Journal Pre-proofs

Identifying new lead structures to enhance tolerance towards drought stress

via high-throughput screening giving crops a quantum of solace

Jens Frackenpohl, Linn Schneider, Luka J.B. Decker, Jan Dittgen, Franz
Fenkl, Christian Fischer, Jana Franke, Joerg Freigang, Rahel Getachew,
Susana M. Gonzalez Fernandez-Nino, Hendrik Helmke, Martin J. Hills,
Sabine Hohmann, Jochen Kleemann, Karoline Kurowski, Gudrun Lange,
Peter Luemmen, Nicole Meyering, Fabien Poree, Dirk Schmutzler, Sebastian
Wrede

PII: S0968-0896(19)31027-2
DOI: https://doi.org/10.1016/j.bmc.2019.115142
Reference: BMC 115142

To appear in: Bioorganic & Medicinal Chemistry

Received Date: 12 July 2019

Revised Date: 19 September 2019
Accepted Date: 25 September 2019

Please cite this article as: J. Frackenpohl, L. Schneider, L.J.B. Decker, J. Dittgen, F. Fenkl, C. Fischer, J. Franke,
J. Freigang, R. Getachew, S.M. Gonzalez Fernandez-Nino, H. Helmke, M.J. Hills, S. Hohmann, J. Kleemann, K.
Kurowski, G. Lange, P. Luemmen, N. Meyering, F. Poree, D. Schmutzler, S. Wrede, Identifying new lead
structures to enhance tolerance towards drought stress via high-throughput screening giving crops a quantum of
solace, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115142

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Identifying new lead structures to enhance Leave this area blank for abstract info.
tolerance towards drought stress via high-
throughput screening giving crops a
quantum of solace
Jens Frackenpohla,, Linn Schneiderb, Luka J. B. Deckera, Jan Dittgena, Franz Fenkla, Christian
Fischera, Jana Frankea, Joerg Freigangc, Susana M. Gonzalez Fernandez-Ninoa, Hendrik Helmkea,
Martin J. Hillsa, Sabine Hohmanna, Rahel Getachewa, Jochen Kleemannc, Karoline Kurowskia, Gudrun
Langea, Peter Luemmena, Nicole Meyeringb, Fabien Poreea, Dirk Schmutzlera and Sebastian Wredeb
a
Research & Development, Weed Control - Bayer AG, Crop Science Division, Industriepark Höchst, D-65926 Frankfurt am Main
b
Research & Development, Lead Discovery – Bayer AG, Pharmaceutical Division, Aprather Weg 18a, D-42096 Wuppertal
c
Research & Development - Bayer AG, Crop Science Division, Gebäude 6240, Alfred-Nobel-Straße 50 ,D-40789 Monheim

High-throughput screening
 Bioorganic & Medicinal Chemistry

Identifying new lead structures to enhance tolerance towards drought stress via high-
throughput screening giving crops a quantum of solace
Jens Frackenpohla, , Linn Schneiderb, Luka J. B. Deckera, Jan Dittgena, Franz Fenkla, Christian Fischera,
Jana Frankea, Joerg Freigangc, Rahel Getachewa, Susana M. Gonzalez Fernandez-Ninoa, Hendrik Helmkea,
Martin J. Hillsa, Sabine Hohmanna, Jochen Kleemannc, Karoline Kurowskia, Gudrun Langea, Peter
Luemmena, Nicole Meyeringb, Fabien Poreea, Dirk Schmutzlera and Sebastian Wredeb
a
Research & Development, Weed Control - Bayer AG, Crop Science Division, Industriepark Höchst, D-65926 Frankfurt am Main
b
Research & Development, Lead Discovery – Bayer AG, Pharmaceutical Division, Aprather Weg 18a, D-42096 Wuppertal
c
Research & Development - Bayer AG, Crop Science Division, Alfred-Nobel-Straße 50 ,D-40789 Monheim

ARTICLE INFO ABSTRACT

Article history: Novel synthetic lead structures interacting with RCAR/(PYR/PYL) receptor proteins were
Received identified based on the results of a high-throughput screening campaign of a large compound
Received in revised form library followed by focused SAR studies of the three most promising hit clusters. Whilst
Accepted indolinylmethyl sulfonamides 8y,z and phenylsulfonyl ethylenediamines 9y,z showed strong
Available online affinities for RCAR/ (PYR/PYL) receptor proteins in wheat, thiotriazolyl acetamides 7f,s
exhibited promising efficacy against drought stress in vivo (wheat, corn and canola) combined
Keywords: with confirmed target interaction in wheat and arabidopsis thaliana. Remarkably, binding
phytochemistry affinities of several representatives of 8 and 9 were on the same level or even better than the
drought stress essential plant hormone abscisic acid (ABA).
high throughput screening
aryl sulfonamides 2009 Elsevier Ltd. All rights reserved.
thiotriazolyl acetamides

1. Introduction proteins as soluble ABA-receptors.10,11 It was shown via crystal

Agricultural crops face tremendous losses due to biotic stress
such as pests, diseases, and weed damages, but abiotic stress
adversely affects crop production even further in various parts of
the world, decreasing average yields for most of the crops
significantly.1 Among various abiotic stresses affecting
agricultural production, drought stress is considered to be the
main source of crop losses around the globe.2 There are various
strategies for reducing the impact that drought has on crop yield.
These include exploiting beneficial effects of crop protection
agents,3 developing drought tolerant crops through transgenic
approaches or breeding,4,5 but also exploring novel chemical
active ingredients inspired by naturally occurring plant
hormones. Abscisic acid (1, S-(+)-ABA), a chiral sesquiterpenoid
first discovered in the 1960s,6-9 is one the most important plant
hormones regulating signals in plants such as seed maturation,
dormancy or water use. It has been commercialized, e.g. for
enhancing colour development in red table grapes, but it also
mediates the adaption of plants to different types of
environmental abiotic stress such as drought, heat or salinity
stress. Studies on ABA mediated signaling have progressed
———
rapidly since the recent discovery of RCAR/(PYR/PYL) receptor
structural analysis that binding of ABA to AtRCAR12 (cf.
AtPYL1) induces a conformational change in the highly
conserved ABA receptor proteins giving rise to an interaction
with phosphoprotein phosphatases 2C (PP2Cs) thereby inhibiting
their activity. Hence, these findings have granted new insights
into the structure activity relationship (SAR) of ABA whilst its
catabolism had been well explored prior to identifying the
RCAR/(PYR/PYL) receptor proteins.
ABA’s principal pathway of oxidation is through
monooxygenase-mediated hydroxylation, i.e. by the CYP707A-
family, of the 8’-methyl group affording 8’-hydroxy-ABA which
is in equilibrium with the cyclized form phaseic acid. 12-15 Whilst
stabilizing or further substituting the cyclohexenone moiety has
thus attracted significant interest,16-22 surprisingly few approaches
have been made to identify open-chain analogues of ABA with
promising target affinity. Recently, it was shown that cyano-
cyclopropyl groups in 2a-c served as suitable replacements of the
cyclohexenone moiety leading to ABA analogues with strong
activity in vitro and in vivo.23 Furthermore, the first synthetic
compounds interacting with RCAR/(PYR/PYL) receptor proteins
have been prepared.24-26 In our view this indicates that we can

 Corresponding author. Building G836 Industriepark Höchst, D- 65926 Frankfurt am Main; E-mail: jens.frackenpohl@bayer.com
 6
8´ 9´
5
3
1´
2
5´ 6´
4
1
4´

2´ 7´
3´
3a X = CH, R1 = Br 5a R1 = n-Pr, R2 = Me
(S)-(+)-ABA 1 3b X = N, R1 = F 5b R1 = allyl, R2 = Me
5c R1 = F3C-CH2-, R2 = Cl/CN

2a R2 = Et, R3 = H 4a X = C-CH3, Y = S, R1 = Br 6a X = Me, R1 = n-propyl, R2 = Me

2b R2 = Et, R3 = CH3 4b X = N-Et, Y = CH, R1 = H 6b X = OMe, R1 = c-Pr-CH2-, R2 = Me
2c R2 = CF3, R3 = Me

Figure 1. Abscisic acid and selected terpenes, sulfonamides and phosphonamides interacting with RCAR/(PYR/PYL) receptor proteins

identify ABA analogues with more profound structural changes

that exhibit good receptor binding and promising in vivo efficacy.
Substituted aryl sulfonamides, e. g. pyrabactin 3a,27,28 closely
related pyrimidine 3b, as well as thiazoles and pyrazoles 4a-b,29
have shown promising initial receptor affinity and beneficial
effects in in vivo tests (Fig. 1). More recently, tetrahydro-
quinolinyl sulfonamides 5a-c have shown improved in vitro
activity.30-33 Interestingly, it was further demonstrated that the
sulfonamide unit in these hit structures could be replaced by
phosphonamides 6a-b preserving binding affinity and thus
emphasizing opportunities to introduce further structural motifs
into ABA-related structures.34 Herein, we outline how we
identified new lead structures interacting with RCAR/(PYR/
PYL) receptor proteins via high-throughput screening (HTS)
using a wheat RCAR and subsequent focused SAR studies.
2. Results and discussion
2.1. Receptor protein identification and isolation
In earlier studies we solved crystal structures with close
analogs of ABA (1) in complex with receptor protein AtRCAR11
(cf. AtPYR1) and the PP2C phosphatase AtABI1 isolated from
Arabidopsis thaliana (At).35 In line with results observed within
our in vivo screening campaign of terpenoid-based ABA-analogs
it was shown that structural variations (i) of the diene side chain
or (ii) of key elements responsible for receptor binding (e.g. the
cyclohexenone carbonyl group) had a stronger impact on receptor
binding in AtRCAR11 than changes in the absolute configuration
of stereocentres in the cyclohexanone moiety. In order to explore
unprecedented synthetic lead structures we decided not to use the
model plant Arabidopsis thaliana, but to investigate RCAR
receptor proteins in a relevant target crop as the basis for a high-
throughput screening campaign, i.e. wheat (Triticum aestivum,
Ta). Therefore, the orthologous genes of AtRCAR11 and AtABI1
coding for the proteins were identified in Triticum aestivum. To
do so, a reciprocal BLAST search was performed using the
Arabidopsis sequences as the query. TaABI1 (AB238930) and
TaRCAR14 (KD239761) were identified. Synthetic coding genes
were ordered and cloned in the expression vector pQE70 (Life
Techn.). Proteins were produced in Escherichia coli and
subsequently purified. Thereafter, their activity was measured
and compared to the activity of the corresponding Arabidopsis
protein as previously described.35 Likewise, the effects of a
selected set of compounds having emerged from our recent
synthesis programmes23, 29, 32, 33, 35 in the field of ABA signaling
were tested. Significant differences could not be observed
between both series of proteins. Thus, TaABI1 and TaRCAR14
isolated from wheat could be used to start the high-throughput
screening campaign.
2.2. High-throughput screening
A biochemical assay was developed, probing for compounds
inducing the inhibitory complex of wheat phosphatase TaABI1
and co-regulator TaRCAR14. TaABI1 activity was detected by
dephosphorylation of 4-methylumbelliferyl phosphate (MUP).
The assay was successfully adapted to the 1536 well plate format
and fully automated processing with the robotic screening
system. Sensitivity towards compounds inducing the inhibitory
complex was assessed using ABA as the reference compound,
which was also continuously used to monitor the assay
performance and the constancy of the primary screening
campaign. On each robotic test day full kinetic traces were
determined to confirm the overall enzymatic activity. A total of
3,691,096 tested compounds were classified by the change in
fluorescence intensity of the substrate from an initial
measurement directly after pipetting (M1, Figure 2b) to an end
point of the enzymatic reaction after 80 min at 32 °C (M2).
Change in fluorescence intensity in the presence of reference
compound ABA or the test compounds (termed M2-M1) was
normalized by the negative control DMSO, which is set to 100 %
(Figure 2a). Primary hits were selected by activity values below
DMSO scattering with a z-score < -8 (Figure 2a). This hit limit is
also annotated in the dose response curve (DRC) of ABA (Figure
2c). With the selected hit limit, the high assay sensitivity allowed
to resolve an activity of about 15 nM ABA, a concentration
eliciting ~20 % of the maximum response.
A B
C
Figure 2. Primary HTS scatter plot: Phosphatase activity, determined by
change in fluorescence intensity of the substrate from an initial measurement
directly after pipetting to an end point of the reaction after 80 min at 32 °C
(M2-M1) (B), is plotted on the y-axis of the scatter plot (A). Values were
normalized to DMSO, which is set to 100. Compounds were tested at 10 or 5
µM and the reference compound ABA was included as DRC. For hit
selection, a hit limit was set to z-score < -8. This hit limit is also annotated in
ABA DRCs of the robotic run (C) (brown dotted line). Compounds affecting
the fluorescence intensity independent of the enzymatic activity were
 deselected by the change in initial fluorescence intensity plotted at the x-axis
of the scatter plot (A).
As a result of the in-depth hit evaluation, three promising hit
clusters were identified with several closely related members
Among these hits a set of compounds affecting the (clusters I – III, Figure 4). Compounds from clusters II and III
fluorescence intensity independent of enzymatic activity was showed strong initial binding affinities, whereas moderate
deselected by the respective increase or decrease of the initial affinities were observed for the members of cluster I. These
measurement directly after pipetting (M1 % Figure 2a). findings prompted us to investigate structural features of the
Additionally, obvious frequent hitters were excluded based on scaffolds and their respective in vitro and in vivo efficacies in
their uHTS data base profile, indicating non-specific broad detail.
activity on biochemical assays. In total, 3,908 compounds were Cluster I Cluster III
selected for secondary HTS.
For the selected hit compounds EC50 values were determined.
Based on a compound concentration of 10 µM, as used for
primary screening, an effect above 20% of the maximum
response could be confirmed for 2,988 compounds. This re- 7a R1 = Et, R2 = H, R3 = o-Cl 9a R1 = CF3, R2 = H
confirmation rate of 76 % evaluates the quality and precision of 7b R1 = c-Pr, R2 = Me, R3 = p-CO2Me
9b R1 = CF3, R2 = p-F
7c R1 = p-MeO-Bn, R2 = Me, R3 = m-SMe
the uHTS campaign. To distinguish compounds inducing the 9c R1 = H, R2 = p-Br
9d R1 = CF3, R2 = p-OMe
TaRCAR-TaABI1 inhibitory complex from complex-indepen- 9e R1 = CF3, R2 = m-Cl
Cluster II
dent compound effects, like direct inhibition of TaABI1 9f R1 = CF3, R2 = o-Cl
phosphatase activity, a counter assay on TaABI1 in the absence 9g R1 = CF3, R2 = m-F
9h R1 = CF3, R2 = m-OMe
of TaRCAR14 was performed. Figure 3 shows the correlation of
EC50 values in presence and absence of TaRCAR14. Most
compounds directly inhibit TaABI1 phosphatase activity, leading
8a R1 = Cl, R2 = Me
to comparable potency in both assay formats (Figure 3). Final 8b R1 = Br, R2 = Et
hits were selected by EC50 values below 10 µM in the presence of 8c R1 = Cl, R2 = Ph
8d R1 = Br, R2 = Ph
TaRCAR14 and no detectable direct effect on phosphatase
activity. The inset of Figure 3 shows the DRCs for an exemplary Figure 4. Promising hit clusters (all EC50 values below 10 µM@TaRCAR14)
hit compound. The final hit list with 127 compounds was then with selected representative members chosen for further exploration.
submitted to chemical inspection and subsequent hit evaluation.

