LEGO-Inspired Drug Design: Unveiling a Class of Benzo[d]thiazoles Containing a 3,4-Dihydroxyphenyl Moiety as Plasma Membrane H+-ATPase Inhibitors

Article abstract of DOI:10.1002/cmdc.201700635

The fungal plasma membrane H⁺-ATPase (Pma1p) is a potential target for the discovery of new antifungal agents. Surprisingly, no structure–activity relationship studies for small molecules targeting Pma1p have been reported. Herein, we disclose a LEGO-inspired fragment assembly strategy for the design, synthesis, and discovery of benzo[d]thiazoles containing a 3,4-dihydroxyphenyl moiety as potential Pma1p inhibitors. A series of 2-(benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones was found to inhibit Pma1p, with the most potent IC₅₀ value of 8 μm in an in vitro plasma membrane H⁺-ATPase assay. These compounds were also found to strongly inhibit the action of proton pumping when Pma1p was reconstituted into liposomes. 1-(3,4-Dihydroxyphenyl)-2-((6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)ethan-1-one (compound 38) showed inhibitory activities on the growth of Candida albicans and Saccharomyces cerevisiae, which could be correlated and substantiated with the ability to inhibit Pma1p in vitro.

Full text of DOI:10.1002/cmdc.201700635

Accepted Article
Title: LEGO-inspired drug design: unveiling of the novel class of
benzo[d]thiazole containing a 3,4-dihydroxyphenyl moiety as
plasma membrane H+-ATPase inhibitors
Authors: Truong Thanh Tung, Dao Trong Tuan, Marta Grifell Junyent,
Michael Palmgren, Thomas Günther-Pomorski, Anja Thoe
Fuglsang, Søren Brøgger Christensen, and John Nielsen
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201700635
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700635
A Journal of
www.chemmedchem.org

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
LEGO-inspired drug design: unveiling of the novel class of
benzo[d]thiazole containing a 3,4-dihydroxyphenyl moiety as
plasma membrane H+-ATPase inhibitors
Truong-Thanh Tung,[a] Dr. Trong T. Dao,[a][b] Marta G. Junyent,[b] Prof. Dr. Michael Palmgren,[b] Prof. Dr.
Thomas Günther-Pomorski,[b] Assoc. Prof. Dr. Anja T. Fuglsang,[b] Prof. Dr. Søren B. Christensen*[a]
and Prof. Dr. John Nielsen*[a]
[a]
[b]
T. T. Tung, Dr. T. T. Dao, Prof. Dr. S. B. Christensen, Prof. Dr. J. Nielsen
Department of Drug Design and Pharmacology
University of Copenhagen
Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark
E-mail: soren.christensen@sund.ku.dk (SBC), john.nielsen@sund.ku.dk (JN)
Dr. T. T. Dao, M. G. Junyent, Prof. Dr. M. Palmgren, Prof. Dr. T. G. Pomorski, Assoc. Prof. Dr. A. T. Fuglsang
Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Department of Plant and Environmental Sciences
University of Copenhagen
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Supporting information for this article is given via a link at the end of the document.
Abstract: The fungal plasma membrane H+-ATPase (Pma1p) is a
potential target for the discovery of new antifungal agents.
Surprisingly, no structure-activity relationship studies for small
molecules targeting Pma1p have been reported. Herein, we disclose
a LEGO-inspired fragment assembly strategy for design, synthesis
and discovery of benzo[d]thiazoles containing a 3,4-dihydroxyphenyl
moiety as potential Pma1p inhibitors. A series of 2-(benzo[d]thiazol-
2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones was found to inhibit
Pma1p with the most potent IC50 of 8 µM in an in vitro plasma
membrane H+-ATPase assay. These compounds also strongly
inhibited the action of proton pumping when Pma1p was reconstituted
into liposomes. Compound 38 showed inhibitory activities on the
growth of Candida albicans and Saccharomyces cerevisiae, which
could be correlated and substantiated to the ability to inhibit Pma1p in
vitro.
too toxic for systemic administration, are used topical: 1)
allylamines and thiocarbamates like naftifine and tolnaftate
possessing the same mechanism of action as terbenafine, 2)
nystatin that has the same mechanism of action as amphotericin
and 3) amorolfine, which inhibit sterol reductase. All of these
drugs, however, cause severe side effects and resistant strains
have developed. Consequently, antifungal agents with higher
selectivity and less prone to resistant are urgently needed.[2]
The plasma membrane proton pump (Pma1p) that is exposed on
the surface of fungal cells is essential for the survival of fungi.[3]
Pma1p is the housekeeper enzyme responsible for maintaining
the transmembrane electrochemical gradient by pumping protons
to the extracellular matrix.[3b] Compounds blocking Pma1p on the
surface of
a fungal cell will perturb the transmembrane
electrochemical gradient across the plasma membrane and
thereby compromise and eventually kill the fungus.[4] The
members of the Pma1p family (P3A-ATPases) are solely found in
fungi and plants and not in mammalian cells. Since these pumps
exhibit low homology to human P-type ATPases,[5] it might be
possible to develop agents, which selectively block Pma1p
without affecting mammalian ATPases as well as other enzymes
or receptors.[4a, 4c]
Introduction
Fungal infections are of major importance for the production,
storage, distributions and consumer use of plants, plant material,
vegetable-based fodder, and crops. It is much less realized that
millions of patients worldwide are at risk of fungal infections, many
of which may be life threatening if not treated appropriately.[1]
Presently, four main classes of antifungal drugs for systemic
administration are used, 1) a polyene antibiotic like amphotericin,
which interacts with ergosterol, the primary sterol in fungal cell
membranes, 2) azoles like fluconazole and ketoconazole, which
deactivate cytochrome P450 (CYP51A1) and thereby inhibit C-14
demethylation of lanosterol and consequently causes ergosterol
depletion, 3) 5-fluorocytosine that cause misconduct of coding
and thereby inhibits DNA synthesis and 4) allylamines like
terbenafine, which inhibits oxosqualene cyclase and thereby
reduces ergosterol formation. In addition, some drugs which are
Surprisingly, very few studies developing Pma1p inhibitors have
been reported (vide infra). Omeprazole,
a selective and
irreversible gastric proton pump inhibitor and top-selling antiulcer
drug, strongly inhibits human gastric H+/K+-ATPase (a P2C-
ATPase) and is also reported to inhibit Pma1p activity.[6]
Nevertheless, in our screening system, omeprazole showed very
weak inhibitory effect at 500 µM (data not show). Ebselen, an
organo-selenium drug that is used to treat reperfusion injury and
stroke, inhibits Pma1p by forming covalent bonds to the cystein
thiols group in the protein.[7] However, due to their reactivity and
associated general toxicity, ebselen and its analogues represent
a poor starting point for discovery and development of new drugs.
