Antimicrobial benzodiazepine-based short cationic peptidomimetics

Article abstract of DOI:10.1002/psc.2771

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic 'back', and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 μg/ml against Staphylococcus aureus and 25 μg/ml against Escherichia coli, similar to the well-known antimicrobial peptide MSI-78. In contrast to MSI-78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications.

Full text of DOI:10.1002/psc.2771

Research Article
Received: 18 October 2014
Revised: 11 February 2015
Accepted: 18 February 2015
Published online in Wiley Online Library: 25 March 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2771
Antimicrobial benzodiazepine-based
short cationic peptidomimetics
Galina M. Zats,a,b Marina Kovaliov,a,b Amnon Albecka* and Shimon Shatzmillerb
Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the
synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydro-
phobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiaz-
epine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic ‘back’, and two
arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed
us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was
discovered which has MICs of 12.5μg/ml against Staphylococcus aureus and 25μg/ml against Escherichia coli, similar to the well-
known antimicrobial peptide MSI-78. In contrast to MSI-78, the above mentioned compound has lower lytic effect against
mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics
of AMPs for therapeutic and biomedical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s website.
Keywords: antimicrobial peptide; benzodiazepine scaffold; cationic peptides; drug design; peptidomimetics
Herein, we present the design and synthesis of a series of anti-
bacterial compounds, SMAMPs, that are based on short cationic
Introduction
Antimicrobial peptides (AMPs) play an essential protective role
in the immune system in various kingdoms of living organisms
such as animals, fungi, bacteria, and amphibians [1–5]. They
share several common features, i.e. positive charge domains
(contain lysines (Lys) and arginines (Arg)), hydrophobic amino
acids (AAs) [valine (Val) and phenylalanine (Phe) and amphi-
pathic structures [6,7]. Most native AMPs have no cytolytic
activity against normal mammalian cells at their minimal antimi-
crobial inhibitory concentrations (MICs) [8]. The advantage of
AMPs over conventional antibiotics is their operating mecha-
nism, which refers to the interaction between the bacteria’s
membrane and the peptide, resulting in the disintegration of
the cell wall [9–11]. This fact makes it much more difficult for
the microorganisms to develop resistance to AMPs. However,
natural AMPs have high molecular weight, low stability in the
blood serum, low selectivity, and high synthetic cost [12–15].
For these reasons, the pharmaceutical industry shows interest
in synthetic mimics of antimicrobial peptides (SMAMPs). For a
number of years, several research groups, including ours, have
focused on the development of very short SMAMPs [14–20].
These studies have shown that the presence of two cationic
units and a minimum of one lipohylic and bulky unit is required
for antibacterial activity [21–28].
antibacterial peptides that flank a BDZ scaffold, providing drug-like
properties.
Results and Discussions
Design
The design strategy was based on modification of AA sequences as
well as their chemical nature. Furthermore, the AA themselves were
replaced by a BDZ scaffold. In these series of SMAMPs, hydrophobic
groups, the BDZ scaffold, and a positive charge were all present
(Figure 1). These aspects have been suggested to be the minimum
requirement for antimicrobial activity [14,22]. To evaluate the effect
of different non-polar and polar groups on biological properties,
the AAs were varied. Lys and Histidine (His) represent basic,
naturally occurring AAs. Isoleucine (Ile), leucine (Leu), valine (Val),
and Phe were included to establish the effect of hydrophobicity.
Cysteine was used as a polar uncharged AA, containing a thiol
*
Correspondence to: Amnon Albeck, Department of Chemistry, Bar-Ilan University,
Ramat Gan 52900, Israel. E-mail: amnon.albeck@biu.ac.il
a Department of Chemistry, Bar-Ilan University, Ramat Gan, 52900, Israel
b Department of Biological Chemistry, Ariel University, Ariel, 40700, Israel
We used benzodiazepine (BDZ) derivatives, which received sig-
nificant attention, as their core is indeed a ‘privileged scaffold’
found in active compounds against a variety of target types
[29,30]. BDZ are known as the core structure of pharmaceutically
significant agents, are widely used as enzyme inhibitors [31,32],
possess antiproliferative properties on tumor cell [29], and have
been proven to be efficient peptidomimetics [33]. Moreover, BDZ
was reported as an antimicrobial compound against gram-positive
and gram-negative bacteria [34–36].
