Synthesis of α-Branched Acyclic Nucleoside Phosphonates as Potential Inhibitors of Bacterial Adenylate Cyclases

Article abstract of DOI:10.1002/cmdc.201700715

Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC₅₀>10 μm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC₅₀ values ranging from 55 to 362 nm.

Full text of DOI:10.1002/cmdc.201700715

Accepted Article
Title: Synthesis of α-branched acyclic nucleoside phosphonates as
potential inhibitors of bacterial adenylate cyclases
Authors: Jan Frydrych, Jan Skácel, Markéta Šmídková, Helena
Mertlíková-Kaiserová, Martin Dračínský, Ramachandran
Gnanasekaran, Martin Lepšík, Monica Soto-Velasquez, Val J.
Watts, and Zlatko Janeba
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10.1002/cmdc.201700715
ChemMedChem
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Synthesis of α-Branched Acyclic Nucleoside Phosphonates as
Potential Inhibitors of Bacterial Adenylate Cyclases
Jan Frydrych,[a] Jan Skácel,[a] Markéta Šmídková,[a] Helena Mertlíková-Kaiserová,[a] Martin Dračínský,[a]
Ramachandran Gnanasekaran,[a],† Martin Lepšík,[a] Monica Soto-Velasquez,[b] Val J. Watts,[b] Zlatko
Janeba*[a]
[a]
J. Frydrych, J. Skácel, Dr. M. Šmídková, Dr. H. Mertlíková-Kaiserová, Dr. M. Dračínský, Dr. R. Gnanasekaran, Dr. M. Lepšík, Dr. Zlatko Janeba
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic
E-mail: janeba@uochb.cas.cz
ORCID ID Zlatko Janeba: 0000-0003-4654-679X
[b]
M. Soto-Velasquez, Dr. V. J. Watts
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN
47907, USA
†
Current address: Department of Chemistry, Pondicherry University, Puducherry, 605014, India
Supporting information for this article is given via a link at the end of the document.
Abstract: Inhibition of Bordetella pertussis adenylate cyclase toxin
(ACT) and Bacillus anthracis edema factor (EF), the key virulence
factors with adenylate cyclase (AC) activity, represents a potential
method how to treat or prevent toxemia related to whooping cough
and anthrax, respectively. A novel series of α-branched acyclic
nucleoside phosphonates (ANPs) having a hemiaminal ether moiety
was synthesized as potential inhibitors of bacterial adenylate cyclases.
ANPs prepared as bisamidates were not cytotoxic but did not
exhibited any profound activity (IC50 > 10 µM) towards ACT in J774A.1
macrophage cells. The apparent lack of activity of the bisamidates
was speculated to be due to the inefficient formation of the biologically
active species (ANPpp) in the cells. On the other hand, two 5-
haloanthraniloyl-substituted ANPs in the form of diphosphates
(ANPpp) were shown to be potent ACT and EF inhibitors with IC50
values ranging from 55 to 360 nM.
Bacillus anthracis is a Gram-positive spore-forming bacterium and the
etiologic agent of anthrax, a highly contagious disease.[7] Anthrax is a
common disease in livestock, only occasionally affecting the humans,
but it may spread as a result of biological warfare. B. anthracis
secretes three main virulence factors, protective antigen (PA), lethal
factor (LF) and edema factor (EF).[8] EF, just as B. pertussis ACT,
exhibits adenylate cyclase activity and is responsible for the rapid ATP
break down in infected cells leading to enormously high levels of cyclic
AMP (cAMP) and, subsequently, to cell homeostasis disruption.[5a]
Although B. pertussis[9] and B. anthracis[10] are, in general, sensitive
to antibiotics, antibiotic treatment does not affect the bacterial
toxaemia.[11] In addition, several antibiotic-resistant B. pertussis[12] and
B. anthracis[13] strains have been reported. Thus, a development of
novel strategies for prophylaxis and/or treatment of B. pertussis and
B. anthracis infections is urgently needed.
Acyclic nucleoside phosphonates (ANPs)[14] are structural analogues
of natural nucleotides that are known especially for their antiviral,[15]
antibacterial,[16] and antiparasitic[17] properties. Adefovir dipivoxil
(bis(POM)PMEA, I, Fig. 1), for example, is an efficient antiviral drug
currently approved for treatment of hepatitis B infections.[18] In 1999
Shoshani et al.[19] reported that adefovir diphosphate (PMEApp, II, Fig.
1), the active metabolite of adefovir dipivoxil, is a powerful inhibitor of
rat brain adenylate cyclase. Later, it was found that PMEApp is also a
potent inhibitor of bacterial adenylate cyclases,[20] and crystal
structures of EF-calmodulin[20] and ACT-calmodulin[21] with PMEApp
were resolved. Recently, Česnek et al.[22] studied a series of amidate
prodrugs of PMEA and selected the corresponding isopropyl ester L-
phenylalanyl derivative III (Fig. 1) as a promising type of prodrug
considering its in vitro activity, bioavailability and synthetic
accessibility. Later, a series of C-2 substituted PMEA analogues was
studied by our research group in order to improve the ACT inhibitory
properties of these nucleotide analogues.[23]
Introduction
Pertussis, also called whooping cough, is a severe disease of the
respiratory tract caused by Bordetella pertussis, characterized by
persistent cough. Despite a relatively high level of vaccination
coverage, pertussis remains a serious health problem, especially for
children in developing countries.[1,2] B. pertussis is a Gram-negative
pleomorphic, aerobic coccobacillus[3] with strictly human
pathogenicity.[4] Pertussis symptoms are a result of cooperation of
several B. pertussis virulence factors.[5] Adenylate cyclase toxin
(ACT)[5a] – a member of the RTX (repeat in toxin) family of bacterial
pore-forming toxins – represents one of the key B. pertussis virulence
factors.[6]
1
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Studies carried out by the Seifert group[24] revealed that O-anthraniloyl
(ANT) and O-(N-methylanthraniloyl) (MANT) derivatives of nucleoside
triphosphates (NTPs), i.e. ANT-NTPs and MANT-NTPs, respectively,
had low micromolar to submicromolar inhibitory activity on B.
pertussis ACT. Furthermore, Geduhn et al.[24b] studied a large series
of mono- and bis-ANT-substituted NTPs and identified the first potent
ACT inhibitor bis-Cl-ANT-ATP (IV, Fig. 2) with high selectivity relative
to mammalian ACs. In addition to that, numerous ANT-NTPs and
MANT-NTPs derivatives were shown to be potent competitive EF
inhibitors.[25]
part of a hemiaminal ether moiety. Although such compounds were
expected to be considerably less stable and their synthesis more
demanding compared to standard ANPs, the hemiaminal ether linker
may represent a better mimic of the (deoxy)riboside moiety of natural
NTPs than simple N9-alkyl chain of classic ANPs. The new ANPs have
two aliphatic arms, one bearing the phosphonate group and the
second bearing various substitutions, including the 5-haloanthraniloyl
moiety attached either through the ester or amide bond.
Results and Discussion
Chemistry
Commercially available 1,3-dioxolanes 1 and 2 were chosen as
convenient starting materials. First, (1,3-dioxolan-2-yl)methanol (1,
Scheme 1) was treated with benzylbromide and sodium hydride in
DMF to give 2-(benzyloxymethyl)-1,3-dioxolane (3) in a 65% yield.
Compound 3 was then used as a model compound for optimization of
alkylation of purines with 1,3-dioxolane 1. Reaction of compound 3
with 6-chloropurine, pre-treated with sodium hydride in DMF, afforded
alkylated 6-chloropurine derivative 4 (Scheme 1) in a 52% yield.
Unfortunately, subsequent attempts to alkylate intermediate 4 at the
free hydroxyl group with diisopropyl (bromomethane)phosphonate
were not successful and only decomposition of starting material 4 was
observed. Instead, benzoyl-protected adenine derivative 5 (Scheme
1) was prepared by the reaction of N6-benzoyladenine (N-(9H-purin-
6-yl)benzamide) with compound 3 in a 30% yield. Subsequently, the
Figure 1. Structures of adefovir dipivoxil (I), adefovir diphosphate (II), and
PMEA bisamidate prodrug III.
Fluorescent (M)ANT-NTPs were also shown to be suitable candidates
for detailed studies of their binding interactions with ACT using direct
fluorescence and/or fluorescence resonance energy transfer
(FRET),[24a] but clearly were not therapeutically applicable due to their
rapid degradation in vivo. For this reason, and in order to increase the
flexibility of the ribose moiety of natural NTPs, novel fluorescent 5-
chloroanthraniloyl-substituted ANPs were recently synthesized either
as their diphosphates, i.e. Cl-ANT-ANPpp analogues (e.g. compound
V, Fig. 2) or as their bisamidate prodrugs to improve their
bioavailability.[26] These compounds were shown to be potent
competitive ACT and EF inhibitors with sub-micromolar potency (IC50
values: 11–622 nM).[26]
alkylation
of
adenine
derivative
5
with
diisopropyl
(bromomethane)phosphonate and sodium hydride in DMF afforded
phosphonate 6 (in 13% yield), that was further converted by
methanolysis to the desired adenine compound 7 (Scheme 1) in a
53% yield.
Scheme 1. Optimization of alkylation of the purine bases. Reagents and
conditions: a) benzylbromide, NaH, DMF, 0 °C to rt, 24 h; b) TMSI, cyclohexene,
-78 °C to -60 °C, then a mixture of 6-chloropurine, NaH, DMF, -60 °C to rt; c)
TMSI, cyclohexene, -78 °C to rt, then a mixture of N6-benzoyladenine, NaH,
DMF, -60°C to rt; d) BrCH2P(O)(OiPr)2, NaH, DMF 0 °C to rt, 24 h; e) 1 M
NaOMe/MeOH, MeOH, rt, 24 h.
The original goal was to develop an efficient synthesis of PMEA
derivatives branched at the C-1’ position (α-position) of the aliphatic
linker (general structure VI, Fig. 2). The synthesis started with
alkylation of 2-(bromomethyl)-1,3-dioxolane 2 (Scheme 2) with
diisopropyl (hydroxymethane)phosphonate and sodium hydride in
DMF to obtain phosphonate 8 in a 47% yield. An attempt to prepare
Figure 2. Structures of bis-Cl-ANT-ATP[24b] (IV), Cl-ANT-ANPpp derivative
(V),[26] and new target molecules VI.
Herein, we report a synthesis of novel halo-ANT-ANPs as acyclic
analogues of above mentioned halo-ANT-NTPs.[24,25] Compared to
recently reported β-branched (branched at C-2’ position) analogues
(e.g. compound V, Fig. 2),[26] the current series represents α-branched
ANPs (general structure VI, Fig. 2), that are branched at C-1’ position
of the acyclic linker connecting adenine and the phosphonate moiety
in the form of the bisamidate prodrug. In this case, the C-1’ atom is a
acyclic nucleoside phosphonate
9 (Scheme 2) directly from
compound 8 using previously reported conditions (treatment of 2-
(benzyloxymethyl)-1,3-dioxolane with trimethylsilyl iodide (TMSI) in
cyclohexene at -78 °C, then addition of the mixture to the sodium salt
2
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of 6-chloropurine in dry DMF at -60 °C)[27] was not successful. Albeit
TMSI is commonly used for the preparation of free phosphonic acids
from the corresponding dialkyl esters,[28] it was anticipated that
decomposition of diisopropyl phosphonate moiety under the above
conditions (-60 °C)[27] would be prevented, or at least slowed down.
Unfortunately, this was not the case and decomposition of compound
8 led to formation of a complex reaction mixture and the desired
product 9 was not observed.
azido derivative 16 with hydrogen on Pd/C in methanol at room
temperature yielded 84% of desired amino derivative 17 (Scheme 3).
Scheme 3. Synthesis of α-branched ANPs and their anthraniloyl
derivatives. Reagents and conditions: a) AcCl, pyridine, 0 °C to rt, 2 h; b) TsCl,
DMAP, pyridine, 0 °C to rt, 12 h; c) MsCl, pyridine, -15 °C to 0 °C, 2 h; d) NaN3,
DMF, rt, 96 h; e) H2 (760 torr), Pd/C (10%), MeOH, rt, 96 h; f) 5-bromoisatoic
anhydride, TEA, DMF, 50 °C, 1.5 h; g) 5-chloroisatoic anhydride, TEA,
DCM/DMF, rt, 8 h.
Once we had synthesized compounds 12 and 17 we were able to
proceed with the synthesis of target fluorescent halo-anthraniloyl-
substituted acyclic nucleoside phosphonates (halo-ANT-ANPs).
Amino derivative 17 was converted to compound 18 (64%) by its
treatment with 5-bromoisatoic anhydride and triethylamine in DMF at
50 °C. Product 19 (Scheme 3), containing an ester bond, was
prepared in the same yield (64%) by the reaction of compound 12 with
Scheme 2. Synthesis of α-branched ANPs. Reagents and conditions: a)
HOCH2P(O)(OiPr)2, NaH, DMF, -15 °C to rt, 16 h; b) TMSI, cyclohexene, -78 °C
to -60 °C, then a mixture of 6-chloropurine, NaH, DMF, -60 °C to rt; c) Ac2O,
H2SO4 (cat.), rt, overnight; d) N6-benzoyladenine, SnCl4, MeCN, 2 h; e) 1 M
NaOMe/MeOH, MeOH, rt, 24 h.
To overcome this problem, we decided to employ a modified
methodology for synthesis of α-branched acyclic nucleosides
developed by Zavgorodnii et al.[29] First, 1,3-dioxolane derivative 8
(Scheme 2) was – after some optimization of previously reported
acetolysis of cyclic acetals[30] – converted into acyclic diacetylated
phosphonate 10 in a 44% yield. An application of the modified
Zavgorodnii conditions,[29] i.e. treatment of N6-benzoyladenine and
diacetyl intermediate 10 with tin(IV) chloride in acetonitrile at room
temperature, afforded fully protected phosphonate 11 in a 51% yield.
The removal of both acyl groups from phosphonate 11 using a
catalytic amount of sodium methoxide in anhydrous methanol yielded
desired acyclic nucleoside phosphonate 12 (Scheme 2) in 91%.
Key intermediate 12 was then converted to the corresponding acetyl,
tosyl, and mesyl derivatives 13 (28%), 14 (58%), and 15 (76%),
respectively (Scheme 3). Both tosyl and mesyl derivatives 14 and 15,
respectively, were utilized for the preparation of amino derivative 17
via azido compound 16 (Scheme 3). The azidation showed to be quite
a problematic step due to a cleavage of one isopropyl group from the
diisopropyl phosphonate moiety during heating of compound 14 or 15
with sodium azide in DMF, as reported before by Holý.[31] Thus, the
azidation conditions had to be optimized and the best results were
obtained when compound 14 or 15 was treated with sodium azide in
DMF at room temperature for a prolonged period of time (96 h). These
reaction conditions afforded azido derivative 16 (Scheme 3) in high
yields: 64% from 14 and 84% from 15. Clearly, the reaction sequence
exploiting mesyl derivative 15 is advantageous since both reaction
steps, i.e. mesylation and azidation, are more efficient and provided
azido compound 16 in higher overall yield. Subsequent treatment of
5-chloroisatoic
dichloromethane/DMF mixture at room temperature.
Finally, the selected diisopropyl phosphonates were converted into
the corresponding bisamidates using transsilylation
anhydride
and
triethylamine
in
a
(trimethylsilylbromide (TMSBr) in pyridine at room temperature) and
subsequent treatment of silyl esters with L-phenylalanine isopropyl
ester under standard reaction conditions (trimethylamine, 2,2′-
dipyridyldisulfide (aldrithiol-2), triphenylphosphine, pyridine, 55 °C)
developed in our laboratory.[32] Since our α-branched ANPs with
hemiaminal ether moiety were expected to be labile under acidic
conditions, pyridine is the preferred solvent over acetonitrile during
the bisamidate formation as pyridine is able to scavenge HBr which
may be present in commercial TMSBr and is released during the
reaction course. Under the above reaction conditions, phosphonates
9, 11, 13, 18 and 19 (Scheme 4) afforded final bisamidate prodrugs
20, 21, 23, 25 and 26 in 17-35% yields. Analogous treatment of
compounds 12 and 17 did not afford desired bisamidates 22 and 24
(Scheme 4), respectively. High reactivity of the free hydroxyl and
amino groups might be the reason for the failure to prepare
compounds 22 and 24 by the above procedure and another synthetic
approach had to be developed.
3
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arisen as a result of nucleophilic attack of pyridine-2-sulfide (released
from the aldrithiol-2/triphenylphosphine complex) on an activated
intermediate during the reaction course. The azido analogue 29
(Scheme 5) was successfully prepared in a 40% yield directly from
bisamidate 22 by its treatment with diphenoxyphosphoryl azide
(DPPA)[34] under the Mitsunobu reaction conditions.[35]
Scheme 6. Synthesis of bisamidate prodrugs of α-branched ANPs.
Reagents and conditions: a) TMSBr, pyridine, rt, 12 h, then L-phenylalanine
isopropyl ester hydrochloride, Et3N, 2,2′-dipyridyldisulfide, PPh3, pyridine, 55 °C,
24 h.
In order to study inhibitory potential of novel halo-ANT-ANPs directly
on ACT and EF enzymes, the selected analogues 18 and 19 were
converted to their diphosphate derivatives (halo-ANT-ANPpp), i.e.
analogues of natural NTPs. Since the standard morpholidate
methodology for the NTPs synthesis[36] failed, the method using 1,1′-
carbonyldiimidazole (CDI) was employed instead.[37] Thus, diisopropyl
phosphonates 18 and 19 (Scheme 7) were stepwise treated with
TMSBr in pyridine (to obtain silyl esters), with tetraethylammonium
bicarbonate (TEAB) buffer (to form tetraethylammonium salts), CDI in
DMF (to form activated esters), and finally with tetrabutylammonium
pyrophosphate in DMF to obtain, after purification on DOWEX
(50WX8 Na+) and lyophilisation, desired NTP analogues 31 and 32 as
Scheme 4. Synthesis of bisamidate prodrugs of α-branched ANPs.
Reagents and conditions: a) TMSBr, pyridine, rt, 12 h, then L-phenylalanine
isopropyl ester hydrochloride, Et3N, 2,2′-dipyridyldisulfide, PPh3, pyridine, 55 °C,
24 h. Compounds 22 and 24 were not obtained.
In order to prepare the final product 22, t-butyldimethylsilyl (TBDMS)
group was selected as suitable protecting group of the hydroxyl in
derivative 12, as TBDMS can be efficiently removed later under non-
acidic conditions. Thus, compound 12 was treated with TBDMSCl and
imidazole in DMF at room temperature to give TBDMS derivative 27
(66%), which was then converted by the standard procedure[32] to
bisamidate 28 in a 35% yield (Scheme 5). The target hydroxyl
derivative 22 (Scheme 5) was obtained in a 46% yield upon removal
of the TBDMS group from compound 28 using silica gel-supported
tetrabutylammonium fluoride (TBAF) in dry DMF.
sodium salts in about 20% overall yields.
Scheme 7. Synthesis of phosphonato-diphosphates. Reagents and
conditions: a) TMSBr, pyridine, rt, 12 h, then 2 M TEAB buffer; b) CDI, DMF, rt,
1 h; c) (Bu3N)2P2O7, DMF, rt, 12 h; d) DOWEX (50WX8 Na+).
Scheme 5. Synthesis of bisamidate prodrugs of α-branched ANPs.
Reagents and conditions: a) TBDMS-Cl, imidazole, DMF, rt, 12 h; b) TMSBr,
pyridine, rt, 12 h, then L-phenylalanine isopropyl ester hydrochloride, Et3N, 2,2′-
dipyridyldisulfide, PPh3, pyridine, 55 °C, 24 h; c) TBAF on silica gel (~1.5 mmol/g
F- loading), DMF, rt, 12 h; d) PPh3, DIAD, DPPA, THF, 1 °C to -15 °C, 20 min,
0 °C to rt, 12 h.
Biological activity
Inhibition of ACT in the cell-based assay
All prepared bisamidate prodrugs were tested for their ability to inhibit
ACT activity in J774A.1 macrophage cells. Murine macrophage cells
J774A.1 were preincubated with various concentrations of the tested
compounds for 5 h and subsequently exposed to Bordetella pertussis
ACT for 0.5 h. The cells were lysed, and the amount of cAMP was
determined. None of the final prodrugs tested (20 - 26, 29, and 30)
showed any appreciable activity towards ACT (IC50 > 10 µM) and none
exhibited any cytotoxic effect in J774A.1 cells at concentrations of 10
µM for 5 h.
An attempt to prepare bisamide prodrug directly from the azido
derivative 16 (Scheme 6) was not successful. Instead, under the
standard reaction conditions for the formation of bisamidate
prodrugs,[32] only amino derivative 24 (2%) and 2-(pyridin-2-ylthio)
analogue 30 (5%) were isolated in low yields. Compound 24 was most
likely formed by Staudinger[33] reduction of azido group with
triphenylphosphine (or its complex) and side product 30 probably
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Inhibition of ACT activity in the cell-free assay
Molecular modelling
The triphosphate analogues 31 and 32 were tested, along with
PMEApp as a control, for their inhibitory activity towards recombinant
adenylate cyclase toxin from B. pertussis provided by two distinct
suppliers (Sigma and Enzo Life Sciences), as well as towards edema
factor (EF) from Bacillus anthracis (Table 1). Compounds 31 and 32
inhibited both ACTs with IC50 values in a range of 100 – 360 nM and
had slightly better inhibitory activity towards EF with IC50 values 55
and 81 nM, respectively.
Crystal Structure Reinterpretation and Docking. The crystal
structure of ACT with PMEApp bound (PDB code: 1ZOT)[21] was re-
interpreted due to several problems in the active site caused by low
resolution and weak electron density in that region. First, the purine
core of PMEApp was shifted to form two H-bonds with Val271:N,O
backbone and still fit in the electron density visualised in Coot.[39]
Second, Mg903 was fitted to the electron density map and the
PMEApp linker (up to the phosphonate group) was optimized by
quantum mechanics to relieve any clashing and to maintain a good
magnesium coordination geometry. Third, several active-site residues
(Arg41, Lys58, Lys65) were fitted to the 2FoFc and FoFc electron
density maps to achieve good β- and γ-phosphate coordination.
Furthermore, the Asn304 and His298 side chains were flipped and
Mg902 was changed to a water molecule to comply with H-bonding
and magnesium coordination requirements. This re-interpreted crystal
structure was used to redock the PMEApp ligand. The redocked
Table 1. IC50 values of halo-ANT-ANPpp for ACT and EF.
IC50 [nM][a]
Compound
ACT Sigma
ACT Enzo
EF
PMEApp
16.0 ± 0.2
214 ± 26
103 ± 30
13.6 ± 4.7
102 ± 17
362 ± 36
11.5 ± 2.6
55.1 ± 4.5
81.2 ± 25.8
31
32
[a] Data are the mean ± SD of at least three independent experiments.
Inhibition of mACs
Furthermore, the ability of the prepared bisamidate prodrugs (21 – 24,
26 and 30) to inhibit host mammalian adenylate cyclases (mACs) was
examined (Table 2). The assays were carried out using HEK293 cells
stably expressing mAC1, mAC2, or mAC5, and each compound was
tested in two independent experiments at 30 μM. The mACs tested
are representatives of the three major mAC families. Specific
activation of the mACs overexpressed in the cells was accomplished
by previously reported methodology,[38] in which the calcium
ionophore A23187 was employed to stimulate AC1, a PKC activator
– phorbol 12-myristate 13-acetate (PMA) – was used to stimulate AC2,
and a low forskolin concentration was used to selectively stimulate
AC5. Most compounds had no or minimal effects on the activity of the
mACs. Exceptions included compound 22, which inhibited AC2 by
50% and for two compounds that slightly potentiated the selectively-
stimulated cAMP response at AC5 (compound 26) and at both AC2
and AC5 (compound 30, Table 2).
ligand closely mimicked the re-interpreted crystal structure, which
validated the procedure (Fig. 3a).
Table 2. Mammalian AC1, AC2 and AC5 inhibition with prodrugs at 30 μM.
Response % of Control[a]
Compound
AC1
67 ± 11
72 ± 11
80 ± 8
AC2
AC5
Figure 3. Docking studies of PMEApp and compound 32 to ACT. Picture
description: Red sphere - Mg902 changed to a water molecule; Brown spheres
- Mg901 and Mg903 ions; Purple - optimized position of original PMEApp; Cyan
- docking results a) redocked PMEApp with optimized docking setup; b) first
representative docking pose of 32; c) second non-chelating docking pose of 32;
d) second chelating pose of 32.
21
119 ± 4
50 ± 7
125 ± 1
105 ± 6
123 ± 3
144 ± 3
-1 ± 5
116 ± 2
102 ± 6
116 ± 5
113 ± 4
137 ± 10
137 ± 21
107 ± 7
70 ± 4
22
23
24
26
79 ± 18
105 ± 15
70 ± 1
Subsequently, docking studies were performed to elucidate the
binding properties of the ANT moiety in compound 32. For that
purpose, the template docking protocol was used due to the close
structural similarity of the main aliphatic chains of PMEApp and
compound 32. This approach helped to eliminate potential false-
positive results arising from the combination of quite a large and
flexible ligand on one side, and a bulky binding site of ACT on the
other. First, we attempted to figure out which of the two enantiomers
30
SKF83566[b]
SQ22536[c]
77 ± 7
64 ± 5
47 ± 8
[a]Data are the mean ± SEM relative to the control response (100%) in two
independent experiments. [b]SKF83566 is a selective inhibitor AC2.[38]
[c]SQ22536 is a non-selective P-site inhibitor.
5
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of 32 would be preferred by the enzyme. Although it was difficult to
absolutely discriminate between the two isomers due to the high
flexibility of these molecules, (S)-32 exhibited better ability to relax in
the ACT binding site than (R)-32. The docking of (S)-32 revealed two
possible binding modes in the ACT active site: a) one depicts
presumed orientation of the ANT moiety in the pocket adjacent to the
purine base without any specific interaction (Fig. 3b); b) another
shows the ANT moiety positioned in the space nearby magnesium ion
Mg903 in non-chelating (Fig. 3c) or chelating (Fig. 3d) orientations,
which cannot be excluded or considered as false-positive because of
the inhibitor structure. Again, because of the significant flexibility of
the acyclic part of inhibitor 32, it is difficult to determine the
predominant pose.
In order to reveal potential selectivity of the prepared compounds,
they were also evaluated as potential inhibitors of selected
mammalian adenylate cyclases (mACs). No significant inhibition of
mAC1, mAC2, and mAC5 in cell-based assays was observed for the
novel ANPs, except for compound 22, which selectively inhibited
mAC2 by 50%. Two other compounds were shown to slightly
potentiate some of the mACs, namely mAC5 (compound 26) and both
mAC2 and mAC5 (compound 30).
In summary, variously modified ANPs represent excellent tools to
study the molecular interactions between nucleotide analogues and
their target enzymes, and they are potentially valuable agents with
promising biological properties. This study is a part of broader
structure-activity relationship (SAR) study of modified ANPs as potent
inhibitors of bacterial ACs. The compounds designed as
phosphonato-diphosphate derivatives were shown to be potent
inhibitors of both ACT and EF in the range of hundreds of nM, but in
the form of their bisamidate prodrugs they lacked activity in the cell-
based assays. Thus, although the ANPs reported herein lack the
potential for further development as therapeutic agents, the
nucleoside triphosphate analogues are valuable compounds for
further studies of their interactions with adenylate cyclases on
molecular basis.
Conclusions
We have designed and synthesized a novel series of acyclic
nucleoside phosphonates (ANPs) as potential inhibitors of bacterial
adenylate cyclases (ACs), namely adenylate cyclase toxin (ACT) from
Bordetella pertussis and edema factor (EF) from Bacillus anthracis.
The structural design of target ANPs was based on previously
reported[24] 5-haloanthraniloyl derivatives of nucleoside triphosphates
(halo-ANT-NTPs) as potent ACT inhibitors with high selectivity relative
to mammalian ACs (mACs). It was expected that the attached
aromatic halo-ANT moiety could increase the inhibitory properties of
halo-ANT-ANPs due to its potential interactions with the hydrophobic
pocket of bacterial adenylate cyclases.
Experimental Section
General chemistry.
Starting compounds and other chemicals were purchase from
commercial suppliers or prepared according to the published
procedures. Solvents were dried by standard procedures. Solvents
were evaporated at 40 °C/2 kPa. Analytical TLC was performed on
plates of Kieselgel 60 F 254(Merck). NMR spectra were recorded on
Bruker Avance 500 (1H at 500 MHz, 13C at 125.8 MHz, 31P at 202.4
MHz,) spectrometer with TMS or dioxane (3.75 ppm for 1H, 67.19 ppm
for 13C NMR) as internal standard or referenced to the residual solvent
signal. The numbering system for 1H NMR and 13C NMR spectra of
the prepared compounds are shown on compounds 5, 12, 19, 25, and
26 (see Supplementary Information).
The synthesis of the desired compounds was a challenging task
because of the presence of the hemiaminal ether moiety (an analogy
of N-glycosidic bond of natural nucleotides) that made the target
compounds considerably less stable compared to standard ANPs.
The methodology consisting of condensation of suitable acetal
derivative, namely compound 10, with N6-benzoyladenine in the
presence of SnCl4 enabled us to prepare nine final bisamidates,
including the key halo-ANT-ANP derivatives 25 and 26, for the cell-
based assays and two phosphonato-diphosphates 31 and 32
(analogues of natural NTPs) for enzymatic assays.
The bisamidates tested did not exhibited the ability to significantly
inhibit ACT activity in J774A.1 macrophage cells (IC50 > 10 µM). The
lack of potency of the prepared compounds was speculated to be due
to insufficient phosphorylation in the cells to form the active species
(ANPpp) or their poor stability. Therefore, we decided to prepare two
analogues of natural NTPs, namely 31 and 32 (Scheme 7), to verify
their direct inhibitory potential in the enzymatic assays. Both
compounds were shown to be potent inhibitors of all ACTs and EF
tested (IC50s in a range of 55 – 360 nM). Furthermore, the known
crystal structure of ACT with bound PMEApp was re-interpreted in
order to obtain more reliable data from molecular modelling. The
thorough docking studies based on this improved model suggested
several possible docking poses for (S)-32, but it was not possible to
determine the predominant pose with certainty.
HR MS spectra were taken on a LTQ Orbitrap XL spectrometer. Flash
chromatography on normal phase and deionization on reversed
phase were performed on a Reveleris Flash Chromatography System.
Final purification was performed on a Redisep Rf Gold C18 Teledyne
ISCO column. Preparative HPLC purification of triphosphate
analogues was performed on a column packed with POROS®HQ 50
mm (50 mL) with use of a gradient of TEAB in water (0.05-0.5 M). The
purity of the tested compounds was determined by HPLC (H2O-
CH3CN,linear gradient) and was higher than 95%.
Molecular Modelling.
Crystal Structure Reinterpretation. Crystal structure of Bordetella
pertussis adenylate cyclase toxin (ACT) with calmodulin (CaM) and
6
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10.1002/cmdc.201700715
ChemMedChem
FULL PAPER
bound PMEApp (PDB ID:1ZOT, resolution 2.2 Å)[21] was used for
molecular modelling. A close inspection of ACT active site using Coot
(version 0.8.7)[39] revealed several obvious problems with the ligand,
Mg2+ ions and active-site residues due to low resolution and weak
electron density in this region. We have therefore used model building
in Coot and quantum mechanical (QM) optimizations. For the latter,
we have used fast and reliable corrected semiempirical QM method
PM6-D3H4[40] coupled with implicit solvent model COSMO[41] with εr =
78.4 using MOPAC2016 program[42] within Cuby4 framework.[43]
Optimization convergence criteria were the following: the maximal
energy change was below 0.006 kcal/mol, the maximum gradient
change was below 1.2 kcal/mol/Å and the maximal root-mean-square
of the gradient was below 0.6 kcal/mol/Å.
according to the manufacturer’s instructions. Fluorescence signal was
acquired using an Infinite M1000 plate reader (Tecan Systems Inc.,
San Jose, CA, USA).
Inhibition of ACT – cell-free assay. In a cell-free assay, AC
enzymatic activity was measured by conversion of [3H] ATP to
o
[3H]cAMP. The reaction was carried out at 30 C for 30 min, with a
final reaction volume of 50 µl. Each assay mixture contained 3 µM
BSA, 20 mM HEPES (pH 7.4), 10 mM MnCl2, 1 mM EDTA, 1 µM CaCl2,
0.1 mM cold ATP, 20 µCi [2,8-3H]ATP (ARC, St. Louis, MO, USA;
specific activity 20 Ci/mmol), 1.2 µM calmodulin and tested compound
at concentrations of 0 – 100 µM. Inhibition of AC activity was
determined in the presence of 3 different enzymes ACT (Sigma,
specific activity 65 µmol/min/mg), ACT (Enzo, specific activity 115
µmol/min/mg) and EF (LBL, specific activity 830 µmol/min/mg) with
the final enzyme concentration of 1.1 nM , 0.67 nM and 0.12 nM ,
respectively. The incubation was carried out for 30 min at 30 oC, in a
final reaction volume of 50 µl. A 2 µL aliquot of the assay mixture was
spotted on a polyethylenimine chromatographic sheet, and developed
in 4M LiCl:1 M acetic acid (1:4). After developing, the spots containing
ATP and cAMP were quantified using Radio-TLC scanner RITA
(RAYTEST, Germany) with evaluation software GINA STAR TLC.
Data were calculated from the percentage conversion of [3H]ATP to
[3H]cAMP. Ki values were calculated using the Graphpad Prism 5
software (San Diego, CA, USA). All assays were performed in
duplicate with three independent repetitions. For statistical analysis,
Student's t test (two-sided) was used. Results are given as means ±
SD.
Docking. The pre-processed protein was prepared with MOE
Structure Preparation and Protonate 3D tools with the default setup.
Both enantiomers of compound 32, (R)-32 and (S)-32, were properly
protonated and minimized to RMS gradient of 0.001 kcal/mol. For
docking study, the Template docking protocol was selected with
substructure setup where all atoms of PMEApp except hydrogens in
the C-1’ position (aliphatic α-carbon atom) were selected as the Query
in the setup. Bond rotation of ligands was allowed and default
placement and refinement methods were used with 50 retained
structures after the placement and 30 retained structures after the
refinement. For all calculations Amber12:EHT mixed force-field was
used with R-Field solvent model.
Biological assays
Effect on the viability of J774A.1 cells. J774A.1 cells were plated
onto white 96-well assay plates at 5 x 104 cells per well and allowed
to attach overnight. Cells were then washed with HBSS and treated
with 10 µM compounds for 5 h. Cell viability was then assessed with
a Cell Titer-Glo Luminescent Cell Viability assay (Promega, Madison,
WI, USA) according to the manufacturer’s instructions. Measurement
of luminescence signal was performed by use of a GENios microplate
reader (Tecan Systems). Data was expressed as percentage of the
control, represented by untreated cells.
Assays with mACs. HEK cells stably expressing AC1, AC2, or AC5
were cultured and frozen as previously described.[38,44] Cells were
thawed and plated in a white bottom 384-well plate (PerkinElmer,
Shelton, CT), and incubated for 1 hour at 37 °C with 5% CO2. Inhibitor
compounds (for a final concentration of 30 μM) were added to cells
and incubated at room temperature for 30 min prior to the addition of
the specific mAC stimulator (final concentrations: 3 M A23187 for
AC1, 100 nM PMA for AC2, and 300 nM forskolin for AC5) in 500 µM
3-isobutyl-1-methylxanthine (IBMX). Cells were incubated at room
temperature for 1 h and cAMP accumulation was measured using
Cisbio’s dynamic 2 kit (Cisbio Bioassays, Bedford, MA) according to
Inhibition of ACT – cell-based assay. J774A.1 cells were seeded in
a 96-well plate at 5x104 cells per well and left to attach overnight. Prior
to the experiment, cells were washed with HBSS (135 mM NaCl, 5.9
mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 25 mM glucose, 10 mM
HEPES [pH 7.4]) and pre-incubated with compounds at
concentrations of 0.001–30 µM for 5 h. After that, cells were exposed
to ACT (2 nM) from B.pertussis (Enzo Life Sciences, Palo Alto, CA;
SA=115 µmol/min/mg) for 30 min. Finally, the cAMP content was
determined by using the CatchPoint cAMP immunoassay kit
(Molecular Devices, Wokingham, UK). After the addition of the lysis
buffer (50 µL per well) provided by the manufacturer, the cellular
content was extracted by shaking the plate at 250 rpm for 10 min. The
plate was centrifuged to remove cell debris, the supernatant was
replaced to the assay plate, and immunoassays were carried out
the
manufacturer’s
instructions.
Materials:
3-Isobutyl-1-
methylxanthine (IBMX), and A23187 were purchased from Sigma-
Aldrich (St. Louis, MO). Phorbol 12-myristate 13-acetate (PMA), and
forskolin were purchased from Tocris Bioscience (Ellisville, MO). Opti-
MEM, antibiotic-antimycotic 100x solution, and Dulbecco’s modified
Eagle’s medium (DMEM) were purchased from Life technologies
(Grand Island, NY). FetalClone I serum and bovine calf serum, were
purchased from Hyclone (Logan, UT). G418 was purchased from
InvivoGen (San Diego, CA). The homogenous time-resolved
fluorescence (HTRF) cAMP kits were purchased from Cisbio
Bioassays (Bedford, MA).
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700715
ChemMedChem
FULL PAPER
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Acknowledgements
This work was supported by the subvention for development of
research organization (Institute of Organic Chemistry and
Biochemistry, RVO 61388963), by the Ministry of Interior of the Czech
Republic (VG20102015046) and Gilead Sciences (Foster City, CA,
USA). We acknowledge the financial support of the Czech Science
Foundation (P208/12/G016), US National Institutes of Health (NIH)
(MH101673) and MCMP Research Enhancement Award. This work
was also supported by the Ministry of Education, Youth and Sports
from the Large Infrastructures for Research, Experimental
Development and Innovations project “IT4Innovations National
Supercomputing Center – LM2015070” and NPU I project No.
LO1302.
ChemMedChem 2017, 12, 1133; l) M. Lukáč, D. Hocková, D. T. Keough, L. W.
Guddat, Z. Janeba, Tetrahedron 2017, 73, 692.
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Shiffman, L. Jeffers, Z. Goodman, M. S. Wulfsohn, S. Xiong, J. Fry, C. L.
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Conflict of interest
[19] I. Shoshani, W. H. Laux, C. Périgaud, G. Gosselin, R. A. Johnson, J. Biol.
Chem. 1999, 274, 34742.
The authors declare no conflict of interest.
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C. R. Wang, C. S. Gibbs, W. J. Tang, Proc. Natl. Acad. Sci. 2004, 101, 3242.
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Dračínský, T. F. Brust, P. Pávek, F. Trejtnar, V. J. Watts, Z. Janeba,
ChemMedChem, 2015, 10, 1351.
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Keywords: acyclic nucleoside phosphonates • adenylate cyclase
toxin • bisamidate • Bordetella pertussis • prodrugs
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Entry for the Table of Contents
Fighting bacterial toxemia, is where inhibitors of adenylate cyclases (ACs), the key virulence factors of many bacteria, may find their clinical
potential. Novel α-branched acyclic nucleoside phosphonato-diphosphates (nucleoside triphosphate analogues), with a hemiaminal ether
moiety and a 5-haloanthraniloyl substituent in the aliphatic part of the molecule are potent bacterial ACs inhibitors with IC50 values in the range
of 55 and 360 nM.
9
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