 163.9, 150.5, 135.6, 111.8, 85.2, 82.2,
TFA (eluent A) and 0.05% TFA in CH3CN (eluent B) were used with a
60.5, 51.9, 37.4, 12.7. MS (MALDI-TOF): calcd for C10H12N8O3: 292.1,
flow rate of 5.0 mL/min at 25 ꢀC.
found 293.1 (M þ H)þ.
4.2. 1-((2R,4S,5R)-4-azido-5-(azidomethyl)tetrahydrofuran-2-yl)-
4.3. 1-((2R,4S,5R)-4-azido-5-(azidomethyl)tetrahydrofuran-2-yl)-
5-methylpyrimidine-2,4(1H,3H)-dione(5)
5-methyl-4-(1H-1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (6)
To the stirred solution of 30-Azido-30-deoxythymidine, 4 (10 g,
37.4 mmol) in dichloromethane (100 mL) at 0 ꢀC, was added TEA
(10.4 mL, 74.8 mmol), DMAP (0.45 g, 3.7 mmol) and tosyl chloride
(7.1 g, 37.4 mmol) and stirred for 15 h at room temperature. Then
the reaction mixture was diluted with dichloromethane (100 mL)
and organic layer was washed with NaHCO3 (30 mL) solution, brine
H2O (30 mL) and dried over Na2SO4. Concentrated and flash chro-
matographic separation afforded O-tosylated compound as white
solid. This tosyl AZT compound was dissolved in 80 mL of DMF, to
which NaN3 was added (14.4 g, 222.3 mmol) and heated at 60 ꢀC for
12 h. Then the reaction was quenched by the addition of water
(200 mL) and extracted in ethyl acetate (2 x 150 mL). The combined
organic layer was dried over Na2SO4, concentrated and purified by
column chromatography to get pure product 5 (9.6 g, 88% yield) as a
To a ice-cold stirring solution of POCl3 (5.1 mL, 54.7 mmol), TEA
(57 mL, 410.9 mmol) and 1,2,4-triazole (17.0 g, 246.5 mmol) in
anhydrous acetonitrile (150 mL) under N2 atmosphere was added
compound 5 (8 g, 27.3 mmol) in 80 mL of anhydrous acetonitrile in
dropwise. Then the reaction mixture was stirred at room temper-
ature for 20 h. It was then extracted with ethyl acetate (200 mL x 2).
The combined organic phase was washed with saturated NaHCO3
solution (200 mL), brine (150 mL) and dried over Na2SO4, concen-
trated and purified by column chromatography to afford pure
product 6 (9.2 g, 98% yield) as a white solid. 1H NMR (500 MHz,
CDCl3)
d
9.27 (s, 1H), 8.15 (s, 1H), 8.11 (s, 1H), 6.15 (dd, J ¼ 6.7, 4.7 Hz,
1H), 4.18 (q, J ¼ 6.8 Hz, 1H), 4.07e4.02 (m, 1H), 3.90 (dd, J ¼ 13.5,
3.1 Hz, 1H), 3.67 (dd, J ¼ 13.5, 3.2 Hz, 1H), 2.79e2.70 (m, 1H),
2.49e2.43 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 158.5, 153.7, 153.7,

8
S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
146.3, 145.2, 106.0, 87.3, 82.8, 59.4, 51.4, 38.7, 17.5. MS (MALDI-TOF):
4.5.2. Synthesis of tert-butyl (tert-butoxycarbonylamino)(2-(5-
methyl-2-oxo-1-((2R,4S,5R)-4-(4-phenylbutanamido)-5-((4-
phenylbutanamido)methyl)tetrahydrofuran-2-yl)-1,2-
dihydropyrimidin-4-ylamino)ethylamino)methylenecarbamate
(10b)
calcd for C12H13N11O2: 343.1, found 344.1 (M þ H)þ.
10b was synthesized from 4-phenylbutyric acid (9b) (130 mg,
0.79 mmol) and diamine (0.2 g, 0.38 mmol) using HOBt, EDC.HCl,
and DIPEA by following the general procedure A as a white solid
4.4. N1-(1-((2R,4S,5R)-4-azido-5-(azidomethyl)tetrahydrofuran-2-
yl)-5-methyl-4-(tert-butyl ((tert-butoxycarbonyl)amino)-N2-
methylene)carbamate) ethane-1,2-diamine) pyrimidin-2(1H)-one
(8)
230 mg (77%). 1H NMR (500 MHz, CDCl3):
d 11.52 (s, 1H), 8.72 (t,
J ¼ 6.4 Hz, 1H), 8.24 (d, J ¼ 5.7 Hz, 1H), 7.35 (t, J ¼ 4.9 Hz, 1H),
7.25e7.21 (m, 6H), 7.17e7.12 (m, 6H), 6.33 (t, J ¼ 6.6 Hz, 1H),
4.18e4.09 (m, 1H), 3.96e3.90 (m, 1H), 3.66e3.59 (m, 3H),
3.53e3.35 (m, 3H), 2.66e2.55 (m, 5H), 2.43e2.30 (m, 2H),
2.30e2.24 (m, 2H), 2.10e2.02 (m, 1H), 2.01e1.92 (m, 7H), 1.48 (s,
To a stirred solution of aminoguanidine 7 (2.6 g, 8.7 mmol) and
K2CO3 (2.4 g, 17.4 mmol) in acetonitrile (40 mL), compound 6 (2 g,
5.8 mmol) was added and stirred for 24 h at room temperature. The
reaction mixture was quenched by addition of water (30 mL) and
extraction with 2% methanol in ethyl acetate (20 mL x 3). The
combine organic layer was washed with brine (30 mL), dried over
Na2SO4 and concentrated. The crude was purified by flash chro-
matography to obtain pure compound 8 as a white solid (3.2 g, 96%
9H), 1.47 (s, 9H). 13C NMR (100 MHz, CDCl3):
d 174.6, 173.5, 163.5,
163.0, 158.6, 156.5, 153.1, 141.8, 141.6, 135.9, 128.6, 128.5, 128.4,
128.4,126.0,125.9,103.4, 85.9, 84.4, 83.9, 79.9, 52.2, 44.8, 42.4, 42.2,
39.2, 36.8, 36.1, 35.5, 35.4, 35.4, 28.3, 28.1, 27.3, 13.8. MS (MALDI-
TOF): calcd for C43H60N8O8: 816.4; found 839.3 (M þ Na)þ.
yield). 1H NMR (500 MHz, CDCl3)
1H), 7.23 (s, 1H), 7.18e7.12 (m, 1H), 6.20 (t, J ¼ 6.2 Hz, 1H), 4.16 (q,
J ¼ 6.6 Hz,1H), 3.94e3.86 (m,1H), 3.72e3.60 (m, 6H), 2.56e2.43 (m,
1H), 2.37e2.26 (m, 1H), 1.92 (s, 3H), 1.48 (s, 9H), 1.46 (s, 9H). 13C
d
11.49 (s, 1H), 8.74 (t, J ¼ 6.0 Hz,
4.5.3. Synthesis of tert-butyl (tert-butoxycarbonylamino)(2-(1-
((2R,4S,5R)-4-(cyclohexanecarboxamido)-5-
(cyclohexanecarboxamidomethyl)tetrahydrofuran-2-yl)-5-methyl-
2-oxo-1,2-dihydropyrimidin-4-ylamino)ethylamino)
methylenecarbamate (10c)
NMR (100 MHz, CDCl3)
d 163.4, 163.1, 158.5, 156.0, 153.1, 136.2,
102.9, 85.9, 83.9, 81.9, 79.7, 60.4, 51.9, 44.8, 39.3, 38.1, 28.3, 28.1,
13.8. MS (MALDI-TOF): calcd for C23H36N12O6: 576.6; found 599.2
(M þ Na)þ.
10c was synthesized from cyclohexanecarboxylic acid (9c)
(154 mg, 1.2 mmol) and diamine (0.3 g, 0.57 mmol) using HOBt,
EDC.HCl, and DIPEA by following the general procedure A as a white
solid (310 mg, 74%). 1H NMR (500 MHz, CDCl3)
d 11.51 (s, 1H),
8.84e8.73 (m, 1H), 7.42 (brs, 1H), 7.29e7.24 (m, 2H), 7.05e6.96 (m,
1H), 6.26e6.15 (m, 1H), 4.20e4.12 (m, 1H), 3.89e3.84 (m, 1H),
3.76e3.70 (m, 2H), 3.67e3.62 (m, 2H), 3.59e3.53 (m, 1H),
3.51e3.45 (m, 1H), 2.64e2.53 (m, 1H), 2.24e2.10 (m, 3H), 1.95 (s,
3H), 1.85e1.72 (m, 7H), 1.67e1.61 (m, 2H), 1.49e1.41 (m, 22H),
4.5. General procedure A
4.5.1. Synthesis of tert-butyl (tert-butoxycarbonylamino)(2-(5-
methyl-1-((2R,4S,5R)-4-(2-(naphthalen-1-yl)acetamido)-5-((2-
(naphthalen-1-yl)acetamido)methyl)tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-ylamino)ethylamino)methylenecarbamate
(10a)
1.31e1.14 (m, 8H). 13C NMR (100 MHz, CDCl3)
d 177.9, 176.9, 163.0,
162.9,158.5,153.1,136.5,103.0, 85.4, 84.0, 83.9, 80.0, 51.0, 45.5, 45.0,
41.2, 39.3, 37.4, 29.8, 29.7, 29.5, 28.3, 28.1, 27.9, 25.9, 25.8, 25.8, 25.7,
13.9. MS (MALDI-TOF): calcd for C37H60N8O8: 744.9; found 768.3
(M þ Na)þ.