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
From random screening, some natural products (chebulagic acid,
tellimagrandin II, dichamanetin) were reported to have inhibitory
activity towards Pma1p, but they also damage the cell membrane
Due to the limited structural studies of Pma1p, the design of
Pma1p inhibitors has to emerge from an empirical starting point.
Inspired from the LEGO concept, all LEGO products were created
from the same and simple bricks. Thus, we hypothesize that the
same type or family of the protein target will share some similarity
structures of its inhibitors. We propose a strategy taking
advantage of combining divergent synthesis and rational drug
design. We suggest the HFSA-SDOS strategy in order to find new
inhibitors of less-well studied targets. HFSA is initiated by
selecting fragments (as the chemical LEGO bricks), which relate
or hypothetically relate to a specific target/biological system, in
this case: Pma1p. Assembling of the selected fragments is
hypothesized to give a specific biological relevant scaffold (SBS),
which is intended to interact specifically with the desired biological
target (building with the chemical LEGO bricks). The second step
of HFSA-SDOS is taking advantage of diversity-oriented
synthesis (DOS) to construct SBS-based DOS (SDOS) library of
relevant compounds in order to understand the topography of the
binding site (building a diverse number of LEGO figures). This
specific biological relevant scaffold-based diversity-oriented
synthesis (SDOS) was hypothesized to result in higher success
rate of finding inhibitors toward the desired targets. Finally, the
new hits from the SDOS approach went through a rational design
process to progress hit-to-leads and provided structure−activity
relationship (SAR) information (refine the LEGO figures). For
more details see the description below and Figure 1 and Figure 2.
[8]
or block other pathways in fungi.[9] By screening 1.8 million
octapeptides, Monk et al.[10] discovered two D-peptides, BM0 and
BM2, acting as Pma1p inhibitors, which are expected to bind to
the extracellular side of the Pma1p pump. However, the general
haematolytic activity of the peptides in the active concentration
range limits their clinical value.[10]
In a recent study ≈ 191,000 commercially available compounds in
a small-molecule library were screened for their ability to inhibit
Saccharomyces cerevisiae plasma membranes containing
Pma1p.[11] In this study, we have developed a new strategy, which
combines hypothesis-based fragment selection and assembly
(HFSA), specific-biological-relevant-scaffold based diversity-
oriented synthesis (SDOS) and rational-design (termed HFSA-
SDOS) to construct small molecule libraries. We demonstrate that
such library compounds can be used for drug discovery and for
creating tool compounds for studying the functions of Pma1p.
Results and Discussion
HFSA-SDOS: from hypothesis-based fragment selection and
assembly (HFSA), specific biological relevant scaffold-based
diversity oriented synthesis (SDOS) to rational design.
Figure 1. Description of HFSA-SDOS work flow. A. The Scope of HFSA-SDOS; B. Scope of HFSA-SDOS applied to find Pma1p inhibitors.
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
Figure 2. Outline of research work-flow: the combination of hypothesis-based fragment collections and assembly (HFSA) of specific biological relevant scaffold
(SBS), specific biological relevant scaffold-based diversity oriented synthesis (SDOS) and rational drug design. Fragment selection: Hypothesis-based selection of
SBS scaffold (HFSA) for library compounds towards inhibitors of the H+-ATPse Pma1p.
Hypothesis-based Fragment Selection and Assembly (HFSA). containing 3,4-dihydroxyphenyl moiety (Figure 2) was used as a
For evolutionary reasons, proteins with similar functions are
generally structurally related and share some homology in both
primary, secondary and tertiary structure. Since all P-type
ATPases belong to the same superfamily, they all are also
constructed over a common scaffold.[4a] In spite of this, inhibitors
like thapsigargin or cardiac glycosides selectively inhibit only one
ATPase subfamily such as the sarco/endoplasmic reticulum Ca2+-
ATPase (SERCA) or the Na+/K+-ATPase, respectively. Recently,
thapsigargin also has been shown to be an inhibitor of the SPCA
Ca2+-ATPase although three orders of magnitudes are less potent
towards SERCA. The close relationship between the SERCA
pumps and the SPCA, however, only emphasizes a surprising
selectivity of thapsigargin.[12] In terms of finding inhibitors for less
studied P-type pumps like the Pma1p, we hypothesize that
selecting the most common fragments from inhibitor libraries of
other well-studied P-type as well as other type pumps would
enhance the likelihood of identifying new hit compounds. Unlike
biology-oriented synthesis (BIOS),[13] which is focused on
selecting the core skeleton/scaffold from existing bioactive natural
products library (vide infra), using our HFSA methodology, we first
choose the small fragments, then subsequently form the SBS
scaffold from those fragments. Herein, we will demonstrate that
our strategy can lead to discoveries of hit compounds for targets
with limited number of inhibitors available or when the topography
of the target molecule is unknown. In case of Pma1p, a careful
analysis of broad class inhibitors of P-type ATPases revealed a
presence of sulfur-derived functional groups in a number of
inhibitors,[4a] e.g. omeprazole as earlier mentioned (the first LEGO
brick).[6] In addition, a common fragment in many naturally
occurring ATPase pump inhibitors is a 3,4-dihydroxyphenyl
group[14] as evidenced in a screening of 160 natural products
selected from our in-house proprietary collection.[15] This
screening revealed that demethoxycurcumin and analogues were
found to inhibit the ATPase activity of Pma1p with IC50 values of
around 30 µM. In addition, curcuminoids are known to inhibit
SERCA pump.[15] Based on these observations the scaffold
starting point (the second LEGO brick).
Specific biological relevant scaffold-based diversity oriented
synthesis in selection of R groups (SDOS).
As illustrated in Figure 2, we aim at constructing a diverse library
of compounds, which mainly focuses on varying the R-fragments
(the A region, the third LEGO brick) and the 3,4-dihydroxyphenyl
moiety (the B region) of the SBS scaffold. As a first attempt, we
designed a number of R-fragments based on DOS concepts.
Originally, DOS methodology was elegantly developed by
Schreiber[16] with an emphasis on increasing diversity within
molecular libraries to increase the potential for multiple protein
targets. Several studies have demonstrated that DOS library can
function as a powerful tool in the exploration of chemical and
biological spaces even for challenging targets.[17] More recently,
Park et. al proposed the privileged-substructure based diversity
oriented synthesis (pDOS) strategy which used privileged
substructures in library construction.[17e,
18]
Applying pDOS
methodology has increased the success rate for finding hit
compounds in several biological systems.[17e, 19] In case of BIOS,
the starting point emphasizes the selection of the biological
relevant scaffold, especially natural product, to construct a
focused small molecule library.[13] Overall, DOS and pDOS can
target multiple biological systems from one structurally diverse
collection of small molecule libraries. In BIOS, the scaffold is
chosen from natural products or collection of several hit
compound libraries, which are useful when dealing with
biomolecules with known structure but provide limited application
for new and unexplored targets. Those ideas encouraged us to
develop the concept of so called ´´specific biological relevant
scaffold-based diversity oriented synthesis´´ which emerged by
elaborating advantages of pDOS and BIOS concepts and
combining those with HFSA (Figure 1). Using HFSA-SDOS, we
have designed and selectivity selected 15 R-groups which
afforded the molecular shape diversity of the library compounds
as demonstrated in supplementary Fig. S3. The synthetic routes
are depicted in Scheme 1.
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
Scheme 1. Synthetic routes for the preparation of the first compound libraries 1 to 15. a) 2-chloro-1-(3,4-dihydroxyphenyl)ethanone, acetonitrile, DBU 10 mol-%,
MW, 90 °C, 30min.
Figure 3. A) Screening for Pma1p inhibition candidates from the first compound library. Pma1p ATPase activity was measured in the presence of 30 µM of the
compounds 1-15 or vanadate, a P-type ATPase inhibitor. B) Inhibition effects of the substituted phenyl moiety from the second library on Pma1p ATPase activity.
The activity was measured in the presence of 30 µM of the compounds 16-30 or vanadate (technical triplicates, n=2). Data are expressed relative to the control (i.e
without inhibitors).
As shown in Scheme 1, the microwave-irradiation synthesis
methodology was used and proved to be an expeditious and
effective way of preparing the desired compounds. Compounds
1-16 were prepared via the reaction of 2-chloro-1-(3,4-
dihydroxyphenyl)ethanone with the appropriately substituted thiol
in acetonitrile as solvent in the presence of 10 mol-% of 1,8-
compound 2 and 5, which inhibited the enzyme activity more than
90 % and 65 %, respectively. In contrast to the two hits, 2 and 5,
all the poorly active compounds did not contain fused aromatic
systems. Interestingly, 2 and 5 sharing the same molecular shape
diversity points as depicted in supplementary Fig. S3. This result
substantiates our strategy using HFSA to construct SDOS library,
which successfully finds interesting hits from a small number of
compounds.
diazabicyclo[5.4.0]undec-7-ene
(DBU)
under
microwave
irradiation to enhance the reaction rate. After heating to 90 °C for
15 minutes, the target compounds could be isolated in excellent
yields. The methodology can be applied for gram scale synthesis.
Purification was accomplished simply from precipitation after
adding concentrated HCl, followed by several washes with
various solvents and recrystallization from ethanol to give the
compounds in a pure state (see experimental section).
The role of 3,4-dihydroxyphenyl moiety.
After having selected the
R
group as quinolone and
benzo[d]thiazole, we subsequently focused on optimizing of the
3,4-dihydroxyphenyl moiety. An array of 15 compounds with
different substitution patterns in the benzoyl moiety was prepared
(Scheme 2).
Compounds 1-15 were screened in an in vitro Pma1p ATPase
assay at 30 µM (Figure 3A) and two hits were disclosed:
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
Scheme 2. Synthetic routes for the preparation of the second screening library 16 to 30. a) CuBr2, EA/DCM = 1/1 (v/v), MW, 60 °C 30 min; b) mercapto heterocyles,
rt, 1h.
-
Scheme 3. Synthetic routes for the preparation of the focused benzo[d]thiazole-containing library 32 to 40. a) EtOS2 K+, DMF, MW, 160 oC; 30 min to 1h; b) 2-
chloro-1-(3,4-dihydroxyphenyl)ethanone, DBU 10 mol-%, acetonitrile, MW, 90 °C, 30min.
The 2-bromo-1-(substituted-phenyl)ethanone intermediates were
prepared in situ directly from bromination of 1-(substituted
phenyl)ethan-1-one using CuBr2 as reagent. Also in this case
irradiation with microwaves at 60 °C for 15 minutes promoted the
reaction. Compounds 16-30 were prepared by reacting
thiolobenzo[d]thiazole or 2-thioloquinoline with appropriately
substituted 2-bromoacetophenones intermediates at room
temperature in acetonitrile (Scheme 2). The obtained library
consisting of 16-30 was screened for Pma1p inhibitory activity
(Figure 3B).
the 3,4-dihydroxy moiety unchanged and further modulate the
decoration pattern in the fused aromatic ring (the A area).
In vitro effects of 2-(substituted benzo[d]thiazol-2-ylthio)-1-
(3,4-dihydroxyphenyl)ethanones on Pma1p ATPase activity.
The prepared substituted benzo[d]thiazoles moiety are presented
in Scheme 3. Intermediates of general structure 31 were obtained
in excellent yield (>80 %) via reaction of substituted 2-
chloroaniline or 2-bromoaniline with potassium ethyl
xanthogenate in DMF at 160 °C for only 30 minutes under
microwave condition. Compounds 32-46 were subsequently
All analogues not possessing a 3,4-dihydroxyphenyl moiety
showed poor inhibition suggesting a pivotal role of this fragment
for interaction with Pma1p. Conjointly, the screening results for
compounds 1-30 disclosed that the optimal inhibitory activity
against Pma1p appears when the R-groups consist of two fused
aromatic rings (quinolone or benzo[d]thiazole) and a 3,4-
dihydroxyphenyl moiety. As a consequence, we decided to keep
synthesized
dihydroxyphenyl)ethanone using
described for compounds 1-16 (Scheme 3).
Compounds 2 and 32-46 inhibited the ATPase activity of Pma1p
in a concentration-dependent manner (Table 1).
from
31
and
2-chloro-1-(3,4-
a
method similar to that
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
Compound 47-49 were synthesized from commercially available
Table 1. IC50 values for compounds 2 and 32-46 determined from Pma1p
1H-benzo[d]imidazole-2-thiol,
benzo[d][1,3]selenazole-2-thiol
benzo[d]xazole-2-thiol
and
ATPase activity measurements.
with 2-chloro-1-(3,4-
dihydroxyphenyl)ethanone using microwave-assisted synthesis
similar to that described for the preparation of 1-16 (Scheme 4).
Entry
1
R
Compounds
IC50 (µM, mean±SD)
28.2 ± 1.0
>100
Table 2. Inhibitory activity of compounds 47 – 49.
H
2
2
5-F
6-F
4-Cl
5-Cl
6-Cl
5-Br
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
3
10.9 ± 1.1
19.3 ± 1.2
20.8 ± 1.1
27.8 ± 1.1
>100
Entry
Compounds
X
% Inhibition
at 30 µM
IC50 (µM)
4
1
2
3
47
48
49
NH
O
<10
<10
>99
ND[a]
ND
5
6
Se
19.0
7
[a] ND, Not Determined.
8
6-CF3
11.9 ± 1.1
11.5 ± 1.1
8.2 ± 1.1
12.7 ± 1.1
29.1 ± 1.2
36.8 ± 1.1
9.2 ± 1.1
>100
The biological results (Table 2) reveal that only replacement of
the sulfur atom with a selenium atom afforded a compound with
affinity for the Pma1p as 49 (IC50 of 19 µM) slightly more potent
than compound 2 (IC50 of 28 µM).
9
4-Methyl
6-isopropyl
4,6-dimethyl
6-MeO
10
11
12
13
14
15
16
6-EtO
6-OCF3
6-NO2
6-NH2
>100
Compound 2, 33-36 and 38-44 possessed strong inhibitory
activity with IC50 values ranged from 8 µM (40) to 36 µM (43). The
most potent compound 40 (IC50: 8 ± 1 µM) displayed an almost
four folds increase in the inhibitory properties compared with the
starting compound
benzo[d]thiazole ring (IC50: 28 ± 1 µM).
2
without any substituents on the
Figure 4. Overview of the structure-activity relationship. Impact on the structural
modification on the ATPase activity of Pma1 are indicated.
Except for 5-fluoro (32), 5-bromo (37), 6-nitro (45) and 6-amino
(46) (benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)-ethanone
all compounds showed modest to good activity as inhibitors of
Pma1p (Table 1). The IC50 values of both 6-isopropyl and 6-
trifluoromethoxy(benzo[d]thiazol-2-ylthio)-1-(3,4-
dihydroxyphenyl)-ethanones (40 and 44, respectively) were
below 10 µM.
The role of sulfur atom in the benzo[d]thiazole moiety
The sulfur atom in the benzo[d]thiazole moiety was displaced by
N, O and Se in order to understand the role of the heteroatom.
The synthetic routes are showed in Scheme 4.
The present structure-activity relationship study for Pma1p
inhibitors, shown in Figure 4, provides essential information which
may pave the road for design new tool compounds as well as
second generation inhibitors of Pma1p.
Mechanism and model of inhibition
In order to determine the mode of action, the four strongest
inhibitors 38-40 and 44 were used for enzyme kinetic studies.
Initialy, the enzyme activities were plotted as a function of subtrate
ATP concentration using Hanes-Woolf analysis followed by fitting
to the appropriate Michaelis-Menten inhibition equation. The
apparent Km values for ATP increased along with concentrations
of the tested compounds, whereas the Vmax values remained at a
constant level (Figure 5 and Table 3). Consequently, the
compounds behave as competitive inhibitors of ATP as the
inhibitors compete with the substrate (ATP) and bind to the
binding site of Pma1p with inhibition constant (Ki) values
calculated to be 9 µM, 11 µM, 8 µM and 6 µM for 38-40 and 44,
respectively.
Scheme 4. Synthesis route for preparation of compounds 47-49. a) 2-chloro-1-
(3,4-dihydroxyphenyl)ethanone, DBU 10 mol-%, ACN, MW, 90 °C, 30min.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
4
3
2
1
0
4
3
2
1
0
Control
Control
5 M comp. 40
7.5 M comp. 40
10 M comp. 40
5 M comp. 44
7.5 M comp. 44
10 M comp. 44
Competitive inhibition
Ki = 6.23 M
Competitive inhibition
Ki = 8.75 M
0
2
4
6
8
0
2
4
6
8
[ATP] mM
[ATP] mM
4
Control
Control
4
3
2
1
0
5 M comp. 38
7.5 M comp. 38
10 M comp. 38
5 M comp. 39
7.5 M comp. 39
10 M comp. 39
3
2
1
0
Competitive inhibition
Ki = 9.34 M
Competitive inhibition
Ki = 11.82 M
0
2
4
6
8
0
2
4
6
8
[ATP] mM
[ATP] mM
Figure 5. Kinetic study of Pma1p-ATPase inhibition by compounds 38-40 and 44. ATP hydrolysis rate was plotted as function of [ATP] in the presence of the
compounds. Error bars represent standard errors of the mean for three independent experiments.
Table 3. Kinetic constants (Vmax, µmol Pi min-1(mg protein)-1; Km, mM) for Pma1p-ATPase in the presence of compounds 38-40 and 44.
Concentration
38
39
40
41
(µM)
V
K
V
K
V
K
V
K
m
max
m
max
m
max
m
max
0
5
5.92
6.38
4.45
9.29
4.28
3.65
3.48
5.23
5.14
5.73
4.01
8.92
4.62
4.01
3.72
7.97
7.5
10
5.46
5.77
17.95
42.49
5.10
16.43
48.77
4.08
4.37
12.99
29.73
2.62[a]
4.60
8.06
8.55[a]
32.82
[a] No significant difference was observed with other Vmax values of the compound (P < 0.05).
Figure 6. Dose-dependent inhibition of proton transport into reconstituted vesicles by compound 40. Lecithin vesicles reconstituted with Pma1p were incubated with
different concentrations of the compound as indicated. ATP stimulated proton pumping was initiated upon addition of MgSO4, and the proton gradient was collapsed
by adding nigericin. Traces from one representative of three independent experiments are shown (A). Proton transport activity was determined as initial rates of the
fluorescence quenching of ∆pH sensor ACMA (B). Values are mean ± S.E. (n = 3). Student’s t tests:*, p < 0.05; ***, p < 0.001 relative to the control (i.e. without
inhibitors).
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
Figure 7. Profiles of fungal cell growth inhibition in the presence of compounds 38 and 40. Growth curves of S. cerevisiae (A, B) and C. albicans (C, D). Cells were
grown in 96-well plates and treated with eight different concentrations of compounds 38 and 40 or 1.4 % DMSO as a solvent control. OD600 was recorded every 10
min during 20 h at 30 °C with continuous shaking by a micro-plate reader.
Effect
of
1-(3,4-dihydroxyphenyl)-2-((6-
derivatives and thereby generate Pma1p inhibitors for developing
antifungal agents.
isopropylbenzo[d]thiazol-2-yl)thio)ethan-1-one
proton transport in plasma membrane.
(40)
on
The active proton transport across yeast cell membrane is tightly
coupled to the energy of ATP hydrolysis.[20] Consequently, we
examinated the effect in proton pumping activity using 40 as a
model compound. Liposomes were prepared by manual extrusion,
reconstituted with purified Pma1p. The ATP-driven proton
pumping activity was determined by the fluorescence quenching
of ACMA (9-amino-6-chloro-2-methoxyacridine), a dye that
accumulates inside the vesicles upon protonation. Pre-incubation
of Pma1p reconstituted liposomes with compound 40 resulted in
reduction of the ATP-driven H+-pumping activity (Figure 6).
Since the activity of Pma1p is essential for fungal cell growth, S.
cerevisiae and C. albicans cell growth were analysed in the
presence of inhibitors. As shown in Figure 7, cell growth of both
S. cerevisiae and C. albicans is reduced after incubation with
compound 38. At concentration above 100 µM greater than 50 %
growth inhibition is observed. Even though compound 40
possesss the potent pump inhibitory ability in vitro, it exhibits
limited effects towards S. cerevisiae and no effects toward C.
albicans growth. As the binding site for the inhibitors is on the
intracellular side of the pump (they compete with ATP, Figure 5),
a poor ability to penetrate the cell wall and cell membrane might
prevent the compounds from reaching their target. The presence
of a trifluoromethyl substituent in 38 might enable this compound
to penetrate the cell membrane more easily than 40. Furthermore,
compound 40 might be excluded from the cell by an efflux
transporter, this is well known drug tolerance mechanism.[21] An
encouraging observation for these agents is the poor toxicity
towards mammalian cells as illustrated by the absence of
cytotoxicity in concentration below 90 µM (see supplementary
information). The present study encourages future investigation
with the goal of improving membrane permeability of these
Conclusions
By using a combination of diversity-oriented synthesis and
rational drug design with the supporting of hypothesis-based
fragment selection and assembly to choose the specific biological
relevant scaffold (HFSA-SDOS work-flow), we have successfully
discovered compounds that in low micromolar concentrations
inhibit ATP hydrolytic activity as well as proton pumping by Pma1p.
This is the first report of a DOS directed towards a specific target.
Our proposed LEGO method has revealed itself as promising
towards reducing the number of compounds required in the initial
biological screening as we obtained a success rate of 13 %. In
another screen independently performed at the same time, about
191,000 commercially available compounds were screened with
the success rate of 0.2 %.[11]
The SAR suggested a crucial role of the 3,4-dihydroxyphenyl
moiety and a fused ring system like benzo[d]thiazole for the
inhibitory activity. Kinetic studies revealed that the inhibitors
competitively bind to the ATP binding-site of Pma1p. In spite of
the decent IC50 values as inhibitor of Pma1p, the compounds only
modestly inhibit the growth of C. albicans and S. cerevisiae in
fungal growth curve assay. These findings are probably caused
by limited access to the intracellular binding site, either the
compounds get caught in the cell wall matrix or they exhibit poor
ability to penetrate the fungal plasma membrane. As the
compounds inhibit Pma1p activity both when assaying ATP
hydrolytic activity as well as H+ pumping, we are confident that
they are Pma1p inhibitors and not acting as Pan-Assay
Interference Compounds (PAINS).[22]
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
°
203.9-205.0 C; LRMS(ESI) m/z [M+H]+ found 282.0; HRMS (ESI) m/z
In conclusion, our present work demonstrated a library of 2-
(benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones as
potential fungal proton pump (Pma1p) inhibitors and paved a new
route for drug discovery toward targets with limited structural
information. Overall, we believe the workflow described for our
approach could be a guideline for the medicinal chemist in other
studies of drug discovery and development pursuing less well-
described targets.
[M+H]+, calcd for C12H12NO3S2+ 282.0259, found 282.0260.
For compounds 2-15: see SI.
General synthetic procedure for compounds 16-30. A mixture of 1-
(substituted phenyl)ethan-1-one (1 eq.), copper (II) bromide (1 eq.), 8 ml
of mixture of ethyl acetate : dichloromethane = 1:1 was added to a 20 mL
microwave reaction vial (Biotage). The vial was sealed and irradiated in a
microwave apparatus at 60 °C, high absorption for 15 min. After the
completion of reaction by TLC, 30 ml of water was added and extracted
with 2 x 100 ml ethyl acetate. The organic layer was evaporated under low
pressure to afford crude intermediates which is used in the next step
without purification.
Experimental Section
Chemistry. All reagents, solvents were purchased from commercial
suppliers (TCI, Aldrich) and used without further purification. Microwave
reactions were carried out in a Biotage® Initiator. NMR-spectra were
recorded using a 400 or 600 MHz Bruker Avance III HD equipped with a
cryogenically cooled 5 mm dual probe optimized for 13C and 1H. Samples
were dissolved in one of following solvent: DMSO-d6, CDCl3, MeOD and
analyzed at 300 K. 1H, COSY, HMBC, and HSQC spectra were recorded
at 400 MHz or 600 MHz using internal standard reference. 13C spectra
were acquired at 101 MHz or 151 MHz. Chemical shifts (δ) are reported in
ppm relative to the residual solvent peak (1H NMR) or the solvent peak
(13C NMR) as the internal standard. The coupling constant (J) are reported
in hertz. Please see SI for numbering. Solvents used for the synthesis were
of analytical grade, dried over activated 4 Å molecular sieves when
necessary (all solvents used under dry conditions had a water content <25
ppm). Analytical TLC was performed using pre-coated silica gel 60 F254
plates and visualized using UV light. Flash column chromatography was
performed using Merk silica 60. Melting points were determined on a
Mettler Toledo MP70 Melting Point system. All tested compounds possess
≥95 % purity (purified by preparative RP-HPLC for compounds less than
90 % purity). Purity determinations were performed on a Waters 2795
system equipped with a Waters 996 PDA detector and a Waters Symmetry
C18 Column (2.1 × 50 mm, 3.5 lm) with a flow of 0.2 ml min−1. 100 % →
0 % A 0–10 min (A: 0.1 % aq formic acid, B: 95 % CH3CN in 0.05 % aq
formic acid). High-resolution mass spectral (HRMS) were performed with
an Agilent 1200 series instrument (Santa Clara, CA) consisting of a
quaternary pump, a degasser, a thermostated column compartment, a
photodiode-array detector, a high-performance auto sampler, and a
fraction collector, all controlled by Agilent ChemStation ver. B.03.02
software and equipped with a reversed phase Luna C18(2) (Phenomenex,
150 × 4.6 mm, 5 μm, 100 Å) maintained at 40 °C. The aqueous eluent (A)
consisted of water/acetonitrile (95:5, v/v), and the organic eluent (B)
consisted of water/acetonitrile (5:95, v/v), both acidified with 0.1 % formic
acid.
The crude product above was dissolved in 20 ml of acetonitrile and the
appropriately substituted heterocyclic thiol (1 eq.) was added and stirred
at room temperature for 30 min until the appearance of a precipitate solid.
The precipitated was then filtered and fast washed 3 x 10 ml with each
acetonitrile, ethanol, hot acetone then recrystallization in ethanol to afford
clean 16 – 30.
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(quinolin-2-ylthio)ethan-1-
one (16). 1.05 g, yield 90 %; Light yellow solid; 1H NMR (600 MHz, CDCl3)
δ 7.91 (d, J = 8.6 Hz, 1H, H10), 7.81 (d, J = 8.2 Hz, 1H, H6), 7.71 (br, 2H,
H1, H20), 7.70 (d, J = 2.1 Hz, 1H, H3), 7.61 (t, J = 7.0 Hz, 1H, H2), 7.41 (t,
J = 7.0 Hz, 1H, Ar-H, H16), 7.28 (d, J = 8.6 Hz, 1H, qui-H, H9), 6.94 (d, J
= 8.9 Hz, 1H, Ar-H, H17), 4.80 (s, 2H, CH2), 4.33 (m, 2H, H23), 4.29 (m,
2H, H22); 13C NMR (151 MHz, CDCl3) δ 193.1(C=O,C-13), 157.4(C-8),
148.3(C-18), 147.8(C-4), 143.4(C-19), 135.9(C-10), 130.1(C-14), 129.8(C-
2), 127.6(C-1), 127.6(C-6), 126.1(C-5), 125.5(C-16), 122.9(C-3), 120.6(C-
9), 118.2(C-20), 117.3(C-17), 64.7(C-23), 64.1(C-22), 36.6(CH2); Melting
°
point 227.3-228.0 C; LRMS(ESI) m/z [M+H]+ found 338.0; HRMS (ESI)
m/z [M+H]+, calcd for C19H16NO3S+, 338.0851, found 338.0851.
For compounds 17-30: see SI
General synthetic procedure for compounds 32-46. Synthesis of
intermediates 31a-g: A mixture of substituted 2-chloro- (or 2-bromo-)
aniline (1g,
1 eq.), potassium ethyl xanthogenate (2 eq.), and
dimethylformamide (10 ml) was added to a 20 ml microwave reaction vial
(Biotage). The vial was sealed and irradiated in a microwave apparatus at
160 °C, using the “high absorption” mode for 30 min. After the completion
of reaction, the vial was cooled, de-capped and poured into 200 ml of water.
The precipitated was filtered and washed several times with water to afford
31a-g in medium to high yield.
Synthetic procedures for some representative compounds are provided.
6-Fluorobenzo[d]thiazole-2(3H)-thione (31b). 1.10 g, yield 87 % from 1g
of 2-chloro-4-fluoroaniline; Red brown solid; 1H NMR (600 MHz, DMSO-
d6) δ 13.8 (s, 1H, NH), 7.61 (dd, J = 8.5 Hz, J = 2.6 Hz, 1H, H6), 7.27 (dd,
J = 8.9 Hz, J = 4.7 Hz, 1H, H3), 7.21-7.24 (td, J = 2.6 Hz, J = 2.6 Hz, 1H,
H1); 13C NMR (151 MHz, DMSO-d6) δ 190.4(C-8), 158.8-160.4(d, J = 242
Hz, C-2), 138.4(C-4), 131.2(d, J = 11.0 Hz, C-5), 115.2(d, J = 24.4 Hz, C-
3), 113.8(d, J = 9.0 Hz, C-1), 109.3(d, J = 27.4 Hz, C-6).
General synthetic procedure for compounds 1-16: A mixture of the thiolo
substituted
heterocycle
(0.5
g,
1
eq.),
2-chloro-1-(3,4-
dihydroxyphenyl)ethanone (1.5 eq.), 10 mol-% of DBU, acetonitrile (8 ml)
was added to a 20 mL microwave reaction vial (Biotage). The vial was
sealed and irradiated at 90 °C, high absorption for 30 min. The reaction
mixture was then cooled to room temperature and conc. HCl was added
until a precipitate appeared. The precipitate was filtered and washed 3
times x 10 ml with acetonitrile, ethanol, hot acetone then recrystallization
in ethanol to afford clean 1 – 16 as HCl salt.
For compounds 31a, 31c-o: See SI.
Synthesis of 32 to 46: Compounds 32 to 46 were synthesized using the
same procedure as described for 1-16 from 31a-o using microwave
methodology (exception to workup procedure: acidified to pH 4 by conc.
HBr instead of HCl and were collected as HBr salt).
1-(3,4-Dihydroxyphenyl)-2-((4-methylthiazol-2-yl)thio)ethan-1-one (1).
0.99 g, yield 82 %; Yellow solid; 1H NMR (600 MHz, DMSO-d6) δ 10.07 (s,
2H, 2OH), 7.44 (dd, J = 8.3 Hz, J = 2.2 Hz, 1H, benzene-H, H7), 7.41 (d,
J = 2.2 Hz, 1H, benzene-H, H11), 7.19 (d, J = 1.1 Hz, 1H, thiazole-H, H16 ),
6.89 (d, J = 8.3 Hz, 1H, benzene-H, H8), 4.86 (s, 2H, CH2), 2.29 (d, J = 1.1
Hz, 3H, CH3); 13C NMR (151 MHz, DMSO-d6) δ 191.5(C=O, C-4), 163.7(C-
1), 151.9(C-9), 151.8(C-15), 145.8(C-10), 127.5(C-5), 122.5(C-7),
115.8(C-16), 115.7(C-8), 115.2(C-11), 41.9(CH2), 16.9(CH3); Melting point
1-(3,4-Dihydroxyphenyl)-2-((5-fluorobenzo[d]thiazol-2-yl)thio)ethan-
1-one (32). 0.89 g, yield 79 %; Brown solid; 1H NMR (600 MHz, DMSO-d6)
δ 8.05 (dd, J = 8.9 Hz, J = 5.5 Hz, 1H, H9), 7.65 (d, J = 7.3 Hz, 1H, H6),
7.51 (d, J = 8.2 Hz, 1H, H16), 7.43 (br, 1H, H20), 7.24-7.28 (br, 1H, H8),
6.88 (d, J = 8.1 Hz, 1H, H17), 5.06 (s, 2H, CH2); 13C NMR (151 MHz,
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
DMSO-d6) δ 191.1(C=O, C-13), 169.9(C-2), 160.9-162.5(d, J = 241 Hz, C-
7), 153.9(d, J = 12.1 Hz, C-4), 151.9(C-18), 145.8(C-19), 130.9(C-5),
127.5(C-15), 123.5(d, J = 10.0 Hz, C-9), 122.5(C-16), 115.7(C-17),
115.17(C-20), 113.1(d, J = 24 Hz, C-8), 107.9(d, J = 24.5 Hz, C-6),
medium. 95 μL of the dilution was distributed into 96-well plates together
with the control well containing an equal amount of DMSO. Yeast cells,
including S. cerevisiae (wild type, ATCC 9763) and C. albicans (SC5314
from ATCC), were cultured overnight and diluted with fresh medium to
obtain optical density at 600 nm (OD600) of 1.2 and 0.4, respectively. Cell
suspensions (5 µL) were then inoculated to the test and control wells (final
OD600 of 0.06 and 0.02, respectively). The plates were covered by a
sealing membrane (Breathe Easier; Sigma-Aldrich) to avoid evaporation,
and OD600 was recorded every 10 min during 20 h at 30 °C with continuous
shaking by a SpectraMax M5 microplate reader.
°
41.3(CH2); Melting point 91-93 C; LRMS(ESI) m/z [M+H]+ found 336.0;
+
HRMS (ESI) m/z [M+H]+, calcd for C15H11FNO3S2 336.0164, found
336.0167.
For compounds 33-46: see SI.
Synthesis of compounds 47-49: Compounds 47 to 49 were synthesized
using the same procedure as 1-16 using microwave methodology
(exception to workup step: acidified to pH 4 by Conc. HBr and were
collected as HBr salt).
Acknowledgements
This work was supported by the University of Copenhagen´s
Excellency Program for Interdisciplinary Excellence (UC2016).
FTP project COMBAT to A.T.F.
2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(3,4-dihydroxyphenyl)ethan-1-
one (47). 1.00 g, yield 91 %; white solid((HCl salt)); H NMR (600 MHz,
1
DMSO-d6) δ 10.19 (br, 1H, OH), 9.53 (br, 1H, OH), 7.61 (q, J = 3.2 Hz, J
= 2.9 Hz, 2H, H18, H21), 7.53 (dd, J = 8.3 Hz, J = 2.2 Hz, 1H, H7), 7.44 (d,
J = 2.2 Hz, 1H, H11), 7.36 (q, J = 3.1 Hz, J = 2.9 Hz, 2H, H19, H20), 6.91
(d, J = 8.3 Hz, 1H, H8), 5.25 (s, 2H, CH2); 13C NMR (151 MHz, DMSO-d6)
δ 190.8(C=O,C-4), 151.8(C-19), 150.5(C-1), 145.6(C-10), 134.9(C-15,C-
16), 126.8(C-5), 124.0(C-19,C-20), 122.3(C-7), 115.4(C-8), 115.4(C-11),
Keywords: Fungal plasma membrane H+-ATPase (Pma1p) •
antifungal • drug design • benzo[d]thiazole
°
References:
113.4(C-18,C-21), 41.1(CH2); Melting point 224-226 C; LRMS(ESI) m/z
[M+H]+ found 301.0; HRMS (ESI) m/z [M+H]+, calcd for C15H13N2O3S+
318.0647, found 301.0650.
[1]
[2]
[3]
G. D. Brown, D. W. Denning, N. A. Gow, S. M. Levitz, M. G. Netea, T. C.
White, Sci Transl Med 2012, 4, 165rv113.
M. Gupte, P. Kulkarni, B. N. Ganguli, Appl Microbiol Biotechnol 2002, 58,
46-57.
For compounds 48-49: see SI.
a) M. B. Baudouin Michelet, Plant Physiol. 1995, 108, 69-73; b) R.
Serrano, M. C. Kielland-Brandt, G. R. Fink, Nature 1986, 319, 689-693;
c) A. Ambesi, M. Miranda, V. V. Petrov, C. W. Slayman, J Exp Biol 2000,
203, 155-160; d) F. Portillo, Biochim Biophys Acta 2000, 1469, 31-42.
a) L. Yatime, M. J. Buch-Pedersen, M. Musgaard, J. P. Morth, A. M. Lund
Winther, B. P. Pedersen, C. Olesen, J. P. Andersen, B. Vilsen, B. Schiott,
M. G. Palmgren, J. V. Moller, P. Nissen, N. Fedosova, Biochim Biophys
Acta 2009, 1787, 207-220; b) B. C. Monk, D. S. Perlin, Crit Rev Microbiol
1994, 20, 209-223; c) R. Tada, J. P. Latge, V. Aimanianda, Curr Pharm
Des 2013, 19, 3738-3747.
Microorganisms and Culture Media. Candida albicans (SC5314 from
ATCC) and Saccharomyces cerevisiae (wild type strain 9763 from ATCC)
used in this study were routinely cultured in YPD medium [1 % (w/v) yeast
extract, 2 % (w/v) peptone, 2 % (w/v) d-glucose] at 30 °C. The S. cerevisiae
strain RS-72 was used for expression of Pma1p (pMP 400), and cultured
as described previously.[15] Media and reagents were obtained from
Sigma-Aldrich unless otherwise indicated.
[4]
Pma1p ATPase Assay. Over expression of Pma1p and the inhibition
assays were carried out as previously described.[15] In short, plasma
membranes were purified from transformed yeast cells S. cerevisiae RS72,
and suspended in GTED20 buffer (20 % [v/v] glycerol, 10 mM Tris-HCl pH
7.5, 1 mM EDTA pH 8.0, 1 mM DTT). Pma1p ATPase activity was
performed with a total of 300 μL volumes containing 5 mM ATP. IC50 values
for compound inhibition were obtained by fitting the data to a dose-
dependent curve using the equation:
[5]
[6]
P. Morsomme, C. W. Slayman, A. Goffeau, Biochim Biophys Acta 2000,
1469, 133-157.
a) B. C. Monk, A. B. Mason, G. Abramochkin, J. E. Haber, D. Seto-Young,
D. S. Perlin, Biochim Biophys Acta 1995, 1239, 81-90; b) B. C. Monk, A.
B. Mason, T. B. Kardos, D. S. Perlin, Acta Biochim Pol 1995, 42, 481-
496; c) D. Grigore, J. C. Meade, J Eukaryot Microbiol 2006, 53, 157-164.
a) B. Billack, M. Pietka-Ottlik, M. Santoro, S. Nicholson, J. Mlochowski,
C. Lau-Cam, J Enzyme Inhib Med Chem 2010, 25, 312-317; b) B. Billack,
M. Santoro, C. Lau-Cam, Microb Drug Resist 2009, 15, 77-83; c) G. Chan,
D. Hardej, M. Santoro, C. Lau-Cam, B. Billack, J Biochem Mol Toxicol
2007, 21, 252-264.
[7]
100
퐴 =
(
)
−Log퐶 ×퐻
1 + 10 LogIC
50
where A is the observed % Pma1p ATPase activity in the presence of
compound; C, the compound concentration; IC50, the concentration that
caused 50 % inhibition of the maximal compound effect; and H, the slope
factor. For kinetic studies, the concentration of ATP substrate was varied
between 0.5 and 8 mM using an ATP regenerating system (5 mM
phosphoenolpyruvate and 50 µg/mL pyruvate kinase).
[8]
[9]
K. T. Kongstad, S. G. Wubshet, A. Johannesen, L. Kjellerup, A. M.
Winther, A. K. Jager, D. Staerk, J Agric Food Chem 2014, 62, 5595-5602.
K. T. Kongstad, S. G. Wubshet, L. Kjellerup, A. M. Winther, D. Staerk,
Fitoterapia 2015, 105, 102-106.
[10] B. C. Monk, K. Niimi, S. Lin, A. Knight, T. B. Kardos, R. D. Cannon, R.
Parshot, A. King, D. Lun, D. R. Harding, Antimicrob Agents Chemother
2005, 49, 57-70.
Proton Transport Measurement. Preparation of lecithin liposome-
reconstituted Pma1p and proton transport assay were performed as
previously described[23] by monitoring fluorescence quenching of 9-amino-
[11] L. Kjellerup, S. Gordon, K. O. Cohrt, W. D. Brown, A. T. Fuglsang, A. L.
Winther, Antimicrob Agents Chemother 2017, 61.
°
[12] J. Chen, J. De Raeymaecker, J. B. Hovgaard, S. Smaardijk, I.
Vandecaetsbeek, F. Wuytack, J. V. Moller, J. Eggermont, M. De Maeyer,
S. B. Christensen, P. Vangheluwe, J Biol Chem 2017, 292, 6938-6951.
[13] S. B. Wetzel, R. S.; Kumar, K; Waldmann, H., Angew Chem Int Ed 2011,
50, 10800–10826.
6-chloro-2-methoxyacridine (ACMA). The assay was conducted at 22 C
in total volumes of 1 mL with a medium buffer containing 20 mM MES-KOH,
pH 6.0, 40 mM K2SO4, 2 mM ATP, 1 μM ACMA and 60 nM valinomycin.
Proton pumping reactions were started by adding a final concentration of
2 mM MgSO4, and the gradient was collapsed by the addition of nigericin
to a final concentration of 10 μM.
[14] a) F. Michelangeli, J. M. East, Biochem Soc Trans 2011, 39, 789-797; b)
L. Olbe, E. Carlsson, P. Lindberg, Nat Rev Drug Discov 2003, 2, 132-
139.
Fungal Growth Study. Tested compounds were dissolved in DMSO to 20
mM as stock solutions and diluted to various concentrations with fresh YPD
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
[15] T. T. Dao, P. Sehgal, T. T. Tung, J. V. Møller, J. Nielsen, M. Palmgren,
S. B. Christensen, A. T. Fuglsang, PLoS ONE 2016, 11, e0163260.
[16] S. L. Schreiber, Science 2000, 287, 1964-1969.
[17] a) D. S. Tan, Nat Chem Biol 2005, 1, 74-84; b) B. M. Ibbeson, L. Laraia,
E. Alza, C. J. O' Connor, Y. S. Tan, H. M. L. Davies, G. McKenzie, A. R.
Venkitaraman, D. R. Spring, Nat Commun 2014, 5; c) A. Ambesi, M.
Miranda, V. V. Petrov, C. W. Slayman, J Exp Biol 2000, 203, 155-160; d)
C. Bathula, S. Singh, S. Sen, Syst Synth Biol
2015, 9, 49-53; e) C. J.
O' Connor, H. S. G. Beckmann, D. R. Spring, Chem Soc Rev 2012, 41,
4444-4456.
[18] a) S. Oh, S. B. Park, Chem Com 2011, 47, 12754-12761; b) H. Kim, T.
T. Tung, S. B. Park, Org Let 2013, 15, 5814-5817; c) J. Kim, H. Kim, S.
B. Park, J Am Chem Soc 2014, 136, 14629-14638.
[19] a) M. Zhu, B. J. Lim, M. Koh, S. B. Park, ACS Comb Sci 2012, 14, 124-
134; b) J. Kim, W. S. Lee, J. Koo, J. Lee, S. B. Park, ACS Comb Sci 2014,
16, 24-32; c) M. Zhu, M. H. Kim, S. Lee, S. J. Bae, S. H. Kim, S. B. Park,
J Med Chem 2010, 53, 8760-8764; d) S. Oh, H. J. Nam, J. Park, S. H.
Beak, S. B. Park, ChemMedChem 2010, 5, 529-533.
[20] a) D. P. Briskin, Arch Biochem Biophys 1986, 248, 106-115; b) M. J.
Buch-Pedersen, E. L. Rudashevskaya, T. S. Berner, K. Venema, M. G.
Palmgren, J Biol Chem 2006, 281, 38285-38292.
[21] R. Prasad, M. K. Rawal, A. H. Shah, Adv Exp Med Biol 2016, 892, 351-
376.
[22] C. Aldrich, C. Bertozzi, G. I. Georg, L. Kiessling, C. Lindsley, D. Liotta, K.
M. Merz, A. Schepartz, S. Wang, J Med Chem 2017, 60, 2165-2168.
[23] a) S. Veshaguri, S. M. Christensen, G. C. Kemmer, G. Ghale, M. P.
Moller, C. Lohr, A. L. Christensen, B. H. Justesen, I. L. Jorgensen, J.
Schiller, N. S. Hatzakis, M. Grabe, T. G. Pomorski, D. Stamou, Science
2016, 351, 1469-1473; b) J. T. Pedersen, J. Falhof, K. Ekberg, M. J.
Buch-Pedersen, M. Palmgren, J Biol Chem 2015, 290, 20396-20406.
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700635
ChemMedChem
FULL PAPER
Entry for the Table of Contents
A LEGO-inspired fragment assembly strategy for drug design, synthesis and discovery is described. Using our method, a series of
benzo[d]thiazoles containing a 3,4-dihydroxyphenyl moiety was found to inhibit Pma1p with the most potent IC50 of 8 µM (Ki = 6 µM) in
an in vitro assay. The SAR studies have been established. In addition, the LEGO design method reported will open new ways for the
discovery of novel inhibitors for less studied targets
12
This article is protected by copyright. All rights reserved.