Abbreviations: AA, amino acid; AMPs, antimicrobial peptides; BDZ, benzodiaze-
pine scaffold; Boc, t-butyloxycarbonyl; tBu, tert butyl; DCM, dichloromethane; DIEA,
N,N-diisopropylethylamine; DMF, dimethylformamide; Fmoc, 9-fluorenylmethox-
ycarbonyl; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi-
nium 3-oxid hexafluorophosphate; MIC, minimum inhibitory concentration; NMP,
N-methyl-2-pyrrolidone; SMAMPS, synthetic mimics of antimicrobial peptides; SPPS,
solid-phase peptide synthesis; TIPS, triisopropyl silane.
J. Pept. Sci. 2015; 21: 512–519
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ANTIMICROBIAL BENZODIAZEPINE-BASED PEPTIDOMIMETICS
Figure 1. Design and structure of SMAMs.
functionality, which was reported as an important determinant of
antibiotic activity against bacteria [37–40]. The C terminal was
blocked as an amide to prevent metabolic degradation.
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, toluene,
85% yield; (b) ammonium acetate, hexamethylenetetramine, EtOH, 95%
yield; (c) t-butyl bromoacetate, NMP, CsCO3, 98% yield; (d) NaCNBH3,
AcOH, 98% yield; (e) CH2Cl2, DIEA, Fmoc chloride, 62% yield; and (f) 4 N
HCl/dioxane, 78% yield.
The BDZ scaffold was used throughout this study because of its
special features: (a) a wide range of biological activities displayed
by BDZ-derived compounds, including the antimicrobial field; (b)
a very effective platform for the development of new biological
compounds; (c) the molecule contains two arms for further
modifications and the scaffold is a mimic of two AAs. Figure 1
shows the design of a BDZ scaffold. This structure contains a
glycyl–glycine dipeptide mimic that confers some flexibility, an
aromatic hydrophobic ‘back’, and two arms used to incorporate
the charged and hydrophobic AAs. With this aim, nine SMAMP
compounds based on the BDZ scaffold were prepared.
yielded a racemic mixture 4, which was subsequently protected
with Fmoc-Cl to provide compound 5 as a racemic mixture.
Finally, hydrolysis of ester 5 yielded the desired product 6 as a
racemic mixture, which was subjected to subsequent SPPS. In
order to study the structure of the final protected monomer 6,
several NMR experiments were performed (see Scheme 1). These
experiments revealed two rotamers in 55 to 45% ratio,
representing G° ≅ 15.9 0.2 kJ (Table 1) [45,46].
Each compound was designed to test various effects: com-
pounds P7 and P8 and the compounds P3 and P5, which contain
the same AA sequence but of different stereochemical configura-
tions, may provide correlation between the absolute configuration
of AAs and biological activity. Moreover, the previous sequences,
which do not include a hydrophobic AA, examine the importance
of the hydrophobicity effect. Compounds P4 and P9, with His and
Lys, respectively, examine the effect of the identity of the positively
charged AA. Compounds P2 and P6, consist of the same AAs but in
deferent order, shed light on the importance of the location of the
positively charged AA along the peptide sequence. Compounds P9,
P2, and P7, each including the same order of the positively charged
AAs but various types of hydrophobic AAs, examine the effect of
the hydrophobic AAs on biological activity. Finally, P1, which
consists of only two AAs, positive and hydrophobic, examines the
importance of the peptide general charge as well as the location
of the charged AA.
SPPS: The syntheses of all the SMAMPs were carried out
according to Scheme 2, applying a manual solid-phase method
Table 1. 1H NMR and 13C NMR data of the BDZ scaffold (BDZ) 6
Atom No.
1H-NMR
13C NMR
(DMSO, δ, ppm)
(DMSO, δ, ppm)
Majora
Minorb
Majora
Minorb
4
—
—
—
—
131.9
141.3
61.7
132.8
141.3
62.1
(a) Synthesis. The synthesis of the new peptidomimetic
compounds consists of two parts: (a) a multi-step synthesis of
the BDZ scaffold and (b) solid-phase peptide synthesis (SPPS)
of the BDZ containing peptides. The scaffold itself contained
two functional groups for the attachment of the AAs.
5
7
6.29
~3.43
—
6.18
~3.43
—
9
49.9
49.9
10
13
15
18
17
21
22
23,26
24,25
170.1
165.6
47.6
170.1
165.1
47.5
—
—
3.52; 4.55
—
3.55; 4.39
—
Benzodiazepine synthesis: The synthesis of the BDZ scaf-
fold was achieved by six steps, as shown in Scheme 1.
Aminobenzophenone was acylated with chloroacetyl chloride,
affording 2-chloroacetamido-benzophenone 1 as previously re-
ported [41]. It was then followed by modification of the known
hexamethylenetetramine-based cyclization reaction, developed
by Blazevic and Kajfez, resulting in 2 [42]. The final scaffold con-
struction steps were designed to include two glycine AAs and
two functional groups for the attachment of the flanking AAs.
Thus, the first arm for contact was installed by alkylation of BDZ
2 with t-butyl bromoacetate to generate 3 in 98% yield after crys-
tallization [41,43]. The second arm for contact was installed by
imine reduction with sodium cyanoborohydride [44], which
153.9
140.2
67.4
154.3
140.5
67.9
—
—
4.41; 4.59
4.38
—
4.25; 4.49
4.20
—
46.8
46.6
143.7
140.7
143.7
140.7
—
—
aMajor rotamer 55%.
bMinor rotamer 45%.
cMajor and minor may be altered. Assignment of the aromatic
protons (7.0–8.0 ppm) and carbons (120–124 ppm) is too
complex to analyze.
J. Pept. Sci. 2015; 21: 512–519
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G. M. ZATS ET AL.
Scheme 2. Solid-phase synthesis of the SMAMP P2.
on Rink-amide resin using Fmoc strategy [21,27]. After the intro-
duction of the first two AAs, the BDZ scaffold Fmoc-6-OH was
coupled through its acidic arm. Fmoc deprotection (20%
piperidine/DMF), followed by coupling of the next N-
protected AA (at 40 °C using HATU, which suites coupling to
secondary amines) and finally deprotection and cleavage
provided the BDZ scaffold-based pentapeptide mimetics. In
most cases, the last coupling was repeated twice to give the
final SMAMPs P(1-9). The overall yield of the peptidomimetic
compounds was 16–29%, in 90–95% purity.
and one Lys as positively charged AAs, had no antibacterial activity.
In the contrary, the P2, P7, and P9 compounds, which contain
two Lys in a sequence similar to that of P4, inhibited the growth
of gram-positive and gram-negative bacteria. However, se-
quences P3 and P5, which also contain two charged Lys resi-
dues, showed no activity. This probably stems from the total
length of the peptide, which is one AA shorter, and from the dis-
tance between the two charged AAs. Our results indicate that
the minimum length required for antibacterial activity is five
AAs (considering the BDZ to mimic the Gly–Gly dipeptide). Com-
parison of peptides P2 and P6, which share the same total
length and AA composition, shows that the positions of the polar
uncharged AA and/or the distance between the positively
charged residues influence the antimicrobial activity. Because
of the antibacterial properties of thiol, the presence of Cys in
P2 appears fundamental, but also its position, considering that
P6 did not display any antibacterial activity. Sequences P3, P5,
P7, and P8 are pairs of enantiomers that show very similar
activity against both types of bacteria, indicating the lack of
stereoselective interactions with the biological target. To con-
clude, a hydrophobic AA, in particular, a polar uncharged AA
such as Cys, in the fourth position (after the Gly–Gly dipeptide
of the BDZ) increases the antibacterial activity, if the SMAMPs in-
clude two charged Lys residues. Moreover, variability in the hy-
drophobic AA in the fourth position does not influence the
antibacterial activity.
(b) Antimicrobial activity. The antimicrobial activity of the
peptidomimetic compounds against bacteria was tested by
two methods: the disk diffusion method and the dilution
method to determine MIC [47], summarized in Table 2. Both
methods showed similar results, although the first method
was less effective to SMAMPs because of the mechanism of ac-
tion of anti-bacterial peptides [10]. Thus, they may have suf-
fered diffusion limitation, which prevented the compounds
from reaching a critical concentration of activity (Figure 2). In
general, all compounds shown in Table 2 have MICs compara-
ble to the magainin analogue MSI-78, an antimicrobial R-
helical peptide with peptide sequence G-I-G-K-F-L-K-K-A-K-K-
F-G-K-A-F-V-K-I-L-K-K-NH2. They show better activity against
gram-positive (Staphylococcus aureus) than gram-negative
bacteria (Escherichia coli) [48].
One of the major disadvantages associated with AMPs is that
they are prone to protease degradation. This is also reflected in
the loss of activity in the presence of blood plasma. [49] We
tested the antimicrobial activity of our most active compound
P2 against S. aureus in 50% blood plasma, and no loss of activity
Among the peptidomimetic compounds prepared for this study,
peptide P2 exhibited the highest activity against both gram-
positive and gram-negative bacteria strains with MIC values of 12
and 25 μg/ml respectively. The nine sequences showed the follow-
ing trend: P1 with just one basic residue and P4, containing one His
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 21: 512–519

ANTIMICROBIAL BENZODIAZEPINE-BASED PEPTIDOMIMETICS
Table 2. Antimicrobial activity against gram-negative and gram-positive bacteria and hemolytic activity of the BDZ
peptidomimetic compounds
Compound
SMAMPs
MIC (μg/ml)
Hemolysis (%)
HC50 (μg/ml)
E. coli
S. aureus*
P1
6#-Ile-Lys-NH2
>200
25
>200
12.5
1.8
1.2
2.3
1.2
2.2
1.2
2.2
2.0
2.1
NDc
NDc
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
120b
P2
Lys-6#-Cys-Lys-NH2
D-Lys-6#-D-Lys-NH2
Lys-6#-Val-His-NH2
Lys-6#-Lys-NH2
Lys-6#-Lys-Cys-NH2
Lys -6#-Phe-Lys-NH2
D-Lys -6#-D Phe-D-Lys-NH2
Lys -6#-Leu-Lys-NH2
—
P3
>200
>200
>200
>200
100
>200
>200
>200
100
P4
P5
P6
P7
100
P8
100
100
P9
100
100
MSI-78
vancomycin
16-32a
NDc
8–16a
0.9c
—
NDc
*Methicillin-resistant S. aureus.
aLiterature values obtained from reference [48].
bLiterature values obtained from reference [50].
cLiterature values obtained from reference [15].
dND stands for ‘not determined’.
Figure 2. Results of the disk diffusion assay. Left: No antibacterial activity of P1 against E. coli. Right: the most active P2 against methicillin-resistant S. aureus.
(12.5 μg/ml) was observed following 3 h preincubation, which is
a physiologically relevant time frame. However slight loss of
activity was observed after 6 h (25 μg/ml). (Figure S7 in the
supporting information).
to MSI-78. [50] Indeed, the HC50 (μg/ml) of P2 was less toxic
toward mammalian cells compared with recently published
SMAMPs [14,15].
The main goal of this manuscript was to report the design and
synthesis of a series of peptidomimetic antibacterial compounds
that were based on short cationic antibacterial peptides,
flanking a BDZ scaffold, in order to provide drug-like properties.
The concept of using short peptides with cationic AAs is not new
nor is the BDZ scaffold, but we thought that the combination of
the two would provide these compounds with drug-like proper-
ties. In addition, these antibacterial peptidomimetic compounds
could solve almost all problems of AMPs, which suffer from high
molecular weight, low stability, poor selectivity, high synthetic
cost, and toxicity. We believe that our lead compound, P2, and
its future improved and modified forms should be tested
in vivo for antimicrobial activity. Currently, we are developing
Hemolytic Activity
In order to evaluate the cytotoxicity of these compounds toward
mammalian cells, the ability to induce lysis in human erythro-
cytes was measured. None of the peptidomimetics displayed
significant hemolytic activity (<3%) at concentrations up to
1000 μg/ml. It is important to mention that the P2 lead com-
pound shows antimicrobial values similar to the well-known
AMP, MSI-78, but it displays a higher MIC value than vancomycin
[15]. P2 does not display any significant hemolytic activity (1.2%)
at concentrations up to 1000 μg/ml, which is in a sharp contrast
J. Pept. Sci. 2015; 21: 512–519
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G. M. ZATS ET AL.
SAMP compounds with fluorescent markers for the detection
and study of in vivo blood brain barrier penetration and antibac-
terial activity. These studies will be reported in due course.
toluene (3 × 10 ml). The purified product was dried at 105 °C for 5 h
to yield 2 (15.3 g, 95% yield) as colorless crystals. HPLC: λ260-96.8% pu-
rity. M.p: >210 °C 1H-NMR (300 MHz CDCl3): δ 4.12 (s, 2H, CH2), 7.17–
7.62 (m, 9H, Ar) 10.53 (s, 1H, NH). IR (KBr): ν 3057, 2900, 2252, 1669,
1584, 1482, 1433, 1380, 1242, 1217, 1178 cm-1. MS ES+: m/z 234 (MH+).
Conclusions
Tert-butyl 2-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4] diazepin-1-yl)
acetate (3)
We have reported here the synthesis of a new BDZ scaffold suitably
designed for incorporation into peptidomimetic compounds by
SPPS. This work combines both solution and solid-phase synthesis.
The BDZ scaffold was selected because of its biological properties
and its structure, with a carboxylate and Fmoc-protected amine
arms (which mimic the C-termini and N-termini of a dipeptide) for
linking. Although the exact conformational aspects responsible
for the activity of SMAMPs are not known, all these compounds
resemble AMPs in terms of their charge and amphiphilicity. Among
the nine prepared compounds, P2 (Lys–BDZ–Cys–Lys–NH2)
showed the best antibacterial activity with MIC values of 12.5μg/
ml against S. aureus and 25μg/ml against E. coli. The other SMAMPs
showed MIC values of 100 μg/ml and higher. All the SMAMPs dem-
onstrated no direct lytic effect against mammalian red blood cells.
Admittedly, further investigations are necessary to evaluate the im-
portance of the molecular size and the number of positive charges
before these new mimetic peptide compounds can be transformed
into drugs useful for systemic application. In particular, the required
studies include synthesis of additional peptidomometic com-
pounds with modification of additional AAs, study of the mecha-
nism of action of these compounds, and most importantly in vivo
studies. We believe that these relatively small and easy to prepare
molecules can serve as a basis for further refinement toward lead
compounds for novel antibacterial agents, and these preliminary
but important studies could guide and encourage further studies
in the field.
A round bottomed flask, equipped with a magnetic stirring bar and
nitrogen inlet, was sequentially charged with 5-phenyl-1H-benzo[e]
[1,4]diazepin-2(3H)-one 2 (5.7g, 0.02 mol) NMP (16.2ml), tert-butyl
bromoacetate (2.62 ml, 1.42 eq), and cesium carbonate (5.2 g,
1.42eq). After stirring overnight at ambient temperature, the reac-
tion mixture was diluted with water (100 ml) and extracted with
EtOAc (3× 30 ml). The combined organic phase was washed with
water (4× 15 ml) and brine (10 ml), dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure. This ma-
terial was crystallized from hexanes/ethyl acetate to provide 3
(3.59 g, 98% yield) as a white crystalline solid. HPLC: λ260-99.8% pu-
rity M.p: >200 °C. 1H-NMR (300 MHz CDCl3): δ 1.45 (s, 9H, t-Bu), 3.85
(d, J = 11.9 Hz, 1H, CH2), 4.21 (d, J = 17.8 Hz, 1H, CH2), 4.48 (d,
J = 11.9Hz, 1H, CH2), 4.53 (d, J = 17.8 Hz, 1H, CH2), 7.21–7.64 (m,
9H, Ar). IR (KBr): ν 3330, 3057, 1669, 1584, 1482, 1493, 1380, 1249,
1217, 1121 cmÀ1
.
Tert-butyl 2-(2-oxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[e] [1,4] diaze
pin-1-yl) acetate (4)
Tert-butyl 2-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e] [1,4] diazepin-
1-yl) acetate 3 (3.54 g, 0.01 mol) was dissolved in acetic acid (195 ml)
and cooled to À10 °C. Sodium cyanoborohydride (1.25 g, 2 eq) was
added to the yellow solution all at once. After stirring for 15 min at
10 °C, the mixture was diluted with H2O (200 ml), made basic with
saturated Na2CO3 (aq.), and extracted with EtOAc (2 × 480 ml). The
combined organic phase was washed with brine, dried over MgSO4,
filtered, and evaporated to dryness in vacuo. The residue was
chromatographed (7 : 3 petroleum ether: EtOAc) to provide 4
(3.5 g, 98% yield) as white crystalline solid. HPLC: λ260-98.8% purity.
Materials and Methods
Solution Synthesis
1
N-(2-benzoylphenyl)-2-chloroacetamide (1)
M.p: >200 °C. H-NMR (300 MHz CDCl3): δ 1.48 (s, 9H, t-Bu), 3.40 (d,
J = 19.1Hz, 1H, CH2), 3.46 (d, J = 19.1Hz, 1H, CH2), 4.37 (d,
J = 17.9Hz, 1H, CH2), 4.47 (d, J = 17.9 Hz, 1H, CH2) 5.70 (s, 1H), 6.67
(d, J=7.8Hz, 1H, Ar), 7.01–7.60 (m, 8H, Ar). 13C NMR (CDCl3): δ 28.3,
50.0, 50.8, 59.4, 82.4, 121.3, 126.9, 128.5, 128.6, 128.9, 129.6, 134.7,
141.0, 142.7, 168.1, 171.1. IR (KBr): ν 3257, 3057, 1868, 1686, 1610,
A solution of chloroacetyl chloride (8.4 ml, 11.93 g, 1.06 eq) in toluene
(20 ml) was added dropwise at 10 ºC during 30 min to a solution of 2-
amino-benzophenone (20 g, 0.1 mol) in toluene (200 ml). The
reaction mixture was then stirred at room temperature for 3 h. The
resulting mixture was evaporated to dryness. The crude product
was crystallized by stirring in 96% ethanol (100 ml) at room temper-
ature for 20 h. The crystals were separated by filtration, washed with
96% ethanol (3 × 10 ml), and dried at 50 °C for 20 h to yield 1 (23.6 g,
85% yield) as colorless crystals. HPLC: λ260-99% purity. M.p: 120 °C. 1H-
NMR (300 MHz CDCl3): δ 4.20 (s, 2H, CH2), 7.37–7.41 (m, 8H, Ar), 8.61
(d, J=7.8Hz, 1H, Ar) 11.62 (s, 1H, NH) MS ES+: m/z 274 (MH+).
1517, 1479, 1367, 1318, 1334, 1023 cmÀ1
.
(9H-Fluoren-9-yl)methyl-1-(2-tert-butoxy-2-oxoethyl)-2-oxo-5-phenyl-2,3-
dihydro-1H benzo[e] [1,4] diazepine-4(5H)-carboxylate (5)
tert-Butyl 2-(2-oxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[e] [1,4]
diazepin-1-yl) acetate 4 (2.55 g, 7.28mmol) was dissolved in DCM
(40ml) and FmocCl (2.56 g, 7.28mmol) and DIEA (14 ml, 8 eq) were
added at 0 °C. After 30 min, the cold bath was removed and the
solution was stirred overnight. The reaction mixture was diluted
with citric acid (10%). The organic extracts were washed with water
(4 × 15 ml) and brine (10 ml), dried over MgSO4, and evaporated to
dryness. The crude product was crystallized from EtOAc/hexane to
give white crystalline solid (2.59 g, 62% yield). HPLC: λ260-100% pu-
rity. M.p: >200 °C. 1H-NMR (300 MHz CDCl3): Two rotamers (55 : 45
ratio): (major) δ 1.48 (s, 9H, t-Bu), 3.46 (d, J = 16.2Hz, 1H, CH2CO2H),
3.70 (d, J = 16.2 Hz, 1H, CH2CO2H), 4.05; 4.63, (m, 3H, 2 × CH2), 4.76
(d, J = 16.2 Hz, 1H, CH2), 6.15 (s, 1H, CH), 6.96 (d, J = 8.5Hz, 1H, Ar),
7.15–7.79 (16H, Ar). (minor) δ 1.48 (s, 9H, t-Bu), 3.04 (d, J = 16.2Hz,
5-Phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one (2)
Hexamethylenetetramine (14.8 g, 2.2 eq) and ammonium acetate
(8.22 g, 2.2 eq) were added to a solution of N-(2-benzoylphenyl)-2-
chloroacetamide 1 (18.6g, 0.079 mol) in 96% ethanol (300 ml).
The reaction mixture was stirred at reflux temperature for 6 h. Then
the reaction mixture was evaporated to dryness. Distilled water
(200 ml) was added, and the resulting suspension was stirred at
60 °C for 0.5h. The suspension was cooled to 15 °C and filtered.
The crude product was dried at 105 °C for 5 h. It was then
suspended in toluene (50 ml) at 70 °C for 0.5h and then cooled to
10 °C, and the obtained crystals were filtered and washed with cold
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J. Pept. Sci. 2015; 21: 512–519

ANTIMICROBIAL BENZODIAZEPINE-BASED PEPTIDOMIMETICS
1H, CH2CO2), 3.63 (d, J = 16.2Hz, 1H, CH2CO2), 4.05–4.63, (m, 3H,
2 × CH2), 4.71 (d, J = 16.2Hz, 1H, CH2), 6.40 (s, 1H, CH), 6.96 (d,
J = 8.5 Hz, 1H, Ar), 7.15–7.79 (16H, Ar) 13C NMR (CDCl3): (major + mi-
inor) δ 28.2, 48.3, 50.6, 51.2, 62.5, 65.4, 82.5, 120.3, 124.9, 125.4,
127.2, 127.4, 127.8, 128.6, 130.5, 131.7, 132.8, 141.5, 141.7, 142.0,
143.8, 144.0, 155.3, 167.6, 168.5. MS ES+: m/z 575 (MH+).
Lys-BDZ-Phe-Lys-NH2 (P7). HPLC: λ260-95% purity. M.S ES+: m/z
699.1 (MH+).
D-Lys-BDZ-D-Phe-D-Lys-NH2 (P8). HPLC: λ260-92% purity. M.S
ES+: m/z 699.1 (MH+).
Lys-BDZ-Leu-Lys-NH2 (P9). HPLC: λ260-95% purity. M.S ES+: m/z
665.3 (MH+).
2-(4(((H-Fluoren-9-yl)methoxy)carbonyl)-2-oxo-5-phenyl-2,3,4,5-tetrahy
dro-1Hbenzo[e][1,4] diazepin-1-yl) acetic acid (6)
Antimicrobial Activity
The peptidomimetic compounds (P1–P9) were subjected to MIC
testing, identifying the lowest concentration of antimicrobial
compounds that inhibit the growth of a microorganism after 24 h
incubation at 37 °C. Microplate autoreader E1309 Bio-tek Instrument
was used to measure absorption at 690 nm. MIC values for gram-
positive (methicillin-resistant S. aureus) and gram negative (E. coli)
bacteria were determined. The concentrations of the antimicrobial
peptidomimetics in the microplates were 400, 200, 100, 50, 25, 12.5,
6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, and 0.05 μg/ml. The MIC values
and errors are reported as averages and standard errors of mean of
tree independent experiments (each experiment was performed in
triplicates) respectively. The error of the experiments is less than 5%.
HCl (20 ml, 4 N in dioxane) was added to a solution of 9H-zfluoren-
9-yl)methyl 1-(2-tert-butoxy-2-oxoethyl)-2-oxo-5-phenyl 2,3-dihydro-
1H-benzo[e][1,4]diazepine-4(5H)-carboxylate 5 (1g, 1.74 mmol) in
dioxane (30 ml) at 4 °C. After 30 min, the cold bath was removed
and the solution was stirred overnight. The organic layer was evap-
orated to dryness, and the crude product was crystallized from
EtOAc/hexane to give 6 as white crystalline solid (78% yield). HPLC:
λ
260-93% purity. M.p: >200 °C. 1H-NMR (700 MHz DMSO): Two
rotomers (55 : 45 ratio): (major) δ 3.43 (2H, CH2CO2H), 4.55;3.52
(2H, CH2), 4.59;4.41 (2H, CO2CH2), 4.36 (1H, CH), 6.29 (1H, CH),
7.0–8.0 (16H, Ar). (minor) δ 3.43 (2H, CH2CO2H), 4.39;3.55, (2H,
CH2), 4.49;4.25, (2H, CO2CH2), 4.20 (1H, CH), 6.18 (1H, CH), 7.0–8.0
(16H, Ar). 13C NMR (CDCl3): (major) δ 46.8, 47.5, 61.7, 67.4, 131.9,
140.2, 140.7, 141.3, 143.7, 165.6, 170.1. (minor) δ 46.6, 47.4, 49.7,
62.1, 67.9, 132.3, 140.5, 140.7, 141.3, 143.3, 165.1, 170.1. M.S ES+:
m/z 519 (MH+), 541 (MNa+), 557 (MK+).
Antimicrobial Activity in Plasma
Fresh human blood cells were centrifuged at 3000 rpm for 5 min.
The plasma, which separated from hRBCs, was collected. P2 was
dissolved in water and then diluted twofold into plasma solution
so that final concentration of the compound was 800μg/ml. This
sample was preincubated for 0, 3, or 6 h. Then the MIC values for
methicillin-resistant S. aureus were determinate as mentioned be-
fore in antimicrobial assay.
General Procedure for SPPS
Rink amide resin (0.4mmol) was shaken with DCM: NMP 1 : 1 (3ml)
for 24 h. in a Merrifield flask. Solvent was filtrated under vacuum,
and the resin was treated with 20% piperidine in DMF (v/v) to
deprotect the amino groups (2× 10 min). Then the solid was
washed five times with NMP and three times with DCM. Positive
Kaiser test indicated successful removal of the Fmoc protecting
group. A solution of absolute DMF (3ml), HATU (4eq), Fmoc-AA
(0.4 mmol), and DIEA (3eq) was added to the resin, and the mixture
was agitated for 3 h. During the coupling to the secondary amine of
the BDZ scaffold, the agitating was performed for 2 h at 40 °C and
the next AA was added twice. At the end of the peptide synthesis,
the peptide-loaded resin was dried in vacuum for 2 h. The peptide
was then cleaved by addition of a solution of TFA: H2O: TIS
(95: 2.5: 2.5, 4 ml), which turned the solid and the solution deep
red. After agitating for 2 h, the solution was pressed out of the Mer-
rifield flask, vacuum filtered, and evaporated under nitrogen. The
resulting highly viscous liquid/oil was precipitated from cold diethyl
ether (6ml) and lyophilized. The SMAMPs were finally purified by
solid-phase extraction pack (RP-18), first washed with water and
then extracted with acetonitrile. Their purity was determined by
HPLC (RP-18, CH3CN/0.1% TFA (aq.), 2 : 1).
Hemolytic Activity
The hemolytic activity of each peptidomimetic compound was
tested against human red blood cells (hRBCs), obtained from healthy
volunteers. Fresh heparinized hRBCs were rinsed three times with
phosphate buffered saline (PBS) buffer (35 mM, 150 mM NaCl,
pH 7.4) by centrifugation (10 min, 1500 rpm), followed by resuspen-
sion and dilution in PBS (10% hematocrit). Various concentrations
of the peptidomimetics were then dissolved in PBS buffer and added
to the hRBC solution, yielding a final erythrocyte concentration of 1%
v/v. The suspensions were incubated under agitation for 1 h at 37 °C,
followed by centrifugation (5 min, 4000 rpm). The release of hemo-
globin was monitored by measuring the absorbance of the superna-
tant at 450 nm. Negative controls for zero hemolysis and positive
controls (100% hemolysis) consisted of hRBC suspended in PBS
and Triton 1%, respectively. The degree of hemolysis is defined as
the ratio of the optical density (OD) of the peptidomimetic sample
relative to the OD of the difference between the positive and nega-
tive controls for hemolysis.
BDZ-Ile-Lys-NH2 (P1). HPLC: λ260 -95% purity. M.S ES+: m/z
537.3 (MH+).
Acknowledgements
Lys-BDZ-Cys-Lys-NH2 (P2). HPLC: λ260-95% purity. M.S ES+:
m/z 655.3 (MH+).
Our special thanks go to Dr. Hugo Gottlieb for the NMR determina-
tion of the final BDZ scaffold 6.
D-Lys-BDZ-D-Lys-NH2 (P3). HPLC: λ260-93% purity. M.S ES+:
m/z 575.4 (MNa+).
Lys-BDZ-Val-His-NH2 (P4). HPLC: λ260-90% purity. M.S ES+: m/z
660.1 (MH+).
References
Lys-BDZ-Lys-NH2 (P5). HPLC: λ260-95% purity. M.S ES+: m/z
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Supporting Information
Additional supporting information may be found in the online ver-
sion of this article at the publisher’s website.
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