33 mg of 5% (Pd/C) was added to the stirred solution of 8
(328 mg, 0.57 mmol) in methanol (15 mL) and stirred for 6 h under
H2. After completion of reaction, filtered through celite pad and
concentrated to get crude diamine compound, which was pro-
ceeded to next step. To a stirred solution of 1-naphthaleneacetic
acid (9a) (224 mg, 1.2 mmol) in DMF (6 mL) was added HOBt
(232 mg,1.7 mmol), EDC.HCl (330 mg,1.7 mmol) and DIPEA (0.5 mL,
2.8 mmol) diamine (300 mg, 0.57 mmol) in DMF (5 mL) under N2
and stirred for 10 h at room temperature. The reaction mixture was
diluted with CH2Cl2 (60 mL) and washed with 1N HCl (20 mL), H2O
(20 mL), 5% aq. NaHCO3 soln. (20 mL), and brine. The combined
organic layer was dried (Na2SO4) and concentrated, and the residue
was purified by column chromatography using 2% MeOH: CH2Cl2 to
obtain pure product 10a as white solid (370 mg, 76% yield). 1H NMR
4.5.4. Synthesis of tert-butyl(tert-butoxycarbonylamino)(2-(1-
((2R,4S,5R)-4-hexanamido-5-(hexanamidomethyl)tetrahydrofuran-
2-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-ylamino)
ethylamino)methylenecarbamate (10d)
10d was synthesized from hexanoic acid (9d) (140 mg,
1.2 mmol) and diamine (0.3 g, 0.57 mmol) using HOBt, EDC.HCl, and
DIPEA by following the general procedure A as a white solid
(260 mg, 63%). 1H NMR (500 MHz, CDCl3):
d 11.52 (s, 1H), 8.81 (s,
1H), 7.68e7.36 (m, 2H), 7.29 (s, 1H), 7.20e7.13 (m, 1H), 6.29e6.19
(m, 1H), 4.20e4.12 (m, 1H), 3.98e3.91 (m, 1H), 3.77e3.64 (m, 4H),
3.59e3.48 (m, 2H), 2.65e2.55 (m, 1H), 2.31e2.25 (m, 2H),
2.25e2.21 (m, 2H), 2.19e2.12 (m, 1H), 1.97 (s, 3H), 1.67e1.59 (m,
4H), 1.49 (s, 9H), 1.48 (s, 9H), 1.33e1.26 (m, 8H), 0.90e0.85 (m, 6H).
(500 MHz, CDCl3): d 11.48 (s, 1H), 8.56e8.47 (m, 1H), 8.05 (brs, 1H),
8.00e7.96 (m, 1H), 7.94 (d, J ¼ 8.2 Hz, 1H), 7.88e7.84 (m, 1H), 7.78
(d, J ¼ 7.9 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.65 (d, J ¼ 7.8 Hz, 1H),
7.50e7.47 (m, 2H), 7.44e7.35 (m, 4H), 7.31e7.26 (m, 2H), 7.18 (s,1H),
7.15 (brs, 1H), 6.91 (t, J ¼ 5.2 Hz, 1H), 6.20 (t, J ¼ 6.5 Hz, 1H), 4.13 (s,
2H), 4.01e3.96 (m, 1H), 3.91e3.82 (m, 2H), 3.79e3.74 (m, 1H),
3.58e3.52 (m, 1H), 3.36e3.20 (m, 5H), 2.47e2.38 (m, 1H), 1.97e1.91
(m, 1H), 1.84 (s, 3H), 1.49 (s, 9H), 1.45 (s, 9H). 13C NMR (100 MHz,
13C NMR (100 MHz, CDCl3):
d 175.0, 174.0, 163.5, 163.0, 158.6, 156.5,
153.1, 136.0, 103.3, 85.8, 84.3, 83.9, 79.9, 51.9, 44.8, 42.1, 39.2, 37.0,
36.8, 36.2, 31.6, 31.6, 28.3, 28.1, 25.6, 25.5, 22.5, 22.5, 14.1, 13.8. MS
(MALDI-TOF): calcd for C35H60N8O8: 720.9; found 744.3 (M þ Na)þ.
4.5.5. Synthesis of tert-butyl(tert-butoxycarbonylamino)(2-(1-
((2R,4S,5R)-4-(adamantaneacetamido)-5-
CDCl3):
d
172.4, 171.6, 163.4, 163.0, 158.4, 156.6, 153.0, 136.1, 133.9,
(adamantaneacetamidomethyl)tetrahydrofuran-2-yl)-5-methyl-2-
oxo-1,2-dihydropyrimidin-4-ylamino)ethylamino)
methylenecarbamate (10e)
10e was synthesized from 1-adamanatanacetic acid (9e)
(230 mg, 1.2 mmol) and diamine (0.3 g, 0.57 mmol) using HOBt,
133.9, 132.4, 132.2, 131.5, 128.8, 128.6, 128.3, 128.2, 128.0, 126.4,
126.3, 126.0, 125.8, 125.7, 125.6, 124.3, 124.1, 103.2, 86.3, 84.5, 83.8,
79.8, 52.1, 44.6, 42.3, 41.4, 40.6, 38.9, 37.1, 28.3, 28.1, 13.6. MS
(MALDI-TOF): calcd for C47H56N8O8: 860.4; found 883.3 (M þ Na)þ.

S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
9
EDC.HCl, and DIPEA by following the general procedure A, as a
white solid (340 mg, 68%). 1H NMR (500 MHz, CDCl3)
11.52 (s, 1H),
29.2, 29.1, 29.0, 25.4, 25.4, 25.2, 25.2, 12.5. MS (MALDI-TOF): calcd
d
for C27H44N8O4: 544.3, found 545.3 (M þ H)þ.
8.80 (brs, 1H), 7.44 (brs, 1H), 7.30 (s, 1H), 7.09e6.97 (m, 2H), 6.25 (t,
J ¼ 6.3 Hz, 1H), 4.19e4.11 (m, 1H), 3.99e3.92 (m, 1H), 3.76e3.70 (m,
2H), 3.69e3.63 (m, 2H), 3.62e3.56 (m, 1H), 3.49e3.43 (m, 1H),
2.66e2.48 (m, 1H), 2.16e2.09 (m, 1H), 2.00e1.92 (m, 13H),
4.6.4. 4.6.4 Synthesis of AMG-1d
AMG-1d was synthesized from 10d (110 mg, 0.1527 mmol) using
TFA and DCM mixture by following the general procedure B as a
1.70e1.65 (m, 6H), 1.64e1.57 (m, 18H), 1.49 (s, 9H), 1.48 (s, 9H). 13
C
white solid (56 mg, 71%).1H NMR (800 MHz, DMSO‑d6)
d 8.24 (d,
NMR (100 MHz, CDCl3)
d
172.7, 171.8, 163.5, 163.1, 158.5, 156.5, 153.1,
J ¼ 7.4 Hz, 1H), 7.96 (t, J ¼ 5.1 Hz, 1H), 7.77 (brs, 1H), 7.73 (t,
J ¼ 5.2 Hz, 1H), 7.59 (s, 1H), 6.98 (brs, 2H), 6.12 (t, J ¼ 5.8 Hz, 1H),
4.21e4.18 (m, 1H), 3.76e3.73 (m, 1H), 3.44e3.37 (m, 2H), 3.37e3.33
(m, 1H), 3.31e3.28 (m, 1H), 3.28e3.22 (m, 2H), 2.17e2.10 (m, 2H),
2.09e2.03 (m, 4H), 1.91 (s, 3H), 1.52e1.44 (m, 4H), 1.29e1.17 (m,
8H), 0.85 (t, J ¼ 6.4 Hz, 3H), 0.82 (t, J ¼ 6.4 Hz, 3H). 13C NMR
135.9, 103.4, 85.6, 84.7, 83.9, 79.9, 52.3, 51.7, 50.8, 50.7, 44.9, 42.7,
42.7, 42.3, 39.2, 37.0, 36.9, 36.8, 33.1, 32.9, 28.8, 28.7, 28.3, 28.2, 28.1,
13.8. MS (MALDI-TOF): calcd for C47H72N8O8: 876.5; found 899.4
(M þ Na)þ.
4.6. Synthesis of AMG-(1a-1e)
General procedure B
(200 MHz, DMSO‑d6) d 174.4, 172.2, 158.4, 157.2, 138.3, 116.6, 102.3,
84.7, 82.8, 50.4, 41.2, 39.9, 39.0, 36.8, 35.3, 35.2, 30.8, 30.8, 24.9,
24.9, 21.8, 21.8, 13.8, 13.8, 12.5. MS (MALDI-TOF): calcd for
C
25H44N8O4: 520.3, found 543.3 (M þ Na)þ.
4.6.1. Synthesis of AMG-1a
50% TFA in CH2Cl2 (4 mL) was added to a stirred solution of 10a
(150 mg, 0.174 mmol) in CH2Cl2 (4 mL) at 0 ꢀC and the temperature
was increased to room temperature, and the resulting solution was
stirred for 4 h. Then the reaction mixture was concentrated under
vacuum and residue was washed with cold Et2O. The resultant
crude product was purified by RP-HPLC to give final compound
AMG-1a (101 mg, 88%) as a white solid. 1H NMR (900 MHz,
4.6.5. Synthesis of AMG-1e
AMG-1e was synthesized from 10e (170 mg, 0.1940 mmol) using
TFA and DCM mixture by following the general procedure B as a
white solid (130 mg, 99%). 1H NMR (800 MHz, DMSO‑d6)
d 8.19 (d,
J ¼ 7.1 Hz, 1H), 7.94 (brs, 1H), 7.87 (t, J ¼ 5.7 Hz, 1H), 7.68 (t,
J ¼ 5.7 Hz, 1H), 7.66 (s, 1H), 6.97 (brs, 2H), 6.14 (t, J ¼ 6.7 Hz, 1H),
4.20e4.17 (m,1H), 3.80e3.76 (m,1H), 3.46e3.40 (m, 2H), 3.39e3.34
(m, 1H), 3.32e3.26 (m, 3H), 2.17e2.12 (m, 2H), 1.92 (s, 3H),
1.92e1.89 (m, 3H), 1.87e1.85 (m, 3H), 1.84e1.83 (m, 4H), 1.66e1.60
(m, 6H), 1.59e1.55 (m, 9H), 1.53e1.50 (m, 9H). 13C NMR (200 MHz,
DMSO‑d6)
d
8.75 (d, J ¼ 7.3 Hz, 1H), 8.41 (t, J ¼ 5.8 Hz, 1H), 8.07 (d,
J ¼ 8.3 Hz, 1H), 8.03 (d, J ¼ 8.3 Hz, 1H), 7.94 (brs, 1H), 7.91 (d,
J ¼ 8.0 Hz,1H), 7.88 (d, J ¼ 8.0 Hz,1H), 7.81 (d, J ¼ 7.8 Hz,1H), 7.79 (d,
J ¼ 7.7 Hz, 1H), 7.71 (t, J ¼ 5.8 Hz, 1H), 7.55 (s, 1H), 7.54e7.51 (m, 1H),
7.50e7.48 (m, 1H), 7.47e7.38 (m, 6H), 7.01 (brs, 2H), 6.23 (t,
J ¼ 6.7 Hz,1H), 4.28e4.24 (m,1H), 3.95e3.86 (m, 5H), 3.47e3.36 (m,
4H), 3.28 (q, J ¼ 6.4 Hz, 2H), 2.23e2.15 (m, 2H), 1.69 (s, 3H). 13C NMR
DMSO‑d6)
d 170.1, 170.0, 158.3, 157.2, 138.5, 116.2, 102.3, 84.9, 83.0,
50.5, 50.0, 49.7, 42.1, 42.0, 41.2, 40.0, 39.9, 39.8, 39.7, 39.6, 39.0, 36.7,
36.4, 32.3, 32.2, 28.0, 12.5. MS (MALDI-TOF): calcd for C37H56N8O4:
676.4, found 677.4 (M þ H)þ.
(225 MHz, DMSO‑d6)
d 170.4, 170.2, 158.3, 157.2, 138.5, 133.3, 133.2,
132.7, 132.6, 131.9, 131.9, 128.3, 128.2, 127.7, 127.7, 127.1, 127.0, 126.0,
125.7, 125.6, 125.5, 125.5, 125.4, 124.3, 124.1, 116.2, 102.5, 84.9, 82.8,
50.9, 41.5, 40.1, 39.9, 39.8, 39.0, 36.5, 12.3. MS (MALDI-TOF): calcd
for C37H40N8O4: 660.3, found 660.6 (M)þ.
4.7. Di-tert-butyl(((1-((2R,4S,5R)-4-azido-5-(azidomethyl)
tetrahydrofuran-2-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)
azanediyl)bis(propane-3,1-diyl))dicarbamate (12)
To a stirred solution of N1,N9-bis-Boc-norspermidine 11, (2.3 g,
7.0 mmol) in acetonitrile (30 mL) was added K2CO3 (2.4 g,
17.4 mmol) followed by compound 6 (2 g, 5.8 mmol) at room
temperature. Then the reaction mixture was stirred for 24 h at
40 ꢀC. After completion of reaction, solvent was removed through
vacuum. The H2O (20 mL) was added to the crude product and
extracted with 2% methanol in ethylacetate (20 mL x 3). The
combine organic layer was washed with brine (30 mL), dried over
Na2SO4 and concentrated, and purified by flash chromatography to
obtain pure compound 12 (3.0 g, 87% yield) as white sticky solid. 1H
4.6.2. Synthesis of AMG-1b
AMG-1b was synthesized from 10b (79 mg, 0.096 mmol) using
TFA and DCM mixture by following the general procedure B as a
white solid (59 mg, 99%). 1H NMR (900 MHz, DMSO‑d6)
d 8.27 (d,
J ¼ 7.4 Hz, 1H), 8.01 (t, J ¼ 5.8 Hz, 1H), 7.87 (brs, 2H), 7.68 (t,
J ¼ 6.0 Hz, 1H), 7.63 (s, 1H), 7.27 (t, J ¼ 7.6 Hz, 2H), 7.24 (t, J ¼ 7.5 Hz,
2H), 7.20e7.11 (m, 6H), 6.97 (brs, 2H), 6.13 (t, J ¼ 6.6 Hz, 1H),
4.24e4.20 (m, 1H), 3.79e3.76 (m, 1H), 3.45e3.39 (m, 2H),
3.38e3.31 (m, 2H), 3.29e3.23 (m, 2H), 2.56e2.53 (m, 2H),
2.53e2.50 (m, 2H), 2.18e2.12 (m, 2H), 2.12e2.06 (m, 4H), 1.90 (s,
NMR (500 MHz, CDCl3)
d
7.35 (s, 1H), 6.11 (t, J ¼ 6.0 Hz, 1H), 5.05
3H), 1.81e1.74 (m, 4H). 13C NMR (225 MHz, DMSO‑d6)
d
172.1, 171.9,
(brs, 2H), 4.20 (q, J ¼ 6.6 Hz, 1H), 3.95e3.89 (m, 1H), 3.79e3.74 (m,
158.3,157.2, 141.7,141.7, 138.4,128.3,128.2,128.2,128.2,125.7,125.7,
116.3, 102.4, 84.8, 82.8, 50.4, 41.2, 40.0, 39.9, 39.8, 39.0, 36.7, 34.8,
34.7, 34.5, 34.5, 27.0, 26.9, 12.5. MS (MALDI-TOF): calcd for
1H), 3.65e3.53 (m, 5H), 3.17e3.05 (m, 4H), 2.57e2.47 (m, 1H),
2.47e2.36 (m, 1H), 2.16 (s, 3H), 1.87e1.74 (m, 4H), 1.42 (s, 18H). 13
C
NMR (100 MHz, CDCl3)
d 164.3, 156.3, 154.1, 141.3, 102.8, 86.2, 82.2,
C
33H44N8O4: 616.3, found 639.3 (M þ Na)þ.
79.4, 60.3, 51.8, 47.5, 38.1, 38.1, 29.2, 28.5, 19.0. MS (MALDI-TOF):
calcd for C26H43N11O6: 605.3; found 606.2 (M þ H)þ.
4.6.3. Synthesis of AMG-1c
AMG-1c was synthesized from 10c (160 mg, 0.215 mmol) using
TFA and DCM mixture by following the general procedure B as a
4.8. Synthesis of 13 (aee)
white solid (110 mg, 94%). 1H NMR (900 MHz, DMSO‑d6)
d
8.14 (d,
General procedure C
J ¼ 7.4 Hz, 1H), 7.85 (t, J ¼ 5.8 Hz, 1H), 7.78 (brs, 1H), 7.71 (t,
J ¼ 6.0 Hz, 1H), 7.57 (s, 1H), 6.97 (brs, 2H), 6.12 (t, J ¼ 6.6 Hz, 1H),
4.20e4.17 (m, 1H), 3.73e3.71 (m, 1H), 3.40 (q, J ¼ 6.6 Hz, 2H),
3.35e3.31 (m, 1H), 3.29e3.23 (m, 3H), 2.14e2.06 (m, 4H), 1.91 (s,
4.8.1. Synthesis of 13a
Synthesis of tert-butyl 3,3’-(5-methyl-1-((2R,4S,5R)-4-(2-(naph-
thalen-1-yl)acetamido)-5-((2-(naphthalen-1-yl)acetamido)methyl)
tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylazanediyl)
bis(propane-3,1-diyl)dicarbamate (13a)
3H), 1.72e1.58 (m, 10H), 1.34e1.26 (m, 4H), 1.23e1.10 (m, 6H). 13
C
NMR (225 MHz, DMSO‑d6)
d 175.4, 175.3, 158.3, 157.2, 138.2, 116.6,
102.3, 84.6, 82.8, 50.2, 43.9, 40.9, 39.9, 39.8, 39.8, 39.0, 36.8, 29.2,
Compound 12 (544 mg, 0.9 mmol) was dissolved in methanol

10
S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
(20 mL), to which 54 mg of 5% (Pd/C) was added and stirred for 6 h
under H2 atm. After completion of reaction, filtered through celite
pad and concentrate residue to get crude diamine compound which
was proceeded to next step directly. To a stirred solution of 1-
naphthaleneacetic acid (9a) (350 mg, 1.89 mmol) in dime-
thylformamide (10 mL) was added subsequently HOBt (370 mg,
2.7 mmol) and EDC.HCl (520 mg, 2.7 mmol) under N2 atm. After
stirring 10 min, diamine (0.5 g, 0.9 mmol) in dimethylformamide
(5 mL) and DIPEA (0.78 mL, 4.5 mmol) was added and stirred for
10 h at room temperature. After dilution with CH2Cl2 (50 mL), the
mixture was washed with 1N HCl (15 mL), H2O (15 mL), 5% aq.
NaHCO3 soln. (15 mL), and brine. The combined organic layers were
dried (Na2SO4) and concentrated, and the crude residue was puri-
fied by column chromatography using 2% MeOH:CH2Cl2 to afford
the pure product 13a as a white solid (680 mg, 85% yield). 1H NMR
4.8.4. Synthesis of tert-butyl 3,3’-(1-((2R,4S,5R)-4-hexanamido-5-
(hexanamidomethyl) tetrahydrofuran-2-yl)-5-methyl-2-oxo-1,2-
dihydropyrimidin-4-ylazanediyl)bis(propane-3,1-diyl)dicarbamate
(13d)
13d was synthesized from diamine (0.4 g, 0.72 mmol) and
hexanoic acid (9d) (176 mg, 1.5 mmol) using HOBt, EDC.HCl, and
DIPEA by following the general procedure C as a white solid 500 mg
(93%). 1H NMR (500 MHz, CDCl3)
d 7.70 (brs, 1H), 7.35 (s, 1H), 7.23
(brs, 1H), 6.15 (t, J ¼ 6.2 Hz, 1H), 5.05 (brs, 2H), 4.22e4.12 (m, 1H),
3.98e3.88 (m, 1H), 3.63e3.48 (m, 6H), 3.21e3.12 (m, 4H),
2.68e2.53 (m, 1H), 2.26e2.17 (m, 8H), 1.84e1.74 (m, 4H), 1.67e1.57
(m, 4H), 1.41 (s, 18H), 1.32e1.24 (m, 8H), 0.86 (t, J ¼ 6.8 Hz, 6H). 13
C
NMR (100 MHz, CDCl3) d 174.9, 174.1, 164.3, 156.2, 154.4, 141.3, 103.1,
85.9, 84.0, 79.4, 51.3, 47.6, 41.6, 38.1, 37.3, 36.8, 36.3, 31.6, 31.6, 29.2,
28.5, 25.5, 22.5, 18.9, 14.1. MS (MALDI-TOF): calcd for C38H67N7O8:
749.5; found 772.5 (M þ Na)þ.
(500 MHz, CDCl3) d 8.07e7.87 (m, 3H), 7.84e7.79 (m, 1H), 7.76e7.72
(m, 2H), 7.67e7.63 (m, 1H), 7.46e7.42 (m, 2H),7.40e7.33 (m, 4H),
7.31e7.27 (m, 3H), 7.11e6.99 (m, 1H), 6.02 (t, J ¼ 6.2 Hz, 1H), 4.82
(brs, 2H), 4.02e3.93 (m, 3H), 3.92e3.80 (m, 2H), 3.76 (q, J ¼ 5.4 Hz,
1H), 3.55e3.48 (m, 1H), 3.46e3.33 (m, 5H), 3.01e2.92 (m, 4H),
2.49e2.37 (m, 1H), 2.13e2.03 (m, 1H), 2.01 (s, 3H), 1.63 (p,
4.8.5. Synthesis of tert-butyl 3,3’-(5-methyl-2-oxo-1-((2R,4S,5R)-4-
(1-adamanataneacetamido)-5-((4-1-adamanataneacetamido)
methyl)tetrahydrofuran-2-yl)-1,2-dihydropyrimidin-4-ylazanediyl)
bis(propane-3,1-diyl)dicarbamate (13e)
J ¼ 6.8 Hz, 4H), 1.39 (s, 18H). 13C NMR (100 MHz, CDCl3)
d
172.1,
13e was synthesized from diamine (0.9 g, 1.6 mmol) and 1-
adamanataneacetic acid (9e) (670 mg, 3.4 mmol) using HOBt,
EDC.HCl and DIPEA by following the general procedure C as a white
171.7, 164.4, 156.1, 154.6, 141.6, 133.9, 133.9, 132.3, 132.2, 131.7, 131.5,
128.8, 128.7, 128.4, 128.3, 128.1, 128.0, 126.5, 126.3, 126.0, 125.9,
125.7, 124.1, 102.9, 86.4, 84.0, 79.4, 53.5, 51.5, 47.3, 41.8, 41.4, 40.7,
38.0, 37.3, 29.0, 28.5, 18.6. MS (MALDI-TOF): calcd for C50H63N7O8:
889.4; found 912.4 (M þ Na)þ.
solid 1.2 g (82%). 1H NMR (500 MHz, CDCl3)
d 7.46 (brs, 1H), 7.34 (s,
1H), 7.18 (brs, 1H), 6.25e6.12 (m, 1H), 5.07 (brs, 2H), 4.19e4.11 (m,
1H), 4.00e3.93 (m, 1H), 3.64e3.54 (m, 5H), 3.46e3.39 (m, 1H),
3.15e3.07 (m, 4H), 2.20e2.12 (m, 4H), 2.01e1.96 (m, 3H), 1.95e1.91
(m, 6H), 1.82e1.75 (m, 4H), 1.69e1.54 (m, 26H), 1.42 (s, 18H). 13C
NMR (100 MHz, CDCl3) d 172.6,171.8,164.4,156.3,154.8,141.0,103.1,
4.8.2. Synthesis of tert-butyl 3,3’-(5-methyl-2-oxo-1-((2R,4S,5R)-4-
(4-phenylbutanamido)-5-((4-phenylbutanamido)methyl)
tetrahydrofuran-2-yl)-1,2-dihydropyrimidin-4-ylazanediyl)
bis(propane-3,1-diyl)dicarbamate (13b)
85.7, 84.4, 79.4, 51.8, 51.7, 50.8, 47.5, 42.7, 42.0, 38.1, 37.3, 36.9, 36.8,
33.0, 32.9, 29.2, 28.8, 28.7, 28.5, 19.0. MS (MALDI-TOF): calcd for
C
50H79N7O8: 905.6; found 928.5 (M þ Na)þ.
13b was synthesized from diamine (0.6 g, 1.08 mmol) and 4-
phenylbutyric acid (9b) (374 mg, 2.27 mmol) using HOBt,
EDC.HCl, and DIPEA by following the general procedure C as a white
4.9. Synthesis of ADL 3(aee)
General procedure D
solid 900 mg (94%). 1H NMR (500 MHz, CDCl3)
d 7.32 (s, 1H),
7.25e7.19 (m, 5H), 7.17e7.12 (m, 6H), 6.15 (t, J ¼ 6.2 Hz, 1H), 4.99
(brs, 2H), 4.21e4.11 (m, 1H), 3.96e3.89 (m, 1H), 3.62e3.46 (m, 6H),
3.17e3.0 (m, 4H), 2.65e2.58 (m, 4H), 2.30e2.24 (m, 4H), 2.21e2.12
(m, 4H), 1.99e1.91 (m, 4H), 1.80e1.72 (m, 4H), 1.42 (s, 18H). 13C NMR
4.9.1. Synthesis of ADL-3a
Boc-protected intermediate 13a (100 mg, 0.1123 mmol) was
dissolved in 1.25M HCl in MeOH (3 mL) and stirred at 50 ꢀC for 1 h.
The reaction mixture was concentrated under reduced pressure.
The crude was washed with 10% ethyl acetate in hexane and pu-
rified by RP-HPLC to get the product ADL-3a as white solid (70 mg,
(100 MHz, CDCl3)
d 174.4, 173.6, 164.4, 156.2, 154.6, 141.8, 141.6,
141.2, 128.6, 128.5, 128.5, 128.4, 126.1, 126.0, 103.1, 85.9, 84.0, 79.4,
51.5, 47.5, 41.8, 38.1, 37.2, 36.2, 35.7, 35.4, 35.4, 29.2, 28.5, 27.3, 27.3,
18.9. MS (MALDI-TOF): calcd for C46H67N7O8: 845.5; found 868.4
(M þ Na)þ.
90%). 1H NMR (900 MHz, DMSO‑d6)
d
8.76 (d, J ¼ 7.1 Hz, 1H), 8.42 (t,
J ¼ 5.8 Hz, 1H), 8.07 (d, J ¼ 8.3 Hz, 1H), 8.03 (d, J ¼ 8.4 Hz, 1H),
7.92e7.88 (m, 6H), 7.82 (d, J ¼ 7.9 Hz, 1H), 7.80e7.79 (m, 1H),
7.54e7.51 (m, 2H), 7.50e7.48 (m, 1H), 7.46e7.38 (m, 6H), 6.21 (t,
J ¼ 6.7 Hz,1H), 4.26e4.24 (m,1H), 3.93e3.87 (m, 6H), 3.53e3.45 (m,
4H), 3.41e3.38 (m, 2H), 2.90 (brs, 1H), 2.82e2.77 (m, 4H), 2.21e2.15
(m, 2H), 1.96 (s, 3H), 1.85e1.81 (m, 4H). 13C NMR (225 MHz,
4.8.3. Synthesis oftert-butyl 3,3’-(1-((2R,4S,5R)-4-
(cyclohexanecarboxamido)-5-(cyclohexanecarboxamidomethyl)
tetrahydrofuran-2-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-
ylazanediyl)bis(propane-3,1-diyl)dicarbamate (13c)
DMSO‑d6)
d 170.4, 170.2, 163.9, 158.2, 153.5, 142.0, 133.3, 133.2,
13c was synthesized from diamine (0.5 g, 0.9 mmol) and
cyclohexanecarboxylic acid (9c) (243 mg, 1.9 mmol) using HOBt,
EDC.HCl, and DIPEA by following the general procedure C as a white
132.7, 132.5, 131.9, 128.3, 128.2, 127.7, 127.7, 127.1, 127.0, 126.0, 125.7,
125.6, 125.5, 125.5, 125.4, 124.2, 124.1, 116.5, 102.1, 84.8, 82.8, 50.9,
45.7, 41.6, 36.7, 36.4, 26.2, 23.9,17.2,12.4. MS (MALDI-TOF): calcd for
solid 650 mg (93%). 1H NMR (500 MHz, CDCl3)
d
7.31 (s, 1H), 7.03
C
40H47N7O4: 689.3, found 690.3 (M þ H)þ.
(brs, 1H), 6.11 (t, J ¼ 6.0 Hz, 1H), 5.09 (brs, 2H), 4.22e4.11 (m, 1H),
3.88 (q, J ¼ 5.6 Hz, 1H), 3.64e3.49 (m, 6H), 3.16e3.04 (m, 4H),
2.82e2.66 (m, 1H), 2.62e2.49 (m, 1H), 2.22e2.09 (m, 6H), 1.86e1.72
(m, 12H), 1.67e1.61 (m, 2H), 1.47e1.37 (m, 23 H), 1.29e1.16 (m, 6H).
4.9.2. Synthesis of ADL-3b
ADL-3b was synthesized from 13b (150 mg, 0.193 mmol) using
1.25M HCl in MeOH by following general procedure D as a white
13C NMR (100 MHz, CDCl3):
d 177.8, 176.9, 164.5, 156.3, 154.7, 141.1,
102.9, 85.4, 83.9, 79.4, 51.0, 47.5, 45.6, 45.1, 41.3, 38.1, 37.5, 29.8, 29.7,
29.7, 29.5, 29.2, 25.8, 25.8, 25.8, 25.7, 18.9. MS (MALDI-TOF): calcd
for C40H67N7O8: 773.5; found 796.5 (M þ Na)þ.
solid (90 mg, 79%). 1H NMR (900 MHz, CDCl3)
1H), 8.05e8.00 (m, 1H), 7.90 (brs, 6H), 7.63 (s, 1H), 7.29e7.22 (m,
2H), 7.19e7.12 (m, 6H), 6.10 (t, J ¼ 6.5 Hz, 1H), 4.23e4.18 (m, 1H),
d
8.29 (d, J ¼ 7.3 Hz,

S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
11
3.79e3.76 (m, 1H), 3.55e3.47 (m, 3H), 3.38e3.33 (m, 2H),
2.84e2.76 (m, 3H), 2.56e2.51 (m, 4H), 2.17 (s, 3H), 2.16e2.12 (m,
1H), 2.11e2.08 (m, 4H), 1.96e1.92 (m, 1H), 1.86e1.82 (m, 3H),
vacuum, dissolved in 1.25M HCl in MeOH (8 mL), and stirred for
1 h at 0 ꢀC. After that cold ethyl acetate (20 mL) was added. The
white precipitate was filtered off and purified by RP-HPLC to give
the final compound ADG-2a as a white solid (67.3 mg, 67%). 1H NMR
1.80e1.75 (m, 4H). 13C NMR (225 MHz, DMSO‑d6)
d 172.1, 171.9,
164.0, 158.2, 153.5, 142.0, 141.7, 141.7, 128.3, 128.2, 128.2, 128.2,
125.7, 125.7, 116.6, 102.1, 84.7, 82.9, 50.4, 45.8, 39.8, 36.4, 34.8, 34.7,
34.5, 34.5, 27.0, 27.0, 26.2, 17.5, 12.6. MS (MALDI-TOF): calcd for
(900 MHz, DMSO‑d6)
d
8.78 (d, J ¼ 7.2 Hz,1H), 8.43 (t, J ¼ 5.8 Hz,1H),
8.39 (brs, 2H), 8.07 (d, J ¼ 8.3 Hz,1H), 8.01 (d, J ¼ 8.4 Hz,1H), 7.92 (d,
J ¼ 7.9 Hz,1H), 7.87 (d, J ¼ 8.0 Hz,1H), 7.82 (d, J ¼ 7.8 Hz,1H), 7.79 (d,
J ¼ 7.8 Hz, 1H), 7.54e7.48 (m, 3H), 7.46e7.37 (m, 8H), 7.29 (brs, 5H),
6.24 (t, J ¼ 6.7 Hz, 1H), 4.25e4.22 (m, 1H), 3.94e3.86 (m, 5H),
3.50e3.41 (m, 5H), 3.40e3.36 (m, 1H), 3.16e3.07 (m, 4H), 2.23e2.14
(m, 2H), 1.87 (s, 3H), 1.85e1.76 (m, 4H). 13C NMR (225 MHz,
C
36H51N7O4: 645.4, found 646.4 (M þ H)þ.
4.9.3. Synthesis of ADL-3c
ADL-3c was synthesized from 13c (150 mg, 0.193 mmol) using
1.25M HCl in MeOH by following general procedure D as a white
DMSO‑d6) d 170.4,170.2,162.9,158.4,156.9,154.4,141.7,133.3,133.2,
132.7, 132.5, 131.9, 131.9, 128.3, 128.2, 127.8, 127.7, 127.1, 127.0, 126.0,
125.7, 125.6, 125.5, 125.5, 125.4, 124.3, 124.1, 116.5, 102.9, 84.8, 83.0,
50.9, 46.9, 41.5, 39.9, 39.8, 38.9, 36.7, 27.5, 17.1. MS (MALDI-TOF):
calcd for C42H51N11O4: 773.4, found 774.5 (M þ H)þ.
solid (110 mg, 99%). 1H NMR (900 MHz, DMSO‑d6)
d 8.16 (d,
J ¼ 7.3 Hz, 1H), 7.91 (brs, 6H), 7.88e7.86 (m, 1H), 7.60 (s, 1H), 6.10 (t,
J ¼ 6.5 Hz, 1H), 4.19e4.17 (m, 1H), 3.75e3.72 (m, 1H), 3.55e3.49 (m,
4H), 3.42 (brs, 1H), 3.33e3.29 (m, 2H), 2.90 (brs, 1H), 2.83e2.77 (m,
4H), 2.18 (s, 3H), 2.12e2.07 (m, 4H), 1.97e1.93 (m, 1H), 1.86e1.83
(m, 4H), 1.71e1.63 (m, 10H), 1.33e1.28 (m, 4H), 1.23e1.16 (m, 5H).
4.10.2. Synthesis of ADG-2b
13C NMR (225 MHz, DMSO‑d6)
d
175.4, 175.3, 164.0, 158.2, 153.5,
ADG-2b was synthesized from ADL-3b (150 mg, 0.23 mmol) and
1H-Pyrazole-1-carboxamidine hydrochloride (102 mg, 0.69 mmol)
by following general procedure E as a white solid (114 mg, 67%). 1H
116.6, 102.1, 84.7, 82.9, 50.3, 45.8, 43.9, 43.8, 41.0, 39.9, 36.9, 36.4,
36.2, 29.2, 29.1, 29.1, 29.0, 26.2, 25.4, 25.4, 25.2, 25.2, 25.2, 23.9,17.5,
12.6. MS (MALDI-TOF): calcd for C30H51N7O4: 573.4, found 574.4
(M þ H)þ.
NMR (900 MHz, DMSO‑d6)
d
8.39 (brs, 2H), 8.29 (d, J ¼ 7.2 Hz, 1H),
8.03 (t, J ¼ 5.8 Hz, 1H), 7.67 (s, 1H), 7.40 (brs, 4H), 7.27 (t, J ¼ 7.5 Hz,
2H), 7.24 (t, J ¼ 7.4 Hz, 2H), 7.19e7.11 (m, 6H), 6.13 (t, J ¼ 6.4 Hz, 1H),
4.21e4.17 (m, 1H), 3.81e3.76 (m, 1H), 3.55e3.45 (m, 4H), 3.40e3.32
(m, 2H), 3.17e3.08 (m, 4H), 2.55 (t, J ¼ 7.6 Hz, 2H), 2.51 (t, J ¼ 7.6 Hz,
4.9.4. Synthesis of ADL-3d
ADL-3d was synthesized from 13d (95 mg, 0.126 mmol) using
1.25M HCl in MeOH by following general procedure D as a white
2H), 2.21e2.13 (m, 5H), 2.12e2.06 (m, 4H), 1.88e1.73 (m, 8H). 13
C
NMR (225 MHz, DMSO‑d6)
d 172.1, 171.9, 163.0, 158.5, 156.9, 154.4,
solid (59 mg, 85%). 1H NMR (800 MHz, DMSO‑d6)
d
8.25 (d,
141.8, 141.7, 141.7, 128.3, 128.2, 128.2, 128.2, 125.7, 125.7, 116.5, 102.9,
84.8, 83.0, 50.4, 46.9, 41.2, 39.9, 39.8, 38.8, 36.8, 34.8, 34.6, 34.5,
34.5, 27.5, 27.0, 26.9, 17.5. MS (MALDI-TOF): calcd for C38H55N11O4:
729.4, found 730.4 (M þ H)þ.
J ¼ 7.2 Hz,1H), 7.99e7.93 (m, 5H), 7.61 (s, 1H), 6.12 (t, J ¼ 6.7 Hz,1H),
4.20e4.16 (m, 1H), 3.78e3.74 (m, 1H), 3.43 (brs, 2H), 3.36e3.30 (m,
2H), 2.99 (brs, 2H), 2.91 (brs, 4H), 2.16e2.12 (m, 2H), 2.06 (q,
J ¼ 7.4 Hz, 4H), 1.97e1.94 (m, 2H), 1.93 (s, 3H), 1.85 (p, J ¼ 6.8 Hz,
2H), 1.51e1.45 (m, 4H), 1.29e1.17 (m, 9H), 0.85 (t, J ¼ 7.2 Hz, 3H),
4.10.3. Synthesis of ADG-2c
0.82 (t, J ¼ 7.2 Hz, 3H). 13C NMR (200 MHz, DMSO‑d6)
d 172.4, 172.3,
158.4, 138.1, 116.7, 102.4, 84.7, 82.8, 50.4, 44.4, 43.9, 41.2, 36.8, 36.2,
35.3, 35.2, 30.8, 30.8, 25.6, 24.9, 24.8, 23.9, 21.8, 21.8, 13.8, 13.8, 12.6.
ADG-2c was synthesized from ADL-3c (100 mg, 0.174 mmol) and
1H-Pyrazole-1-carboxamidine
0.522 mmol) by following general procedure E as a white solid
(114 mg, 77%). 1H NMR (900 MHz, DMSO‑d6)
8.38 (brs, 2H), 8.15 (d,
hydrochloride
(76.4
mg,
MS (MALDI-TOF): calcd for
C28H51N7O4: 549.4, found 550.4
(M þ H)þ.
d
J ¼ 7.3 Hz, 1H), 7.87 (t, J ¼ 5.8 Hz, 1H), 7.63 (s, 1H), 7.32 (brs, 8H), 6.11
(t, J ¼ 6.5 Hz, 1H), 4.19e4.15 (m, 1H), 3.76e3.74 (m, 1H), 3.53e3.49
(m, 4H), 3.34e3.29 (m, 2H), 3.16e3.11 (m, 4H), 2.19 (s, 3H), 2.14 (t,
J ¼ 6.6 Hz, 2H), 2.11e2.07 (m, 2H), 1.82 (p, J ¼ 6.8 Hz, 4H), 1.71e1.58
(m, 10H), 1.34e1.27 (m, 4H), 1.23e1.10 (m, 6H). 13C NMR (225 MHz,
4.9.5. Synthesis of ADL-3e
ADL-3e was synthesized from 13e (150 mg, 0.1657 mmol) using
1.25M HCl in MeOH by following general procedure D as a white
solid (110 mg, 91%). 1H NMR (800 MHz, DMSO‑d6)
d
8.21 (d,
DMSO‑d6) d 175.4, 175.3, 163.0, 158.5, 156.9, 154.4, 141.7, 102.9, 84.7,
J ¼ 7.2 Hz,1H), 7.92e7.88 (m, 5H), 7.64 (s, 1H), 6.12 (t, J ¼ 6.6 Hz,1H),
4.19e4.16 (m, 1H), 3.79e3.77 (m, 1H), 3.55e3.49 (m, 4H), 3.40e3.36
(m, 1H), 3.30e3.27 (m, 1H), 2.83e2.78 (m, 4H), 2.19 (s, 3H),
2.16e2.12 (m, 2H), 1.91e1.89 (m, 4H), 1.86e1.84 (m, 6H), 1.66e1.62
83.1, 50.2, 47.0, 43.9, 43.8, 41.0, 39.9, 39.8, 38.8, 36.9, 29.2, 29.2, 29.1,
29.0, 27.6, 25.4, 25.4, 25.2, 25.2, 25.2, 17.6. MS (MALDI-TOF): calcd
for C32H55N11O4: 657.4, found 658.4 (M þ H)þ.
(m, 6H), 1.58e1.52 (m, 22H). 13C NMR (200 MHz, DMSO‑d6)
d
170.1,
4.10.4. Synthesis of ADG-2d
170.0, 158.2, 153.5, 142.1, 116.6, 102.0, 84.8, 83.0, 50.5, 50.0, 49.7,
45.7, 42.1, 42.0, 39.8, 36.4, 32.3, 32.2, 28.0, 26.2, 17.5, 12.7. MALDI-
TOF): calcd for C40H63N7O4: 705.5, found 706.5 (M þ H)þ.
ADG-2d was synthesized from ADL-3d (109 mg, 0.2 mmol) and
1H-Pyrazole-1-carboxamidine hydrochloride (92 mg, 0.6 mmol) by
following general procedure E as a white solid (128 mg, 76%). 1H
4.10. Synthesis of ADG-2(aee)
General procedure E
NMR (800 MHz, DMSO‑d6)
d
8.39 (brs, 2H), 8.26 (d, J ¼ 7.2 Hz, 1H),
7.97 (t, J ¼ 5.8 Hz, 1H), 7.65 (s, 1H), 7.36 (brs, 8H), 6.12 (t, J ¼ 6.5 Hz,
1H), 4.19e4.16 (m, 1H), 3.79e3.76 (m, 1H), 3.54e3.49 (m, 4H),
3.36e3.32 (m, 2H), 3.14 (q, J ¼ 5.8 Hz,4H), 2.19 (s, 3H), 2.18e2.14 (m,
2H), 2.07 (q, J ¼ 7.2 Hz,4H),1.82 (p, J ¼ 6.8 Hz,4H),1.51e1.45 (m, 4H),
4.10.1. Synthesis of ADG-2a
1.28e1.17 (m, 8H), 0.85 (t, J ¼ 7.2 Hz, 3H), 0.82 (t, J ¼ 7.2 Hz, 3H). 13
C
1H-Pyrazole-1-carboxamidine
hydrochloride
(38
mg,
NMR (200 MHz, DMSO‑d6) d 172.4, 172.3, 163.0, 158.5, 156.9, 154.4,
0.26 mmol) was added to a solution of ADL-3a (60 mg, 0.087 mmol)
and DIPEA (0.09 mL, 0.52 mmol) in dimethylformamide (1 mL), and
stirred for overnight. The reaction mixture was concentrated under
141.8, 116.9, 102.9, 84.8, 84.3, 50.4, 47.0, 41.2, 39.9, 39.8, 39.7, 38.8,
36.9, 35.3, 35.2, 30.8, 27.6, 24.9, 24.8, 21.8, 21.8, 17.5, 13.8, 13.8. MS
(MALDI-TOF): calcd for C30H55N11O4: 633.4, found 634.5 (M þ H)þ.

12
S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
4.10.5. Synthesis of ADG-2e
cell viability was normalized to the control group.
ADG-2e was synthesized from ADL-3e (120 mg, 0.17 mmol) and
1H-Pyrazole-1-carboxamidine hydrochloride (75 mg, 0.51 mmol)
by following general procedure E as a white solid (103 mg, 77%). 1H
4.11.3.2. Wound healing and migration assays
MDA-MB-231 cell was cultured on 24 well cell culture plates for
18 h. The confluent monolayer cells were scratched with a yellow
tip, and each well was washed with PBS to remove nonadherent
NMR (800 MHz, DMSO‑d6)
d
8.38 (brs, 2H), 8.21 (d, J ¼ 7.1 Hz, 1H),
7.89 (t, J ¼ 5.8 Hz, 1H), 7.68 (s, 1H), 7.35 (brs, 6H), 6.14 (t, J ¼ 6.6 Hz,
1H), 4.18e4.14 (m, 1H), 3.81e3.78 (m, 1H), 3.54e3.48 (m, 4H),
3.44e3.40 (m, 1H), 3.30e3.25 (m, 1H), 3.14 (q, J ¼ 5.8 Hz, 4H), 2.20
(s, 3H), 2.19e2.14 (m, 2H), 1.91e1.81 (m, 13H), 1.66e1.50 (m, 25H).
cells. The cells were treated with ADG-2e (20, 40 mM) and then
incubated for up to 48 h. The perimeter of the area with a central
cell-free gap was confirmed at the time intervals 0, and 48 h under
a Cellomics TM microscope (Thermo Fisher Sci., Waltham, MA,
USA).
13C NMR (200 MHz, DMSO‑d6)
d 170.1, 170.0, 163.0, 158.4, 156.9,
154.4, 141.9, 116.4, 102.9, 84.8, 83.1, 50.6, 50.0, 49.7, 47.0, 42.1, 42.0,
41.2, 39.9, 39.8, 39.7, 39.6, 38.8, 36.7, 36.4, 32.3, 32.2, 28.0, 17.6. MS
(MALDI-TOF): calcd for C42H67N11O4: 789.5, found 790.5 (M þ H)þ.
4.11.3.3. Transwell chamber invasion assay
A Transwell permeable support system with polycarbonate
translucent inserts with a pore size of 8.0 mm was used for the cell
4.11. Biological test methods
invasion assay. MDA-MB-231 cells (1 ꢁ 105 cells/well) were incu-
bated in the Matrigel-coated upper chamber with ADG-2e in 300 mL
4.11.1. Antibacterial activity
of serum-free medium for 24 h, whereas the lower chamber was
loaded with DMEM medium containing serum to attract invasive
cells. After removal of the noninvasive cells, the invasive cells were
fixed in 4% paraformaldehyde for 15 min, stained with 0.2% crystal
violet, and captured by a microscope at a 100 ꢁ magnification.
The bacterial strains Escherichia coli (KCTC 1682) and Pseudo-
monas aeruginosa (KCTC 1637), Bacillus subtilis (KCTC 3068),
Staphylococcus aureus (KCTC 1621), three methicillin-resistant
Staphylococcus aureus (CCARM 3089, 3090 and 3095) and two
multidrug-resistant Pseudomonas aeruginosa (CCARM 2095 and
2109) were employed in the experiment. The antimicrobial activ-
ities of the small molecule compounds were determined by the
broth microdilution method as described in the previously re-
ported paper [44]. Briefly, the grown bacteria to mid-logarithmic
phase in LB broth were diluted to 2 ꢁ 106 CFU/mL in 1% peptone
and added to serial two-fold dilutions of different small molecule
4.11.3.4. Time-lapse cell tracking analysis
MDA-MB-231 cells (6 ꢁ 103 cells/100
mL) were seeded in m-Slide
I, ibiTreat (ibidi, Martinsried, Germany) and incubated for the
adherence of the cells. After 1 h, 500 mL of DMEM medium was
added into both reservoirs and incubated overnight. ADG-2e with
the indicated concentrations was treated and directly transferred to
the HoloMonitor M4 time-lapse cytometer (Phase Holographic
Imaging) kept in a 37 ꢀC incubator. Live cells were captured every
15 min for 12 h, and the migration was analyzed with the Holo-
Studio M4 software. Migration is the value indicating the shortest
distance between the starting point of cell tracking and the current
point. Motility represents how far the cells have moved during a
total analysis time. Motility speed is the total cell movement per
hour.
compounds ranging from 1 to 64 mg/mL. Subsequently, these plates
were incubated at 37 ꢀC for 18e22 h in a shaking incubator. The
minimum inhibitory concentration (MIC) was confirmed by the
absence of bacterial growth by visually and measured by spectro-
photometrically by OD600 readings recorded by a microplate ELISA
reader (Molecular Devices, Sunnyvale, CA, USA). All the measure-
ments were performed using three independent measurements.
4.11.2. Hemolytic activity
4.11.3.5. Immunostaining and analysis
MDA-MB-231 cells (6 ꢁ 103 cells/100
mL) were seeded in 13 mm
Hemolytic activity was performed by following the protocol
described in our previous article [29]. Using phosphate-buffered
saline (PBS), the Sheep red blood cells (sRBCs) were washed three
times and centrifuged at 1000 rpm for 5 min and diluted to a
cover glass which was coated with poly-l-L lysine (Merck, Darm-
stadt, Germany) and incubated for the adherence of the cells. After
6 h or 9 h, cells were fixed and then stained F-actin with Texas Red-
Phalloidin (Cell signaling Technology, Danvers, MA, USA). Confocal
images were acquired using a Zeiss LSM 710 system mounted on a
Zeiss Axiovert 100M microscope (Carl Zeiss MicroImaging). The
images of unsaturated fluorescence signals were acquired with the
same laser intensity at 512 ꢁ 512 pixels and 12- bit resolution.
Fluorescence image for an entire cell was determined after sub-
traction of the background signal intensity using Zeiss AIM confocal
software.
concentration of 4% in PBS. Further, 4% sRBC (100
molecule compounds (100 L) were added simultaneously to each
well of a 96-well plate, and incubated at 37 ꢀC for 2 h. After the plate
L) from
mL) and small
m
was centrifuged at 120ꢁg for 5 min, the supernatant (100
m
each well was transferred to a new 96-well plate and measured by a
microplate ELISA reader (Molecular Devices, Sunnyvale, CA, USA) at
414 nm. Zero and 100% hemolysis were determined in PBS and 0.1%
Triton-X 100, respectively.
4.11.3. Cancer metastasis
4.11.3.6. 4.11.3.6 Statistical analysis
Values are presented as mean standard deviation (SD) of three
or more experiments. Data were analyzed using Student’s t-test or
one-way analysis of variance for comparisons between two mean
values. A value of P < 0.05 was considered significant.
4.11.3.1. Cell viability assay
MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum, 100
units/mL penicillin, and 100 m
g/mL streptomycin at 37 ꢀC with 5%
CO2 in a humidified atmosphere. Each cell (4 ꢁ 103 cells/well) were
seeded in 96-well cell culture plates overnight and treated in
triplicate with various concentrations of ADG-2e. After 48 h of in-
Declaration of competing interest
cubation, 10
m
L of EZCytox cell viability assay solution (Daeil Lab
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.
service) was directly added and incubated at 37 ꢀC for 2 h. The
absorbance was measured using a microplate reader at 450 nm. The

S. Chirumarry et al. / European Journal of Medicinal Chemistry 193 (2020) 112233
ꢁ ꢁ
13
ꢁ
ꢀ
ꢂ ꢁ
ꢀ
J. Pospísil, O. Benada, T. Krízek, D. Sedlak, P. Bartunek, L. Krasný, D. Rejman,
Lipophosphonoxins II: design, synthesis, and properties of novel broad
spectrum antibacterial agents, J. Med. Chem. 60 (2017) 6098e6118.
[21] K. Campbell, F. Rossi, J. Adams, I. Pitsidianaki, F.M. Barriga, L. Garcia-Gerique,
E. Batlle, J. Casanova, A. Casali, Collective cell migration and metastases
induced by an epithelial-to-mesenchymal transition in Drosophila intestinal
tumors, Nat. Commun. 10 (2019) 2311.
Acknowledgments
This work was supported by the R&D Convergence Program
(CAP-16-03-KRIBB, J.K.B), the KRIBB Research Initiative Program
and the National Research Foundation of Korea (NRF) grant funded
by the Korea government (MSIT) (2020R1A2C1009289 to J. K. B and
2018R1A2B6003250 to S.Y.S.)
[22] T.N. Seyfried, L.C. Huysentruyt, On the origin of cancer metastasis, Crit. Rev.
Oncol. 18 (2013) 43e73.
[23] A. Diz-Munoz, D.A. Fletcher, O.D. Weiner, Use the force: membrane tension as
~
an organizer of cell shape and motility, Trends Cell Biol. 23 (2013) 47e53.
[24] M.M. Zegers, P. Friedl, Translating membrane tension into cytoskeletal action
by FBP17, Dev. Cell 33 (2015) 628e630.
Appendix A. Supplementary data
[25] T. Masuda, K. Baba, T. Nomura, K. Tsujita, T. Murayama, T. Itoh, T. Takatani-
Nakase, M. Sokabe, N. Inagaki, S. Futaki, An influenza-derived membrane
tension-modulating peptide regulates cell movement and morphology via
actin remodeling, Commun. Biol. 2 (2019) 243.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112233.
[26] P. Gunasekaran, m. Fan, E.Y. Kim, J.H. Shin, J.E. Lee, E.J. Son, J. Kim, E. Hwang,
M.S. Yim, E.-H. Kim, Y.-J. Choi, Y.-H. Lee, Y.-H. Chung, H.N. Kim, E.K. Ryu,
S.Y. Shin, E.-K. Kim, J.K. Bang, Amphiphilic triazine polymer derivatives as
antibacterial and anti-atopic agents in mice model, Sci. Rep. 9 (2019) 15161.
[27] M. Ahn, B. Jacob, P. Gunasekaran, R.N. Murugan, E.K. Ryu, G.-H. Lee, J.-K. Hyun,
C. Cheong, N.-H. Kim, S.Y. Shin, J.K. Bang, Poly-lysine peptidomimetics having
potent antimicrobial activity without hemolytic activity, Amino Acids 46
(2014) 2259e2269.
[28] R.N. Murugan, B. Jacob, E.-H. Kim, M. Ahn, H. Sohn, J.-H. Seo, C. Cheong, J.-
K. Hyun, K.S. Lee, S.Y. Shin, J.K. Bang, Non hemolytic short peptidomimetics as
a new class of potent and broad-spectrum antimicrobial agents, Bioorg. Med.
Chem. Lett 23 (2013) 4633e4636.
[29] M. Ahn, P. Gunasekaran, G. Rajasekaran, E.Y. Kim, S.-J. Lee, G. Bang, K. Cho, J.-
K. Hyun, H.-J. Lee, Y.H. Jeon, N.H. Kim, E.K. Ryu, S.Y. Shin, J.K. Bang, Pyrazole
derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm
activity, Eur. J. Med. Chem. 125 (2017) 551e564.
[30] A. Kolocouris, K. Dimas, C. Pannecouque, M. Witvrouw, G.B. Foscolos,
G. Stamatiou, G. Fytas, G. Zoidis, N. Kolocouris, G. Andrei, R. Snoeck, E. De
Clercq, New 2-(1-adamantylcarbonyl) pyridine and 1-acetyladamantane thi-
osemicarbazonesethiocarbonohydrazones: cell growth inhibitory, antiviral
and antimicrobial activity evaluation, Bioorg. Med. Chem. Lett 12 (2002)
723e727.
References
[1] B. Ding, Q. Guan, J.P. Walsh, J.S. Boswell, T.W. Winter, E.S. Winter, S.S. Boyd,
C. Li, P.B. Savage, Correlation of the antibacterial activities of cationic peptide
antibiotics and cationic steroid antibiotics, J. Med. Chem. 45 (2002) 663e669.
[2] P.B. Savage, C. Li, U. Taotafa, B. Ding, Q. Guan, Antibacterial properties of
cationic steroid antibiotics, FEMS Microbiol. Lett. 217 (2002) 1e7.
[3] B. Ding, U. Taotofa, T. Orsak, M. Chadwell, P.B. Savage, Synthesis and charac-
terization of peptideꢂcationic steroid antibiotic conjugates, Org. Lett. 6 (2004)
3433e3436.
[4] P. Gunasekaran, G. Rajasekaran, E.H. Han, Y.-H. Chung, Y.-J. Choi, Y.J. Yang,
J.E. Lee, H.N. Kim, K. Lee, J.S Kim, H.J. Lee, E.J. Choi, E.-K. Kim, S.Y. Shin,
J.K. Bang, Cationic amphipathic triazines with potent anti-bacterial, anti-in-
flammatory and anti-atopic dermatitis properties, Sci. Rep. 9 (2019) 1292.
[5] K. Matsuzaki, Why and how are peptideelipid interactions utilized for self-
defense? Magainins and tachyplesins as archetypes, BBA-Biomembranes
1462 (1999) 1e10.
[6] A.K. Marr, W.J. Gooderham, R.E. Hancock, Antibacterial peptides for thera-
peutic use: obstacles and realistic outlook, Curr. Opin. Pharmacol. 6 (2006)
468e472.
[7] R.E. Hancock, K.L. Brown, N. Mookherjee, Host defence peptides from
invertebrateseemerging antimicrobial strategies, Immunobiology 211 (2006)
315e322.
[8] J.A. Jones, K.G. Virga, G. Gumina, K.E. Hevener, Recent advances in the rational
design and optimization of antibacterial agents, MedChemComm 7 (2016)
1694e1715.
[9] C. Ghosh, J. Haldar, Membrane-active small molecules: designs inspired by
antimicrobial peptides, ChemMedChem 10 (2015) 1606e1624.
[10] L.D. Andrews, T.R. Kane, P. Dozzo, C.M. Haglund, D.J. Hilderbrandt, M.S. Linsell,
T. Machajewski, G. McEnroe, A.W. Serio, K.B. Wlasichuk, D.B. Neau,
S. Pakhomova, G.L. Waldrop, M. Sharp, J. Pogliano, R.T. Cirz, F. Cohen, Opti-
mization and mechanistic characterization of pyridopyrimidine inhibitors of
bacterial biotin carboxylase, J. Med. Chem. 62 (2019) 7489e7505.
[11] K. Pyta, A. Janas, M. Szukowska, P. Pecyna, M. Jaworska, M. Gajecka, F. Bartl,
P. Przybylski, Synthesis, docking and antibacterial studies of more potent
amine and hydrazone rifamycin congeners than rifampicin, Eur. J. Med. Chem.
167 (2019) 96e104.
[12] A. Hammad, N.S. Abutaleb, M.M. Elsebaei, A.B. Norvil, M. Alswah, A.O. Ali,
J.A. Abdel-Aleem, A. Alattar, S.A. Bayoumi, H. Gowher, M.N. Seleem,
A.S. Mayhoub, From phenylthiazoles to phenylpyrazoles: broadening the
antibacterial spectrum toward carbapenem-resistant bacteria, J. Med. Chem.
62 (2019) 7998e8010.
[31] O. Kouatly, A. Geronikaki, C. Kamoutsis, D. Hadjipavlou-Litina, P. Eleftheriou,
Adamantane derivatives of thiazolyl-N-substituted amide, as possible non-
steroidal anti-inflammatory agents, Eur. J. Med. Chem. 44 (2009) 1198e1204.
~
[32] M.H.S. Ramada, G.D. Brand, F.Y. Abrao, M. Oliveira, J.L.C. Filho, R. Galbieri,
K.P. Gramacho, M.V. Prates, C. Bloch Jr., Encrypted antimicrobial peptides from
plant proteins, Sci. Rep. 7 (2017) 13263.
[33] W.H. Organization, Antimicrobial Resistance Global Report on Surveillance:
2014 Summary, World Health Organization, 2014.
[34] M.R. Felício, O.N. Silva, S. Gonçalves, N.C. Santos, O.L. Franco, Peptides with
dual antimicrobial and anticancer activities, Front. Chem. 5 (2017) 5.
[35] D.W. Hoskin, A. Ramamoorthy, Studies on anticancer activities of antimicro-
bial peptides, BBA-Biomembranes 1778 (2008) 357e375.
[36] M. Riggi, K. Niewola-Staszkowska, N. Chiaruttini, A. Colom, B. Kusmider,
V. Mercier, S. Soleimanpour, M. Stahl, S. Matile, A. Roux, R. Loewith, Decrease
in plasma membrane tension triggers PtdIns(4,5)P2 phase separation to
inactivate TORC2, Nat. Cell Biol. 20 (2018) 1043e1051.
[37] D. Raucher, M.P. Sheetz, Cell spreading and lamellipodial extension rate is
regulated by membrane tension, J. Cell Biol. 148 (2000) 127e136.
[38] K. Keren, Z. Pincus, G.M. Allen, E.L. Barnhart, G. Marriott, A. Mogilner,
J.A. Theriot, Mechanism of shape determination in motile cells, Nature 453
(2008) 475e480.
[39] N.C. Gauthier, M.A. Fardin, P. Roca-Cusachs, M.P. Sheetz, Temporary increase
in plasma membrane tension coordinates the activation of exocytosis and
contraction during cell spreading, Proc. Natl. Acad. Sci. 108 (2011)
14467e14472.
[40] S. Boulant, C. Kural, J.-C. Zeeh, F. Ubelmann, T. Kirchhausen, Actin dynamics
counteract membrane tension during clathrin-mediated endocytosis, Nat. Cell
Biol. 13 (2011) 1124e1131.
[41] E.L. Batchelder, G. Hollopeter, C. Campillo, X. Mezanges, E.M. Jorgensen,
P. Nassoy, P. Sens, J. Plastino, Membrane tension regulates motility by con-
trolling lamellipodium organization, Proc. Natl. Acad. Sci. 108 (2011)
11429e11434.
[13] E. De Clercq, Anti-HIV drugs: 25 compounds approved within 25 years after
the discovery of HIV, Int. J. Antimicrob. Agents 33 (2009) 307e320.
[14] G.C. Jagetia, R. Aruna, Correlation of micronuclei-induction with the cell sur-
vival in HeLa cells treated with a base analogue, azidothymidine (AZT) before
exposure to different doses of g-radiation, Toxicol. Lett. 139 (2003) 33e43.
[15] R.M. da Rosa, B.C. Piccoli, F.D.A. da Silva, L. Dornelles, J.B.T. Rocha, M.S. Sonego,
K.R. Begnini, T. Collares, F.K. Seixas, O.E.D. Rodrigues, Synthesis, antioxidant
and antitumoral activities of 50-arylchalcogeno-3-aminothymidine (ACAT)
derivatives, MedChemComm 8 (2017) 408e414.
[16] E.M. Guantai, K. Ncokazi, T.J. Egan, J. Gut, P.J. Rosenthal, P.J. Smith, K. Chibale,
Design, synthesis and in vitro antimalarial evaluation of triazole-linked
chalcone and dienone hybrid compounds, Bioorg. Med. Chem. 18 (2010)
8243e8256.
[17] A. Dalpiaz, G. Paganetto, B. Pavan, M. Fogagnolo, A. Medici, S. Beggiato,
D. Perrone, Zidovudine and ursodeoxycholic acid conjugation: design of a new
prodrug potentially able to bypass the active efflux transport systems of the
central nervous system, Mol. Pharm. 9 (2012) 957e968.
[18] Y. Jin, R. Xin, L. Tong, L. Du, M. Li, Combination anti-HIV therapy with the self-
assemblies of an asymmetric bolaamphiphilic zidovudine/didanosine pro-
drug, Mol. Pharm. 8 (2011) 867e876.
[42] S. Futaki, I. Nakase, Cell-surface interactions on arginine-rich cell-penetrating
peptides allow for multiplex modes of internalization, Acc. Chem. Res. 50
(2017) 2449e2456.
[43] A. Mishra, G.H. Lai, N.W. Schmidt, V.Z. Sun, A.R. Rodriguez, R. Tong, L. Tang,
J. Cheng, T.J. Deming, D.T. Kamei, G.C. Wong, Translocation of HIV TAT peptide
and analogues induced by multiplexed membrane and cytoskeletal in-
teractions, Proc. Natl. Acad. Sci. 108 (2011) 16883e16888.
[44] E.Y. Kim, G. Rajasekaran, S.Y. Shin, LL-37-derived short antimicrobial peptide
KR-12-a5 and its d-amino acid substituted analogs with cell selectivity, anti-
biofilm activity, synergistic effect with conventional antibiotics, and anti-
inflammatory activity, Eur. J. Med. Chem. 136 (2017) 428e441.
[19] J.M. Thomson, I.L. Lamont, Nucleoside analogues as antibacterial agents, Front.
Microbiol. 10 (2019) 952.
[20] G. Seydlova, R. Pohl, E. Zborníkova, M. Ehn, O. Simak, N. Panova, M. Kolar,
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K. Bogdanova, R. Vecerova, R. Fiser, H. Sanderova, D. Vítovska, P. Sudzinova,