2.3. Modeling studies

Figure 3. EC50 correlation and HTS overview: For EC50 determination
hits were retested in 8 concentrations in the primary HTS assay format and in
absence of TaRCAR14 to deselect compounds with direct effects on
phosphatase activity. All in all, 127 final hits were selected, showing no
direct phosphatase inhibition. The inset shows DRCs of an exemplary final
hit.
As a starting point for our SAR investigations selected
members of the three most potent hit clusters were docked into
the ABA binding site of the crystal structure of ABA-bound
AtRCAR11 and related phosphatase AtHAB1. Resulting poses
were then scored using the HYDE scoring function.36
Unfortunately, for hit cluster I no convincing docking poses
were identified. In contrast, for hit clusters II and III, reasonable
docking poses were obtained. The carbonyl group within the
indolinyl amide moiety of hit structure 8b forms an H-bond to
the conserved water molecule at the interface between
AtRCAR11 and AtHAB1 adopting the role of the carbonyl
oxygen in ABA. This essential water molecule forms all four
possible H-bonds including H-bonds to the backbone oxygen of
proline 115 and backbone amide of arginine 143 from
AtRCAR11 and NE1 of tryptophan 385 from AtHAB1, thus
representing a key element in the protein–protein interaction

a c

b e

10 11 12a-i 7d-x

12a R = CH3, R = CH3

1 2
f h
12b R1 = CH3, R2 = i-Pr
12c R1 = c-Bu, R2 = CH3
12d R1 = CF3, R2 = CH3
12e R1 = furan-2-yl, R2 = CH3
12f R1 = 4-Cl-Ph, R2 = c-Pr g
12g R1 = CH3, R2 = furan-2-ylmethyl
12h R1 = H, R2 = furan-2-ylmethyl
12i R1 = c-Pr, R2 = CH3

Scheme 1. Synthesis of triazolyl acetamides 7; a) 1. (COCl)2,or SOCl2, N,N-DMF, 0 °C – r.t., 2. N2H4-H2O; b) EtOH, 80 °C; c) 1. aq. NaOH (1M), reflux, 2.
aq. HCl (1M); d) R3-Ph-NH2, DMA, 0 °C, r.t.; e) MeCN, K2CO3, reflux; f) MeCN, K2CO3, g) LiOH, H2O; h) R3-Ph-NH2, HOBt, EDC, (i-Pr)2NEt CH2Cl2.
 a c

13 14 15

d d

h i e

17a-f 16
g j

f
17a R2 = CH3
17b R2 = Et
17c R2 = Ph

17d R2 = 2-Me-prop-1-yl
17e R2 = i-Pr
17f R2 = 2,2,2-trifluoroethyl

8e-z

Scheme 2. Synthesis of indolinylmethyl sulfonamides 8; a) formic acid (5 equiv.), toluene, reflux, 5h; b) conc. H2SO4, 0 °C – r.t., 18h; c) aq. HCl (1M),
MeOH, reflux, 4h; d) Et3N, DMAP, CH2Cl2, 0 °C – r.t., 4h; e) N2H4-H2O, CH2Cl2/MeOH, r.t., 12h; f) pyridine (3 equiv.), MeCN, 70 °C, 5h; g) formylation
failed; h) 1. N-Bromosuccinimide, CH2Cl2, r.t., 4h; 2. CuCN, CuI, N,N-DMF, reflux, 4d; i) H2 (40 bar), Raney-Ni/H2O, NH3 in MeOH, r.t.; j) NaBH3CN

between the RCARs and the corresponding phosphatases.

Furthermore, the sulfonamide moiety in 8b adopts the role of
ABA’s terminal carboxylic acid group with one of the SO2-
oxygens forming a second H-bond to a crucial water molecule
(Figure 5a).
5b)
5a)
9b
8b
Figure 5. Superposition of the best ranked docking poses of sulfonamides
8b and 9b (purple) with ABA (green) bound to AtRCAR11 and AtABI1
[special colour: O(red), S(yellow), N(blue), F(light green)].
A similar binding mode can be observed for the cyanophenyl
aminoethyl sulfonamide 9b (from cluster III), with the nitrile
moiety of 9b forming an H-bond to the essential water molecule
at the interface between AtRCAR11 and AtHAB1 adopting the
role of the carbonyl oxygen in ABA or the amide C=O group in
8b from hit cluster II (Figure 5b). Interestingly, the free electron
pairs serving as the H-bond donors in ABA and 9b, i.e. the
carbonyl oxygen of ABA’s cyclohexenone moiety and the nitrile
group in 9b, are not located at the same place due to differences
in binding geometry. The docking poses suggest in both cases
that there is additional space around the phenyl or heteroaryl
moiety for further exploration. In addition, there is some space
for small hydrophobic moieties at position R2 for compound
cluster II and at position R1 for III, respectively (cf. Fig. 4).
The beneficial impact of p-cyano aniline moieties on the
interaction of agonists with ABA receptor proteins has also been
observed in a structural analysis of the binding mode of
sulfonamide-based agonist cyanabactin with PYR1.37, 38 In good
correlation with X-ray analyses of cyano cyclopropyl analogues
of ABA (cf. 2a-c),23 this study showed that a 3-cyclopropyl-4-
cyanoaniline moiety mimics the oxygen of ABA’s
cyclohexenone engaging the key components required to stabilize
the activated receptors.

a b

18 9i-z

c d e

19 20a-h

Scheme 3. Synthesis of phenylsulfonyl ethylenediamines 9 and 20; a) 2-aminoethyl amine, Et3N, MeCN, 50 °C, 4h; b) Arylsulfonylchloride (1 equiv.), (i-
Pr)2NEt, CH2Cl2, r.t., 20h; c) 2-bromoethyl amine, Et3N, CH2Cl2, 5h; d) KOH, H2O, toluene, 1.5h; e) aniline (1 equiv.), SiO2, H2O, 3d.

2.4. Chemistry
Based on the results from high-throughput screening and
modeling studies versatile synthetic approaches were investigated
with emphasis on structural variations of key moieties crucial for
good receptor binding. Substituted thiotriazolyl acetamides
which share key structural features with hit structures 7a-c (hit
cluster I) have been described in earlier studies as active
ingredients against insect odorant sensory receptors. 39 We thus
explored and optimized the established general synthetic
approach to prepare a diverse set of thiotriazolyl acetamides 7.
Starting from readily available carboxylic acids intermediate
1,2,4-triazole-3-thiols 12a-i were obtained in 4 steps via oxalyl
chloride-mediated formation of hydrazides 10, followed by
coupling with a suitable isothiocyanate and subsequent base-
mediated cyclization of the thiosemicarbazides 11. In our hands,
cyclizations worked best using aq. KOH or NaOH at elevated
temperatures affording desired intermediates 12a-i in 75 to 94%
yield. 1,2,4-Triazole-3-thiols 12a-i were then converted into
desired thiotriazolyl acetamides 7d-x via (i) direct base-mediated
coupling of a freshly prepared halogen acetanilide, preferably
using K2CO3 or Cs2CO3 as base or via (ii) formation of a suitable
triazolylthio acetic acid ester with subsequent ester cleavage and
amide formation. Following the second approach good yields
were obtained uing HOBt, EDC and Hünig’s base as amide
coupling reagents (Scheme 1).40
Indolinylmethyl sulfonamides which had emerged within hit
cluster II of our high throughput screening were synthesized
based on two complementing routes starting from commercially
available indoline 13 (Scheme 2). After introducing a N-formyl
group to protect the indoline nitrogen via formic acid-mediated
formylation, the indolinylmethyl phthalimide 14 was formed via
Tscherniac-Einhorn reaction using concd sulfuric acid as
catalyst.41,42 Subsequent indoline N-deprotection, N-acylation
and hydrazine-mediated phthalimide cleavage afforded the key
indolinylmethyl amine intermediates 17a-f, albeit in low overall
yields (26-41%). Alternatively, indolinylmethyl amines 17 could
be obtained with improved yield (up to 57%) using an optimized
four-step protocol via N-acylation of 13, followed by
bromination, Cu-salt mediated cyanation and Ni-mediated
hydrogenation in NH3/MeOH under elevated pressure (40 bar). A
further approach via formylation and reductive amination did not
afford desired key intermediates 17. With indolinylmethyl
Table 1. SAR-results of selected thiotriazolyl acetamides 7
7d-v
Substituents[a] In vitro activity – AtABI1/TaABI1[b]
En- No. R1 R2 R3 Activity
try (TaRCAR14)
[%] 5 M
1 7d CH3 CH3 4-CN + ++
2 7e CH3 i-Pr 4-CN + -
3 7f c-Bu CH3 4-CN +++
amines 17a-f in hand, a set of desired sulfonamides 8e-z was
prepared using pyridine in acetonitrile as the base.43
Substituted phenylsulfonyl ethylenediamines could be easily
prepared using two alternative approaches, (i) via nucleophilic
aromatic substitution and subsequent formation of the
sulfonamide or (ii) via formation of an aziridinyl sulfonamide,
followed by ring opening using a substituted aniline.44 The first
approach proved to be particularly suitable for 2-trifluoromethyl-
4-fluorobenzonitrile, which reacted easily with 2-aminoethyl
amine to afford intermediate 18. The desired 4-cyanophenyl
aminoethylsulfonamides 9i-z were then obtained in good yields
using the corresponding sulfonyl chloride with diisopropyl ethyl-
amine as base. In our hands, the second pathway turned out to
work well with anilines carrying less electron-withdrawing
groups enabling us to explore a wider range of substituents. SiO2-
mediated ring opening of intermediate aziridinyl sulfonamides 19
in water afforded phenylsulfonyl ethylenediamines 20a-h as the
target compounds, thus broadening our SAR-study (Scheme 3).
2.5. SAR study
All compounds prepared to explore the SAR of hit clusters I-II
were tested for target affinity, as well as for beneficial effects in
vivo under drought stress conditions upon foliar application on
crops. Within cluster III selected representatives were tested in
dedicated greenhouse trials. Wheat and Canola were chosen as
model plants for monocotyledonous and dicotyledonous species,
whereas in vitro tests were carried out for thiotriazolyl
acetamides 7 both at the ABA-receptor system AtRCAR11 and
the orthologous ABA-receptor system in wheat, i.e. TaRCAR14.
As these results were nicely correlating, measurements for
compounds 8 and 9 were only carried out on TaRCAR14.
Good receptor affinities could be observed for substituted
thiotriazolyl acetamides carrying 4-cyano phenyl acetamide
moieties combined with small alkyl and haloalkyl substituents in
the triazolyl unit, albeit with lower pI50 values than abscisic acid
1 as the internal standard (Table 1). Other anilide substituents
afforded weaker target affinities emphasizing the role of the 4-
cyano group in receptor binding. In particular, target compounds
carrying a 4-CN-phenyl acetamide moiety together with c-Pr (7x,
Table 1, entry 21), c-Bu (7f, entry 3) and CF3-substituents (7j,
entry 7) at position R1 showed good target affinities together with
good efficacy against drought stress in vivo, particularly in wheat.
In vivo efficacy vs. drought stress
[250 g/ha][c]
Activity pI50 Wheat[c] Canola[c]
(AtRCAR11)
[%] 5 M
4.2 ++ +
n.d. ++ ++
+++ 4.7 +++ ++
4 7g c-Bu CH3 2,4-F2 + - n.d. ++ 0

5 7h CF3 CH3 3-Cl - + n.d. + ++

6 7i CF3 CH3 2,4-F2 + + n.d. ++ ++

7 7j CF3 CH3 4-CN ++ +++ 4.8 +++ ++

8 7k Furan-2-yl CH3 3-CF3 + + n.d. + +

9 7l Thiophen-2-yl CH3 4-CN + + n.d. ++ +

10 7m 4-Cl-C6H4 c-Pr 4-CN n.d. ++ n.d. ++ +

11 7n 4-Cl-C6H4 c-Pr 2-Cl + ++ n.d. ++++ ++

12 7o CH3 Furan-2-ylmethyl 4-Cl + + n.d. ++ +

13 7p CH3 Furan-2-ylmethyl 4-CN +++ + n.d. ++ +

14 7q H Furan-2-ylmethyl 4-Cl +++ +++ 4.6 + +++

15 7r H Furan-2-ylmethyl 3-CN n.d. + n.d. + +

16 7s H Furan-2-ylmethyl 4-CN ++++ ++++ 5.3 ++ +++

17 7t H Furan-2-ylmethyl 3-Cl ++ ++ 4.5 + +

18 7u H Furan-2-ylmethyl 2,4-Cl2 ++ +++ 4.4 ++ ++

19 7v H Furan-2-ylmethyl 2-Cl - - n.d. + 0

20 7w c-Pr c-Pr 2-Cl - - n.d. + 0

21 7x c-Pr c-Pr 4-CN ++ + 4.2 +++ +

22 1 Abscisic acid ++++ ++++ 7.1 +++ +++

a
c-Pr = cyclopropyl, c-Bu = cyclobutyl, i-Pr = iso-propyl; b a final expert assessment of target affinity was made according to the following classification: “-”: <
30% ; “+” = 30% < activity < 50%; “++” = 50% < activity < 70%; “+++” = 70% < activity < 90%; “++++” = activity > 90%; c standard application rate for crop
protection greenhouse trials; d a final expert assessment of efficacies was made according to the following classification: “0” = no effect, “+” = slight beneficial
effect, “++” = significant beneficial effect, “+++” = strong beneficial effect against drought stress, “++++” = very strong beneficial effect superior to internal
standard ABA (comparative visual assessment of greenmass)

Whilst other variations at R1 such as methyl, furan-2-yl,
thiophen-2-yl or 4-Cl-phenyl groups afforded weaker effects,
both in vitro and in vivo (cf. Table 1, entries 1, 8, 9, 10),
thiotriazolyl acetamide 7n carrying a p-Cl-phenyl group at R1
together with a c-Pr group at R2 showed strong effects against
drought stress in wheat. Hence, a focused variation of R2 showed
that furan-2ylmethyl groups had a beneficial impact on binding
affinity with 7s (entry 16) exhibiting the best target affinity of all
thiotriazolyl acetamides investigated within our SAR study.
Figure 6. Advanced drought stress trials with 7n (application rate: 25 g/ha
with replicates) in wheat and barley under moderate water stress conditions,
i.e. 42% damage for barley and 48% for wheat, respectively in comparison
with untreated, unstressed control plants.
Interestingly, a broader variety of anilide substituents R3 was
tolerated in combination with furan-2ylmethyl groups at R2 and
the hydrogen at position R1 as also 4-Cl-, 3-Cl- and 2,4-Cl2-
anilides 7q, 7t, 7u exhibited good receptor affinities in wheat and
Arabidopsis thaliana (cf. Table 1, entries 14, 17, 18). To further
validate our initial in vivo screening results further, we selected
7n (with moderate target affinity, but surprisingly strong efficacy
in wheat) for advanced greenhouse trials (i.e. more replicates,
additional crops such as corn and barley, different corn varieties
and moderate stress levels). Accordingly, significant beneficial
effects against drought stress were observed in wheat compared
to untreated controls upon treatment with 7n (Figure 6).
Exploring hit cluster II gave a similar picture as observed for
the SAR study of cluster I. In line with results observed for
thiotriazolyl acetamides 7d-7x, receptor binding affinities of
indolinylmethyl sulfonamides 8e-8z varied strongly upon
changes in substituents R1 and R2 giving rise to a narrow SAR.
Halogen-substituted thiophen-2-yl moieties as sulfonyl
substituents R1 proved to afford the best binding affinities when
combined with small alkyl groups at R2. 5-Me-, 5-Br-, 5-Cl- and
4,5-dibromo substituents in the thiophen-2-ylsulfonamide moiety
were well tolerated since indolinylmethyl sulfonamides 8h, 8i,
8k, and 8r (Table 2, entries 4, 5, 7, and 8) showed good in vitro
activity, as well as promising in vivo efficacy against drought
stress in wheat. Interestingly, compounds 8e and 8g bearing 4-
cyano- and 4-bromo phenyl groups as R1 substituents still gave
good results in vivo, but showed weaker target affinity.
Furthermore, replacing halogen thiophenyl groups by 4-CF3- and
4-MeO-phenyl groups led to significantly weaker target affinities
combined with a loss of efficacy in vivo against drought stress
(cf. cpds. 8l, 8n, Table 2, entries 8, and 10). These observations
are in line with results from dedicated modeling studies
indicating the privileged role of halogen-substituted thiophene
moieties in receptor binding of indolinylmethyl sulfonamides 8.
Introducing a benzthiophen-2-yl group as part of the sulfonyl
moiety also afforded weaker effects in vitro and in vivo (8m,
entry 9, table 2).
Whilst small alkyl groups such as methyl (cf. 8r, Table 2,
entry 14,), ethyl (e.g. 8h, entry 4) and iso-propyl (e.g. 8p, entry
12) were well tolerated as substituents R2, introducing haloalkyl
or phenyl groups led to a loss of target affinity, and of in vivo
efficacy against drought stress.

Table 2. SAR-results of indolinylmethyl sulfonamides 8. Table 3. SAR-results of phenylsulfonyl ethylenediamines.

Substituentsa In vitro
Substituents a
In vitro In vivo activity b
activity b efficacy TaABI1
TaABI1 vs. drought
stress En- No. R1 R2 R3 TaRCAR14b
try [%] [5 M]
En- No. R1 R2 TaRCAR14 b Wheatc
try [%] [5 M] [250 g/ha] d 1 9i CF3 2-Cl-C6H4 CN ++++

1 8e 4-CN-C6H4 Et ++ ++ 2 9j CF3 4-Cl-C6H4 CN ++++

2 8f thiophen-2-yl Et + + 3 9k CF3 4-Me-C6H4 CN ++++

3 8g 4-Br-C6H4 Et + ++ 4 9l H 4-Cl-C6H4 CN +++

4 8h 5-Cl-thiophen-2-yl Et +++ ++ 5 9m H 4-F-C6H4 CN +++

5 8i 4,5-Br2-thiophen-2-yl Et +++ + 6 9n H 3-F-C6H4 CN +

6 8j 4-Cl-C6H4 Et + + 7 9o MeO 4-F-C6H4 CN ++++

7 8k 5-Me-thiophen-2-yl Et +++ + 8 9p MeO 4-CH3-C6H4 CN ++++

8 8l 4-CF3-C6H4 Et + 0 9 9q Cl 4-Cl-C6H4 CN ++++

9 8m benzthiophen-2-yl Et + + 10 9r Cl 2-Cl-C6H4 CN ++++

10 8n 4-MeO-C6H4 Et + 0 11 9s Cl 4.H3CO-C6H4 CN ++++

11 8o thiophen-2-yl i-Pr ++ + 12 9t Me 4-H3CO-C6H4 CN ++++

12 8p 5-Br-thiophen-2-yl i-Pr +++ ++ 13 9u Me 2-Cl-C6H4 CN ++++

13 8q 5-Cl-thiophen-2-yl i-Pr ++ + 14 9v Me 3-Cl-C6H4 CN ++++

14 8r 5-Br-thiophen-2-yl CH3 +++ ++ 15 9w Me 4-Cl-C6H4 CN ++++

15 8s thiophen-2-yl F3C-CH2- + 0 16 9x Me 4-F-C6H4 CN ++++

16 8t 5-Br-thiophen-2-yl F3C-CH2- ++ ++ 17 9y Me 4-Me-C6H4 CN ++++

18 9z Me 1-methyl-1H- CN ++++
17 8u 5-Cl-thiophen-2-yl F3C-CH2- + +
imidazol-4-yl
18 8v 5-Me-thiophen-2-yl F3C-CH2- + 0
19 20a CF3 4-Cl-C6H4 F ++
19 8w 5-Me-thiophen-2-yl CH3 ++ +
20 20b CF3 4-CH3-C6H4 F +++
20 8x 5-Me-thiophen-2-yl C6H5 + 0
21 20c CF3 4-H3CO-C6H4 F +++
21 8y 5-Br-thiophen-2-yl c-Pr-CH2 +++ ++
22 20d CF3 2-F-C6H4 F +++
22 8z 5-Cl-thiophen-2-yl c-Pr-CH2 +++ +
23 20e CF3 4-Cl-C6H4 H 0
a
Me = CH3, Et = ethyl, i-Pr = iso-propyl, c-Pr = cyclopropyl; b a final expert
assessment of target affinity was made according to the following 24 20f CF3 4-H3CO-C6H4 Cl +
classification: “-”: < 30%; “+” = 30% < activity < 50%; “++” = 50% <
activity < 70%; “+++” = 70% < activity < 90%,; “++++” = activity > 90%; c 25 20g CF3 3-Cl-C6H4 Cl 0
standard application rate for crop protection greenhouse trials; d a final expert
assessment of efficacies was made according to the following classification: 26 20h CF3 4-F-C6H4 OMe 0
“0” = no effect; “+” = slight beneficial effect; “++” = significant beneficial a
effect; “+++” = strong beneficial effect against drought stress; “++++” = very Me = CH3, Et = ethyl, i-Pr = iso-propyl; b a final expert assessment of target
strong beneficial effect superior to internal standard ABA (comparative visual affinity was made according to the following classification: “-”: < 30% ; “+”
assessment of greenmass). = 30% < activity < 50%; “++” = 50% < activity < 70%; “+++” = 70% <
activity < 90%; “++++” = activity > 90%.
 Interestingly, cyclopropylmethyl groups at R 2 afforded some
of the best in vitro results observed for indolinylmethyl
sulfonamides when combined with halogen-substituted thiophen-
2-ylsulfonyl moieties (cf. 8y, 8z, Table 2, entries 21, 22,).
Compound 8y also exhibited promising efficacy against drought
stress in wheat.
The detailed SAR study of phenylsulfonyl ethylenediamines 9
and 20 (Table 3) afforded highly consistent results nicely
complementing results obtained for our largest hit cluster III. As
observed for thiotriazolyl acetamides 7d-7x, anilines 9 carrying a
p-CN group at R3 showed high target affinities (e.g. 9i, 9j, 9k,
and 9s, entries 2, 3, 4, and 11), albeit a second substituent R1 in
the 3-position of the aniline appeared to be mandatory for strong
in vitro activity. This observation is based on weaker binding
affinities of compounds 9l, 9m, and 9n bearing a hydrogen atom
at R1. Furthermore, p-cyano aniline moieties with an additional
substituent at R1 such as CF3, methyl or methoxy exhibited a
good tolerance of changes in substitution of the sulfonamide
moiety (i.e. R2). In particular, 2-Cl-, 3-Cl- and 4-Cl-phenyl
sulfonyl ethylene-diamines 9u, 9v and 9w gave consistently high
target affinities. Remarkably, phenylsulfonyl ethylenediamines 9j
and 9k exhibited higher target affinities (pI50 7.32 and 7.27) than
reference compound ABA (pI50 7.11). To validate our promising
in vitro screening results, we selected 9j and 9k for advanced
greenhouse trials. Accordingly, beneficial effects against drought
stress were observed in wheat and canola compared to untreated
controls upon treatment, e.g. results for 9j: wheat(250 g/ha) ++,
canola(250 g/ha) +++; for 9k: wheat(250 g/ha) ++, canola(250
g/ha) ++.
3. Conclusions
We have identified and confirmed three unprecedented lead
structures interacting with RCAR/(PYR/PYL) receptor proteins
in wheat via high-throughput screening of our corporate
compound library and subsequent dedicated SAR studies.
Receptor protein TaRCAR14 and the PP2C phosphatase TaABI1
were isolated from wheat and chosen for the screening campaign.
Independently, a transgenic approach has demonstrated that ABA
receptor overexpression in wheat improves grain production
under drought underlining the potential for the use of new
chemical lead structures that interact with relevant receptor
proteins in wheat.45 Whilst phenylsulfonyl ethylenediamines (e.g.
9j and 9k) showed consistent strong target affinities in vitro on
the same level or even better than the plant hormone abscisic acid
(ABA), several thiotriazolyl acetamides (e.g. 7f and 7s) exhibited
promising efficacy against drought stress mainly in wheat
combined with good target interaction. In all three compound
classes particular structural features such as 4-cyanoaniline or 5-
halogenthiophen-2-yl moieties were explored which had an
essential impact on target affinity in vitro and in vivo efficacy.
Our results demonstrate that p-cyano aniline moieties are
effective bioisosters of the carbonyl group in ABA’s
cyclohexenone headgroup and in tetrahydroquinolinyl
sulfonamides. Furthermore, indolinylmethyl sulfonamides 8 and
phenylsulfonyl ethylenediamines 9 represent two novel classes of
sulfonamides interacting with RCAR/(PYR/PYL) receptor
proteins, thus nicely complementing earlier work in this field.
4. Experimental
4.1. Biology
4.1.1. In vivo studies - Seeds of monocotyledonous and
dicotyledonous crop plants were laid out in sandy loam in wood-
fiber pots, covered with soil and cultivated in a greenhouse under
good growth conditions. The test plants were treated at the first
leaf stage (BBCH10 – BBCH13). To ensure uniform water
supply before commencement of drought stress, the potted plants
were supplied with the maximum amount of water immediately
beforehand by dam irrigation and, after application, transferred
into plastic inserts in order to prevent subsequent, excessively
rapid drying. The respective compounds, formulated in the form
of wettable powders (WP) or emulsion concentrates (EC), were
sprayed onto the green parts of the plants as an aqueous
suspension at an equivalent water application rate of 600 l/ha
with addition of 0.2% wetting agent (agrotin). Substance
application is followed immediately by drought stress treatment
of the plants. Drought stress was induced by gradual drying out
under the following conditions: “day” = 14 hours with
illumination at 26 °C; “night” = 10 hours without illumination at
18 °C. The duration of the respective stress phases was guided
mainly by the state of the untreated (= treated with blank
formulation but without test compound), stressed control plants
and thus varied from wheat to canola. It was ended (by re-
irrigating or transfer to a greenhouse with standard growth
conditions) as soon as irreversible damage was observed on the
untreated, stressed control plants. In the case of dicotyledonous
crops, for example canola, the duration of the drought stress
phase varied between 3 and 5 days; in the case of
monocotyledonous crops, for example wheat, it varied between 6
and 10 days. The end of the stress phase was followed by an
approx. 5-7-day recovery phase, during which the plants were
once again kept under good growth conditions in a greenhouse.
In order to rule out any influence of the effects observed by any
fungicidal action of the test compounds, it was additionally
ensured that the tests proceeded without fungal infection and
without infection pressure. After the recovery phase had ended,
the intensities of damage were rated visually in comparison to
untreated, unstressed controls of the same age (in the case of
drought stress) or the same growth stage (in the case of cold
stress). The intensity of damage was first recorded as a
percentage (100% = plants have died, 0% = like control plants).
These values were then used to calculate the efficacy of the test
compounds (= percentage reduction in the intensity of damage as
a result of substance application), and a final assessment of the
respective efficacy was made, i.e. “0” = no effect, “+” = slight
beneficial effect, “++” = significant beneficial effect, “+++” =
strong beneficial effect against drought stress, “++++” = very
strong beneficial effect superior to internal standard ABA.
4.1.2. In vitro - AtABI1-(AtRCAR11) / TaABI1-(TaRCAR14) -
The assays described hereinafter utilize the inhibition of the
phosphatase AtABI1 via the co-regulator RCAR11/PYR1 from
Arabidopsis thaliana or the inhibition of the wheat TaABI1 via
co-regulator RCAR14 from Triticum aestivum. Expression and
purification of RCARs and PP2Cs was performed as described.10
A concise overview of the correlation between PYR/PYL and
RCAR numbering can be found in reference [46] by Grill et al. 46
For the determination of activity, the dephosphorylation of 4-
methylumbelliferyl phos-phate (MUP) was measured at 460 nm.
The in vitro assay was conducted in Greiner 96-well, 384-well or
1536-well PS microplates, using two controls: a) dimethyl
sulfoxide (DMSO) 0.5% (f.c.) and b) 5 M (f.c.) abscisic acid
(ABA). For high throughput screening 1 µg/ml ABI1 and 0,5
µg/ml RCAR11 were incubated with 100 µM MUP in reaction
buffer (50 mM Tris/HCl pH 7.8, 50 mM NaCl, 0.3 mM MnCl2,
0.01 % Tween, 0.01 % BSA) at 32°C for 80 minutes.
Fluorescence intensity was detected by a BMG Labtech Pherastar
using a 340 nm excitation filter and a 460 nm emission filter. For
follow up compound characterization the assay was conducted
with substance concentrations of the appropriate chemical test
substances in a concentration range of 0.1 M to 100 M in a
solution of DMSO and water. The substance solution thus
obtained, if necessary, was stirred with esterase from porcine
liver (EC 3.1.1.1) at room temperature for 3 h and centrifuged at
4000 rpm for 30 min. A total volume of 45 l was introduced
into each cavity of the microplate, having the following
composition: 1. 5l of substance solution, i.e. a) DMSO 5% or b)
abscisic acid solution or c) the corresponding example compound
of the general formula (I) dissolved in 5% DMSO. 2. 20l of
enzyme buffer mix, composed of a) 40% by vol. of enzyme
buffer [10 ml contain equal proportions by volume of 500 mM
Tris-HCl pH8, 500 mM NaCl, 3.33 mM MnCl2, 40 mM
dithiothreitol (DTT)], b) 4% by vol. of AtABI1 or TaABI1
dilution (protein stock solution was diluted so as to give, after
addition, a final concentration in the assay of 0.15 g
ABI1/well), c) 4% by vol. of AtRCAR11 or TaRCAR14 dilution
(enzyme stock was diluted so as to give, on addition of the
dilution to the enzyme buffer mix, a final concentration in the
assay of 0.30 g enzyme/well), d) 5% by vol. of Tween20 (1%),
e) 47% by vol. H2O bi-dist. 3. 20l of substrate mix, composed
of a) 10% by vol. of 500 mM Tris-HCl pH8, b) 10% by vol. of
500 mM NaCl, c) 10% by vol. of 3.33 mM MnCl 2, d) 5% by vol.
of 25 mM MUP, 5% by vol. of Tween20 (1%), 60% by vol. of
H2O bi-dist. Enzyme buffer mix and substrate mix were made up
5 minutes prior to the addition and warmed to a temperature of
35°C. On completion of pipetting of all the solutions and on
completion of mixing, the plate was incubated at 35°C for 20
minutes. Finally, a relative fluorescence measurement was made
at 35°C with a BMG Labtech "POLARstar Optima" microplate
reader using a 340/10 nm excitation filter and a 460 nm emission
filter. An expert assessment of target affinity was made according
to the following classification: “-”: < 30% , “+” = 30% < activity
< 50%, “++” = 50% < activity < 70%,, “+++” = 70% < activity <
90%,, “++++” = activity > 90%.
4.2. Chemistry
4.2.1. General – All reagent-grade solvents and chemicals
were purchased from standard commercial suppliers and used
without further purification. All non-aqueous reactions were
carried out under anhydrous conditions using dry solvents.
Reactions were monitored by LC-MS or TLC carried out on 0.25
mm silica gel plates (60F-254). TLC plates were visualized using
UV light. Flash chromatography was carried out using Biotage
Isolera One systems with pre-packed column cartridges (Biotage
KP-Sil [40+M] or KP-Sil [25+M]). The 1H NMR, 13C NMR and
19
F NMR spectroscopy data which are reported for the chemical
examples described below (400 MHz for 1H NMR and 150 MHz
for 13C NMR and 375 MHz for 19F NMR, solvent: CDCl3,
CD3OD or d6-DMSO, internal standard: tetramethylsilane δ =
0.00 ppm), were obtained on a Bruker instrument, and the signals
listed have the meanings given below: br = broad; s = singlet, d =
doublet, t = triplet, dd = doublet of doublets, ddd = doublet of a
doublet of doublets, m = multiplet, q = quartet, quint = quintet,
sext = sextet, sept = septet, dq = doublet of quartets, dt = doublet
of triplets. The abbreviations used for chemical groups or
dedicated hydrogen atoms are defined as follows: Me = CH3, Et
= CH2CH3, t-Hex = C(CH3)2CH(CH3)2, t-Bu = C(CH3)3, n-Bu =
unbranched butyl, n-Pr = unbranched propyl, c-Hex =
cyclohexyl, ArH = aromatic hydrogen, HetH = heteroaromatic
hydrogen. In the case of diastereomeric mixtures, either the
significant signals for each of the diastereomers or the
characteristic signal of the main diastereomer is/are reported.
4.2.2. General Procedure A for the preparation of thio-
triazolyl acetamides 7d-x. Step a: The appropriate carboxylic
acid ester (1.0 equiv.) was dissolved either in N,N-DMF of in
ethanol (2 ml/mmol), and hydrazine hydrate (5 equiv) was added.
The resulting reaction mixture was heated under reflux
conditions for 3-5 h. After cooling to room temperature, ethanol
was removed carefully under reduced pressure, followed by
addition of dichloromethane and thorough extraction. The
combined organic layer was dried over magnesium sulfate and
concentrated under reduced pressure to afford the desired acid
hydrazide.
Step b: The requisite acid hydrazide (1.0 equiv) was dissolved
or suspended in ethanol (1.5 mL/mmol) at r.t. The appropriate
isothiocyanate (1.0 equiv) was added in one portion. The reaction
flask was immersed in an oil bath and stirred at 80 °C until the
reaction was completed (2-3 h, TLC: DCE/MeOH 5:1). After
cooling to room temperature, the product precipitated, which was
filtered off, washed with cold ethanol (1 mL/mmol) and dried
thoroughly under reduced pressure. The product was TLC pure
(average yield: 65-85%).
Step c: The appropriate thiosemicarbazide derivative (1.0
equiv) was suspended thereafter in 1M aq. NaOH solution (1
mL/mmol). The mixture was heated at reflux temperature until
the starting thiosemicarbazide derivative was no longer
detectable by TLC (n-heptane/EtOAc 1:1 or CHCl3:MeOH 10:1).
After completion of the reaction, the mixture was cooled to 5 °C
and neutralized with 1M aq. HCl solution to pH ~6-7. The
precipitated product was filtered off, washed with water (2×1
mL/mmol) and dried in a vacuum desiccator over P2O5/KOH
(average yield: 65-95%). Only in few cases the resulting 1,2,4-
triazole-3-thiol had to be purified via flash chromatography
(gradient EtOAc/n-heptane).
Step d: DMA (3.3 mL) was cooled to 0 °C, then
chloroacetylchloride (10 mmol, 1.0 equiv) was added and the
solution was stirred for 10 minutes at 0 °C. A solution of the
appropriate aniline (10 mmol, 1.0 equiv) in DMA (3.3 mL) was
added dropwise and the reaction mixture was stirred for 2-4
hours at rt. The reaction was monitored by TLC (n-
heptane:EtOAc 1:1). Water (12 mL) was added and the reaction
mixture became a thick crystalline slurry. The crude product was
isolated by filtration, washed with water (10 mL) and then dried
at 50 °C. Purification via flash chromatography afforded the
desired chloro-acetamide in average yield from 60 to 90%.
Step e: The appropriate 1,2,4-triazole-3-thiol derivative (1.0
equiv), acetonitrile (4 mL/mmol) and freshly powdered
potassium carbonate (2.0 equiv) were placed in a 5 mL vial. The
appropriate chloro-acetanilide (1.07 equiv) was then added in one
portion at r.t. The reaction mixture was heated at 80 °C for 1-2 h
(TLC: CHCl3/MeOH 5:1). After cooling to r.t., the mixture was
concentrated, dissolved in dichloromethane (4.0 mL/mmol) and
extracted with water (2×2 mL/mmol). The phases were separated,
and the organic layer was dried, filtered and evaporated.
Purification by flash chromatography or prep HPLC afforded the
desired S-alkylated 1,2,4-triazole derivative (av. yield: 30-70%).
Step f: The appropriate 1,2,4-triazole-3-thiol derivative (1.0
equiv), acetonitrile (4 mL/mmol) and freshly powdered
potassium carbonate (2.0 equiv) were placed in a 5 mL vial. The
appropriate chloro- or bromo acetic acid ester (1.07 equiv) was
then added in one portion at r.t. The reaction mixture was stirred
at r.t. for 3-4 h, then concentrated under reduced pressure,
dissolved in dichloromethane (4.0 mL/mmol) and extracted
thoroughly with water (3×2 mL/mmol). The combined organic
layer was dried, filtered and evaporated under reduced pressure.
Purification by flash chromatography or prep HPLC afforded the
desired substituted 1,2,4-triazolylthio acetic acid ester derivative
(average yield: 65-90%).
 Step g: The appropriate 1,2,4-triazol ethyl ester (1.0 equiv)
was diluted with ethanol (2 mL), then water (1 mL) and solid
NaOH or LiOH (1.0 equiv) were added. The solution was stirred
at r.t. for 2-4 h (TLC: DCM/EtOH 5:1). Ethanol was removed
under reduced pressure, the remaining residue was diluted with
water (ca. 2 mL) and acidified with 10% aq. HCl solution to pH
~1. The precipitated carboxylic acid product was filtered off,
washed with water (2×2 mL) and dried. The crude product was
purified by flash chromatography or preparative HPLC to give
the desired product (average yield: 45-75%).
Step h: The appropriate 1,2,4-triazolylthio acetic acid (1
equiv.) was dissolved in dry dichloromethane (7 mL/mmol) and
1-hydroxy-1H-benzotriazol (1.3 equiv.), as well as 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (1.3 equiv.) were added. The
resulting reaction mixture was stirred at r.t. for 10 min, followed
by addition of the corresponding substituted aniline (1.1 equiv.).
After further stirring at r.t. for 10 min triethyl amine (3 equiv.)
was added, and the reaction mixture was stirred at r.t. for 12 h.
Thereafter, the reaction mixture was filtered, diluted with CH2Cl2
and water, followed by thorough extraction of the aq. layer with
CH2Cl2 (3×10 mL/mmol). The combined organic layer was dried,
filtered and evaporated under reduced pressure. Purification by
flash chromatography or prep HPLC afforded the desired
thiotriazolyl acetamide 7d-x.
4.2.2.1 N-(4-cyanophenyl)-2-{[4,5-dimethyl-4H-1,2,4-triazol-
3-yl]sulfanyl}acetamide (7d). Waxy colourless solid; yield 72%;
1
H NMR (600 MHz, CDCl3)  11.23 (s, 1H, NH), 7.73 (d, J = 2
Hz, 2H, ArH), 7.58 (d, J = 2 Hz, 2H, ArH), 3.91 (s, 2H, CH2),
3.50 (s, 3H, CH3), 2.47 (s, 3H, CH3). 13C NMR (150 MHz,
CDCl3) 166.79, 153.09, 148.18, 143.04, 133.35, 119.19, 118.97,
105.19, 37.66, 30.03, 10.49. LCMS (ESI, m/z): [M+H]+ 288.3.
HRMS (ESI, m/z): calcd. for C13H14N5OS, 288.0919 [M+H]+;
found 288.0924.
4.2.2.2 N-(4-cyanophenyl)-2-{[4-(prop-2-yl)-5-methyl-4H-
1,2,4-triazol-3-yl]sulfanyl}acetamide (7e). Waxy colourless
solid; yield 72%; 1H NMR (600 MHz, CDCl3)  11.23 (s, 1H,
NH), 7.74 (d, J = 2 Hz, 2H, ArH), 7.58 (d, J = 2 Hz, 2H, ArH),
4.42 (sept, 1H, i-Pr), 3.93 (s, 2H, CH2), 2.53 (s, 3H, CH3), 1.55
(d, J = 3 Hz, 6H, CH3). 13C NMR (150 MHz, CDCl3) 167.43,
152.67, 150.67, 142.51, 133.15, 119.64, 119.04, 106.82, 48.94,
36.94, 21.23, 12.37. LCMS (ESI, m/z): [M+H] + 288.3. HRMS
(ESI, m/z): calcd. for C15H18N5OS, 288.0919 [M+H]+; found
288.092
4.2.2.3 N-(4-cyanophenyl)-2-{[4-methyl-5-cyclobutyl-4H-
1,2,4-triazol-3-yl]sulfanyl}acetamide (7f). Waxy colourless solid;
yield 77%; 1H NMR (600 MHz, CDCl3)  11.29 (s, 1H, NH),
7.76 (d, J = 2 Hz, 2H, ArH), 7.59 (d, J = 2 Hz, 2H, ArH), 3.97 (s,
2H, CH2), 3.56-3.51 (m, 1H, c-Bu), 3.42 (s, 3H, CH3), 2.56-2.50
(m, 2H, c-Bu), 2.48-2.43 (m, 2H, c-Bu), 2.21-2.13 (m, 1H, c-Bu),
2.09-2.04 (m, 1H, c-Bu). 13C NMR (150 MHz, CDCl3) 167.36,
159.36, 151.73, 142.49, 133.14, 119.77, 119.04, 106.90, 36.74,
30.28, 30.01, 26.58, 18.91. LCMS (ESI, m/z): [M+H] + 328.2.
HRMS (ESI, m/z): calcd. for C16H18N5OS, 328.1232 [M+H]+;
found 328.1227.
4.2.2.4 N-(2,4-difluorophenyl)-2-{[4-methyl-5-cyclobutyl-4H-
1,2,4-triazol-3-yl]sulfanyl}acetamide (7g). Waxy colourless
solid; yield 82%; 1H NMR (400 MHz, CDCl3)  10.42 (s, 1H,
NH), 7.96 (m, 1H, ArH), 7.65 (d, J = 2 Hz, 1H, ArH), 7.44-7.37
(m, 1H, ArH), 3.99 (s, 2H, CH2), 3.54-3.50 (m, 1H, c-Bu), 3.46
(s, 3H, CH3), 2.53-2.48 (m, 2H, c-Bu), 2.46-2.41 (m, 2H, c-Bu),
2.22-2.15 (m, 1H, c-Bu), 2.07-2.01 (m, 1H, c-Bu). LCMS (ESI,
m/z): [M+H]+ 338.4.
4.2.2.5 N-(3-chlorophenyl)-2-{[4-methyl-5-trifluoromethyl-
4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7h). Waxy colourless
solid; yield 31%; 1H NMR (400 MHz, CDCl3)  9.98 (s, 1H,
NH), 7.73 (s, 1H, ArH), 7.36 (d, J = 2 Hz, 1H, ArH), 7.20 (m,
1H, ArH), 7.08 (m, 1H, ArH), 4.00 (s, 2H, CH2), 3.70 (s, 3H,
CH3). LCMS (ESI, m/z): [M+H]+ 351.0.
4.2.2.6. N-(2,4-difluorophenyl)-2-{[4-methyl-5-trifluorome-
thyl-4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7i). Waxy
colourless solid; yield 82%; 1H NMR (400 MHz, CDCl3)  10.12
(s, 1H, NH), 7.96 (s, 1H, ArH), 7.65 (d, J = 2 Hz, 1H, ArH), 7.44-
7.37 (m, 1H, ArH), 4.03 (s, 2H, CH2), 3.71 (s, 3H, CH3). LCMS
(ESI, m/z): [M+H]+ 353.2.
4.2.2.7. N-(4-cyanophenyl)-2-{[4-methyl-5-(4trifluoromethyl-
4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7j). Waxy colourless
solid; yield 59%; 1H NMR (400 MHz, CDCl3)  10.47 (s, 1H,
NH), 7.71 (d, J = 2 Hz, 2H, ArH), 7.60 (d, J = 2 Hz, 2H, ArH),
4.01 (s, 2H, CH2), 3.71 (s, 3H, CH3). LCMS (ESI, m/z): [M+H]+
342.1.
4.2.2.8. N-(3-trifluoromethylphenyl)-2-{[4-methyl-5-(furan-2-
yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7k). White solid;
yield 92%; 1H NMR (400 MHz, CDCl3)  10.70 (s, 1H, NH),
8.01 (s, 1H, HetH), 7.70 (m, 1H, ArH), 7.62 (m, 1H, HetH), 7.40
(m, 1H, ArH), 7.33 (m, 1H, ArH), 7.11 (m, 1H, HetH), 6.61 (m,
1H, HetH), 3.99 (s, 2H, CH2), 3.80 (s, 3H, CH3). LCMS (ESI,
m/z): [M+H]+ 383.1.
4.2.2.9. N-(4-cyanophenyl)-2-{[4-methyl-5-(thiophen-2-yl)-
4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7l). White solid; yield
79%; 1H NMR (600 MHz, CDCl3)  11.08 (s, 1H, NH), 7.75 (d, J
= 2 Hz, 2H, ArH), 7.58 (m, 3H, ArH+HetH), 7.48 (m, 1H, HetH),
7.22 (m, 1H, HetH), 3.99 (s, 2H, CH2), 3.73 (s, 3H, CH3). 13C
NMR (150 MHz, CDCl3) 167.03, 152.94, 151.51, 142.34,
133.17, 129.07, 128.33, 128.04, 126.84, 119.75, 118.96, 107.03,
106.88, 36.61, 31.94. LCMS (ESI, m/z): [M+H] + 340.4. HRMS
(ESI, m/z): calcd. for C16H14N5OS2, 356.0640 [M+H]+; found
356.0635.
4.2.2.10. N-(4-cyanophenyl)-2-{[4-cyclopropyl-5-(4-chloro-
phenyl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7m). White
solid; yield 81%; 1H NMR (400 MHz, CDCl3)  11.00 (s, 1H,
NH), 7.74 (br. d, 4H, ArH), 7.59 (d, J = 2 Hz, 2H, ArH), 7.52 (d,
J = 2 Hz, 2H, ArH), 4.01 (s, 2H, CH2), 3.22 (m, 1H, c-Pr), 1.14
(m, 2H, c-Pr), 0.79 (m, 2H, c-Pr). LCMS (ESI, m/z): [M+H]+
410.1.
4.2.2.11. N-(2-chlorophenyl)-2-{[4-cyclopropyl-5-(4-chloro-
phenyl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7n). White
solid; yield 80%; 1H NMR (400 MHz, CDCl3)  9.92 (s, 1H,
NH), 8.32 (m, 1H, ArH), 7.73 (d, J = 2 Hz, 2H, ArH), 7.49 (d, J =
2 Hz, 2H, ArH), 7.32 (m, 1H, ArH), 7.22 (m, 1H, ArH), 7.03 (m,
1H, ArH), 4.17 (s, 2H, CH2), 3.20 (m, 1H, c-Pr), 1.13 (m, 2H, c-
Pr), 0.79 (m, 2H, c-Pr). LCMS (ESI, m/z): [M+H]+ 419.1.
4.2.2.12. N-(4-chlorophenyl)-2-{[4-(2-furylmethyl)-5-methyl-
4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7o). White solid; yield
39%; 1H NMR (400 MHz, CDCl3)  10.22 (s, 1H, NH), 7.54 (d, J
= 3 Hz, 2H, ArH), 7.43 (m, 1H, HetH), 7.23 (d, J = 3 Hz, 2H,
ArH), 6.46 (m, 1H, HetH), 6.37 (m, 1H, HetH), 5.05 (s, 2H,
CH2), 3.92 (s, 2H, CH2), 2.49 (s, 3H, CH3). LCMS (ESI, m/z):
[M+H]+ 363.1.
4.2.2.13. N-(4-cyanophenyl)-2-{[4-(2-furylmethyl)-5-methyl-
4H-1,2,4-triazol-3-yl]sulfanyl}acetamide (7p). White solid; yield
46%; 1H NMR (400 MHz, CDCl3)  11.04 (s, 1H, NH), 7.72 (d, J
= 2 Hz, 2H, ArH), 7.59 (d, J = 2 Hz, 2H, ArH), 7.42 (m, 1H,
HetH), 6.45 (m, 1H, HetH), 6.37 (m, 1H, HetH), 5.05 (s, 2H,
CH2), 3.92 (s, 2H, CH2), 2.51 (s, 3H, CH3),. LCMS (ESI, m/z):
[M+H]+ 354.2.
4.2.2.14. N-(4-chlorophenyl)-2-{[4-(2-furylmethyl)-4H-1,2,4-
triazol-3-yl]sulfanyl}acetamide (7q). White solid; yield 68%; 1H
NMR (600 MHz, CDCl3)  10.46 (s, 1H, NH), 8.19 (s, 1H,
HetH), 7.54 (d, J = 3 Hz, 2H, ArH), 7.42 (m, 1H, HetH), 7.23 (d,
J = 3 Hz, 2H, ArH), 6.45 (m, 1H, HetH), 6.38 (m, 1H, HetH),
5.05 (s, 2H, CH2), 3.94 (s, 2H, CH2). 13C NMR (150 MHz,
CDCl3) 166.43, 151.18, 145.97, 144.54, 144.13, 136.84, 130.51,
128.99, 120.98, 110.93, 110.79, 41.63, 36.92. LCMS (ESI, m/z):
[M+H]+ 349.2. HRMS (ESI, m/z): calcd. for C15H14N4O2SCl,
349.0526 [M+H]+; found 349.0528.
4.2.2.15. N-(3-cyanophenyl)-2-{[4-(2-furylmethyl)-4H-1,2,4-
triazol-3-yl]sulfanyl}acetamide (7r). White solid; yield 73%; 1H
NMR (600 MHz, CDCl3)  10.85 (s, 1H, NH), 8.21 (s, 1H,
HetH), 8.06 (s, 1H, ArH), 7.71 (m, 1H, ArH), 7.43 (m, 1H,
HetH), 7.39-7.35 (m, 2H, ArH), 6.46 (m, 1H, HetH), 6.39 (m,
1H, HetH), 5.06 (s, 2H, CH2), 3.94 (s, 2H, CH2). 13C NMR (150
MHz, CDCl3) 166.88, 151.30, 145.87, 144.60, 144.21, 139.11,
129.69, 127.63, 123.86, 122.84, 118.56, 112.97, 110.96, 110.86,
41.69, 36.78. LCMS (ESI, m/z): [M+H]+ 340.4. HRMS (ESI,
m/z): calcd. for C16H14N5O2S, 340.0868 [M+H]+; found
340.0865.
4.2.2.16. N-(4-cyanophenyl)-2-{[4-(2-furylmethyl)-4H-1,2,4-
triazol-3-yl]sulfanyl}acetamide (7s). White solid; yield 70%; 1H
NMR (600 MHz, CDCl3)  11.01 (s, 1H, NH), 8.21 (s, 1H,
HetH), 7.73 (d, J = 2 Hz, 2H, ArH), 7.59 (d, J = 2 Hz, 2H, ArH),
7.43 (m, 1H, HetH), 6.46 (m, 1H, HetH), 6.39 (m, 1H, HetH),
5.05 (s, 2H, CH2), 3.93 (s, 2H, CH2). 13C NMR (150 MHz,
CDCl3) 166.97, 151.18, 145.97, 144.61, 144.23, 133.17, 119.71,
118.96, 110.96, 110.88, 106.88, 41.70, 36.85. LCMS (ESI, m/z):
[M+H]+ 340.4. HRMS (ESI, m/z): calcd. for C16H14N5O2S,
340.0868 [M+H]+; found 340.0871.
4.2.2.17. N-(3-chlorophenyl)-2-{[4-(2-furylmethyl)-4H-1,2,4-
triazol-3-yl]sulfanyl}acetamide (7t). White solid; yield 49%; 1H
NMR (400 MHz, CDCl3)  10.48 (s, 1H, NH), 8.18 (s, 1H,
HetH), 8.06 (m, 1H, ArH), 7.43 (m, 1H, HetH), 7.38 (m, 1H,
ArH), 7.23 (m, 1H, ArH), 6.47 (m, 1H, HetH), 6.39 (m, 1H,
HetH), 5.05 (s, 2H, CH2), 3.99 (s, 2H, CH2). LCMS (ESI, m/z):
[M+H]+ 349.2. HRMS (ESI, m/z): calcd. for C15H14N4O2SCl,
349.0526 [M+H]+; found 349.0524.
4.2.2.18. N-(2,4-dichlorophenyl)-2-{[4-(2-furylmethyl)-4H-
1,2,4-triazol-3-yl]sulfanyl}acetamide (7u). White solid; yield
65%; 1H NMR (600 MHz, CDCl3)  10.05 (s, 1H, NH), 8.27 (d, J
= 2 Hz, 1H, ArH), 8.18 (s, 1H, HetH), 7.43 (m, 1H, HetH), 7.35
(m, 1H, ArH), 7.21 (m, 1H, ArH), 6.44 (m, 1H, HetH), 6.38 (m,
1H, HetH), 5.03 (s, 2H, CH2), 4.08 (s, 2H, CH2). 13C NMR (150
MHz, CDCl3) 166.87, 150.40, 146.04, 144.68, 144.08, 133.86,
129.46, 129.00, 127.47. 124.72, 123.01, 110.90, 110.69, 41.51,
36.45. LCMS (ESI, m/z): [M+H]+ 383.2. HRMS (ESI, m/z):
calcd. for C15H13N4O2SCl2, 383.0136 [M+H]+; found 383.0130.
4.2.2.19. N-(2-chlorophenyl)-2-{[4-(2-furylmethyl)-4H-1,2,4-
triazol-3-yl]sulfanyl}acetamide (7v). White solid; yield 37%; 1H
NMR (400 MHz, d6-DMSO)  9.94 (s, 1H, NH), 7.79 (m, 1H,
ArH), 7.64 (s, 1H, HetH), 7.53 (m, 1H, HetH), 7.48 (m, 1H,
HetH), 7.33 (m, 1H, ArH), 7.19 (m, 1H, ArH), 6.49 (m, 1H,
HetH), 6.44 (m, 1H, HetH), 5.22 (s, 2H, CH2), 4.12 (s, 2H, CH2).
LCMS (ESI, m/z): [M+H]+ 349.2. HRMS (ESI, m/z): calcd. for
C15H14N4O2SCl, 349.0526 [M+H]+; found 349.0524.
4.2.2.20. N-(2-chlorophenyl)-2-{[4,5-bis-cyclopropyl-4H-
1,2,4-triazol-3-yl]sulfanyl}acetamide (7w). White solid; yield
77%; 1H NMR (400 MHz, CDCl3)  9.96 (s, 1H, NH), 8.26 (m,
1H, ArH), 7.32 (m, 1H, ArH), 7.20 (m, 1H, ArH), 6.99 (m, 1H,
ArH), 4.07 (s, 2H, CH2), 3.05-3.01 (m, 1H, c-Pr), 1.99-1.94 (m,
1H, c-Pr), 1.21-1.11 (m, 6H, c-Pr), 1.09-1.04 (m, 2H, c-Pr).
LCMS (ESI, m/z): [M+H]+ 349.2.
4.2.2.21. N-(4-cyanophenyl)-2-{[4,5-bis-cyclopropyl-4H-
1,2,4-triazol-3-yl]sulfanyl}acetamide (7x). White solid; yield
91%; 1H NMR (400 MHz, CDCl3)  11.11 (s, 1H, NH), 7.71 (d, J
= 3 Hz, 2H, ArH), 7.58 (d, J = 3 Hz, 2H, ArH), 3.91 (s, 2H, CH2),
3.06-3.03 (m, 1H, c-Pr), 2.02-1.98 (m, 1H, c-Pr), 1.23-1.18 (m,
2H, c-Pr), 1.16-1.09 (m, 6H, c-Pr). LCMS (ESI, m/z): [M+H]+
340.1.
4.2.3. General Procedure B for the preparation of
indolinylmethyl sulfonamides 8e-z. Step a: Indoline (2.00 g, 16.7
mmol, 1 eq) was dissolved in abs. toluene (50 mL), followed by
addition of formic acid (3.17 mL, 83.9 mmol, 5 eq). The resulting
reaction mixture was refluxed for 5h using a Dean-Stark-
apparatus. After cooling to room temperature, the solvent was
removed under reduced pressure and chloroform was added.
After washing with NaOH-soln. (2N) the organic layer was dried
over sodium sulfate and concentrated under reduced pressure to
afford 1-formyl indoline (1.81 g, 73%) which was used in the
next step without further purification. 1H-NMR (400 MHz,
CDCl3 , ppm) 8.94 (s, 1H), 7.15-7.26 (m, 3H), 7.00-7.08 (m,
1H), 4.05-4.14 (m, 2H), 3.14-3.22 (m, 2H).
Step b: 1-Formyl indoline (22.52 g, 0.15 mol, 1 eq) was
dissolved in conc. sulfuric acid (200 mL) at 0 °C, and N-
(hydroxymethyl)phthalimide (27.11 g, 153.0 mol) was added
portionwise. The resulting reaction mixture was stirred at 0 °C
for 6h and was allowed to warm to room temperature thereafter
with stirring being continued continued for 12h. Then, the
reaction mixture was poured carefully on ice water. The
precipitate formed was filtered off and dissolved in a mixture of
water and chloroform, followed by adjusting to pH10 by addition
of sodium carbonate. The aqueous layer was extracted thoroughly
with chloroform, and the combined organic layer was dried over
Na2SO4 and concentrated under reduced pressure. The remaining
crude product was dissolved in a mixture of dichloromethane and
acetone (1:1) and refluxed for 15 min. After cooling to room
temperature, a colourless precipitate was formed which was
filtered off, washed with acetone and dried to afford 5-[(1,3-
dioxoisoindolin-2-yl)methyl]-1-formyl indoline 14 (29.8 g, 57%).
1
H-NMR (400 MHz, CDCl3 , ppm) 8.89 (s, 1H), 7.82-7.87 (m,
2H), 7.69-7.74 (m, 2H), 7.26-7.34 (m, 2H), 7.08-7.10 (m, 1H),
4.80 (s, 2H), 4.01-4.11 (m, 2H), 3.09-3.17 (m, 2H).
Step c: 5-[(1,3-Dioxoisoindolin-2-yl)methyl]-1-formyl
indoline 14 (29.8 g, 0.097 mol, 1 eq) was then dissolved in
methanol (300 mL) and HCl (5.5N soln. in iso-propanol, 50 mL)
was added. The reaction mixture was refluxed for 4 h and
concentrated under reduced pressure after cooling to room
temperature. The residue was taken up with satd. sodium
carbonate solution and CH2Cl2, followed by thorough extraction
of the aqueous layer with CH2Cl2. The combined organic layer
was dried over Na2SO4 and concentrated under reduced pressure
to afford 2-(indolin-5-ylmethyl)isoindoline-1,3-dione 15 (25.7 g,
90%) as colourless solid. 1H-NMR (400 MHz, CDCl3 , ppm)
7.80-7.84 (m, 2H), 7.67-7.70 (m, 2H), 7.22-7.26 (m, 1H), 7.11-
7.14 (m, 1H), 6.54-6.56 (d, 1H), 4.73 (s, 2H), 3.50-3.54 (m, 2H),
2.96-3.00 (m, 2H).
Steps d,e: 2-(Indolin-5-ylmethyl)isoindoline-1,3-dione 15 (1.0
g, 3.59 mmol, 1 eq), Et3N (0.75 mL, 5.39 mmol, 1.5 eq) and
DMAP (4 mg, 0.03 mmol, 0.01 eq) were dissolved in CH2Cl2 (25
mL) and cooled to 0 °C. Thereafter, the corresponding
acylchloride (4.31 mmol, 1.2 eq), dissolved in CH2Cl2 (5 mL),
was added dropwise to the reaction mixture at 0 °C. Thereafter,
the reaction mixture was allowed to warm to room temperature.
Stirring was continued for 4h, then water was added, and the
aqueous layer was extracted with CH2Cl2. The combined organic
layer was dried over Na2SO4 and concentrated under reduced
pressure. The remaining crude product was dissolved in Et2O and
refluxed for 10 min. After cooling to room temperature, a
colourless precipitate was formed which was filtered off, washed
with cold Et2O and dried to afford the desired acylated 2-
(indolin-5-ylmethyl)isoindoline-1,3-dione 16 which was then in
dissolved in step e in a mixture of CH2Cl2 and MeOH (5
mL/mmol, 1.25:1). Hydrazine hydrate (3.2 eq) was added
carefully, and the resulting reaction mixture was stirred at room
temperature for 12 h. The precipitate formed was filtered off and
washed with CH2Cl2/MeOH. The filtrate was diluted with CH2Cl2
and washed with water. The combined organic layer was dried
over Na2SO4 and concentrated under reduced pressure to afford
the desired intermediate 17a-f.
Step f: 17a-f (0.77 mmol, 1 eq) and pyridine (0.19 mL, 2.32
mmol, 3 eq) were dissolved in abs. MeCN (10 mL) and the
desired aryl or heteroaryl sulfonyl chloride (0.10 mL, 0.77 mmol,
1 eq) was added. The resulting reaction mixture was stirred at 70
°C for 5h. After cooling to room temperature CH2Cl2 (10 mL)
was added. The organic layer was washed with HCl (10%, 10
mL), dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified via column chromatography
(heptane/EtOAc, gradient 10:0 to 1:1) to afford desired
indolinylmethyl sulfonamide 8e-z.
General Procedure C for the preparation of indolinylmethyl
sulfonamides 8e-z. Step d: Indoline (1 eq), Et3N (1.5 eq) and
DMAP (0.01 eq) were dissolved in CH2Cl2 (2 mL/mmol) and
cooled to 0 °C. Thereafter, the corresponding acylchloride (4.31
mmol, 1.2 eq), dissolved in CH2Cl2 (5 mL), was added dropwise
to the reaction mixture at 0 °C. Thereafter, the reaction mixture
was allowed to warm to room temperature. Stirring was
continued for 4h, then water was added, and the aqueous layer
was extracted with CH2Cl2. The combined organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified via column chromatography
(heptane/EtOAc, gradient 10:0 to 1:1) to afford the acylated
indoline which was then dissolved in dichloromethane (1 eq in
5ml/mmol).
Step h: N-Bromosuccinimide was added (1.1 equiv) and the
resulting reaction mixture was stirred at r.t. for 90 min. Water
was added, and the aqueous layer was extracted with CH2Cl2.
The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure to afford the crude
brominated acyl indoline which was used in the next step without
further purification. The corresponding brominated acyl indoline
(1 eq) was dissolved in abs. N,N-dimethylformamide (4
ml/mmol), Copper(I)cyanide (2.5 eq) and copper(I)iodide (0.1
eq) were added, and the resulting reaction mixture was stirred
under reflux conditions for 4 d. After cooling to r.t. ethylacetate
and water were added, and the aqueous layer was extracted with
ethyl acetate. The combined organic layer was dried over Na2SO4
and concentrated under reduced pressure. The remaining residue
was purified via column chromatography (heptane/EtOAc,
gradient 10:0 to 1:1) to afford the desired acylated cyano
indoline.
Steps i, f: The corresponding acylated cyano indoline (1 eq)
was dissolved in a high pressure reactor in NH3/MeOH (20
ml/mmol) and Raney-Nickel in H2O was added. Then, the reactor
was closed, and a pressure of 40 bar was established by adding
hydrogen gas. The reaction mixture was agitated at 40 bar at a
temperature of 50 °C for 90 min. After cooling to room
temperature and full pressure release the reaction mixture was
filtered and concentrated under reduced pressure. The remaining
residue was purified via column chromatography
(heptane/EtOAc, gradient 10:0 to 1:1) to afford the desired
intermediate 17. 17a-f (0.77 mmol, 1 eq) and pyridine (0.19 mL,
2.32 mmol, 3 eq) were dissolved in dry MeCN (10 mL) and the
desired aryl or heteroaryl sulfonyl chloride (0.10 mL, 0.77 mmol,
1 eq) was added. The resulting reaction mixture was stirred at 70
°C for 5h. After cooling to room temperature CH2Cl2 (10 mL)
was added. The organic layer was washed with HCl (10%, 10
mL), dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified via column chromatography
(heptane/EtOAc, gradient 10:0 to 1:1) to afford desired
indolinylmethyl sulfonamide 8e-z.
4.2.3.1. 4-Cyano-N-[(1-propionyl-2,3-dihydro-1H-indol-5-yl)-
methyl]benzenesulfonamide (8e). White solid; yield 32%; 1H
NMR (400 MHz, CDCl3)  8.12-8.14 (m, 1H), 7.92-7.94 (m,
2H), 7.76-7.79 (m, 2H), 6.98-7.00 (m, 2H), 4.73 (m, 1H), 4.14-
4.16 (d, 2H), 4.04 (m, 2H), 3.10-3.12 (m, 2H), 2.44-2.46 (m, 2H),
1.21-1.26 (m, 3H). LCMS (ESI, m/z): [M+H]+ 370.3. HRMS
(ESI, m/z): calcd. for C19H19N3O3S, 369.4384 [M+H]+; found
369.4379.
4.2.3.2. N-[(1-Propionyl-2,3-dihydro-1H-indol-5-yl)-methyl]-
thiophen-2-ylsulfonamide (8f). White solid; yield 32%; 1H NMR
(400 MHz, CDCl3)  8.09-8.14 (m, 1H), 7.59-7.63 (m, 2H), 7.09-
7.13 (m, 2H), 6.96-7.02 (m, 1H), 4.66-4.73 (m, 1H), 4.16-4.17
(m, 2H), 4.02-4.08 (m, 2H), 3.14-3.18 (m, 2H), 2.41-2.46 (m,
2H), 1.17-1.28 (m, 3H). LCMS (ESI, m/z): [M+H]+ 351.4.
4.2.3.3. 4-Bromo-N-[(1-propionyl-2,3-dihydro-1H-indol-5-yl)-
methyl]benzenesulfonamide (8g). White solid; yield 22%; 1H
NMR (400 MHz, CDCl3)  8.13-8.15 (m, 1H), 7.70 (d, 2H), 7.64
(d, 2H), 6.94-7.00 (m, 2H), 4.67 (m, 1H), 4.09-4.12 (d, 2H),
4.01-4.05 (m, 2H), 3.09-3.13 (m, 2H), 2.42-2.47 (m, 2H), 1.21-
1.26 (m, 3H). LCMS (ESI, m/z): [M+H]+ 422.9/424.9, logp 2.62.
4.2.3.4. 5-Chloro-N-[(1-propionyl-2,3-dihydro-1H-indol-5-
yl)-methyl]-thiophen-2-ylsulfonamide (8h). White solid; yield
29%; 1H NMR (400 MHz, CDCl3)  8.08-8.11 (m, 1H), 7.38 (m,
1H), 7.13 (m, 1H), 6.94 (m, 1H), 6.90 (m, 1H), 4.70 (m, 1H),
4.20 (m, 2H), 4.03-4.09 (m, 2H), 3.15-3.20 (m, 2H), 2.44-2.40
(m, 2H), 1.21-1.26 (m, 3H). LCMS (ESI, m/z): [M+H]+ 384.9.
4.2.3.5. 4,5-Dibromo-N-[(1-propionyl-2,3-dihydro-1H-indol-
5-yl)-methyl]-thiophen-2-ylsulfonamide (8i). White solid; yield
19%; 1H NMR (400 MHz, CDCl3)  8.18 (m, 1H), 7.27-7.30 (m,
1H), 6.98-7.06 (m, 2H), 4.80 (m, 1H), 4.21 (m, 2H), 4.04-4.09
(m, 2H), 3.14-3.20 (m, 2H), 2.42-2.50 (m, 2H), 1.20-1.25 (m,
3H). LCMS (ESI, m/z): [M+2H]+ 508.7.
4.2.3.6. 4-Chloro-N-[(1-propionyl-2,3-dihydro-1H-indol-5-
yl)-methyl]benzenesulfonamide (8j). White solid; yield 54%; 1H
NMR (400 MHz, CDCl3)  8.12-8.14 (m, 1H), 7.78 (d, 2H), 7.48
(d, 2H), 6.95-7.02 (m, 2H), 4.61 (m, 1H), 4.11 (d, 2H), 4.02-4.07
(m, 2H), 3.09-3.14 (m, 2H), 2.41-2.47 (m, 2H), 1.21-1.26 (m,
3H). LCMS (ESI, m/z): [M+H]+ 379.0, logp 2.56.
4.2.3.7. 5-Methyl-N-[(1-propionyl-2,3-dihydro-1H-indol-5-
yl)-methyl]-thiophen-2-ylsulfonamide (8k). White solid; yield
22%; 1H NMR (400 MHz, CDCl3)  8.13-8.15 (m, 1H), 7.44 (m,
1H), 7.11 (m, 1H), 6.97-7.00 (m, 1H), 6.76 (m, 1H), 4.58 (br. t,
1H, NH), 4.14 (d, 2H), 4.02-4.08 (m, 2H), 3.14-3.18 (m, 2H),
2.55 (s, 3H), 2.47-2.41 (m, 2H), 1.21-1.25 (m, 3H). LCMS (ESI,
m/z): [M+H]+ 364.9, logp 2.38.
4.2.3.8. 4-Trifluoromethyl-N-[(1-propionyl-2,3-dihydro-1H-
indol-5-yl)-methyl]benzenesulfonamide (8l). White solid; yield
43%; 1H NMR (400 MHz, CDCl3)  8.11-8.14 (m, 1H), 7.97 (d,
2H), 7.76 (d, 2H), 6.95-7.01 (m, 2H), 4.69 (m, 1H), 4.15 (d, 2H),
4.02-4.07 (m, 2H), 3.08-3.13 (m, 2H), 2.41-2.47 (m, 2H), 1.21-
1.25 (m, 3H). LCMS (ESI, m/z): [M+H]+ 412.1, logp 2.80.
4.2.3.9. N-[(1-Propionyl-2,3-dihydro-1H-indol-5-yl)-methyl]-
benzthiophen-2-ylsulfonamide (8m). White solid; yield 32%; 1H
NMR (400 MHz, CDCl3)  8.10-8.12 (m, 1H), 7.81-7.88 (m,
3H), 7.45-7.52 (m, 2H), 6.99-7.05 (m, 2H), 4.79 (m, 1H), 4.22 (d,
2H), 3.93-3.97 (m, 2H), 2.99-3.05 (m, 2H), 2.38-2.44 (m, 2H),
1.19-1.24 (m, 3H). LCMS (ESI, m/z): [M+H]+ 401.0, logp 2.71.
4.2.3.10. 4-Methoxy-N-[(1-propionyl-2,3-dihydro-1H-indol-5-
yl)-methyl]benzenesulfonamide (8n). White solid; yield 43%; 1H
NMR (400 MHz, CDCl3)  8.11-8.13 (m, 1H), 7.81 (d, 2H), 7.07
(m, 1H), 7.00 (d, 2H), 6.92-6.96 (m, 1H), 4.47 (m, 1H), 4.01-4.08
(m, 4H), 3.88 (s, 3H), 3.12-3.17 (m, 2H), 2.42-2.47 (m, 2H),
1.21-1.26 (m, 3H). LCMS (ESI, m/z): [M+H]+ 375.1, logp 2.18.
4.2.3.11. N-[(1-Isobutyryl-2,3-dihydro-1H-indol-5-yl)methyl]-
thiophene-2-sulfonamide (8o). White solid; yield 39%; 1H NMR
(400 MHz, CDCl3)  8.15-8.17 (m, 1H), 7.60-7.64 (m, 2H), 7.09-
7.13 (m, 2H), 6.96-7.00 (m, 1H), 4.66 (br. t, 1H, NH), 4.10-4.18
(m, 4H), 3.14-3.18 (m, 2H), 2.73-2.81 (m, 1H), 1.20-1.28 (m,
6H). LCMS (ESI, m/z): [M+H]+ 364.9, logp 2.40.
4.2.3.12. 5-Bromo-N-[(1-isobutyryl-2,3-dihydro-1H-indol-5-
yl)-methyl]-thiophen-2-ylsulfonamide (8p). White solid; yield
29%; 1H NMR (400 MHz, CDCl3)  8.17-8.19 (m, 1H), 7.35 (m,
1H), 7.09 (m, 1H), 7.05 (m, 1H), 7.01 (m, 1H), 4.67 (m, 1H),
4.11-4.21 (m, 4H), 3.15-3.19 (m, 2H), 2.78 (m, 1H), 1.17-1.27
(m, 6H). LCMS (ESI, m/z): [M+H]+ 443.0, logp 3.00.
4.2.3.13. 5-Chloro-N-[(1-isobutyryl-2,3-dihydro-1H-indol-5-
yl)-methyl]-thiophen-2-ylsulfonamide (8q). White solid; yield
37%; 1H NMR (400 MHz, CDCl3)  8.16-8.18 (m, 1H), 7.39 (m,
1H), 7.09 (m, 1H), 7.00 (m, 1H), 6.91 (m, 1H), 4.78 (m, 1H),
4.11-4.20 (m, 4H), 3.14-3.18 (m, 2H), 2.79 (m, 1H), 1.21-1.23 (d,
6H). LCMS (ESI, m/z): [M+H]+ 399.1, logp 2.97.
4.2.3.14. 5-Bromo-N-[(1-methyl-2,3-dihydro-1H-indol-5-yl)-
methyl]-thiophen-2-ylsulfonamide (8r). White solid; yield 45%;
1
H NMR (400 MHz, CDCl3)  8.15-8.17 (m, 1H), 7.35 (m, 1H),
7.09 (m, 1H), 7.05 (m, 1H), 7.01 (m, 1H), 4.77 (m, 1H), 4.11-
4.20 (m, 4H), 3.14-3.18 (m, 2H), 2.30 (s, 3H). LCMS (ESI, m/z):
[M+H]+ 414.9, logp 2.29.
4.2.3.15. N-[(1-(3,3,3-Trifluoropropionyl)-2,3-dihydro-1H-
indol-5-yl)methyl]-thiophene-2-sulfonamide (8s). White solid;
yield 13%; 1H NMR (400 MHz, CDCl3)  8.13-8.15 (m, 1H),
7.61-7.65 (m, 2H), 7.16 (m, 1H), 7.12 (m, 1H), 7.01 (m, 1H),
4.67 (br. m, 1H, NH), 4.19 (m, 2H), 4.09-4.14 (m, 2H), 3.30-3.38
(m, 2H), 3.20-3.26 (m, 2H). LCMS (ESI, m/z): [M+H]+ 405.0,
logp 2.35.
4.2.3.16. 5-Bromo-N-[(1-(3,3,3-trifluoropropionyl)-2,3-di-
hydro-1H-indol-5-yl)-methyl]-thiophen-2-ylsulfonamide (8t).
White solid; yield 34%; 1H NMR (400 MHz, CDCl3)  8.16-8.18
(m, 1H), 7.35 (m, 1H), 7.12 (m, 1H), 7.02-7.07 (m, 2H), 4.70 (br.
m, 1H, NH), 4.19 (m, 2H), 4.09-4.14 (m, 2H), 3.30-3.39 (m, 2H),
3.20-3.26 (m, 2H). LCMS (ESI, m/z): [M+H]+ 482.8, logp 2.93.
4.2.3.17. 5-Chloro-N-[(1-(3,3,3-trifluoropropionyl)-2,3-di-
hydro-1H-indol-5-yl)-methyl]-thiophen-2-ylsulfonamide (8u).
White solid; yield 33%; 1H NMR (400 MHz, CDCl3)  8.16-8.18
(m, 1H), 7.40 (m, 1H), 7.14 (m, 1H), 7.06 (m, 1H), 6.93 (m, 1H),
4.70 (br. m, 1H, NH), 4.19 (m, 2H), 4.09-4.14 (m, 2H), 3.29-3.37
(m, 2H), 3.21-3.26 (m, 2H). LCMS (ESI, m/z): [M+H]+ 438.9,
logp 2.90.
4.2.3.18. 5-Methyl-N-[(1-isobutyryl-2,3-dihydro-1H-indol-5-
yl)methyl]-thiophen-2-ylsulfonamide (8v). White solid; yield
54%; 1H NMR (400 MHz, CDCl3)  8.16-8.18 (m, 1H), 7.43 (m,
1H), 7.13 (m, 1H), 6.97-7.00 (m, 1H), 6.75 (m, 1H), 4.56 (br. t,
1H, NH), 4.11-4.17 (m, 4H), 3.14-3.18 (m, 2H), 2.73-2.81 (m,
1H), 2.55 (s, 3H), 1.23 (d, 6H). LCMS (ESI, m/z): [M+H]+ 379.0,
logp 2.68.
4.2.3.19. 5-Methyl-N-[(1-acetyl-2,3-dihydro-1H-indol-5-yl)-
methyl]-thiophen-2-ylsulfonamide (8w). White solid; yield 27%;
1
H NMR (400 MHz, CDCl3)  8.09-8.11 (m, 1H), 7.44 (m, 1H),
7.13 (m, 1H), 6.97-7.00 (m, 1H), 6.76 (m, 1H), 4.61 (br. t, 1H,
NH), 4.13-4.17 (m, 2H), 4.04-4.07 (m, 2H), 3.14-3.19 (m, 2H),
2.55 (s, 3H), 2.22 (s, 3H). LCMS (ESI, m/z): [M+H]+ 351.2, logp
2.06.
4.2.3.20. 5-Methyl-N-[(1-benzoyl-2,3-dihydro-1H-indol-5-yl)-
methyl]-thiophen-2-ylsulfonamide (8x). White solid; yield 25%;
1
H NMR (400 MHz, CDCl3)  7.52-7.56 (m, 2H), 7.44-7.47 (m,
3H), 7.31 (m, 2H), 7.17 (m, 1H), 6.99 (m, 1H), 6.77 (m, 1H),
4.58 (br. t, 1H, NH), 4.14-4.17 (m, 2H), 4.07-4.11 (m, 2H), 3.08-
3.13 (m, 2H), 2.55 (s, 3H). LCMS (ESI, m/z): [M+H]+ 413.0,
logp 2.77.
4.2.3.21. 5-bromo-N-{[1-(cyclopropylacetyl)-2,3-dihydro-1H-
indol-5-yl]methyl}thiophene-2-sulfonamide (8y). White solid;
yield 27%; 1H NMR (400 MHz, CDCl3)  8.16-8.18 (m, 1H),
7.35 (m, 1H), 7.06-7.09 (m, 2H), 7.00-7.04 (m, 1H), 4.74 (br. t,
1H, NH), 4.17 (d, 2H), 4.01-4.05 (m, 2H), 3.13-3.17 (m, 2H),
2.36-2.39 (m, 2H), 1.16-1.21 (m, 1H), 0.59-0.65 (m, 2H), 0.18-
0.23 (m, 2H). LCMS (ESI, m/z): [M+H]+ 454.9, logp 2.97.
4.2.3.22. 5-chloro-N-{[1-(cyclopropylacetyl)-2,3-dihydro-1H-
indol-5-yl]methyl}thiophene-2-sulfonamide (8z). White solid;
yield 27%; 1H NMR (400 MHz, CDCl3)  8.17-8.19 (m, 1H),
7.38 (m, 1H), 7.09 (m, 1H), 7.01 (m, 1H), 6.91 (m, 1H), 4.73 (br.
t, 1H, NH), 4.17 (d, 2H), 4.01-4.05 (m, 2H), 3.13-3.17 (m, 2H),
2.36-2.38 (m, 2H), 1.16-1.20 (m, 1H), 0.61-0.65 (m, 2H), 0.18-
0.23 (m, 2H). LCMS (ESI, m/z): [M+H]+ 411.1, logp 2.93.
4.2.4. General Procedure D for the preparation of
phenylsulfonyl ethylenediamines 9i-z. 2-Aminoethyl amine (1
equiv.) and 2-trifluormethyl-4-fluoro benzonitrile (1 equiv.) were
dissolved under argon in dry MeCN, followed by addition of
Et3N (1.5 equiv). The resulting reaction mixture was stirred at 50
°C for 4h. After cooling to room temperature water and CH 2Cl2
were added, followed by thorough extraction. The combined
organic layer was dried over Na2SO4, filtered and concentrated
under reduced pressure to afford desired 4-[(2-aminoethyl)-
amino]-2-(trifluormethyl)benzonitrile. 4-[(2-aminoethyl)-amino]-
2-(trifluormethyl)benzonitrile (160 mg, 0.69 mmol) was
dissolved in a flame-dried round-bottomed flask in CH2Cl2 (20
mL) under argon and diisopropylethyl amine (0.37 mL, 2.09
mmol) was added. After stirring at room temperature for 5 min a
solution of the corresponding aryl- or hetarylsulfonyl chloride
(0.69 mmol) in dry CH2Cl2 (5 mL) was added, and the resulting
reaction mixture was stirred at room temperature for 20h. H2O
and CH2Cl2 were added, followed by thorough extraction. The
combined organic layer was dried over Na2SO4, filtered,
concentrated under reduced pressure and purified via column
chromatography (heptane/EtOAc, gradient 10:0 to 1:1) to afford
desired N-(2-{[4-cyano-3-(trifluormethyl)phenyl]amino}-ethyl)-
aryl-/hetaryl sulfonamide 9i-z.
4.2.4.1. N-(2-{[4-Cyano-3-(trifluormethyl)phenyl]amino}-
ethyl)-2-chlorobenzene sulfonamide (9i). White solid; yield 49%;
1
H NMR (400 MHz, d6-DMSO)  7.99-7.94 (m, 2H), 7.70 (d,
1H), 7.61-7.57 (m, 2H), 7.54-7.48 (m, 1H), 7.21-7.16 (m, 1H),
6.92 (m, 1H), 6.76 (m, 1H), 3.27-3.22 (m, 2H), 3.03-2.97 (m,
2H). LCMS (ESI, m/z): [M+H]+ 404.0, logp 2.74. HRMS (ESI,
m/z): calcd. for C16H13ClF3N3O2S, 403,0369 [M+H]+; found
403,0307.
4.2.4.2. N-(2-{[4-Cyano-3-(trifluormethyl)phenyl]amino}-
ethyl)-4-chlorobenzene sulfonamide (9j). White solid; yield 49%;
1
H NMR (400 MHz, d6-DMSO)  7.88 (br. t, 1H, NH), 7.78-7.76
(d, 2H), 7.70 (d, 1H), 7.63 (d, 2H), 7.21-7.18 (br. t, 1H, NH),
6.96 (m, 1H), 6.79-6.76 (dd, 1H), 3.27-3.22 (m, 2H), 2.93-2.88
(m, 2H). LCMS (ESI, m/z): [M+H]+ 404.0, logp 2.91.
4.2.4.3. N-(2-{[4-Cyanophenyl]amino}-ethyl)-4-chloroben-
zene sulfonamide (9l). White solid; yield 91%; 1H NMR (400
MHz, d6-DMSO)  7.87-7.74 (br. m, 3H, 2H+NH), 7.65 (d, 2H),
7.42 (d, 2H), 6.65-6.62 (br. t, 1H, NH), 6.55 (d, 2H), 3.19-3.14
(m, 2H), 2.93-2.87 (m, 2H). LCMS (ESI, m/z): [M+H]+ 336.1,
logp 2.98.
4.2.4.4. N-(2-{[4-Cyanophenyl]amino}-ethyl)-4-fluoroben-
zene sulfonamide (9m). White solid; yield 87%; 1H NMR (400
MHz, d6-DMSO)  7.87-7.80 (br. m, 3H, 2H+NH), 7.44-7.38 (m,
4H), 6.67-6.64 (br. t, 1H, NH), 6.55 (d, 2H), 3.19-3.14 (m, 2H),
2.92-2.85 (m, 2H). LCMS (ESI, m/z): [M+H]+ 320.1, logp 2.51.
4.2.4.5. N-(2-{[4-Cyanophenyl]amino}-ethyl)-3-fluoroben-
zene sulfonamide (9n). White solid; yield 81%; 1H NMR (400
MHz, d6-DMSO)  7.89 (m, 1H), 7.69-7.65 (m, 2H), 7.59 (m,
1H), 7.52 (m, 1H), 7.42 (d, 1H), 6.67-6.64 (br. t, 1H, NH), 6.56
(d, 2H), 3.20-3.15 (m, 2H), 2.93-2.88 (m, 2H). LCMS (ESI, m/z):
[M+H]+ 320.1, logp 2.66.
4.2.4.6. N-(2-{[4-Cyano-3-(methoxy)phenyl]amino}-ethyl)-4-
fluorobenzene sulfonamide (9o). White solid; yield 62%; 1H
NMR (400 MHz, d6-DMSO)  7.88-7.82 (m, 3H), 7.45-7.38 (m,
2H), 7.28 (m, 1H), 7.51 (d, 1H), 6.67 (br. t, 1H, NH), 6.19-6.13
(m, 2H), 3.80 (s, 3H), 3.22-3.17 (m, 2H), 2.92-2.87 (m, 2H).
LCMS (ESI, m/z): [M+H]+ 350.0, logp 2.13.
4.2.4.7. N-(2-{[4-Cyano-3-(methoxy)phenyl]amino}-ethyl)-4-
methylbenzene sulfonamide (9p). White solid; yield 78%; 1H
NMR (400 MHz, d6-DMSO)  7.69-7.66 (m, 3H), 7.38 (d, 2H),
7.28 (d, 1H), 6.66 (br. t, 1H, NH), 6.18-6.13 (m, 2H), 3.79 (s,
3H), 3.21-3.16 (m, 2H), 2.90-2.84 (m, 2H), 2.37 (s, 3H). LCMS
(ESI, m/z): [M+H]+ 346.0, logp 2.26.
4.2.4.8. N-(2-{[4-Cyano-3-(chloro)phenyl]amino}-ethyl)-4-
chlorobenzene sulfonamide (9q). White solid; yield 45%; 1H
NMR (400 MHz, d6-DMSO)  7.99-7.94 (m, 2H), 7.86 (m, 1H),
7.78 (d, 2H), 7.51 (d, 1H), 7.01-6.96 (m, 1H), 6.69 (m, 1H), 6.55
(m, 1H), 3.22-3.17 (m, 2H), 2.92-2.87 (m, 2H). LCMS (ESI,
m/z): [M+H]+ 369.9, logp 2.71.
4.2.4.9. N-(2-{[4-Cyano-3-(chloro)phenyl]amino}-ethyl)-2-
chlorobenzene sulfonamide (9r). White solid; yield 38%; 1H
NMR (400 MHz, d6-DMSO)  7.96 (m, 2H), 7.62 (m, 2H), 7.54-
7.49 (m, 2H), 6.96 (m, 1H, NH), 6.66 (d, 1H), 6.52 (m, 1H),
3.20-3.16 (m, 2H), 3.00-2.96 (m, 2H). LCMS (ESI, m/z):
[M+H]+ 369.9, logp 2.56.
4.2.4.10. N-(2-{[4-Cyano-3-(chloro)phenyl]amino}-ethyl)-4-
methoxybenzene sulfonamide (9s). White solid; yield 51%; 1H
NMR (400 MHz, d6-DMSO)  7.72 (d, 2H), 7.57 (br. t, 1H, NH),
7.51 (d, 1H), 7.09 (d, 2H), 6.98 (br. t, 1H, NH), 6.69 (d, 1H),
6.54 (dd, 1H), 3.83 (s, 3H), 3.21-3.15 (m, 2H), 2.85-2.82 (m,
2H). LCMS (ESI, m/z): [M+H]+ 365.1, logp 2.38.
4.2.4.11. N-(2-{[4-Cyano-3-(methyl)phenyl]amino}-ethyl)-4-
methoxybenzene sulfonamide (9t). White solid; yield 93%; 1H
NMR (400 MHz, d6-DMSO)  7.73 (d, 2H), 7.61-7.54 (br. m,
1H, NH), 7.36 (d, 1H), 7.10 (d, 2H), 6.54-6.51 (br. t, 1H, NH),
6.43-6.38 (m, 2H), 3.82 (s, 3H), 3.17-3.12 (m, 2H), 2.87-2.81 (m,
2H), 2.30 (s, 3H).. LCMS (ESI, m/z): [M+H]+ 346.2, logp 2.83.
4.2.4.12. N-(2-{[4-Cyano-3-(methyl)phenyl]amino}-ethyl)-2-
chlorobenzene sulfonamide (9u). White solid; yield 77%; 1H
NMR (400 MHz, d6-DMSO)  7.98-7.95 (m, 2H), 7.65-7.61 (m,
2H), 7.53-7.49 (m, 1H), 7.35 (d, 1H), 6.52-6.49 (br. t, 1H, NH),
6.42-6.36 (m, 2H), 3.17-3.12 (m, 2H), 3.00-2.95 (m, 2H), 2.30 (s,
3H). LCMS (ESI, m/z): [M+H]+ 350.1, logp 3.01.
4.2.4.13. N-(2-{[4-Cyano-3-(methyl)phenyl]amino}-ethyl)-3-
chlorobenzene sulfonamide (9v). White solid; yield 81%; 1H
NMR (400 MHz, d6-DMSO)  7.92 (br. t, 1H, NH), 7.79-7.70
(m, 3H), 7.63-7.60 (m, 1H), 7.36 (d, 1H), 6.55-6.52 (br. t, 1H,
NH), 6.45-6.40 (m, 2H), 3.19-3.14 (m, 2H), 2.94-2.89 (m, 2H),
2.30 (s, 3H). LCMS (ESI, m/z): [M+H]+ 350.1, logp 3.19.
4.2.4.14. N-(2-{[4-Cyano-3-(methyl)phenyl]amino}-ethyl)-4-
chlorobenzene sulfonamide (9w). White solid; yield 81%; 1H
NMR (400 MHz, d6-DMSO)  7.84 (br. m, 1H, NH), 7.78 (d,
2H), 7.65 (d, 2H), 7.36 (d, 1H), 6.53-6.50 (br. t, 1H, NH), 6.44-
6.38 (m, 2H), 3.18-3.13 (m, 2H), 2.93-2.87 (m, 2H), 2.30 (s, 3H).
LCMS (ESI, m/z): [M+H]+ 350.1, logp 3.19.
4.2.4.15. N-(2-{[4-Cyano-3-(methyl)phenyl]amino}-ethyl)-4-
fluorbenzene sulfonamide (9x). White solid; yield 84%; 1H NMR
(400 MHz, d6-DMSO)  7.87-7.84 (m, 2H), 7.79-7.76 (m, 1H),
7.44-7.38 (m, 2H), 7.37-7.34 (m, 1H), 6.54-6.51 (br. t, 1H, NH),
6.43-6.39 (m, 2H), 3.18-3.13 (m, 2H), 2.89-2.84 (m, 2H), 2.30 (s,
3H).. LCMS (ESI, m/z): [M+H]+ 334.2, logp 2.89
4.2.4.16. N-(2-{[4-Cyano-3-(methyl)phenyl]amino}-ethyl)-4-
methylbenzene sulfonamide (9y). White solid; yield 77%; 1H
NMR (400 MHz, d6-DMSO)  7.69-7.63 (m, 3H), 7.38-7.34 (m,
3H), 6.54-6.51 (br. t, 1H, NH), 6.44-6.38 (m, 2H), 3.18-3.12 (m,
2H), 2.86-2.81 (m, 2H), 2.37 (s, 3H), 2.30 (s, 3H). LCMS (ESI,
m/z): [M+H]+ 330.2, logp 3.19.
4.2.4.17. N-[2-(4-Cyano-3-methylanilino)ethyl]-1-methyl-1H-
imidazole-4-sulfonamide (9z). White solid; yield 31%; 1H NMR
(400 MHz, d6-DMSO)  7.78 (s, 1H), 7.73 (s, 1H), 7.53 (br. m,
1H, NH), 7.38 (d, 1H), 6.57-6.53 (br. t, 1H, NH), 6.48 (d, 1H),
6.44-6.40 (dd, 1H), 3.68 (s, 3H), 3.20-3.14 (m, 2H), 2.99-2.94
(m, 2H). LCMS (ESI, m/z): [M+H]+ 319.4, logp 1.88.
4.2.5. Preparation of phenylsulfonyl ethylenediamines 20a-h
via sulfonyl aziridines – all cpds were prepared following the
protocols outlined for the synthesis of 20a,b:
4.2.5.1. N-(2-{[4-Fluoro-3-(trifluormethyl)phenyl]amino}-
ethyl)-4-chlorobenzene sulfonamide 20a. 2-Bromoethyl amine (1
equiv.) and 4-Chlorphenylsulfonylchloride (1 equiv.) were
dissolved in dry CH2Cl2 under argon and triethyl amine (1.5
equiv) was added. The resulting reaction mixture was stirred at
room temperature for 5 h. Then, water and CH 2Cl2 were added,
followed by thorough extraction of the aqueous layer with
CH2Cl2. The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified
via column chromatography (heptane/EtOAc, gradient 10:0 to
1:1) to afford desired N-(2-bromoethyl)-4-chlorobenzene
sulfonamide as intermediate which was dissolved in toluene (15
mL/mmol). Thereafter, a solution of KOH (3 equiv) in water (1
mL/mmol) was added. The resulting reaction mixture was stirred
at room temperature for 90 min. After complete conversion,
water and ethyl acetate were added, followed by thorough
extraction of the aqueous layer with EtOAc. The combined
organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified via column
chromatography (heptane/EtOAc, gradient) to afford desired 1-
[(4-chlorophenyl)sulfonyl]aziridine as intermediate. 1-[(4-
chlorophenyl)sulfonyl]aziridine (150 mg, 0.69 mmol), 4-fluoro-
3-trifluoromethyl aniline (123 mg, 0.69 mmol) and SiO2 (100
mg, 1.66 mmol) were then suspended in H2O (2 mL), and the
resulting reaction mixture was treated with ultra sound for 90 min
and was stirred at room temperature for 20h. Upon addition of
water and ethyl acetate the aqueous layer was extracted
thoroughly with ethyl acetate. The combined organic layer was
dried over Na2SO4 and concentrated under reduced pressure.
Purification of the remaining residue via column chromatography
(heptane/EtOAc, gradient) afforded N-(2-{[4-fluoro-3-(tri-
fluormethyl)phenyl]amino}ethyl)-4-chlorobenzene sulfonamide
20a (210 mg, 77 %) as colourless solid. 1H-NMR (400 MHz,
CDCl3 , ppm) 7.80 (d, 2H), 7.48 (d, 2H), 7.00 (m, 1H), 6.70-
6.64 (m, 2H), 4.92 (br. t, 1H, NH), 4.02 (br. m, 1H, NH), 6.79
(m, 1H), 3.30-3.25 (m, 2H), 3.22-3.17 (m, 2H). LCMS (ESI,
m/z): [M+H]+ 396,7, logp 2.98. HRMS (ESI, m/z): calcd. for
C15H13ClF4N2O2S, 396,0322 [M+H]+; found 396,0341.
4.2.5.2. N-(2-{[4-Fluoro-3-(trifluormethyl)phenyl]amino}-
ethyl)-4-methylbenzolsulfonamide 20b. 2-Bromoethyl amine (1
equiv.) and 4-methylphenylsulfonylchloride (1 equiv.) were
dissolved in dry CH2Cl2 under argon and triethyl amine (1.5
equiv) was added. The resulting reaction mixture was stirred at
room temperature for 5 h. Then, water and CH 2Cl2 were added,
followed by thorough extraction of the aqueous layer with
CH2Cl2. The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified
via column chromatography (heptane/EtOAc, gradient 10:0 to
1:1) to afford desired N-(2-bromoethyl)-4-methylbenzene
sulfonamide (1.00 g, 3.54 mmol) as intermediate which was
dissolved in toluene (100 mL). Thereafter, a solution of KOH
(1.19 g, 21.27 mmol) in water (15 mL) was added. The resulting
reaction mixture was stirred at room temperature for 90 min.
After complete conversion, water and ethyl acetate were added,
followed by thorough extraction of the aqueous layer with
EtOAc. The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified
via column chromatography (heptane/EtOAc, gradient) to afford
desired 1-[(4-methylphenyl)sulfonyl]aziridine (781 mg, 98 %) as
intermediate. 1H-NMR (400 MHz, CDCl3 , ppm) 7.85 (d, 2H),
7.37 (d, 1H), 2.45 (s, 3H), 2.42-2.30 (m, 4H). 1-[(4-
methylphenyl)sulfonyl]aziridine (120 mg, 0.61 mmol), 4-fluoro-
3-trifluoromethyl aniline (109 mg, 0.61 mmol) and SiO2 (110
mg, 1.83 mmol) were then suspended in H2O (2 mL), and the
resulting reaction mixture was stirred at room temperature for
20h. Upon addition of water and ethyl acetate the aqueous layer
was extracted thoroughly with ethyl acetate. The combined
organic layer was dried over Na2SO4 and concentrated under
reduced pressure. Purification of the remaining residue via
column chromatography (heptane/EtOAc, gradient) afforded N-
(2-{[4-fluoro-3-(trifluormethyl)phenyl]amino}ethyl)-4-methyl-
benzene sulfonamide 20b (70 mg, 31 %) as colourless solid. 1H-
NMR (400 MHz, CDCl3 , ppm) 7.75 (d, 2H), 7.31 (d, 2H), 7.00
(m, 1H), 6.68-6.64 (m, 2H), 4.79 (br. t, 1H, NH), 4.05 (br. m, 1H,
NH), 6.79 (m, 1H), 3.25-3.16 (m, 4H), 2.43 (s, 3H). LCMS (ESI,
m/z): [M+H]+ 396,7, logp 2.98. HRMS (ESI, m/z): calcd. for
C16H16F4N2O2S, 376,0869 [M+H]+; found 376,0856.
4.2.5.3. N-(2-{[4-Fluoro-3-(trifluormethyl)phenyl]amino}-
ethyl)-4-Methoxybenzene sulfonamide (20c). White solid; yield
41%; 1H NMR (400 MHz, d6-DMSO)  7.72 (d, 2H), 7.55 (m,
1H), 7.20-7.14 (m, 1H), 7.08 (d, 2H), 6.77-6.73 (m, 2H), 6.00-
5.96 (m, 1H), 3.82 (s, 3H), 3.13-3.08 (m, 2H), 2.85-2.81 (m, 2H).
LCMS (ESI, m/z): [M+2H]+ 394.1, logp 3.09.
4.2.5.4. N-(2-{[4-Fluoro-3-(trifluormethyl)phenyl]amino}-
ethyl)-2-fluorobenzene sulfonamide (20d). White solid; yield
70%; 1H NMR (400 MHz, d6-DMSO)  8.01 (br. t, 1H, NH),
7.82-7.78 (m, 1H), 7.70-7.65 (m, 1H), 7.42-7.35 (m, 2H), 7.20-
7.16 (m, 1H), 6.78-6.73 (m, 2H), 6.01-5.98 (br. t, 1H, NH), 3.16-
3.10 (m, 2H), 3.02-2.97 (m, 2H). LCMS (ESI, m/z): [M+2H]+
382.2, logp 3.07.
4.2.5.5. N-(2-{[3-(Trifluormethyl)phenyl]amino}-ethyl)-4-
chlorobenzene sulfonamide (20e). White solid; yield 63%; 1H
NMR (400 MHz, d6-DMSO)  7.86 (m, 1H), 7.81-7.77 (m, 2H),
7.65-7.61 (m, 2H), 7.28-7.24 (m, 1H), 6.81 (m, 1H), 6.74 (m,
2H), 6.12-6.08 (m, 1H), 3.17-3.12 (m, 2H), 2.91-2.87 (m, 2H).
LCMS (ESI, m/z): [M+H]+ 379.7, logp 3.48.
4.2.5.6. N-(2-{[4-Chloro-3-(trifluormethyl)phenyl]amino}-
ethyl)-4-methoxybenzene sulfonamide (20f). White solid; yield
64%; 1H NMR (400 MHz, d6-DMSO)  7.72 (d, 2H), 7.56 (m,
1H), 7.32 (m, 1H), 7.09 (d, 2H), 6.90 (m, 1H), 6.73 (m, 1H),
6.30-6.25 (m, 1H), 3.82 (s, 3H), 3.16-3.11 (m, 2H), 2.85-2.80 (m,
2H). LCMS (ESI, m/z): [M+2H]+ 410.9, logp 3.33.
4.2.5.7. N-(2-{[4-Chloro-3-(trifluormethyl)phenyl]amino}-
ethyl)-3-chlorobenzene sulfonamide (20g). White solid; yield
42%; 1H NMR (400 MHz, d6-DMSO)  7.89 (m, 1H), 7.78 (m,
1H), 7.74 (m, 1H), 7.70 (m, 1H), 7.60 (m, 1H), 7.33-7.30 (m,
1H), 6.90 (m, 1H), 6.73 (m, 1H), 6.31-6.26 (m, 1H), 3.17-3.12
(m, 2H), 2.94-2.88 (m, 2H). LCMS (ESI, m/z): [M+2H]+ 414.5,
logp 3.75.
4.2.5.8. N-(2-{[4-Methoxy-3-(trifluormethyl)phenyl]amino}-
ethyl)-4-fluorobenzene sulfonamide (20h). White solid; yield
61%; 1H NMR (400 MHz, d6-DMSO)  7.87-8.83 (m, 2H), 7.76
(br. m, 1H, NH), 7.42-7.38 (m, 2H), 7.03-7.00 (m, 1H), 6.74-6.69
(m, 2H), 5.57-5.54 (br. t, 1H, NH), 3.74 (s, 2H), 3.11-3.05 (m,
2H), 2.90-2.85 (m, 2H). LCMS (ESI, m/z): [M+H]+ 393.2, logp
2.90.
4.3. Modelling studies
Modelling was done using the crystal structure of ABA-bound
AtRCAR11 and related phosphatase AtHAB1 (3QN1 – F.
Dupeux et al.).47 Docking poses were generated using SeeSAR
from BioSolveIT and scored using the HYDE scoring function.36
Acknowledgments
We would like to thank Matthias Jank, Susanne Ries, Gudrun
Fey, Peter Zöllner and Martin Annau for valuable analytical
support. Furthermore, we would like to thank David Barber for
fruitful discussions during the preparation of the manuscript.
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Graphical Abstract – J. Frackenpohl

Identifying new lead structures to enhance Leave this area blank for abstract info.
tolerance towards drought stress via high-
throughput screening giving crops a
quantum of solace
Jens Frackenpohla,, Linn Schneiderb, Luka J. B. Deckera, Jan Dittgena, Franz Fenkla, Christian
Fischera, Jana Frankea, Joerg Freigangc, Susana M. Gonzalez Fernandez-Ninoa, Hendrik Helmkea,
Martin J. Hillsa, Sabine Hohmanna, Rahel Getachewa, Jochen Kleemannc, Karoline Kurowskia, Gudrun
Langea, Peter Luemmena, Nicole Meyeringb, Fabien Poreea, Dirk Schmutzlera and Sebastian Wredeb
a Research & Development, Weed Control - Bayer AG, Crop Science Division, Industriepark Höchst, D-65926 Frankfurt am Main
b Research & Development, Lead Discovery – Bayer AG, Pharmaceutical Division, Aprather Weg 18a, D-42096 Wuppertal
c Research & Development - Bayer AG, Crop Science Division, Gebäude 6240, Alfred-Nobel-Straße 50 ,D-40789 Monheim

H 3
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High-throughput screening N N
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R N
2
R

O O
2 S
R N
H N
O
1
R
3
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2
H R
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BMC_2019_046

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential
competing interests: