Aryl Bis-Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum

Article abstract of DOI:10.1002/cmdc.201500382

2-Methylerythritol 2,4-cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti-infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis-sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC₅₀ values in the micromolar range. The ortho-bis-sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC₅₀ values of 1.4 μm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha^-1. Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non-symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double-digit micromolar IC₅₀ range.

Full text of DOI:10.1002/cmdc.201500382

DOI: 10.1002/cmdc.201500382
Full Papers
Aryl Bis-Sulfonamide Inhibitors of IspF from Arabidopsis
thaliana and Plasmodium falciparum
Jonas Thelemann,[a] Boris Illarionov,[b] Konstantin Barylyuk,[a] Julie Geist,[a]
Johannes Kirchmair,[c] Petra Schneider,[d] Lucile Anthore,[a] Katharina Root,[a] Nils Trapp,[a]
Adelbert Bacher,[e] Matthias Witschel,*[f] Renato Zenobi,*[a] Markus Fischer,*[b]
Gisbert Schneider,*[d] and FranÅois Diederich*[a]
2-Methylerythritol 2,4-cyclodiphosphate synthase (IspF) is an
essential enzyme for the biosynthesis of isoprenoid precursors
in plants and many human pathogens. The protein is an attrac-
tive target for the development of anti-infectives and herbi-
cides. Using a photometric assay, a screen of 40000 com-
pounds on IspF from Arabidopsis thaliana afforded symmetrical
aryl bis-sulfonamides that inhibit IspF from A. thaliana (AtIspF)
and Plasmodium falciparum (PfIspF) with IC50 values in the mi-
cromolar range. The ortho-bis-sulfonamide structural motif is
essential for inhibitory activity. The best derivatives obtained
by parallel synthesis showed IC50 values of 1.4 mm against
PfIspF and 240 nm against AtIspF. Substantial herbicidal activity
was observed at a dose of 2 kghaÀ1. Molecular modeling stud-
ies served as the basis for an in silico search targeted at the
discovery of novel, non-symmetrical sulfonamide IspF inhibi-
tors. The designed compounds were found to exhibit inhibito-
ry activities in the double-digit micromolar IC50 range.
Introduction
Plants, many bacteria, and some protozoa, including numerous
human pathogens, use the non-mevalonate pathway for the
biosynthesis of isoprenoids.[1,2] This pathway is absent in
humans and has been clinically validated as a druggable anti-
malarial target.[3,4] Following its discovery in the 1990s,[1,2,5] the
pathway was shown to start with the condensation of pyru-
vate (1) and glyceraldehyde 3-phosphate (2) (Scheme 1). The
consecutive action of six enzymes (IspC–IspH) performs the
conversion into a mixture of isopentenyl diphosphate (IPP, 3)
and dimethylallyl diphosphate (DMAPP, 4). The antepenulti-
mate enzyme in this pathway, IspF, converts diphosphocytidyl-
2-methylerythritol 2-phosphate (5) into 2-methylerythritol 2,4-
cyclodiphosphate (6). This enzyme is active as a homotrimer,
with the three active sites located between adjacent subunits.
Several IspF inhibitors have been reported, including sub-
strate analogue ligands with dissociation constants (Kd) of
15 mm against IspF from Escherichia coli[6] and 70 mm against
IspF of Burkholderia pseudomallei (BpIspF).[7] We previously re-
ported thiazolopyrimidine-derived PfIspF inhibitors, discovered
by high-throughput screening (HTS), with IC50 (median inhibi-
tory concentration) values as low as 9.6 mm.[8]
[a] J. Thelemann, Dr. K. Barylyuk, Dr. J. Geist, L. Anthore, K. Root, Dr. N. Trapp,
Prof. Dr. R. Zenobi, Prof. Dr. F. Diederich
Laboratorium für Organische Chemie, ETH Zürich
Vladimir-Prelog-Weg 3, 8093 Zürich (Switzerland)
Fax: (+41)44-632-1109
E-mail: diederich@org.chem.ethz.ch
Herein we report work on aryl bis-sulfonamides, a new class
of IspF inhibitors, which were identified following the same
HTS approach.[8] Derivatives 7–19 (Tables 1 and 2) of the origi-
nal hit 7a were synthesized, and their interaction with IspF
was analyzed by biochemical, biophysical, and computational
methods. The structure-based rational design and synthesis of
novel aryl sulfonamide inhibitors 20a–g (Table 3) is also report-
ed.
zenobi@org.chem.ethz.ch
[b] Dr. B. Illarionov, Prof. Dr. M. Fischer
Institut für Lebensmittelchemie, Universität Hamburg
Grindelallee 117, 20146 Hamburg (Germany)
E-mail: markus.fischer@chemie.uniso-hamburg.de
[c] Prof. Dr. J. Kirchmair
Zentrum für Bioinformatik, Universität Hamburg
Bundesstr. 43, 20146 Hamburg (Germany)
[d] Dr. P. Schneider, Prof. Dr. G. Schneider
Institut für Pharmazeutische Wissenschaften, ETH Zürich
Vladimir-Prelog-Weg 4, 8093 Zurich (Switzerland)
E-mail: gisbert.schneider@pharma.ethz.ch
Results and Discussion
[e] Prof. Dr. A. Bacher
Binding affinities of aryl bis-sulfonamide ligands
Lehrstuhl für Biochemie, Technische Universität München
Lichtenbergerstr. 4, 85748 Garching (Germany)
HTS on AtIspF using a library of 40000 compounds afforded
the ortho-bis-sulfonamide derivative 7a. The compound
(Table 1) was shown to inhibit AtIspF and PfIspF (see Section S5
in the Supporting Information for a schematic representation
of the active sites of these enzymes) with respective IC50 values
[f] Dr. M. Witschel
BASF SE, Carl-Bosch-Str. 38, 67056 Ludwigshafen (Germany)
E-mail: matthias.witschel@basf.com
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500382.
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of 18 and 1.9 mm, based on ini-
tial rate measurements at a sub-
strate concentration of 0.5 mm.
In an attempt to improve these
values,
a series of symmetric
ortho-bis-sulfonamide derivatives
7–13
(Scheme 2).
was
synthesized
21a–
Diamines
c were treated with the corre-
sponding sulfonyl chlorides to
yield compounds 9–11. R1
groups were subsequently intro-
duced by nitration of 9 to yield
compounds 7a–i, or bromina-
tion to afford compounds 8a–k.
Compound 12 was formed by
cyanation of aryl bromide 8a,[9]
whereas Suzuki cross-coupling
of 8a with phenylboronic acid
yielded inhibitor 13.
Scheme 1. Non-mevalonate pathway of isoprenoid biosynthesis focusing on the transformation catalyzed by IspF.
Table 1. Inhibition of recombinant A. thaliana, P. falciparum, and B. pseudomallei IspF by symmetric aryl bis-sul-
fonamides.
All synthesized compounds 7–
13 were tested using the previ-
ously reported photometric
assay[10] with IspF isoforms from
A. thaliana, P. falciparum, and
B. pseudomallei (Table 1). Typical
inhibition curves are shown in
Section S2 of the Supporting In-
formation. Notably, 8 f inhibits
PfIspF with an IC50 value of
1.4 mm, and 8j inhibits AtIspF
with an IC50 value of 0.240 mm.
Our photometric assay cou-
ples the enzyme activity of IspF
with the oxidation of NADH via
a cascade of auxiliary enzymes
to enable photometric detec-
tion.[10] Most of the studied com-
pounds were verified to not in-
hibit these auxiliary enzymes
(Section S3, Supporting Informa-
tion). To rule out any contamina-
tion of the results via inhibition
of auxiliary enzymes, we used an
HPLC-based assay to directly de-
termine the IspF-mediated con-
version of 5 into 6, without any
auxiliary components.[11] Howev-
er, we emphasize that the HPLC
assay is an end-point assay, with
samples analyzed after a prede-
termined incubation period,
whereas the multicomponent
assay measures initial rates. The
HPLC assay was applied to the
most promising inhibitor/ortho-
logue combinations, with appar-
Compd R1
R2
IC50 [mm][a]
PfIspF
clogP[b] clogD[c]
AtIspF
BpIspF
7a
7b
7c
7d
7e
7 f
7g
7h
7i
NO2
p-tolyl
cPr
nHex
CH2CF3
Ph
4-Br-C6H4
4-OMe-C6H4
4-O(nBu)C6H4
4-CF3-C6H4
18Æ4
1.9Æ0.4
7.7Æ1.8
173Æ11
3.9
0.9
4.3
2.0
3.2
4.6
2.7
4.6
4.2
4.4
0.2
4.7
3.0
3.5
5.5
4.5
7.7
5.4
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
35Æ15
>500
[d]
[d]
–
–
54Æ23
226Æ16
296Æ25
101Æ17
311Æ22
44Æ7
17Æ5
21Æ4
13Æ5
2.7Æ0.5
1.4Æ0.2
4.8Æ0.9
1.4Æ0.2
4.6Æ1
0.69Æ0.3
7.7Æ2
12Æ2
4.6Æ1
123Æ13
8a
8b
8c
8d
8e
8 f
8g
8h
8i
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
p-tolyl
4-Br-C6H4
3-Br-C6H4
2-Br-C6H4
4-O(nBu)C6H4
4-CF3-C6H4
4-OCF3-C6H4
3,5-bis(CF3)C6H4
2-thiophenyl
4-Br-CH2C6H4
3-pyridyl
5.6Æ0.5
0.53Æ0.3
0.25Æ0.05
0.74Æ0.15
1.5Æ1.2
13Æ1
70Æ3
23Æ7
5.7
6.1
6.4
6.3
6.3
6.4
5.7
7.3
4.5
6.4
3.0
6.5
6.7
7.1
5.8
9.4
6.5
7.6
8.4
3.7
6.7
2.1
1.4Æ0.5
3.8Æ0.8
8.3Æ0.08
2.1Æ1.1
1.4Æ0.4
5.6Æ0.7
4.0Æ0.9
22Æ3
[e]
–
–
[e]
61Æ16
21Æ16
1.8Æ0.2
[e]
0.47Æ0.1
0.3Æ0.07
53Æ15
–
–
–
–
[e]
[e]
[e]
8j
8k
0.24Æ0.04
5.5Æ0.5
30Æ3
[e]
>500
–
9a
9b
9c
9d
9e
H
H
H
H
H
p-tolyl
cPr
nHex
CH2CF3
2-thiophenyl
40Æ12
>500
29Æ9
>500
3.2
1.2
4.4
2.1
2.9
4
281Æ24
>500
>500
>500
–
0.7
5.2
1.6
0.9
[d]
[d]
–
–
77Æ21
52Æ10
[e]
[e]
>500
181Æ50
10
p-tolyl
1.4Æ0.4
>500
62Æ10
–
5.7
5.9
[e]
[e]
[e]
11
12
13
Me
CN
Ph
p-tolyl
p-tolyl
p-tolyl
>500
–
–
–
5.1
3.7
6.1
5.9
3.2
7.6
48Æ12
50Æ11
7.8Æ2
22Æ4
[a] Assay buffer: 100 mm Tris·HCl (pH 8.0); error margins correspond to the RMSD of the regression fit (Sec-
tion S2 in the Supporting Information for details). [b] Values were calculated with the ACD/Percepta[43] software
package (GALAS algorithm). [c] Values were calculated with ACD/Percepta[43] at pH 8.0. [d] Data were not inter-
pretable. [e] Inhibition was not determined. Inhibition by compounds 9 f–p was not determined.
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Bis-sulfonamides are known to
chelate metal cations.[13,14] We re-
corded the crystal structures of
free ligand 8a and of its dianion
forming a 2:1 host–guest com-
plex with Zn2+ (Figure 1). The
two sulfonamide moieties of free
8a have torsional angles of N(2)-
S(2)-C(8)-C(9)=1048 and N(1)-
S(1)-C(5)-C(14)=1018, which is
close to the preferred N-S-C-C
torsional angle of 908. As ob-
served before, the nitrogen lone
pair bisects the O-S-O frag-
ment.[15] The four N-S-C-C tor-
sional angles for the complex of
Scheme 2. Synthesis of compounds 7–13. A list of R2 substituents is provided in Table 1. Reagents and conditions:
a) R2SO2Cl, CH2Cl2, pyridine, 258C, 12 h; b) HNO3, AcOH, 608C, 30 min; c) Br2, NaOAc, AcOH, 25–1008C, 1 h;
d) CuCN, DMF, 1208C, 20 h; e) phenylboronic acid, Cs2CO3, [Pd(dppf)Cl2]·CH2Cl2, 1,4-dioxane, 908C, 2 h. Ts=p-tolu-
enesulfonyl; dppf=1,1’-bis(diphenylphosphino)ferrocene.
ent IC50 values in the nanomolar or low single-digit micromolar
range, according to the multicomponent assay. As shown in
Section S4 in the Supporting Information, the apparent IC50
values determined by the two different assay methods were
found to be in agreement, within narrow margins.
The results listed in Table 1 show that nitro or bromo sub-
stituents at the R1 positions of the ligands were conducive to
strong inhibitory activity. Compounds 7a (R1 =NO2) and 8a
(R1 =Br) show IC50 values of 1.9 and 13.1 mm against PfIspF,
whereas 9a (R1 =H) has an IC50 value of 29 mm and compound
11 (R1 =Me) is inactive (IC50 >500 mm). These findings correlate
with the measured pKa1 values (Section S7, Supporting Infor-
mation) for the first sulfonamide NH deprotonation, which is
lower for compounds 7a (pKa1 =3.7) and 8a (pKa1 =5.9) than
for 9a (pKa1 =7.9) and 11 (pKa1 =7.5), suggesting that the bis-
sulfonamides bind in a deprotonated state to the enzyme, as
the activity assays were performed at pH 8.0.
Compound 8b was also assayed for herbicidal activity in the
greenhouse (Section S6, Supporting Information) and showed
very good activity (+ + +) against Setaria viridis, Echinochloa
crusgalli, and Apera spica-venti at a dose of 2 kghaÀ1. Li-
gands 7–9 were tested for antimalarial activity using
a [3H]hypoxanthine incorporation assay,[12] but failed to inhibit
the proliferation of blood-stage P. falciparum (EC50 values
>5 mm).
Scheme 3. Synthesis of non-symmetric sulfonamides 14–19. Reagents and
conditions: a) SO2Cl2, 1508C, 3 h; b) NH3, 1,4-dioxane, 258C, 30 min; c) 4-bro-
mobenzenesulfonyl chloride, CH2Cl2, pyridine, 258C, 12 h; d) R1SO2Cl,
R2SO2Cl, CH2Cl2, pyridine À78–258C, 12 h; e) Br2, NaOAc, AcOH, 25–1008C,
1 h; f) 4-bromobenzenesulfonyl chloride, CH2Cl2, pyridine, 258C; g) Br2,
NaOAc, AcOH, 25–1008C, 1 h; h) 4-toluenesulfonyl chloride, CH2Cl2, pyridine
258C, 12 h.
To determine whether the molecular symmetry of ortho-bis-
sulfonamides is essential for IspF inhibition, we prepared the
non-symmetric derivatives 14–16 (Scheme 3). Moreover, sever-
al monosulfonamides 17–19 were synthesized. Specifically,
compound 21d was treated with chlorosulfonic acid followed
by ammonia to provide sulfonamide 22, which was reacted
with 4-bromobenzenesulfonylchloride to give 14. Amines 21a,
21e, 21 f, and 21g were reacted with the corresponding aryl-
sulfonyl chloride to give 23–26 and 19, respectively. Bromina-
tion provided compounds 15–18.
The non-symmetric bis-sulfonamides 14–16 exhibited weak
activity, and the monosulfonamides 17–19 little or no inhibito-
ry activity (Table 2). Similar to the other sulfonamide ligands re-
ported herein, the solubility of the compounds is high, ena-
bling all the physical studies performed.
8a with Zn2+ are identical at 388. The Zn2+ ion is tetrahedrally
coordinated, with the four Zn···N distances being 2.00 Š.
Isothermal titration calorimetry (ITC) experiments at 303 K in
Tris hydrochloride buffer (0.1m)/(CH3)2SO 2:1 confirmed that
bis-sulfonamides 8a and 8b bind to the Zn2+ ion with appar-
ent association constants (Kapp) of 7.7”106 and 15.6”106 mÀ2
Therefore, it appeared possible that depletion of the essential
Zn2+ cofactor in IspF could cause the observed inhibitory
action of the studied compounds. However, compounds 7a,
7 f, 7g, 8a, 8b, and 8 f caused undiminished IspF inhibition in
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HEPES, 150 mm NaCl, pH 8.0;
T=298 K) revealed a Kd value of
17 mm with fitting parameters
Table 2. Inhibition of recombinant A. thaliana and P. falciparum IspF by the non-symmetric sulfonamides 14–
19.
DHo =À5.9 kJmolÀ1
,
TDSo =21.3 kJmolÀ1
,
and
DGo =À27.2 kJmolÀ1. Complexa-
tion is strongly entropically and
weakly enthalpically driven. The
substantial increase in entropy
hints at the desolvation of ionic
residues in both ligand and
enzyme upon coordination.[16–18]
The stoichiometric factor n was
determined at 2.3 (IspF mono-
mer: 8b), which leads to a stoi-
chiometry of 1.3 ligands per IspF
trimer.
Compd
R1
R2 R3
R4
R5
IC50 [mm][a]
clogP[b]
clogD[c]
AtIspF
PfIspF
14
15
16
17
18
19
Br
Br
Br
Br
Br
NO2
Br
Br
Br
H
H
H
H
Br
Br
H
SO2NH2
NHSO2CF3
NHSO2CH3 4-Br-C6H4
4-Br-C6H4
p-tolyl
301Æ46
23Æ5
42Æ4
14Æ2
56Æ6
3.9
5.6
3.9
4.6
5.4
2.0
3.8
4.7
3.1
4.1
4.4
3.0
>500
OH
4-Br-C6H4
4-Br-C6H4
p-tolyl
133Æ5
>500
272Æ25
>500
>500
177Æ30
H
OMe
NH2
H
[a] Assay buffer: 100 mm Tris·HCl (pH 8.0); error margins correspond to the RMSD of the regression fit (Sec-
tion S2 in the Supporting Information for details). [b] Values were calculated with the ACD/Percepta[43] software
package (GALAS algorithm). [c] Values were calculated with ACD/Percepta[43] at pH 8.0.
ESI-MS binding studies
The binding affinity of 8b for AtIspF was also studied by direct
titration in native ESI-MS (Section S10, Supporting Information).
This method allows evaluation of protein–ligand affinities,
binding stoichiometry, and allosteric effects.[19,20] Advanta-
geously, measurements can be conducted with low ligand and
enzyme concentrations.
Enzymes are believed to largely retain their folded, solution-
like conformation in the gas phase when ionized by ESI under
gentle desolvation and ion-transfer conditions, often referred
to as “native ESI-MS”.[21] Proteins and protein complexes can be
detected as distinct signals to allow direct readout of the com-
plex composition and stoichiometry. Moreover, in the case of
enzyme–inhibitor binding, relative intensities of the free and
ligand-bound enzyme peaks in mass spectra can be treated as
the relative abundances of the respective species in solution.
This gives direct access to solution-phase binding affinity de-
termination by native ESI-MS.[22–24] The binding affinity of 8b
was measured at different ligand concentrations ranging from
1.6 to 50 mm with an enzyme concentration of 4.7 mm
(Figure 2). The observed peak broadening can be attributed to
residual solvent molecules and buffer ions that remain bound
to the protein under the gentle desolvation conditions used.
The peak at 54964 Da (calculated protein mass: 54984 Da) rep-
resents the AtIspF trimer without a bound ligand. The peaks at
55668, 56372, and 57076 Da represent protein molecules
bound to one, two, or three inhibitor molecules, respectively.
No more than three ligands per trimer were observed. As the
ligand concentration increases, the peak intensities of the
enzyme–inhibitor complexes increase. The dissociation con-
stant Kd was determined by applying the Hill equation[25] to
the data on the fraction of bound ligand concentration deter-
mined from the mass spectra (Figure 2). We obtained Kd =
21 mm and a Hill parameter value nH =1.5. The mass spectro-
metric dissociation constant and the value obtained by ITC
(Kd =17 mm) are in remarkably good agreement, but higher
than the calculated Ki value of 0.26 mm using the Cheng–Prus-
Figure 1. A) ORTEP plot at the 50% probability level of the crystal structure
of inhibitor 8a. B) ORTEP plot at the 15% probability level of the crystal
structure of the 2:1 complex of dianionic ligand 8a with Zn2+. One of the
two NBu4 counterions is shown. T=100 K, arbitrary numbering. See Sec-
tion S13 in the Supporting Information for further information.
+
the photometric assay in the presence of Zn(OAc)2 (ligand/
Zn(OAc)2 =2:1, see Table 1SI, Supporting Information). Hence,
the observed inhibition of IspF by bis-sulfonamide compounds
is not a consequence of Zn2+ depletion of the enzyme by the
inhibitors.
Isothermal titration calorimetry (Section S12, Supporting
Information) using 8b and AtIspF in ITC buffer (50 mm
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mode of 8b (Figure 3). The co-crystal structures of A. thaliana
with CDP (PDB ID: 2PMP[28]) and P. falciparum with CMP as
ligand (PDB ID: 4C81[29]) served as templates for the in silico
studies. In light of the ligand’s first pKa value of 5.9, one of the
sulfonamide nitrogen atoms of the symmetric molecule was
assumed to be deprotonated. GOLD[30,31] was used for docking,
ChemPLP[32] for scoring, and MOLOC[33] for geometric optimiza-
tion of the docking poses. Further details are provided in Sec-
tion S8 in the Supporting Information.
Due to steric hindrance, the anionic nitrogen of 8b is
unable to coordinate to the essential Zn2+ ion of IspF. Howev-
er, the docking suggests that 8b could form a salt bridge with
its deprotonated sulfonamide motif to Lys135 (Lys213 for P. fal-
ciparum) at the active site. Furthermore, the second sulfona-
mide moiety can bind with its SO2 group to the Zn2+ ion. It is
known from enzymes containing catalytic Zn2+ ions that the
primary coordination sphere of the metal can be trigonal bi-
pyrimidal or square pyramidal (T5, 44% of all cases[34]). Thus,
the T5 coordination to the catalytic zinc ion of AtIspF, predict-
ed by GOLD, is possible. It was recently well established that
binding to the Zn2+ ion of IspF from different species under
replacement of the fourth (water) ligand only yields a weakly
enhanced binding affinity;[7,35] in other words, the SO2 group
as weakly binding ligand is a good possibility.
Monosulfonamide inhibitors derived by rational design
Figure 2. ESI-MS spectra of AtIspF (at 4.7 mm) titrated with ligand 8b. The
spectral region of the protein trimer is displayed, and the various concentra-
tions of 8b are indicated next to the traces.
Despite the reported poor gain in binding affinity upon substi-
tuting the fourth water coordination site to the Zn2+ ligand by
other donor ligands,[7,35] we wanted to probe this once more
with inhibitors that feature a sterically unencumbered primary
sulfonamide as zinc binding group linked to a cytosine deriva-
tive that docks into Pocket III of the active site.
off equation on the inhibitory data from the photometric
assay.[26,27]
Compounds 20a–g (Scheme 4) were derived by molecular
modeling using MOLOC.[33] Compound 20a (Figure 4) was de-
signed to bind with its 3-aminoisoquinoline ring (similar to li-
gands containing a 2-aminopyridine moiety for occupancy of
the same pocket)[36] to the cytosine binding Pocket III. The het-
erocyclic nitrogen atom was predicted to interact with the
Docking studies
Attempts to obtain a co-crystal structure of an aryl bis-sulfona-
mide inhibitor bound to IspF were unsuccessful. We used auto-
mated ligand docking in an attempt to investigate the binding
Figure 3. Ligand binding poses predicted for 8b using GOLD for docking, ChemPLP for scoring, and MOLOC for optimization. A) AtIspF; B) PfIspF. The docking
poses represent the highest-ranked solutions (ChemPLP scores: 73.62 and 71.77, respectively). Color code: Cenzyme gray, O red, N blue, S yellow, Br dark red,
Cligand green.
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Synthesis and biological activity of designed
monosulfonamide ligands
5-(Chlorosulfonyl)isophthalic acid (27) was converted
into compound 28, then monobrominated to provide
building block 29 (Scheme 4). Precursor 30 was pre-
pared following a published protocol,[38] reduced to
alcohol 31, and brominated to afford building block
32.
3-Aminoisoquinoline precursor 33 was synthesized
according to published procedures[39,40] and Boc pro-
tected to afford compound 34. One-pot borylation,
followed by coupling to 3-bromobenzenesulfona-
mide and deprotection, afforded 20a (Scheme 5).
Borylation of compound 34, coupling to building
block 29 (Scheme 4), and oxida-
Scheme 4. Synthesis of building blocks 29 and 32. Reagents and conditions: a) tBuNH2,
CH2Cl2, 0!238C, 1 h; b) BH3, THF, 0!238C, 15 h; c) NBS, PPh3, THF, 0!258C, 30 min;
d) BH3, THF, 0!238C, 1 h; e) PBr3, CH2Cl2, 238C, 24 h. NBS=N-bromosuccinimide;
THF=tetrahydrofuran.
tion provided aldehyde 35,
whereas borylation of com-
pound 34 and coupling to build-
ing block 32 (Scheme 4) gave
aryl bromide 36. Bromides at
benzylic positions react faster
than aryl bromides;[41] therefore,
the selective coupling of com-
pounds 32 and 34 could be ach-
ieved.
Reductive amination of alde-
hyde 35 with the corresponding
amines afforded inhibitors 20b–
d. Suzuki cross-coupling of com-
pound 36 with the correspond-
ing boronic ester/acid provided,
after deprotection, compounds
20e–g.
Compounds 20b–d, which
Figure 4. Proposed binding mode of 20a in the active site of AtIspF (PDB ID: 2PMP), modeled with MOLOC. Color
code: Cenzyme gray, O red, N blue, S yellow, Cligand green.
had been expected to form a hy-
drogen bond with the backbone
backbone NH of Ile108, (d(N···NHIle108)=2.9 Š), whereas the exo-
cyclic NH2 group should undergo hydrogen bonding to the
backbone C=O groups of Pro106 (d(NH2···O=CPro106)=3.4 Š)
and Leu103 (d(NH2···O=CLeu103)=2.9 Š). Similar to the binding
of inhibitors with a primary sulfonamide to carbonic anhy-
drase,[37] the Zn2+ ion should coordinate to the deprotonated
primary sulfonamide nitrogen atom (Figure 4).
C=O of Phe64 in the hydrophobic Pocket II, showed little to no
biological activity (Table 3). More success was achieved with
those inhibitors that feature hydrophobic substituents to fill
the flexible Pocket II (see Section S5 in the Supporting Informa-
tion for an overlay of various X-ray structures showing the
large conformational flexibility of this pocket). The most active
PfIspF inhibitors developed by rational design were com-
pounds 20 f (R=phenyl) and 20g (R=4-CF3-C6H4), with IC50
values of 39 and 45 mm, respectively.
The moderate activity of compound 20a, with IC50 values of
561Æ92 mm against AtIspF and 287Æ42 mm against PfIspF, led
us to design compounds 20b–g, which feature additional sub-
stituents meta to the sulfonamide group to provide binding to
the flexible hydrophobic Pocket II (Table 3). Compounds 20b–
e were designed to undergo hydrogen bonding to the back-
bone C=O of Phe64 (Figure 4), whereas compounds 20 f and
20g exhibit a hydrophobic group to interact with the hydro-
phobic Pocket II.
Conclusions
We presented aryl bis-sulfonamides as a new class of IspF in-
hibitors, identified by HTS. They are the most active IspF inhibi-
tors reported so far, with IC50 values as low as 240 nm against
AtIspF and 1.4 mm against PfIspF. The inhibition was measured
by an enzyme-coupled photometric assay and was confirmed
for the most active inhibitors by an alternative HPLC assay. The
binding affinities (Kd values) were determined by ITC and ESI-
MS to be 17 mm and 21 mm, respectively. The binding mode of
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Full Papers
bind to the cytosine Pocket III, a terminal sulfona-
mide moiety to coordinate to the Zn2+ ion, and
a vector addressing the region of the flexible Pocke-
t II. Whereas attempted hydrogen bonding to the C=
O group of Phe64 at the entrance of Pocket II was
unsuccessful, significant binding affinity could be
gained by filling the flexible hydrophobic parts of
this pocket, with measured IC50 values down to
39 mm.
Table 3. Inhibition of recombinant A. thaliana, P. falciparum, and M. tuberculosis IspF
by ligands 20a–g.
Compd
R
IC50 [mm][a]
PfIspF
clogP[b] clogD[c]
AtIspF
MtIspF
[d]
20a
20b
H
561Æ92
287Æ42
>1000
–
2.2
2.5
1.9
0.3
[d]
>1000
–
Experimental Section
Biology
[d]
20c
20d
CH2NHEt
CH2NHcPr
>1000
846Æ113
939Æ290
–
–
2.6
2.6
0.3
1.2
[d]
632Æ142
Recombinant IspF proteins from A. thaliana, P. falcipa-
rum, M. tuberculosis and B. pseudomallei, and cytidylate
kinase from E. coli were prepared as reported else-
where.[8,10] Adenylate kinase and lactate dehydrogenase
were purchased from Sigma–Aldrich. NADH was pur-
chased from Acros Organics. See the Supporting Infor-
mation for a description of the biological assays and pKa
measurements.
[d]
20e
296Æ946
170Æ20
–
2.3
1.9
20 f
20g
Ph
>1000
76Æ15
39Æ23 59Æ3
45Æ6 43Æ19
3.6
5.0
3.7
4.7
4-CF3-C6H4
[a] Error margins correspond to the RMSD of the regression fit (see Section S2 in the
Supporting Information). [b] Values were calculated with the ACD/Percepta[43] software
package (GALAS algorithm). [c] Values were calculated with ACD/Percepta[43] at pH 8.0.
[d] Values not determined.
Chemistry
Materials: Experimental details for the synthesis of
ligand 20g are reported below. The synthesis of all other
compounds, ESI-MS and ITC measurements, and details of the mo-
lecular docking studies are provided in the Supporting Information.
Structural models of the protein were visualized in PyMOL.[42]
bis-sulfonamide ligand 8b was investigated by using a docking
approach. In the proposed most favorable geometry, the de-
protonated sulfonamide of the ligand undergoes ion pairing
with the side chain of Lys135 and does not bind to the Zn2+
ion at the active site of IspF. However, the metal ion interacts
with the SO2 group of the second sulfonamide moiety in the
ligand.
N,N-Bis(tert-butoxycarbonyl)-6-bromoisoquinolin-3-amine (34): A
solution of isoquinoline 33 (553 mg, 2.48 mmol) and 4-(dimethyla-
mino)pyridine (31 mg, 0.26 mmol) in CH3CN was cooled to 08C,
treated with di-tert-butyl dicarbonate (1.67 g, 7.67 mmol) and NEt3
(1.70 mL, 12.3 mmol), and stirred for 2 h at 08C and then for 20 h
at 238C. Evaporation and chromatography (SiO2, cyclohexane/
Using a molecular modeling approach, we further developed
a series of inhibitors with a 3-aminoisoquinoline moiety to
Scheme 5. Synthesis of 20a–g. Reagents and conditions: a) Boc2O, DMAP, NEt3, CH3CN, 0–248C, 24 h; b) (BPin)2, KOAc, [Pd(dppf)Cl2]·CH2Cl2, 1,4-dioxane, 808C,
4 h; c) 3-(bromomethyl)benzenesulfonamide, Cs2CO3, [Pd(dppf)Cl2]·CH2Cl2, 1,4-dioxane/H2O (10:1), 808C, 12 h; d) CF3COOH, CH2Cl2, 258C, 12 h; e) 29, Cs2CO3,
[Pd(dppf)Cl2]·CH2Cl2, 1,4-dioxane/H2O (10:1), 808C, 2 h; f) DMP, CH2Cl2, 258C, 5 h; g) RH, NaBH(OAc)3, CH2Cl2, 258C, 80 min; h) CF3COOH, 708C, 90 min; i) 32,
Cs2CO3, [Pd(dppf)Cl2]·CH2Cl2, 1,4-dioxane/H2O (10:1), 808C, 2 h; j) RB(OH)2, Cs2CO3, [Pd(dppf)Cl2]·CH2Cl2, 1,4-dioxane/H2O (10:1), 808C, 50 min. Boc=t-butoxycar-
bonyl; DMAP=4-(dimethylamino)pyridine; Pin=pinacolato; DMP=Dess–Martin periodinane.
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EtOAc 10:1!6:1) afforded 34 (426 mg, 41%) as a yellow resin. Rf =
0.46 (SiO2, cyclohexane/EtOAc 2:1); mp: 82–858C; 1H NMR
(400 MHz, CD3OD): d=1.40 (s, 18H, CH3), 7.73 (brs, 1H, H-C(4)),
7.80 (dd, J=8.8, 1.9 Hz, 1H, H-C(7)), 8.06 (d, J=8.8 Hz, 1H, H-C(8)),
8.21 (d, J=1.6 Hz, 1H, H-C(5)), 9.18 ppm (s, 1H, H-C(1)); 13C NMR
(101 MHz, CD3OD): d=28.11, 84.56, 119.15, 127.22, 127.48, 130.03,
130.66, 132.65, 139.90, 148.60, 152.68, 152.89 ppm; IR (ATR): n˜ =
2979, 2933, 1789, 1752, 1717, 1620, 1367, 1355, 1306, 1272, 1243,
1:1!2:1) afforded 36 (70 mg, 36%) as a yellow oil. Rf =0.32 (SiO2,
1
EtOAc/cyclohexane 1:2); H NMR (400 MHz, CDCl3): d=1.45 (s, 18H,
2CMe3), 4.19 (s, 2H, CH2), 5.00 (brs, 2H, SO2NH2), 7.39 (d, J=8.6 Hz,
1H, H-C(7’)), 7.53–7.59 (m, 2H, H-C(4’, 5’), 7.60 (brs, 1H, H-C(4)),
7.72 (brs, 1H, H-C(2)), 7.91–7.95 (m, 2H, H-C(5, 8’)), 9.11 ppm (s,
1H, H-C(1’)); 13C NMR (101 MHz, CD3OD): d=27.97, 41.64, 83.22,
117.74, 123.30, 125.49, 126.23, 126.58, 127.73, 128.28, 128.77,
136.13, 137.68, 141.65, 143.46, 144.09, 147.47, 151.56, 151.83 ppm;
IR (ATR): n˜ =3329 (brw), 2980 (m), 1781 (m), 1738 (m), 1633 (m),
1566 (w), 1368 (m), 1339 (m), 1280 (m), 1249 (m), 1153 (s), 1104 (s),
948 (m), 912 (m), 729 cmÀ1 (s); HR-ESI-MS: m/z (%): 594.1094 (10,
[M+H]+ calcd for C26H3181BrN3O6S+: 594.1094), 592.1109 (10, [M+
H]+ calcd for C26H3179BrN3O6S+: 592.1111), 437.9942 (100,
[MÀ2C4H8ÀCO2 +H]+ calcd for C17H1581BrN3O4S+: 437.9946),
435.9962 (88, [MÀ2C4H8ÀCO2 +H]+ calcd for C17H1579BrN3O4S+:
435.9967).
1150, 1111, 1059, 1025, 946, 901, 888, 851, 815, 775, 721 cmÀ1; HR-
+
ESI-MS: m/z (%): 425.0890 (10, [M+H]+ calcd for C19H2481BrN2O4
425.0894), 423.0911 (12, [M+H]+ calcd for C19H2479BrN2O4
:
:
+
423.0914), 325.0368 (37, [MÀCH2=CMe2ÀCO2 +H]+ calcd for
C14H1681BrN2O2+: 325.0370), 323.0385 (34, [MÀCH2=CMe2ÀCO2 +
H]+ calcd for C14H1679BrN2O2+: 323.0390), 268.9745 (91, [MÀ2CH2=
CMe2ÀCO2 +H]+ calcd for C10H881BrN2O2+: 268.9744), 266.9764
+
(100, [MÀ2CH2=CMe2ÀCO2 +H]+ calcd for C10H879BrN2O2
266.9764).
5-[(3-Aminoisoquinolin-6-yl)methyl]-4’-(trifluoromethyl)-[1,1’-bi-
3-Bromo-5-(hydroxymethyl)benzenesulfonamide (31): 3-Bromo-
5-sulfamoylbenzoic acid (30, 2.30 g, 8.21 mmol) was dissolved in
dry THF (10 mL), treated with a solution of 1m BH3 in THF
(16.4 mL, 16.4 mmol), and stirred for 20 h at 238C. The mixture was
cooled to 08C and treated with H2O (15 mL). The aqueous layer
was extracted with EtOAc (3”25 mL). The combined organic layers
were dried over Na2SO4, filtered, concentrated, and washed with
toluene (50 mL) to give 31 (2.16 g, 98%) as a white solid. Rf =0.18
(SiO2, CH2Cl2/MeOH 10:1); mp: 115–1168C; 1H NMR (400 MHz,
CD3OD): d=4.66 (s, 2H, CH2), 7.74 (s, 1H, H-C(4)), 7.86 (s, 1H, H-
C(6)), 7.93 ppm (s, 1H, H-C(2)); 13C NMR (101 MHz, CD3OD): d=
63.67, 123.45, 123.86, 128.57, 133.88, 147.00, 147.03 ppm; IR(ATR):
n˜ =3663 (w), 3377 (s), (3263 (s), 3068 (m), 2988 (m), 2915 (m), 1760
(w), 1595 (m), 1568 (m), 1531 (m), 1456 (m), 1430 (m), 1403 (m),
1326 (very s), 1257 (m), 1208 (m), 1148 (very s), 1113 (s), 1104 (s),
1060 (s), 992 (m), 916 (s), 885 (m), 873 (s), 857 (s), 780 (m), 695 (w),
671 (s), 631 cmÀ1 (m); HR-ESI-MS (negative mode): m/z (%):
265.9313 (100, [MÀH]À calcd for C7H781BrNO3SÀ: 265.9314),
263.9334 (100, [MÀH] À calcd for C7H779BrNO3SÀ: 263.9335).
phenyl]-3-sulfonamide (20g):
0.17 mmol), 4-(trifluoromethyl)phenylboronic
A
solution of 34 (100 mg,
acid (39 mg,
0.20 mmol), and Cs2CO3 (165 mg, 0.51 mmol) in 1,4-dioxane (5 mL)
was degassed for 10 min, treated with [PdCl2(dppf)]·CH2Cl2 (14 mg,
0.02 mmol), and stirred for 50 min at 808C. The mixture was fil-
tered over silica, eluting with EtOAc (50 mL) and evaporated. A so-
lution of the residue in CH2Cl2 (4 mL) was cooled to 08C and treat-
ed with TFA (0.15 mL), stirred for 14 h, and evaporated. Chroma-
tography (SiO2, CH2Cl2/MeOH/NH3 95:4:1) afforded 20g (51 mg,
66%) as a white solid. Rf =0.21 (SiO2, CH2Cl2/MeOH/NH3 95:4:1);
1
mp: 223–2248C; H NMR (400 MHz, (CD3)2SO): d=4.20 (s, 2H, CH2),
5.88 (brs, 2H, ArNH2), 6.57 (s, 1H, H-C(4’’)), 7.08 (dd, J=7.4, 1.6 Hz,
1H, H-C(7’’)), 7.40 (brs, 2H, SO2NH2), 7.45 (brs, 1H, H-C(5’’)), 7.71 (d,
J=7.4 Hz, 1H, H-C(8’’)), 7.76 (t, J=1.7 Hz, 1H, H-C(2)), 7.88 (d, J=
8.4 Hz, 2H,H-C(2’, 6’), 7.93 (d, J=8.4 Hz, 2H, H-C(3’, 5’)), 7.96 (t, J=
1.7 Hz, 1H, H-C(4)), 8.02 (t, J=1.7 Hz, 1H, H-C(6)), 8.76 ppm (s, 1H,
H-C(1’’)); 13C NMR (101 MHz, (CD3)2SO): d=41.07, 96.96, 121.29,
1
122.10, 123.17, 123.21, 124.21 (q, J(C,F)=271.9 Hz), 125.43, 126.04
(q, 3J(C,F)=3.7 Hz), 127.63, 128.11, 128.48 (q, 2J(C,F)=31.6 Hz),
130.76, 138.74, 139.45, 142.19, 142.68, 142.89, 145.20, 150.97,
156.64 ppm; 19F NMR (282 MHz, (CD3)2SO): d=À60.98 ppm; IR
(ATR): n˜ =3370 (w), 3312 (w), 1636 (m), 1496 (w), 1447 (w), 1347
(w), 1323 (s), 1148 (m), 1125 (m), 1110 (m), 1062 (w), 899 (w),
840 cmÀ1 (m); HR-ESI-MS: m/z (%): 458.1151 (100, [M+H]+ calcd
for C23H19F3N3O2S+: 458.1145).
3-Bromo-5-(bromomethyl)benzenesulfonamide (32): A suspen-
sion of sulfonamide 31 (1.40 g, 5.26 mmol) in CH2Cl2 (50 mL) was
treated with PBr3 (600 mL, 6.31 mmol) at 238C, stirred for 24 h, di-
luted with H2O (10 mL) and saturated aqueous NaHCO3 (60 mL),
and extracted with EtOAc (3”50 mL). The combined organic layers
were dried over MgSO4, filtered and evaporated to give 32
1
(970 mg, 56%) as a white solid; mp: 120–1218C; H NMR (400 MHz,
CD3OD): d=4.61 (s, 2H, CH2Br), 7.83 (t, J=1.7 Hz, 1H, H-C(4)), 7.92
(t, J=1.7 Hz, 1H, H-C(6)), 7.97 ppm (t, J=1.7 Hz, 1H, H-C(2)); Acknowledgements
13C NMR (101 MHz, CD3OD): d=31.40, 123.58, 126.51, 129.73,
136.39, 143.25, 147.45 ppm; IR (ATR): n˜ =3342 (m), 3249 (m), 1566
We thank the ETH Research Council and the Hans Fischer Gesell-
(w), 1429 (w), 1317 (s), 1297 (m), 1230 (w), 1208 (m), 1157 (s), 1111
(w), 922 (m), 884 (m), 779 (m), 682 cmÀ1 (m); HR-ESI-MS (negative
mode): m/z (%): 329.8457 (50, [MÀH]À calcd for C7H681Br2NO2SÀ:
329.8449), 327.8476 (100, [MÀH]À calcd for C7H681Br79BrNO2SÀ:
327.8471), 325.8498 (44, [MÀH]À calcd for C7H679Br2NO2SÀ:
325.8491).
schaft e.V. for their generous support of this research. We are
grateful to Dr. Bruno Bernet (ETH) for proofreading the Experi-
mental Section and to Anatol Schwab, Dr. Tristan Reekie, and
Oliver Dumele for proofreading the entire manuscript. We are
thankful to Michael Solar for recording the crystal structures.
3-[(3-Aminoisoquinolin-6-yl)methyl]-5-bromobenzenesulfona-
mide (36): A solution of isoquinoline 34 (212 mg, 0.50 mmol),
bis(pinacolato)diboron (135 g, 0.53 mmol), and KOAc (119 mg,
1.50 mmol) in 1,4-dioxane (10 mL) was degassed for 10 min, treat-
ed with [Pd(PPh3)Cl2] (37 mg, 0.05 mmol), and stirred for 4 h at
808C. After addition of Cs2CO3 (1.06 g, 1.50 mmol), degassed H2O
(0.5 mL), and compound 32 (265 mg, 0.60 mmol), stirring was con-
tinued for 2 h. The mixture was filtered over silica, eluting with
EtOAc (50 mL). Chromatography (SiO2, EtOAc/cyclohexane 1:2!
Keywords: bis-sulfonamides · docking · inhibitors · isoprenoid
biosynthesis · non-mevalonate pathway · P. falciparum
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Received: August 28, 2015
Published online on October 5, 2015
ChemMedChem 2015, 10, 2090 – 2098
www.chemmedchem.org
2098
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim