Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase

Article abstract of DOI:10.1021/acs.jmedchem.5b01482

Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions.

Full text of DOI:10.1021/acs.jmedchem.5b01482

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Article
Structure-activity relationships of novel tryptamine-
based inhibitors of bacterial transglycosylase
Izidor Sosic, Marko Anderluh, Matej Sova, Martina Gobec, Irena Mlinaric-Rascan, Adeline
Derouaux, Ana Amoroso, Mohammed Terrak, Eefjan Breukink, and Stanislav Gobec
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01482 • Publication Date (Web): 20 Nov 2015
Downloaded from http://pubs.acs.org on November 23, 2015
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Structureꢀactivity relationships of novel tryptamineꢀ
based inhibitors of bacterial transglycosylase
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Izidor Sosič1, Marko Anderluh1, Matej Sova1, Martina Gobec1, Irena Mlinarič Raščan1, Adeline
Derouaux2, Ana Amoroso2, Mohammed Terrak2, Eefjan Breukink3, Stanislav Gobec1,*
1Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
2Centre d’Ingénierie des Protéines, Université de Liège, Allée de la Chimie, B6a, Bꢀ4000, Sart
Tilman, Liège, Belgium
3Department Biochemistry of Membranes, Bijvoet Centre, Institute of Biomembranes, Utrecht
University, Utrecht 3584 CH, The Netherlands
*Corresponding author: Stanislav Gobec
Faculty of Pharmacy
University of Ljubljana
Aškerčeva 7
1000 Ljubljana, Slovenia
Tel: +386ꢀ1ꢀ476ꢀ9500
Fax: +386ꢀ1ꢀ425ꢀ8031
Eꢀmail: stanislav.gobec@ffa.uniꢀlj.si
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Abstract
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Penicillinꢀbinding proteins represent wellꢀestablished, validated and still very promising targets
for the design and development of new antibacterial agents. The transglycosylase domain of
penicillinꢀbinding proteins is especially important, as it catalyzes polymerization of glycan
chains, using the peptidoglycan precursor lipid II as a substrate. Based on the previous discovery
of a nonꢀcovalent smallꢀmolecule inhibitor of transglycosylase activity, we systematically
explored the structureꢀactivity relationships of these tryptamineꢀbased inhibitors. The main aim
was to reduce the nonꢀspecific cytotoxic properties of the initial hit compound, and concurrently
to retain the mode of its inhibition. A focused library of tryptamineꢀbased compounds was
synthesized, characterized, and evaluated biochemically. The results presented here show the
successful reduction of the nonꢀspecific cytotoxicity, and the retention of the inhibition of
transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II
and to have antibacterial actions.
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1. Introduction
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The rapid development of antibacterial agents in the second half of the 20th century has
resulted in remarkable advances for humanity. The rate of mortality from infectious diseases has
decreased dramatically, and a perception of the final triumph over bacteria has spread to the
public. However, this perception is erroneous, as the current increasing occurrence of resistance
to existing antibiotics and the lack of novel antimicrobials under development are of great
concern. The incidence of soꢀcalled ‘superbugs’ (i.e., organisms that are resistant to most of the
clinically used antibiotics) is rapidly increasing, so new antibiotic drugs that exploit new or soꢀ
farꢀunderexplored targets need to be identified to address this problem.1–3 In the battle against the
antibiotic crisis, alternative therapeutic concepts, such as inhibition of bacterial virulence and
resistance factors, are also being pursued. Of these, different antiꢀadhesion and antiꢀbiofilm
strategies show promise and are receiving a lot of attention. The advantages, disadvantages, and
future prospects of these antibacterial approaches were described recently.4,5
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Peptidoglycan is the major component of the bacterial cell wall. Its polymerꢀlike structure
protects bacterial cells from rupture in challenging environments. Unobstructed synthesis of
peptidoglycan is required for cell viability, and its inhibition leads to cell death. Many enzymes
are involved in the synthesis of peptidoglycan, and most of these are potential targets for
antibacterial agents.6 The last two steps of peptidoglycan biosynthesis are particularly attractive
for potential antibacterial compounds, as they take place on the external surface of the
cytoplasmic membrane, and are therefore more accessible to inhibitors, at least in Gramꢀpositive
bacteria.
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These transglycosylation and transpeptidation reactions are catalyzed by bifunctional
penicillinꢀbinding proteins (PBPs) that have transglycosylase (TG) and transpeptidase (TP)
active sites.7 In the transglycosylation step, the transfer of the sugar moiety from the activated
polymeric peptidoglycan (a glycosyl donor) to the specific 4ꢀhydroxyl group of lipid II (a
glycosyl receptor) is achieved, with the concomitant release of a C55 undecaprenyl
pyrophosphate carrier.8,9 In the subsequent step, transpeptidation occurs, which produces interꢀ
strand peptide crossꢀlinks. Several types of PBPs are present in bacteria, which belong to either
bifunctional PBPs (class A) or monofunctional TP PBPs (class B). Some bacteria also have
monofunctional TGs that form polysaccharide chains, which are then crossꢀlinked by other
PBPs.7
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Most of the antibiotics that tackle peptidoglycan biosynthesis target the peptide crossꢀlinking
step by inhibiting the TP activity of PBPs directly (e.g., βꢀlactams), or by forming a complex
with the DꢀAlaꢀD
ꢀAla residue on the lipid II pentapeptide stem (e.g., vancomycin).10 The TG
activity has been traditionally viewed as very difficult to target, mostly due to the flat and
extended active site topology.11 It has been established that inhibition of the transglycosylation
reaction and, consequently, the disruption of peptidoglycan assembly, can be accomplished
either by compounds binding to the enzyme active site directly (enzyme binders) or by
sequestration of lipid II (substrate binders), thus preventing its use as a substrate.12 A prime
example of a substrate binder is nisin, a naturally occurring lantibiotic that binds to lipid II via
the pyrophosphate moiety, which induces pore formation in the bacterial membrane. The most
extensively investigated TG enzyme binders are moenomycin and its analogs, as well as lipid IIꢀ
based derivatives.
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Recently, significant efforts from academia and industry resulted in the discovery of a few
structurally diverse, lowꢀmolecularꢀweight and nonꢀcarbohydrate classes of TG inhibitors.13
Moenomycin still remains the most potent inhibitor of TGs; unfortunately, despite intensive
research, the greatest disadvantages of this natural product reside in its poor pharmacokinetic
properties, which prevent moenomycin from being clinically useful in humans.14 Therefore, the
discovery of novel compounds with distinct structural features and nonꢀcarbohydrate scaffolds
that can inhibit the TG activity are needed, and could lead to novel antibacterial drugs.
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Recently, we described a hit compound 2ꢀ(1ꢀ(3,4ꢀdichlorobenzyl)ꢀ2ꢀmethylꢀ5ꢀ(methylthio)ꢀ
1Hꢀindolꢀ3ꢀyl)ethanamine (hit compound; Figure 1) and showed that it interacts with the lipid II
substrate via the pyrophosphate motif and inhibits TGs from different bacteria at low micromolar
concentrations. In addition, the initial hit showed antibacterial activity, with MICs ranging from
4ꢀ16 µg/mL.15 This compound, however, has several drawbacks; namely, troublesome synthesis,
chemical instability (towards oxidation) due to the presence of the aromatic methylthio (SMe)
moiety, and more importantly, nonꢀspecific cytotoxic characteristics (e.g., hit compound is
severely cytotoxic on human embryonic kidney [HEK] 293 cells at 8 µg/mL).
Based on previous data15 and cell viability results, we postulated that the initial hit compound
exerts its antibacterial action via two mechanisms: specifically through the inhibition of cellꢀwall
synthesis, and nonꢀspecifically through membrane disruption. Therefore, our main aim in terms
of the optimization of the characteristics of this compound class was to eliminate the structural
features that lead to the cytotoxic effects. Concurrently, we wanted to retain the TG inhibitory
potency of new derivatives, and their ability to bind to lipid II. In this paper, we report on the
design and synthesis of a focused library of tryptamineꢀbased derivatives that were used to
explore the structureꢀactivity relationships (SARs) for inhibition of the TG activity. Our efforts
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have resulted in a small set of novel and potent inhibitors of this very promising target that have
antibacterial activity and very low nonꢀspecific cytotoxic effects.
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Figure 1. Previously published tryptamineꢀbased hit compound (obtained from the National
Cancer Institute) with potent activity against various TGs, and with MICs ranging from 4ꢀ16
ꢁg/mL.15
2. Design and Chemistry
To establish the SARs of tryptamineꢀbased derivatives for inhibition of TG activity and
elimination of nonꢀspecific toxic effects, several approaches were pursued (Figure 2). We
decided to retain the tryptamine core (Figure 2, green) and systematically modify the substituents
of the parent compound. First, incorporation of a guanidine moiety instead of the amine was
chosen based on the results of docking studies.15 The initial hit compound appears to form an
ionic bond with Asp156 and with the pyrophosphate group of lipid II. Therefore, we expected to
boost the interactions with guanidine, as this might form additional Hꢀbonds (besides the ionic
one) with the pyrophosphate and Asp156 when compared to primary amine, and consequently
improve the inhibitory potency. Next, we explored the role of different substituents on the benzyl
ring attached to the nitrogen of the indole. Thirdly, we wanted to determine the importance of the
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methyl group at position 2 of the indole for inhibition of TG activity. Finally, to omit the SMe
group, we synthesized several compounds with a methoxy (OMe) substituent at position 5 of the
indole, and a set of compounds with a 2,5ꢀunsubstituted tryptamine scaffold.
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Figure 2. (A) Schematic representation of the systematic structural modifications to the hit
compound. The tryptamine core is highlighted in green. (B) The benzyl moieties used in the
SAR studies.
To accurately explore the chemical space and include different patterns of scaffold
substitution, two tryptamine derivatives that were commercially available (1 and 2, Scheme 1)
and two that needed to be synthesized (5 and 6, Scheme 1) were used as the core framework. The
2ꢀmethylꢀ5ꢀmethoxytryptamine (5) and 2ꢀmethylꢀ5ꢀmethylthiotryptamine (6) were prepared
according to the previously described Grandberg indole synthesis.16 Briefly, equimolar amounts
of 4ꢀmethoxyphenylhydrazine or 4ꢀthiomethylphenylhydrazine (both obtained from the
corresponding hydrochloride salts 3 and 4 prior to the reaction) and 5ꢀchloroꢀ2ꢀpentanone
(technical grade, 85%) were mixed for 24 h in EtOH. Due to the exothermic nature of the initial
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reaction, the temperature of the mixture during the addition of 5ꢀchloropentanꢀ2ꢀone needed to
be carefully monitored and kept below 40 °C (Scheme 1).
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All of the tryptamine derivatives 1, 2, 5, and 6 were then subjected to phthalimide formation of
the primary amine, by refluxing with phthalic anhydride in toluene using a DeanꢀStark apparatus.
Indole nitrogen benzylation of the phthaloylꢀprotected tryptamines 7ꢀ10 was achieved by treating
these compounds with NaH or Cs2CO3 in DMF or CH3CN, followed by alkylation with various
benzyl bromides, to yield benzylated tryptamines 11ꢀ28 (Scheme 1). This threeꢀstep reaction
sequence was completed with the deprotection of the primary amino group by heating the Nꢀ
phthaloylꢀprotected compounds 11ꢀ28 with excess hydrazine in refluxing EtOH. Finally, these
deprotected amines were converted into their corresponding hydrochloride salts, which yielded
compounds 29ꢀ46 (Scheme 1). In addition, the hit compound was resynthesized and transformed
into its hydrochloride form (compound 46), to improve the chemical stability in comparison with
the free aliphatic amine. Thus, a direct comparison of biological activities between compounds
was possible (see Results and Discussion).
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Scheme 1. Synthesis of the compounds with various substitution patterns on the tryptamine
corea.
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a Reagents and conditions: (a) i. 2 M NaOH, H2O; ii. 5ꢀchloropentanꢀ2ꢀone, EtOH, 35 °C, heat
to 85 °C, 24 h; (b) phthalic anhydride, Et3N, toluene, reflux, 24 h; (c) for compounds 11ꢀ22:
corresponding benzyl bromide (Figure 2B), NaH, DMF, 55 °C, overnight; for compounds 23ꢀ28:
corresponding benzyl bromide (Figure 2B), Cs2CO3, CH3CN, 80 °C, overnight; (d) i.
H2NNH2×H2O, EtOH, reflux, 24 h; ii. 2 M ethanolic HCl, EtOH.
The conversion of the amino moiety into guanidine was performed by applying a previously
described twoꢀstep process,17 in which the amines of a set of desired compounds were initially
converted into the bisꢀBoc protected guanidines 47ꢀ58 using N,N'ꢀdiꢀ(tertꢀbutoxycarbonyl)ꢀSꢀ
methylisothiourea in the presence of HgCl2 (Scheme 2). This was followed by CF3COOHꢀ
mediated Boc deprotection, and then conversion of the final compounds into the corresponding
hydrochlorides 59ꢀ70 (Scheme 2).
Scheme 2. Synthesis of the compounds with the guanidine moietya.
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R4
R4
R3
R3
R5
R5
2,5-unsubstituted-tryptamine
29-37
N
derivatives (
)
MeS
NHBoc
NBoc
N
R1
R1
b
R2
5-methoxy-tryptamine
39-40
R2
derivatives (
)
a
NH2+Cl-
NH2
2-methyl-5-methoxy-tryptamine
NBoc
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HN
HN
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)
derivative (
NHBoc
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4
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(R , R , R , R , R5 = H)
(R , R , R , R , R5 = H)
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(R , R , R , R4 = H, R5 = OMe)
(R , R , R , R4 = H, R5 = OMe)
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(R , R , R3 = H, R4 = OMe, R5 = H)
(R , R , R3 = H, R4 = OMe, R5 = H)
1
1
(R , R2 = H, R3 = OMe, R4, R5 = H)
(R , R2 = H, R3 = OMe, R4, R5 = H)
1
2
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(R , R , R3 = H, R4 = CN, R5 = H)
(R , R , R3 = H, R4 = CN, R5 = H)
1
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3
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3
(R , R , R , R4 = H, R5 = F)
(R , R , R , R4 = H, R5 = F)
1
2
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2
(R , R , R3 = H, R4 = Cl, R5 = H)
(R , R , R3 = H, R4 = Cl, R5 = H)
1
2
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2
(R , R , R3 = H, R4 = F, R5 = F)
(R , R , R3 = H, R4 = F, R5 = F)
1
2
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2
(R , R , R3 = H, R4 = Cl, R5 = Cl)
(R , R , R3 = H, R4 = Cl, R5 = Cl)
(R1 = H, R2 = OMe, R3 = H, R4 = F, R5 = F)
(R1 = H, R2 = OMe, R3 = H, R4 = Cl, R5 = Cl)
(R1 = H, R2 = OMe, R3 = H, R4 = F, R5 = F)
(R1 = H, R2 = OMe, R3 = H, R4 = Cl, R5 = Cl)
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69
(R1 = Me, R2 = OMe, R3 = H, R4 = Cl, R5 = Cl)
(R1 = Me, R2 = OMe, R3 = H, R4 = Cl, R5 = Cl)
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70
a
Reagents and conditions: (a) Et3N, HgCl2/DMF, rt, overnight; (b) i. CF3COOH, CH2Cl2, rt,
overnight; ii. 2 M ethanolic HCl, EtOH (except for compounds 61 and 65).
3. Results and Discussion
3.1. PBP1b Inhibitory Potencies
The synthesized compounds were first evaluated for their relative inhibition of the TG reaction
of PBP1b from E. coli using a fluorescence assay.18 The compounds that showed notable relative
inhibitions at 500 ꢁM were further characterized by determination of their IC50 values (Tables 1
and 2). The exact protocols are described in the Experimental Section. To allow direct
comparisons of new compounds with the initial hit compound, we included the resynthesized hit
in all of the assays performed. We used the hydrochloride salt of the hit compound (compound
46, Table 1) and confirmed that there were no differences in the TG inhibition between these two
forms of the compound (see Table 1 and Figure 1 for IC50 values determined for E. coli PBP1b).
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Table 1. PBP1b inhibitory potencies of tryptamineꢀbased derivatives 29ꢀ46.
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RA (%) of PBP1b (E. coli)a
Compound
IC50 (ꢀM)
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93
80 ±13
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IC50 > 250 ꢀM
62 ±10
IC50 > 250 ꢀM
15 ±4
IC50 = 103 ±23 ꢀM
105 ±27
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24 ±4
40
IC50 = 193 ±7 ꢀM
75 ±29
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118 ±28
28 ±8
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IC50 > 250 ꢀM
5 ±2
IC50 = 104 ±2 ꢀM
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31 ±12
45
IC50 > 250 ꢀM
1 ±1
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(hit compound)
IC50 = 21 ±4 ꢀM
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a RA, relative activity determined at 500 µM for each compound
Table 2. PBP1b inhibitory potencies of compounds with the guanidine moiety 59ꢀ70.
RA (%) of PBP1b (E. coli)a
Compound
IC50 (ꢀM)
73 ±30
67 ±21
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55 ±15
61
IC50 > 250 ꢀM
29 ±5
62
63
64
IC50 > 250 ꢀM
79 ±26
42 ±9
IC50 > 250 ꢀM
15 ±2
65
66
67
68
IC50 = 166 ±39 ꢀM
10 ±1.5
IC50 = 97 ±39 ꢀM
1 ±2
IC50 = 51 ±18 ꢀM
28 ±3
IC50 > 250 ꢀM
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9 ±5
69
70
IC50 = 50 ±17 ꢀM
4 ±3
IC50 = 39 ±9 ꢀM
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a RA, relative activity determined at 500 µM for each compound
The results presented in Tables 1 and 2 allowed us to postulate the structural requirements for
inhibition of TG activity of E. coli PBP1b. First, the results obtained with compounds 29ꢀ45
(Table 1) show that all of the modifications of the substituents on the tryptamine core led to
slightly diminished inhibitory potencies in comparison with the hit compound 46. For example,
compound 44 (IC50 = 104 ±2 ꢁM) showed an approximately 5ꢀfold reduction in inhibition when
compared to 46 (IC50 = 21 ±4 ꢁM), despite only a minor structural change (SMe at position 5 on
the indole ring substituted with an OMe group). In line with this result, the closest analog to
compound 44 without the Me moiety at position 2 on the indole (compound 40) showed a further
decrease in inhibitory potency (IC50 = 193 ±4 ꢁM).
However, when the amine moiety was transformed into guanidine (compounds 59ꢀ70), a
general increase in inhibitory potency was observed, regardless of the indole ring substitutions at
positions 2 and 5. For instance, the guanidineꢀbearing compounds 65ꢀ67 (Table 2) were more
potent than their amine counterparts 35ꢀ37 (Table 1). Similarly, in the 5ꢀmethoxytryptamineꢀ
substituted and the diꢀsubstituted group of compounds, the guanidines 69 (IC50 = 50 ±17 ꢁM,
Table 2) and 70 (IC50 = 39 ±9 ꢁM, Table 2) outperformed the amineꢀbearing analogs 40 (IC50 =
193 ±7 ꢁM, Table 1) and 44 (IC50 = 104 ±2 ꢁM, Table 1).
The results also show that halogens on the benzyl ring attached on the indole nitrogen are
necessary, as these compounds showed more pronounced inhibition. For example, the
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unsubstituted benzyl derivatives (e.g. 29, 41, 45) showed no inhibition, whereas their halogenꢀ
substituted counterparts (37, 44, 46) showed inhibition, with IC50 values in the micromolar range
(Table 1). Chloro atoms attached to the benzyl moiety contributed more to the TG inhibition
when compared with fluoroꢀsubstituted derivatives. This was clearly seen when the
chlorobenzylꢀsubstituted compounds 44 (IC50 = 104 ±2 ꢁM) and 69 (IC50 = 50 ±17 ꢁM) are
compared with fluorobenzylꢀsubstituted tryptamines 43 and 68, which were both inactive (Tables
1 and 2). In addition, two halogens were more beneficial than the monoꢀhalogen substitution
pattern (e.g., compounds 35, 65 versus 37, 67, and 34, 64 versus 36, 66; Tables 1 and 2). All of
the other substituents on the benzyl ring used in our SAR study (i.e., OMe, CN) led to
diminished inhibition of TG activity, regardless of their position on the benzyl ring (compounds
30ꢀ33 and 60ꢀ63; Tables 1 and 2).
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Interestingly, no general SAR rules can be defined for the role of substituents at positions 2 (H
or Me) and 5 (H, OMe, or SMe) on the core indole. For example, the influence of diꢀsubstitution
was in some cases pronounced, as the removal of both moieties led to an approximately 5ꢀfold
reduced inhibition, as observed with the unsubstituted compound 37 (IC50 = 103 ±23 ꢁM; Table
1) and the 2ꢀmethylꢀ5ꢀmethylthioꢀdiꢀsubstituted 46 (IC50 = 21 ±4 ꢁM; Table 1), whereas in some
other cases no significant effects were observed (compare unsubstituted and diꢀsubstituted
compounds 67 and 70, which inhibited TG activity of PBP1b with IC50 values of 51 ±18 ꢁM and
39 ±9 ꢁM, respectively; Table 2).
3.2. Interactions with Lipid II
The inꢀvitro TG activity inhibition assay used measures the overall TG rate, and this can
identify inhibitors that directly inhibit enzyme activity, as well as those that interfere with
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substrate availability. As we have previously shown that binding to lipid II is an important
mechanism of action of the initial hit compound, we decided to determine whether the
compounds used in this study also bind lipid II.
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To test for these specific interactions between selected compounds (66, 67, 69, 70) and lipid II,
a competition assay was set up with nisin, which uses lipid II for its poreꢀforming activity.19,20
Adding nisin to carboxyfluorescein (CF)ꢀloaded vesicles that contain lipid II results in leakage of
the fluorescent dye from the vesicles (due to lipid IIꢀdependent pore formation by nisin). We
assayed inhibition of nisin activity through addition of 20 nM nisin to 20 µM 1,2ꢀdioleoylꢀsnꢀ
glyceroꢀ3ꢀphosphocholine (DOPC) vesicles containing 0.1 mol% lipid II, which gave about 55%
leakage after 1 min. The inhibition by these compounds was determined in triplicate at 3 µM,
and for compounds 69 and 70 also at 1.5 ꢁM (stock solution, 1 mM in DMSO). The compounds
were added to the cuvette with the vesicles 2 min before addition of nisin. The data in Figure 3
were determined 50 s after addition of nisin.
Figure 3. Interactions of the selected compounds with lipid II. The assays were performed at 3
µM compounds, and also 1.5 µM for compounds 69 and 70. The inhibition was determined in
triplicate, and the errors did not exceed 5%. aHit compound.
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In the presence of 3 ꢁM compound 70, more than 90% inhibition of nisin activity was
achieved (i.e., significant reduction in the leakage of CF induced by nisin). These results suggest
that all of these compounds bind to lipid II, and thus prevent its interaction with nisin. Among
these compounds, 69 and 70 showed the most pronounced lipid II binding, which was significant
also at 1.5 ꢁM.
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It was previously established that the amine residue of compound 46 (hit compound) was
crucial in the interaction with the pyrophosphate group of lipid II.15 Based on the results from
Figure 3, it appeared that the guanidine moieties of these compounds can interact with the
pyrophosphate group of lipid II to an even greater extent than the amine group of compound 46.
This is most probably due to more bonds (i.e., Hꢀbonds) formed between the phosphate groups of
lipid II and the basic guanidine moiety. This type of inhibition is reminiscent of several lipid IIꢀ
targeting antibiotics, such as lantibiotic nisin, which binds the pyrophosphate of lipid II,12 as well
as vancomycin and related secondꢀgeneration glycopeptides.21 Moreover, bacitracin also inhibits
peptidoglycan biosynthesis via binding to the pyrophosphate moiety of the lipid carrier.22
3.3. Antimicrobial Activities
For the compounds that showed enzyme inhibition, their inꢀvitro antibacterial activities were
determined using a panel of four different bacterial strains: Staphylococcus aureus, methicillinꢀ
resistant S. aureus (MRSA), Enterococcus faecium, and Escherichia coli (Table 3).
Table 3. Susceptibility of the panel of four bacterial strains to these tryptamineꢀbased
compounds.
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MIC (µg/mL)a
5
6
Compound
S. aureus
E. coli
MRSA
E. faecium
7
ATCC 25923 ATCC 8739 ATCC 43300 ATCC 19434
8
9
32
(100)
64
(198)
16
(45)
32
(83)
64
(174)
8
(20)
32
(92)
16b
(38)
32
(73)
32
(89)
32
(92)
64
(145)
16
(44)
8
(20)
32
(81)
8
(19)
4
32
(100)
64
(198)
16
(45)
64
(166)
64
(174)
8
(20)
128
(369)
256
(616)
32
(73)
> 128
(> 356)
64
(184)
64
(145)
16
(44)
32
(100)
128
(397)
16
(45)
32
(83)
64
(174)
16
(40)
64
(184)
4
(9.6)
64
(146)
32
(100)
64
(198)
4
(11)
32
(83)
64
(174)
8
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12
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45
46
47
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35
36
37
40
43
44
45
(20)
32
(92)
32
(76)
128
(293)
16
(44)
64
(184)
32
(73)
32
(88)
4
(10)
128
(324)
8
(19)
4
(9.1)
46
(hit compound)
61
62
64
65
66
67
68
69
70
32
(89)
128
(369)
64
(145)
32
(88)
8
(20)
64
(162)
8
16
(40)
32
(81)
16
(37)
32
(72)
(19)
4
(9.1)
(9.1)
a In the parentheses, the MIC values are expressed in µM.
b
The free amine form of 46 (hit compound) had a MIC of 4 µg/mL against the S. aureus
ATCC 25923 strain, as determined previously.15 For compound 46, the MIC for the same strain
was 16 µg/mL.
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Compounds 44, 66, 67, 69, and 70 showed the most promising antibacterial activities, with
MICs from 4ꢀ32 µg/mL (Table 3). The bestꢀperforming compound in this series was compound
70, as it showed antibacterial activities with MICs of 4 µg/mL (approx. 9 µM) against S. aureus,
MRSA, and E. faecium. These results represent a clear (4ꢀ to 8ꢀfold) improvement when
compared with the starting compound 46 (initial hit compound). Unfortunately, all of these
compounds (including 69 and 70; Table 3) were less active against Gramꢀnegative E. coli, which
suggested poor permeation through the outer membrane. Very similar effects were also observed
for some other indoleꢀcontaining molecules in preꢀclinical development.23 Nevertheless, this
reduction in activity for compounds 69 and 70 was generally less pronounced than for the parent
compound 46, which showed a 16ꢀfold increase in the MIC when compared with, for example, S.
aureus (Table 3).
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3.4. Cytotoxicity Studies
To rapidly determine the possibility of nonꢀspecific cytotoxic effects of novel tryptamineꢀ
based compounds, we used a HEK 293 cellꢀviability assay. Here, only compounds that showed
either inꢀvitro inhibition of TG activity (Tables 1 and 2) or antibacterial activity (Table 3) were
used (Figure 4). In this cellꢀviability assay, compound 46 (hit compound) showed severe
cytotoxicity against the HEK293 cells (Figure 4, Table S1). This is in agreement with the
previously established data that compound 46 acts as a nonꢀspecific membrane disruptor.15
Interestingly, from a comparison of the cell viability data for compounds 44 and 46, we can
conclude that substitution of SMe with the OMe group at position 5 does not reduce the
cytotoxicity, as both compounds had severe cytotoxic effects at 8 µg/mL (Figure 4). Similarly,
omitting either only the Me group at position 2 (compound 40) or both substituents at positions 2
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and 5 on the indole ring (compound 37) did not reduce the cytotoxicity, as 37 and 40 were still
both severely cytotoxic towards HEK293 cells (Figure 4, Table S1). However, incorporation of
the guanidine moiety instead of the amine almost completely eliminated the cytotoxicity of these
compounds against HEK293 cells, as observed from the comparisons of the cell viabilities with
the amineꢀbearing compounds 37, 40, 44 and the guanidineꢀbased analogs 67, 69, 70 (Figure 4,
Table S1). Moreover, replacement of the two chloro atoms (compound 67) with fluoro atoms
(compound 66) also led to slightly reduced cytotoxic effects at 8 µg/mL (Figure 4). In line with
previous data, omitting the Me group at position 2 on the indole ring only has a minor effect on
cytotoxicity, as compounds 69 and 70 showed very similar effects.
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Figure 4. Cytotoxicity of compounds on HEK293 cells after 24 h treatment. The effects on cell
viability of the compounds at 8 µg/mL were evaluated using the MTS assay. aHit compound.
The cell viability assays shown in Figure 4 indicate that we can modify the starting compound
46 in such a way that its cytotoxicity is significantly reduced. Compound 70 showed the most
promising characteristics in the other assays (IC50 in the low micromolar range, greatest affinity
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for lipid II, good antimicrobial activity), and here it also showed very low cytotoxicity towards
the HEK293 cells.
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Figure 5. Schematic representation of the structural changes and their influence on inꢀvitro
inhibition of TG activity, lipid II binding, antibacterial activity, and toxicity towards HEK293
cells.
4. Conclusions
We synthesized a focused library of tryptamineꢀbased derivatives with the aim to explore the
SARs for effective inhibition of TG activity (Figure 5). As the initial hit compound had several
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drawbacks, the main goal was to reduce the unwanted characteristics of this compound, and
concurrently to retain the desired inhibition of the TG reaction and the antibacterial activity.
Incorporation of structural changes into the core indole structure led to compounds with
significantly better properties, in comparison with the hit compound. The most promising
compounds from this series are compounds 69 and 70, which represent significantly improved
hit compounds for further development of inhibitors of TG activity. Both 69 and 70 are more
drugꢀlike and simpler compared to the known natural products that either target the TG active
site or sequester the substrate lipid II. From the data presented herein, it appears that we are on
the right path to achieve an appropriate balance between substituents that contribute to better
inhibitory potencies and concurrently to lower nonꢀspecific cytotoxicities. Therefore, we believe
that our results represent the basis for additional medicinal chemistry efforts towards more potent
compounds that will show improved inhibition of the TG reaction, increased degree of
specificity, and even better antibacterial activity. As the chemistry of indoleꢀbased derivatives is
well known and highly versatile, variations at positions 3, 6, and 7 should provide additional
chemical space exploration and further optimization.
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5. Experimental Section
5.1. General Chemistry Methods
The reagents and solvents were obtained from commercial sources (Fluka, SigmaꢀAldrich,
Acros Organics, Alfa Aesar, TCI, Fluorochem). DOPC was purchased from Avanti Polar Lipids
Inc. Nisin A was produced, isolated, and purified as described previously.24 Lipid II and dansylꢀ
lipid II were synthesized and purified as described elsewhere.25 The solvents for the chemical
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reactions were distilled before use, while the other chemicals were used as received. Analytical
TLC was performed on Merck silica gel (60F254) preꢀcoated plates (0.25 mm), with the
compounds visualized under UV light and/or stained with the relevant reagent. Flash column
chromatography was performed on Merck silica gel 60 (mesh 70ꢀ230), using the indicated
solvents. Yields refer to the purified products, and they were not optimized. All of the melting
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1
points were determined on a Reichert hotꢀstage apparatus, and are uncorrected. H NMR spectra
were recorded on a Bruker Avance III NMR spectrometer and on a Bruker DPX Avance 400
MHz instrument equipped with a QNP probe. Measurements were made at a temperature of 295
K, and are reported in ppm using tetramethylsilane or solvent as an internal standard (DMSOꢀd6
at 2.50 ppm, CDCl3 at 7.26 ppm). The coupling constants (J) are in Hz, and the splitting patterns
are designated as: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; td, triple doublet; t,
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triplet; dt, double triplet; and m, multiplet. C NMR spectra were recorded on a Bruker Avance
400 DPX spectrometer at 302 K, and are reported in ppm using solvent as internal standard
(DMSO–d6 at 39.5 ppm). Mass spectra data and highꢀresolution mass measurements were
performed on a VGꢀAnalytical Autospec Q mass spectrometer. Infrared spectra were obtained on
a Thermo Nicolet FTꢀIR spectrometer using the attenuated total reflection technique. Analytical
purity of the assayed compounds was ≥95% unless stated otherwise, as determined by HPLC
analyses. HPLC analyses were run on an Agilent 1100 system equipped with a quaternary pump
and a multiple wavelength detector. An Agilent Eclipse C18 column was used (4.6 mm× 50 mm,
5 mm), with a flow rate of 1.0 mL/min, detection at 254 nm, and an eluent system of: A, H2O
with 0.1% TFA; B, MeOH. Different gradients were applied:
Gradient 1: 0ꢀ3 min, 40% B; 3ꢀ18 min, 40% B ꢀ 80% B; 18ꢀ23 min, 80% B; 23ꢀ30 min, 80%
B ꢀ 40% B; run time, 30 min; temperature, 25 °C.
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Gradient 2: 0ꢀ15 min, 20% B ꢀ 80% B; 13ꢀ16 min 80% B ꢀ 90% B; 16ꢀ19 min, 90% B; 19ꢀ
20 min, 90% B ꢀ 10% B; temperature, 25 °C.
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5.2. Protein Expression and Purification
The model protein used in this study (E. coli PBP1b) was produced and purified as previously
described.26
5.3. In-vitro TG Activity and TG Inhibition
TG activity was monitored using the continuous fluorescence assay method.18 For
simultaneous screening of numerous conditions, this technique was adapted to a 96ꢀwell plate
format (Greiner BioꢀOne). The standard reaction was carried out at 30 °C in 50 ꢁL 50 mM
Hepes, pH 7.5, 200 mM NaCl, 0.2% decylꢀPEG, 10 mM CaCl2, 20% DMSO, 10 ꢁM dansylꢀlipid
II (prepared as described previously19), 1 U muramidase (Cellosyl) and 100 nM E. coli PBP1b.
The data were collected for 30 min using a Victor 3 fluorimeter (Perkin Elmer) with excitation at
355 nm and emission at 536 nm.
The IC50 values of the compounds were determined using the same fluorescenceꢀbased assay.
The initial rate of the reaction was determined in the presence of various concentrations of
inhibitor (20ꢀ1500 µM) and plotted versus inhibitor concentration using the SigmaPlot program
(Systas Software). The IC50 value is the inhibitor concentration that decreases the initial rate by a
factor of 2.
5.4. MIC Determination
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Tests were carried out using microtitration plates and 200 µL (final volume) MuellerꢀHinton
broth, following the European Committee on Antimicrobial Susceptibility Testing/ Clinical and
Laboratory Standard Institute recommended procedure. The compounds were dissolved in 100%
DMSO at 10 mg/mL, and 20ꢀfold diluted in MuellerꢀHinton broth just before use. The inocula
were prepared for each strain by suspending isolated colonies from overnight cultured plates.
Equivalents of 0.5 Mac Farland turbidity standard (approximately 1.108 CFU/mL) were prepared
in saline solution (NaCl, 0.085%) and then diluted 100ꢀfold in MuellerꢀHinton broth. The MICs
were determined as the lowest dilution of the product that showed no visual turbidity.
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5.5. Preparation of Large Unilamellar Vesicles
Large unilamellar vesicles (LUVs) containing DOPC and 0.1 mol% lipid II were prepared
essentially as described previously.27 Dried lipid films were hydrated by addition of 50 mM CF
(pH 7.5), followed by vigorous stirring, and these were then submitted to 10 freeze–thaw cycles.
The LUVs were prepared by repeated extrusion through polycarbonate filters with a 200ꢀnm
pore size (Isopore membrane filters, Millipore, Ireland).28 Following extrusion, the vesicles were
passed through a Sephadex Gꢀ50 spin column (equilibrated with 10 mM Tris, 150 mM NaCl, pH
7.5) to remove the nonenclosed probe. The final phospholipid concentration was determined by
phosphate analysis, according to Rouse et al.29
5.6. Carboxyfluorescein Leakage Assay in Large Unilamellar Vesicles
The lipid II binding affinities of the different compounds were determined by measuring their
effects on lipid IIꢀdependent poreꢀformation of the lantibiotic nisin. Any compound binding to
lipid II resulted in reduced induction of CF leakage in response to nisin (20 nM). The compounds
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were preincubated with CFꢀloaded, lipid IIꢀcontaining vesicles (20 ꢁM) for 2 min. None of the
tested compounds induced CF leakage during this preincubation period. Then nisin was added,
and the nisinꢀinduced and lipidꢀIIꢀdependent leakage was followed over time (emission
wavelength 516 nm, excitation wavelength 492 nm). Finally, treatment with Triton Xꢀ100
induced total CF release, which was used to calculate the percentage of CFꢀleakage for that
particular experiment. The potency of the compounds to inhibit the nisin induced leakage (hence
bind to Lipid II) was expressed as % of residual nisin activity. This was calculated by
determining the amount of leakage after 50 s in the presence of compound relative to the amount
of leakage obtained in the absence of compound (determined in a separate control experiment)
which was set to 100%.
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59
60
5.7. Cell Culture
HEK293 cells were obtained from ATCC (LGC Standards, UK) and cultured in Dulbecco’s
modified Eagle’s medium (SigmaꢀAldrich, St. Louis, MO, USA) supplemented with 10% fetal
bovine serum (Gibco, Grand Island, NY, USA), 2 mM Lꢀglutamine, 100 U/mL penicillin, and
100 µg/mL streptomycin. The cells were maintained in a humidified chamber at 37 °C and 5%
CO2.
5.8. Metabolic activity assay
The HEK293 cells were seeded at 2× 104 cells/well in 100 µL medium in 96ꢀwell plates. Stock
solutions of compounds in DMSO were prepared at 8 mg/mL and mixed with fresh medium, to
achieve the final concentration of 8 µg/mL. The cells were allowed to adhere for 24 h. Then, the
medium was removed and replaced with medium containing the compound of interest. The
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control cultures received medium containing the appropriate vehicle. Treatments were performed
in triplicate. The metabolic activities were assessed using the CellTiter 96® Aqueous One
Solution Cell Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer
instructions. For IC50 determinations, various concentrations of the compounds of interest were
used, in triplicate, in 96ꢀwell plates.
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59
60
5.9. Chemical Syntheses
5.9.1. Procedure for preparation of 2,5-disubstituted tryptamines 5 and 6: Grandberg
synthesis.
A suspension of 4ꢀmethoxyphenylhydrazine hydrochloride (3) (1.74 g, 10 mmol) or 4ꢀ
thiomethylphenylhydrazine hydrochloride (4) (1.90 g, 10 mmol) in water (50 mL) was stirred for
10 min, followed by dropꢀwise addition of NaOH (2 M, 5 mL). During the addition, the
corresponding phenylhydrazine began to precipitate. Immediately after completion of addition,
the precipitate was filtered off and used in the next step.
To a solution of 4ꢀmethoxyꢀ or 4ꢀthiomethylphenylhydrazine (1 equiv.) in EtOH (60 mL) at 35
°C, 5ꢀchloroꢀ2ꢀpentanone (85%, 1.4 equiv.) was slowly added, and the temperature of the
reaction mixture was maintained between 35 °C and 40 °C. After the addition, the mixture was
stirred for additional 24 h at 85 °C. The dark reaction mixture was then allowed to cool to room
temperature, and the volatiles were removed in vacuo, yielding a dark brown oil. Water (100
mL) was added to the residue, followed by extraction with CH2Cl2 (5× 100 mL). The combined
organic layers were dried with Na2SO4 and filtered, and the solvent was evaporated under
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reduced pressure to yield a viscous dark oil. The products 5 and 6 were used in the next step
5
6
without purification and spectroscopic characterization.
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59
60
5.9.2. General procedure for synthesis of N-phthaloyl-protected tryptamines 7-10.
To a solution of a desired tryptamine derivative 1, 2, 5, or 6 (1 equiv.) in toluene (100 mL),
phthalic anhydride (1.1 equiv.) and Et3N (1.1 equiv.) were added. The reaction mixture was
heated at reflux for 24 h with continuous removal of water with a DeanꢀStark trap. After the
reaction was complete (as monitored by TLC), the mixture was cooled to room temperature and
washed consecutively with NaOH (1 M, 2× 50 mL), water (1× 50 mL), and HCl (1 M, 2× 50
mL). The organic phase was dried with Na2SO4 and filtered, and the solvent was evaporated
under reduced pressure, to yield a viscous dark orange residue. The compounds 7ꢀ10 were
subsequently purified by column chromatography or crystallization.
5.9.3. General procedures for preparation of benzylated derivatives 11-28.
Procedure A (for compounds 11ꢀ22). To a suspension of NaH (60% wt. in mineral oil, 1.5
equiv.) in dry DMF (5 mL), a solution of the desired Nꢀphthaloylꢀprotected tryptamine 7 or 8 (1
equiv.) in dry DMF (5 mL) was added. After stirring the reaction mixture at room temperature
for 30 min, an appropriate benzyl bromide derivative (1.5 equiv.) was added. The reaction
mixture was heated to 55 °C and stirred overnight under an argon atmosphere, followed by
solvent evaporation under reduced pressure. The residue was dissolved in EtOAc (40 mL) and
extracted consecutively with water (3× 30 mL), saturated aqueous solution of NaHCO3 (1× 40
mL), and brine (1× 40 mL). The organic phase was dried with Na2SO4 and filtered, and
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concentrated to give the crude products 11ꢀ22, which were purified by column chromatography
or crystallization.
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8
9
Procedure B (for compounds 23ꢀ28). To a stirred solution of the desired Nꢀphthaloylꢀprotected
tryptamine 9 or 10 (1 equiv.) in dry CH3CN (20 mL), Cs2CO3 (3 equiv.) was added at room
temperature. After 10 min of stirring, an appropriate benzyl bromide derivative (1.2 equiv.) was
slowly added, and then the reaction mixture was stirred overnight at 80 °C under an argon
atmosphere. After cooling the reaction mixture, the solvent was evaporated and the crude residue
was partitioned between CH2Cl2 (50 mL) and water (50 mL). The organic phase was additionally
washed with brine (2× 50 mL), dried with Na2SO4, filtered, and concentrated, to give crude
products 23ꢀ28 that were purified by column chromatography.
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60
5.9.4. General procedure for removal of the phthaloyl protective group: preparation of
compounds 29-46.
To a suspension of a corresponding Nꢀphthaloylꢀprotected compound 11ꢀ28 (1 equiv.) in EtOH
(10 mL), hydrazine hydrate (5 equiv.) was added dropꢀwise at room temperature. The reaction
mixture was then heated to 90 °C and the resulting solution stirred overnight under reflux. After
the reaction was complete (as monitored by TLC), the mixture was cooled to room temperature
and the white precipitate filtered off. The filtrate was concentrated under reduced pressure,
diluted with aqueous NaOH (1 M, 5 mL), and extracted with EtOAc (3× 20 mL). The combined
organic layers were dried with Na2SO4 and filtered, and the solvent was evaporated under
reduced pressure. The products were subsequently purified by column chromatography using
CH2Cl2/MeOH, 9/1 (saturated with gaseous NH3) as eluent. The purified compounds were
converted into hydrochloride salts 29ꢀ46 by dissolving the oily residues in absolute EtOH (5 mL)
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and treating the solutions with an excess of freshly prepared ethanolic HCl (2 M). After
evaporation of EtOH, the residues were triturated with Et2O (4 mL) and the solids formed were
filtered off to yield pure compounds 29ꢀ46.
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5.9.5. General procedure for synthesis of bis-Boc-protected guanidines 47-58.
To
a
solution of the desired tryptamine (unsubstituted derivatives 29ꢀ37, 5ꢀ
methoxytryptamines 39ꢀ40, and 2ꢀmethylꢀ5ꢀmethoxytryptamine 44) in DMF (5 mL), N,N'ꢀdiꢀ
(tertꢀbutoxycabonyl)ꢀSꢀmethylisothiourea (1 equiv.), Et3N (2 equiv.), and HgCl2 (1 equiv.) were
added consecutively at room temperature. The reaction mixture was stirred at room temperature
overnight. After the reaction was complete (as monitored by TLC), the salt Hg(SMe)Cl was
filtered off, and the filtrate evaporated under reduced pressure. To the residue, Et2O (20 mL) was
added, and the organic phase was extracted with water (3× 10 mL), saturated aqueous solution of
NaHCO3 (3× 10 mL) and brine (1× 10 mL). The organic layer was dried with Na2SO4, filtered,
and concentrated, to give crude products 47ꢀ58, which were purified by column chromatography.
5.9.6. General procedure for synthesis of final compounds 59-70.
The desired bisꢀBocꢀprotected compound 47ꢀ58 (1 equiv.) was dissolved in dry CH2Cl2,
followed by addition of CF3COOH (97%, 10 equiv.). The reaction mixture was stirred overnight
under an argon atmosphere, and then the volatiles were evaporated under reduced pressure, to
yield pure compounds that were converted into hydrochloride salts (59ꢀ60, 62ꢀ64, 66ꢀ70), except
for compounds 61 and 65. The hydrochlorides were obtained by first dissolving the residues in
absolute EtOH (5 mL) and then treating the solutions with an excess of freshly prepared
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