Potent Analogues of Abscisic Acid – Identifying Cyano-Cyclopropyl Moieties as Promising Replacements for the Cyclohexenone Headgroup

Article abstract of DOI:10.1002/ejoc.201701769

Synthetic analogues of plant hormone abscisic acid (ABA) bearing a yet unexplored head group motif were prepared based on a combination of agrochemical experience, in vivo hits and structure-based design. It could thus be explored how modifying key parts of ABA's cyclohexenone unit influenced receptor affinity and in vivo efficacy against drought stress in selected crops. Cyano-cyclopropyl groups proved to be suitable replacements of the cyclohexanone moiety leading to ABA analogues with strong activity in vitro and in vivo. Their efficient and versatile synthesis proceeded via Stille or Sonogashira couplings as the key steps. Combining novel cyano-cyclopropyl headgroups with previously identified substituents in the terpenoid side chain afforded the most promising effects against drought stress in crops, particularly canola and wheat.

Full text of DOI:10.1002/ejoc.201701769

DOI: 10.1002/ejoc.201701769
Full Paper
Abscisic Acid Derivatives | Very Important Paper |
Potent Analogues of Abscisic Acid – Identifying Cyano-
Cyclopropyl Moieties as Promising Replacements for the
Cyclohexenone Headgroup
[a]
[a]
[c]
[a]
[a]
Jens Frackenpohl,* Guido Bojack, Rachel Baltz, Udo Bickers, Marco Busch,
[
a]
[a]
[b]
[d]
[a]
Jan Dittgen, Jana Franke, Jörg Freigang, Erwin Grill, Susana Gonzalez,
[a]
[a]
[a]
[a]
Hendrik Helmke, Martin J. Hills, Sabine Hohmann, Pascal von Koskull-Döring,
[a]
[a]
[a]
[a]
[a]
Jochen Kleemann, Gudrun Lange, Stefan Lehr, Dirk Schmutzler, Arno Schulz,
[a]
[a]
[d]
Kerstin Walther, Lothar Willms, and Christian Wunschel
Abstract: Synthetic analogues of plant hormone abscisic acid suitable replacements of the cyclohexanone moiety leading to
ABA) bearing a yet unexplored head group motif were pre- ABA analogues with strong activity in vitro and in vivo. Their
(
pared based on a combination of agrochemical experience, in efficient and versatile synthesis proceeded via Stille or Sonoga-
vivo hits and structure-based design. It could thus be explored shira couplings as the key steps. Combining novel cyano-cyclo-
how modifying key parts of ABA's cyclohexenone unit influ- propyl headgroups with previously identified substituents in
enced receptor affinity and in vivo efficacy against drought the terpenoid side chain afforded the most promising effects
stress in selected crops. Cyano-cyclopropyl groups proved to be against drought stress in crops, particularly canola and wheat.
Introduction
Abscisic acid [1, S-(+)-ABA], a chiral sesquiterpenoid first discov-
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
[
1]
ered in the 1960s, is one the most important plant hormones
regulating developmental signals in plants such as seed matu-
ration or dormancy. It has been commercialized, e.g. for en-
hancing color development in red-table grapes. Furthermore, it
mediates the adaptation of plants to environmental abiotic
stress such as drought, heat or salinity stress. Several strategies
have been investigated for reducing the impact of drought on
yield, such as exploiting beneficial effects of crop protection
(SAR) of ABA, whilst its catabolism had been well explored prior
to identifying the RCAR/(PYR/PYL) receptor proteins. ABA's prin-
cipal pathway of oxidation is through monooxygenase-medi-
ated hydroxylation, i.e. by the CYP707A-family, of the 8′-methyl
group affording 8′-hydroxy-ABA, which is in equilibrium with
[
5]
the cyclized form phaseic acid. Thus, most chemical modifica-
tions of ABA were dedicated to stabilizing its cyclohexenone
headgroup. It has been shown that metabolism-resistant ana-
logues altered at the C8′ position were more stable towards
[
2]
agents, developing drought tolerant crops through transgenic
[
3]
approaches or breeding, but also exploring novel chemical
entities inspired by naturally occurring plant hormones. Studies
on ABA-mediated signaling have progressed rapidly since the
recent discovery of RCAR/(PYR/PYL) receptor proteins as soluble
[
4]
ABA-receptors. It was shown via crystal structural analysis that
binding of ABA to RCAR12 induced a conformational change in
[
a] Research & Development, Weed Control Bayer AG, CropScience Division
Industriepark Höchst, Frankfurt am Main, 65926, Germany
E-mail: jens.frackenpohl@bayer.com
www.bayer.com
[
b] Research & Development, Research Technology, Bayer AG, CropScience
Division,
Gebäude 6240, Alfred-Nobel-Straße 50, 40789 Monheim, Germany
c] Bayer S.A.S. Centre de Recherche de La Dargoire
[
[
1
4 Impasse Pierre Baizet, 69263 Cedex 09, Lyon France
d] Lehrstuhl für Botanik, Wissenschaftszentrum Weihenstephan,
Technische Universität München
Emil-Ramann-Straße 4, 85354, Freising
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201701769.
Figure 1. The structure of (S)-(+)-abscisic acid (1) with formal atom numbering
and selected closely related analogues with modified headgroup.
Eur. J. Org. Chem. 2018, 1416–1425
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oxidation than ABA itself with 8′-ethynyl-ABA 2a, 8-vinyl-ABA
As a starting point, compound 11 was docked initially into
2
b and 8′-trifluoromethyl-ABA 2c exhibiting promising activity the ABA binding site of the published crystal structure of ABA-
[
6]
in in vitro tests.
bound RCAR12 and related phosphatase ABI1 (PDB: 3 KDJ)
Furthermore, tetralone analogues 3a–b of ABA have been since complex structures of RCAR11 with bound ABA and HAB1
[
7]
[8]
prepared, as well as 3′-sulfanyl-ABA analogues 4a–b, ABA- were not available at that time. Resulting poses were then
[
9]
[18,19]
analogues with modified 7′-Me group 5a–b 3-methyl- and scored using the HYDE scoring function
′-haloalkyl-ABA 6a–b
with the best
[
10]
[11]
3
or cyclohexenol 7 (Figure 1).
Al- ranked pose having an energy of –41 kJ/mol corresponding to
though stabilizing or further substituting the cyclohexenone a binding affinity in micromolar range (Figure 4a, 11 superim-
moiety has attracted significant interest, surprisingly few ap- posed with ABA). The quaternary phosphorus atom of 11 is
proaches have been made to identify open-chain analogues of located at the position of the quaternary carbon of ABA, while
ABA with promising target affinity. The first synthetic com- the P=O moiety replaces the hydroxyl function of ABA forming
pounds interacting with RCAR/(PYR/PYL) receptor proteins have the same H-bond to a water molecule at the protein ligand
[
12]
been recently prepared.
In our view this indicates that we interface. Furthermore, the nitrile moiety of 11 forms an H-
can identify ABA analogues with more profound structural bond to the water molecule at the interface between RCAR12
changes that exhibit good receptor binding and promising in and HAB1 adopting the role of the carbonyl oxygen in ABA.
vivo efficacy. Substituted aryl sulfonamides, e.g. pyrabactin This conserved water molecule forms all four possible H-bonds
[
13]
8
a,
closely related pyrimidine 8b, as well as thiazoles and including H-bonds to the backbone oxygen of proline 115 and
[
14]
pyrazoles 9a–b,
have shown promising initial receptor affin- backbone amide of arginine 143 from RCAR12 and NE1 of
ity and beneficial effects within in vivo tests (Figure 2). More tryptophan 300 from ABI1. It thus represents a key element
recently, tetrahydroquinolinyl sulfonamides have shown im- in the protein–protein interaction between the RCARs and the
[
15]
proved in vitro activity.
Interestingly, it was demonstrated corresponding phosphatases. Interestingly, the nitrile nitrogen
further that the sulfonamide unit in these hit structures could and the carbonyl oxygen are not located at the same place due
be replaced by phosphonamides preserving binding affinity to different localizations of the free electron pair serving as the
and thus emphasizing opportunities to introduce further struc- H-bond donor. The binding mode of 11 was subsequently con-
tural motifs into ABA-related structures.[ Herein, we outline firmed by a co-crystal structure with the closely homologous
how we identified unprecedented potent analogues of ABA car- ABA receptor RCAR11 and the phosphatase HAB1 (Figure 4b).
rying cyano-cyclopropyl moieties as replacements of its cyclo-
16]
hexenone headgroup, and we discuss the structural basis of
their beneficial effects against drought stress observed in the
course of our in vivo-studies.
Figure 4. (a) Superposition of the best ranked docking pose of 11 with ABA
bound to RCAR12 and ABI1. The color code of 11 indicates the stabilization
Figure 2. Selected naphthyl sulfonamides interacting with RCAR/(PYR/PYL)
receptor proteins.
of individual atoms ranging from –3 kJ/mol (dark green) to +3 kJ/mol (dark
red). Common H-bonds are marked in black while the H-bond to the con-
served water molecule is cyan in case of ABA and green in case of 11. (b)
Superposition of the docking model of 11 (green) in the ABA binding site of
RCAR12/ABI1 (cyan) with the crystal structure of 11 (yellow) in complex with
Results and Discussion
RCAR11/HAB1 (orange).
Hit Exploration, Modeling and X-ray Studies
Visual inspection of ABA's superposition and the predicted
binding mode of 11 suggested a chemical entity 16a combin-
ing the diene moiety of ABA with the cyano-cyclopropyl moiety
of 11. The crystal structure of 16a bound to RCAR11 and HAB1
proved its potential to mimic the binding mode of ABA thus
representing an excellent novel synthetic analogue of natural
product ABA (Figure 5). In particular, all H-bonds are conserved
in the crystal structure of 16a bound to RCAR11 and HAB1
including the H-bond to the conserved water molecule that
bridges tryptophan 385 and the backbone of RCAR11 (Fig-
ure 5). Similar to the binding of ABA, space for hydrophobic
Inspired by structural features from earlier agrochemical pro-
jects (e.g. 10) and from in vivo hits 11, 12 showing promising
[
17]
efficacy against drought stress (Figure 3)
we have initiated
modeling studies to identify novel head group motifs with
good binding affinity to the ABA-receptors.
2
substituents of a limited size was identified at position R , and
further analysis of the X-ray structure also indicated space for
1
introducing hydrophobic groups at position R . These findings
Figure 3. In vivo hit structures with interesting structural motifs.
prompted us to start a broad SAR study.
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hands, this methodology proved to be clearly superior towards
related approaches using hydrozirconation and a Negishi-type
[
22]
cross-coupling to establish the (E,Z)-diene side chain.
As a
result, ABA-analogues 16c–16g could be prepared in 5 linear
steps with overall yields in the range of 30–50 % (Scheme 1).
Alkynol 13a also served as a key intermediate to prepare re-
lated enynes 17d–g via Sonogashira coupling. Enyne 17d could
be separated into both enantiomers via prep. HPLC. Interest-
ingly, both enantiomers showed comparable in vitro and in vivo
activity prompting us to use racemates in our SAR study. With
this robust synthetic approach in hand we could prepare a
Figure 5. (a) Details of molecular interaction of 16a bound to RCAR11 and
HAB1. H-bonds are marked in black except for those to the conserved water
molecule which are marked in green. b) Comparison of RCAR11/HAB1 com-
plex structures of 16a (ligand in cyan, protein in green) and ABA (ligand in
orange, protein in yellow).
broad range of compounds with modified head groups
1
(
Scheme 2; entries 2–27, Table 1). The iPr group (R ) could thus
be replaced by a wide variety of alkyl, cycloalkyl and aryl
substituents, such as cyclopropyl, cyclopentyl, tert-butyl, ada-
mantyl, phenyl or tolyl, emphasizing the versatility of our syn-
[
22]
thetic approach (16m, 16u, 16z, Scheme 2, Table 1, Table 2).
Chemistry
Based on our experiences from total synthesis studies in the
field of ABA we decided to apply our versatile approach using
a Stille coupling as the key step to prepare cyano-cyclopropyl
[
20]
analogues of ABA.
LDA-mediated coupling of cyclopropyl
nitrile with a pivaloyl-based Weinreb amide, followed by direct
alkynylation afforded crystalline alkynol 13a in good yield
(
Scheme 1). Subsequently, 13a could then be easily trans-
formed selectively into (E)-configured key intermediate 14a via
Pd(PPh ) Cl -mediated hydrostannylation.
3
2
2
Scheme 2. Cross-coupling-mediated synthesis of cyano cyclopropyl ana-
1
logues of ABA with various substituents R . a) MeO-NHMe, NaHCO
LDA, THF, –78 °C to r.t, 5 h; c) Li-acetylide ethylene diamine complex, THF,
room temp.; d) Bu SnH, Pd(PPh Cl , THF, r.t.; e) 15d, Pd(MeCN) Cl , CuI, DMF,
0 °C; f) Pd(PPh Cl , CuI, diisopropylamine, toluene, r.t.
3
, THF; b)
3
3
)
2
2
2
2
5
3
)
2
2
To broaden our SAR study further we aimed to enlarge the
ring size of the cyano-cycloalkyl unit. Having chosen the cyano-
cyclobutyl moiety as a representative motif, we applied our syn-
thetic approach outlined in Scheme 1 and Scheme 2 to cyclo-
butyl nitrile (Scheme 3) affording (E)-stannane 20a and desired
Scheme 1. Cross-coupling-mediated synthesis of cyano cyclopropyl ana-
1
logues of ABA with R = iPr. a) lithium diisopropylamide (LDA), THF, –78° to
room temp., 5 h; b) Li-acetylide ethylene diamine complex, THF, r.t.; c)
Bu
3
SnH, Pd(PPh
3
)
2
Cl
Cl
2
, THF, r.t.; d) 15c, d, e, f or g, Pd(MeCN)
, CuI, diisopropylamine, toluene, r.t.; f) chiral prep. HPLC.
2 2
Cl , CuI, DMF,
5
0 °C; e) Pd(PPh )
3
2
2
Coupling of (E)-stannane 14a with a suitable freshly pre-
pared vinyl iodide 15c–15g via an optimized Stille-coupling
protocol using Pd(MeCN) Cl and CuI in N,N-DMF at slightly ele-
2
2
Scheme 3. Cross-coupling-mediated synthesis of cyano cyclobutyl analogues
of ABA with R = iPr. a) LDA, THF, –78 °C to room temp., 5 h; b) Li-acetylide
vated temperatures (40–50 °C) afforded desired cyano-cyclo-
1
[
22]
propyl analogues of ABA 16c–16g. (Z)-Vinyl iodides 15c–15g
ethylene diamine complex, THF, r.t.; c) Bu
3
3 2 2
SnH, Pd(PPh ) Cl , THF, r.t.; d) 15b,
were prepared starting from substituted alkynes in two steps
e, Pd(MeCN)
2
Cl
2
, CuI, DMF, 50 °C; e) Pd(PPh Cl , CuI, diisopropylamine, tolu-
3
)
2
2
[
21]
via carboxylation and subsequent hydroiodination.
In our
ene, r.t.
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diene target compounds 21b and 21e in good yield. Likewise, cotyledonous and dicotyledonous species, whereas in vitro
related enynes 22b and 22e were obtained via Sonogashira tests were carried out at the ABA-receptor system RCAR11 in
coupling.
Arabidopsis thaliana. Based on experiences regarding herbicidal
The utility of stannanes 14 as versatile intermediates could antidotes being used as Safeners[24] we also laid emphasis on
be demonstrated further by the preparation of cyclized exploring the impact of esters on in vivo efficacy. In contrast to
analogues via Stille and Suzuki couplings. This enabled us to conclusions from earlier studies, good receptor affinity as well
evaluate the impact of a cyclohexenyl or substituted phenyl as promising efficacy against drought stress in vivo could be
group as rigid replacements for the (Z)-configured double observed for ABA-derivatives containing both, modified non-
[
23]
bond.
cyclic headgroups and a diene side chain with modified substit-
uents at C6.
Firstly, good and consistent binding affinity to RCAR11 was
observed for all ABA analogues bearing cyano cyclopropyl
headgroups with isopropyl [16a–16h (entries 2–9, Table 1)] and
In Vitro and in Vivo SAR Study
All ABA-analogues with modified headgroups were tested both,
for target affinity, as well as for beneficial effects in vivo under
drought stress conditions upon foliar application on plants.
Wheat and Canola were chosen as model plants for mono-
1
cyclopropyl (16r, entry 19) groups at position R . These deriva-
tives showed highly promising effects against drought stress in
wheat and canola in greenhouse experiments. In line with re-
sults from earlier studies (focusing on side chain variations of
ABA) binding affinity to RCAR11 and in vivo efficacy of 16i (en-
try 10) dropped sharply due to the presence of a bulky cyclo-
Table 1. SAR-results of selected cyano cyclopropyl diene-analogues of ABA.
2
[20]
pentyl group at position R in its diene side chain.
Hence,
2
we introduced only the most promising groups at R , such as
methyl, ethyl or trifluoromethyl, to investigate the impact of
structural changes in cyano-cyclopropyl-based headgroups.
Substituents[a]
In vitro activity
In vivo efficacy vs.
drought stress
1
[
b]
Analogues with bulkier secondary substituents R , such as 1-
ABI1-(AtRCAR11)
[c]
ethylprop-1-yl (16n–16q, entries 15–18), cyclopentyl (16s–u,
entries 20–22), and cyclohexyl (16v–16x, entries 23–25) showed
still good receptor affinity, however, in vivo efficacy in wheat
dropped significantly combined with overall weaker efficacy in
canola. Whilst analogues with a quaternary tert-butyl substitu-
[250 g/ha]
Entry No. R1
R2
R3 Activity
pI50
Wheat[c]
Canola[c]
[%] 5 μM
[
e]
+++[d]
++++
++++
+++
++++
+++
++++
+++
+++
+
1
2
3
4
5
6
7
8
9
1
Me
Me
Me
Et
H
H
100
100
7.1
5.9
5.9
6.1
5.8
5.8
6.0
5.9
5.9
4.3
6.1
6.1
6.2
6.1
5.9
5.9
5.9
6.1
6.1
6.3
6.1
6.3
6.0
5.8
6.1
5.1
+++
++++
+++
++++
+++
+++
+++
+++
+++
+
16a iPr
16b iPr
16c iPr
16d iPr
16e iPr
16f iPr
16g iPr
16h iPr
16i iPr
16j tBu
16k tBu
16l tBu
16m tBu
16n iPent Me
16o iPent CF
16p iPent CF
16q iPent Et
16r cPr
16s cPent CF
16t cPent CF
16u cPent Et
16v cHex Me
[
b]
[b]
1
Et 99
100
ent at R (16j–16m, entries 11–14) were highly active in vitro
H
combined with moderate efficacy in vivo, aromatic substituents
[b]
[b]
Et
Me 95
Me 94
nPr 94
Et 99
nPr 99
1
at R (e.g. tolyl, 16y, 16z, entries 25 and 26) led to a sharp loss
[
b]
b]
b]
b]
b]
[b]
CF
Et
3
[
[b]
in in vivo efficacy. Surprisingly, tert-butyl derviatives 16j–16m
consistently showed best effects in wheat rather than in canola.
Likewise, cyano-cyclobutyl analogues afforded good target af-
finities in line with good effects in green house experiments
when bearing small alkyl and cycloalkyl substituents as in com-
pounds 21b and 21e (Scheme 3). However, these results were
on a slightly lower level than for parent cyano-cyclopropyl com-
pounds 16b and 16e (e.g. 21b: pI50 5.6, wheat: +++, canola:
[
[b]
CF
CF
3
[
[b]
3
[
[b]
1
0
cPent Et 72
[b]
[b]
1
1
Me
CF
Et 95
Me 96
Et 94
Me 98
Et 100
Me 99
nPr 99
Me 100
Me 100
Me 95
Et 95
Me 95
Et 100
Me 97
Me 100
Et 84
Me 94
+++
++
++
+++
+
++
++
++
++
+++
++
[
b]
b]
b]
b]
[b]
1
2
3
[
[b]
1
3
CF
Et
3
[
[b]
1
4
[
[b]
1
5
[b]
[b]
1
6
3
+
+
+). As a result, five novel ABA-analogues with cyano-cyclo-
[b]
[b]
1
7
3
+
++
1
[
b]
[b]
propyl headgroup (R = iPr, cPr) were identified exhibiting im-
proved in vivo efficacy than ABA 1 (Entry 1, Table 1) as refer-
ence. Earlier we showed that an enyne side chain is a good
mimic of the corresponding diene units of ABA analogs ensur-
ing the same crucial interactions of cyclohexanone and carb-
oxylate units with RCAR11. To broaden our SAR study further,
as well as to evaluate the impact of the (E)-alkene linker, we
prepared a set of corresponding enynes bearing related modi-
1
8
+
++
[
b]
[b]
1
9
CF
3
+++
+
++++
++
[b]
[b]
2
0
3
[b]
[b]
2
1
3
+
+
[b]
[b]
2
2
+
++
[
b]
[b]
2
3
+
+
+++
++
[
20]
[b]
[b]
2
4
16w cHex CF
16x cHex Et
16y tolyl Me
16z tolyl Et
3
[
b]
[b]
2
5
+
++
[b]
[b]
2
6
+
+
[b]
[b]
27
5.4
+
+
1
2
fied substituents at positions R and R (17a–z, Entries 1–24,
[
1
a] cPr = cyclopropyl, tBu = 1,1-dimethyl-eth-1-yl, cPent = cyclopentyl, iPent =
-ethylprop-1-yl, cHex = cyclohexyl, tolyl = p-CH –C . [b] Target affinity of
Table 2).
3
6 4
H
A similar pattern of in vitro and in vivo activity could be
observed for enynes 17a–z, albeit on a slightly lower level. This
may reflect the lower flexibility of enynes compared to related
dienes. In good accordance with results obtained for diene-ana-
logues with isopropyl and cyclopropyl groups at position R1
(Table 1) enynes 17b, 17c and 17d (Entries 2, 3 and 14, Table 2)
esters was measured after addition of pig liver esterase. [c] Standard applica-
tion 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
effect superior to internal standard ABA (comparative visual assessment of
greenmass). [e] Cyclic cyclohexanone headgroup of ABA.
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Table 2. SAR-results of selected cyano cyclopropyl enyne-analogues of ABA.
to untreated controls upon treatment with 16b and 17d as
representative examples (Figure 6).
Substituents[a]
In vitro activity
ABI1-(AtRCAR11)
In vivo efficacy vs.
drought stress
[b]
[250 g/ha]
Entry No.
R1
R2 R3 Activity
%] 5 μ
pI50
Wheat[c]
Canola[c]
[
M
[
b]
[b]
1
2
3
4
5
6
7
8
9
17a
17b
17d
R-17d iPr
S-17d iPr
17e
17f
17g
17h
17i
17n
17o
17r
17c
17j
Me
iPr
iPr
Me Et
Me Et
Et
Et
Et
79
97
5.1
++
++
[b]
[b]
5.9
++++
++++
+++
++++
+++
+++
+++
+++
++
+
+
++
++
++
++
++
+
+
+++
++
++
+
+++
++++
+++
++++
+++
+++
+++
+++
+++
++
[
b]
[b]
Me 98
Me 93
Me 99
Me 96
nPr 96
5.8
[
b]
b]
b]
b]
b]
[b]
5.4
[
[b]
5.9
[
[b]
iPr
iPr
iPr
iPr
CF
Et
3
5.8
[
[b]
5.8
Figure 6. Advanced drought stress trials with 16b and 17d in wheat, barley
and canola under moderate stress conditions, i.e. 45 % damage for canola,
[
[b]
CF
CF
3
Et
99
nPr 96
5.9
[
b]
[b]
3
5.7
4
2 % damage for barley and 48 % for wheat, respectively.
[
b]
[b]
1
0
iPr
cPr Et
Me Et
99
92
5.9
[
b]
[b]
1
1
iPent
iPent
cPr
cPr
cBu
cBu
5.3
[
b]
[b]
1
2
CF
CF
Et
3
Me 86
Me 99
5.0
++
[
b]
[b]
1
3
3
5.6
+++
++++
+++
++++
++
++
+
++
++
Conclusions
[b]
[b]
1
4
Me 100
5.9
[b]
[b]
1
5
CF
Et
3
Me 99
5.6
We have identified a novel headgroup bearing a cyano-cyclo-
propyl moiety as a key motif, which proved to be a suitable
replacement of ABA's cyclohexenone unit. These findings gave
rise to a series of novel ABA analogues with strong activity in
vitro and in vivo. Our flexible synthetic approach via cross-cou-
pling chemistry enabled us to investigate in vivo activities of a
large set of new ABA-analogues against drought stress. In total,
we have identified seven highly potent analogues of ABA with
improved in vivo efficacy against drought stress in canola and
wheat. Results from in vitro measurements and crystal structure
analyses have confirmed excellent interaction with key func-
tionalities of the ABA-receptor emphasizing the potential of our
cyano-cyclopropyl-based headgroups to act as bioisosters of
[b]
[b]
1
6
17l
17m tBu
Me 100
6.0
[
b]
[b]
1
7
Et
Me 92
5.3
[
b]
[b]
1
8
17u
17v
17q
17s
17x
17y
17z
cPent Et
cHex
Ph
Me 94
5.2
[
b]
[b]
1
9
Me Et
Me Et
CF Me 93
93
5.1
[
b]
[b]
2
0
91
5.5
[
b]
b]
[b]
2
1
Ph
3
5.3
[
[b]
2
2
p-F-Ph Et
tolyl
tolyl
Et
Me Et
Et Me 90
88
5.4
++
++
++
[
b]
[b]
2
3
84
5.1
[b]
[b]
24
5.3
+
[
a] See footnote [a] in Table 1. [b] Target affinity of esters was measured after
addition of pig liver esterase. [c] A final assessment of the respective efficacy was
made according to the following classification: “0“ = no effect, “+” = slight beneficial
effect, “++” = significant beneficial effect, “+++” = strong beneficial effect against
drought stress, “++++” = very strong effect superior to internal standard ABA (comp.
vis. assessment of greenmass).
1
cyclohexenones. The best substituents at R were isopropyl and
cyclopropyl combined with small lipophilic substituents such
as CF and ethyl at ABA's position C6 in the diene side chain.
3
showed the best activities, both in vitro and in vivo. Further- Remarkably, enynes proved to be as strong as related dienes in
1
more, R = cyclobutyl was well tolerated, exhibiting good target in vivo tests.
affinity as well as good effects in vivo against drought stress
(
17j and 17l, entries 15 and 16, Table 2). Derivatives with
bulkier branched alkyl, cycloalkyl and aryl groups gave weaker Experimental Section
results, correlating with what we had observed for related di-
enes with modified R groups. Likewise, cyano cyclobutyl-sub-
stituted enynes afforded good target affinities in line with good
Biology: In Vivo: 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 condi-
1
effects in green house experiments on a slightly lower level tions. The test plants were treated at the early leaf stage (BBCH10–
than related dienes when bearing small alkyl and cycloalkyl BBCH13). To ensure uniform water supply before commencement
substituents as in compounds 22b and 22e (pI50 5.2, wheat: of stress, the potted plants were supplied with the maximum
amount of water immediately beforehand by dam irrigation and,
+
+, canola: ++, Scheme 3).
Interestingly, both enantiomers of 17d proved to be active
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), wettable gran-
ules (WG), suspension concentrates (SC) or emulsion concentrates
in vitro and in vivo. To validate our in vivo screening results
further, we selected certain ABA-analogues for advanced green-
house trials (i.e. more replicates, additional crops such as corn
and barley, different corn varieties and moderate stress levels).
(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
Accordingly, significant beneficial effects against drought stress addition of 0.2 % wetting agent (agrotin). Substance application is
could also be observed in corn, barley and wheat compared followed immediately by drought stress treatment of the plants.
Eur. J. Org. Chem. 2018, 1416–1425
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1420
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Drought stress was induced by gradual drying out under the follow-
ing conditions: “day” = 14 hours with illumination at 26 °C; “night” =
strate mix were made up 5 min prior to the addition and warmed
to a temperature of 35 °C. On completion of pipetting of all the
1
0 hours without illumination at 18 °C. The duration of the respec- solutions and on completion of mixing, the plate was incubated at
tive 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 crop to crop. It was
35 °C for 20 min. 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
(
ended (by re-irrigating or transfer to a greenhouse with good filter.
growth conditions) as soon as irreversible damage was observed on
X-ray Crystallography: For structural studies RCAR11 and an N-
terminally truncated version of HAB1 lacking residues 1–178
the untreated, stressed control plants. In the case of dicotyledonous
crops, for example oilseed rape, the duration of the drought stress
phase varied between 3 and 5 days; in the case of monocotyledon-
ous 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 recov-
ery 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 % = same as 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
(
ΔNHAB1) were expressed and purified according to published pro-
[
26]
tocols.
Prior to crystallization RCAR11, ΔNHAB1 and ABA-ana-
Tris pH 7.5, 150 m NaCl,
ꢀ-mercaptoethanol to final concentrations
of 3 mg/mL, 5 mg/mL and 1 m , respectively. Crystals were ob-
tained by sitting drop vapor diffusion from 20 % Peg 8000, 100 m
Tris pH 8.5, 160 m MgCl , 60 m glycylglycylglycine at 18 °C. Dif-
logue 11 or 16a were mixed in 20 m
MnCl , and 1 m
M
M
1
m
M
M
2
M
M
M
M
2
fraction data were collected on an Xcalibur Nova diffraction system
from Oxford Diffraction. The structures were solved by molecular
[
27]
replacement with MOLREP using the ternary complex of RCAR11,
ΔNHAB1 and ABA (pdb accession code 3QN1) as a search model.
The model was refined through successive rounds of manual model
[
28]
[29]
building using Coot and restrained refinement using RefMac5.
Data processing and refinement statistics are shown in Table S1.
Chemistry: General: All reagent-grade solvents and chemicals were
purchased from standard commercial suppliers and used without
respective efficacy was made, i.e. “0“ = no effect, “+” = slight benefi- further purification. All non-aqueous reactions were carried out un-
cial effect, “++” = significant beneficial effect, “+++” = strong bene-
ficial effect against drought stress, “++++” = very strong effect su-
perior to internal standard ABA.
der anhydrous conditions using dry solvents. Reactions were moni-
tored by LC-MS or TLC carried out on 0.25 mm silica gel plates
(60F-254). TLC plates were visualized using UV light. Flash chroma-
tography was carried out using Biotage Isolera One systems with
pre-packed column cartridges (Biotage KP-Sil [40+M] or KP-Sil
In vitro: ABI1-(AtRCAR11): The assay described hereinafter utilizes
the inhibition of the phosphatase ABI1 via the co-regulator RCAR11/
PYR1 from Arabidopsis thaliana. Expression and purification of
[
25+M]), with typical gradients starting from a ethyl acetate/hept-
1
13
ane ratio of 20:80 to a final ratio of 80:20. The H NMR, C NMR and
[4a,25]
RCARs and PP2Cs was performed as described.
For the deter-
1
9
F NMR spectroscopy data, which are reported for the chemical
mination of activity, the dephosphorylation of 4-methylumbelliferyl
phosphate (MUP) was measured at 460 nm. The in vitro assay was
conducted in Greiner 384-well PS microplates F-well, using two con-
1
examples described below (400 MHz for H NMR and 150 MHz for
1
3
19
C NMR and 375 MHz for F NMR, solvent: CDCl , CD OD or
3
3
[
D ]DMSO, internal standard: tetramethylsilane δ = 0.00 ppm), were
6
trols: a) dimethyl sulfoxide (DMSO) 0.5 % (f.c.) and b) 5 μ
M (f.c.)
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 are defined as follows:
Me = CH , Et = CH CH , tHex = C(CH ) CH(CH ) , tBu = C(CH ) ,
abscisic acid (ABA). The assay described here was generally con-
ducted with substance concentrations of the appropriate chemical
test substances in a concentration range of 0.1 μM to 100 μM 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
3
2
3
3 2
3 2
3 3
3
0 min. A total volume of 45 μL was introduced into each cavity of
the microplate, having the following composition:
1) 5 μL of substance solution, i.e. a) DMSO 5 % or b) abscisic acid
nBu = unbranched butyl, nPr = unbranched propyl, cHex = cyclo-
hexyl. In the case of diastereomeric mixtures, either the significant
signals for each of the diastereomers or the characteristic signal of
the main diastereomer are reported. Further experimental details
are disclosed in the supplementary material, as well as in referen-
(
solution or c) the corresponding example compound of the general
formula (I) dissolved in 5 % DMSO.
(
2) 20 μL of enzyme buffer mix, composed of a) 40 % by vol. of
enzyme buffer [10 mL contain equal proportions by volume of
00 m Tris-HCl pH8, 500 m NaCl, 3.33 m MnCl , 40 m dithio- 1-(3-Hydroxy-4-methylpent-1-yn-3-yl)cyclopropanecarbonitrile
[
22,23]
ces.
5
M
M
M
M
2
threitol (DTT)], b) 4 % by vol. of ABI1 dilution (protein stock solution
(13a): Triethylamine (510 mL, 3.71 mol) and pivaloyl chloride
was diluted so as to give, after addition, a final concentration in the
(106 mL, 1.02 mol) were added to a cooled solution (0 °C) of
assay of 0.15 μg ABI1/well), c) 4 % by vol. of RCAR11 dilution (en- methoxymethylamine hydrochloride (100 g, 0.93 mol) in dichloro-
zyme 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 μg
methane (1 L). The resulting reaction mixture was stirred at room
temp. for 6 h and was diluted with water on completion of the
reaction. Dichloromethane was added, and the aqueous layer was
extracted thoroughly. The combined organic layer was washed with
brine solution, dried with anhydrous sodium sulfate and the solvent
was removed under reduced pressure. The resulting crude product
enzyme/well), d) 5 % by vol. of Tween20 (1 %), e) 47 % by vol. H O
2
bi-distilled.
(
3) 20 μL of substrate mix, composed of a) 10 % by vol. of 500 m
Tris-HCl pH8, b) 10 % by vol. of 500 m NaCl, c) 10 % by vol. of
.33 m MnCl , d) 5 % by vol. of 25 m
1 %), 60 % by vol. of H O bi-distilled. Enzyme buffer mix and sub-
M
M
3
(
M
M
MUP, 5 % by vol. of Tween20 was purified over silica gel column chromatography by eluting with
20 % EtOAc/petroleum ether, and a portion (31 mL, 424 mmol) of
2
2
Eur. J. Org. Chem. 2018, 1416–1425
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1421
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
the resulting product was dissolved in tetrahydrofuran (400 mL)
thereafter. After cooling to –78 °C, a solution of LDA in tetrahydro-
furan (2 M, 250 mL, 501 mmol) was added, and the resulting reac- colorless oil. H NMR (400 MHz, CDCl ): δ = 6.18 (d, 1 H), 6.03 (d, 1
3-hydroxy-4-methyl-1-(tributylstannyl)pent-1-en-3-yl]cyclopropane-
carbonitrile 14a (0.38 mg, 24 % of theoretical) in the form of a
1
3
tion mixture was stirred at room temp. for 4 h. Upon complete
turnover, the reaction mixture was quenched with ammonium
chloride solution and was extracted with ethyl acetate. The com- ppm. HRMS-ESI: calcd. for C H NOSn [M + H] 455.2772, found
H), 2.32 (sept, 1 H), 1.53–1.45 (m, 6 H), 1.35–1.29 (m, 6 H), 1.28 (m,
1 H), 1.14 (m, 1 H), 1.03 (d, 3 H), 0.94–0.88 (m, 18 H), 0.72 (m, 2 H)
+
+
22 42
bined organic layer was washed with brine solution, dried with an-
hydrous sodium sulfate, and the organic solvent was reduced under
reduced pressure. The remaining residue was purified via column
chromatography (silica gel, 20 % EtOAc/petroleum ether as eluent)
to afford 1-isobutyrylcyclopropanecarbonitrile (35.0 g, 66 % of theo-
retical). 1-Isobutyrylcyclopropanecarbonitrile (4.00 g, 29.16 mmol)
was then dissolved in tetrahydrofuran (120 mL) in a round-bottom
flask under argon, cooled to 0 °C, and added dropwise to a solution
of a lithium acetylide-ethylenediamine complex in tetrahydrofuran
455,2784.
1-[(2E)-1-Cyclopentyl-1-hydroxy-3-(tributylstannyl)prop-2-en-1-
yl]cyclopropanecarbonitrile (14c): Tetrakis(triphenylphos-
phine)palladium(0) (464 mg, 0.40 mmol) was initially charged under
argon in a flame-dried round-bottom flask, and tetrahydrofuran
(
30 mL) and 1-(1-cyclopentyl-1-hydroxyprop-2-yn-1-yl)cycloprop-
anecarbonitrile 13c (1900 mg, 10.04 mmol) were added. Stirring at
room temperature for 5 min was followed by the addition of tribu-
tyltin hydride (3.24 mL, 12.05 mmol). The resulting reaction mixture
was stirred at room temperature for 1 h and then water was added.
The aqueous phase was repeatedly extracted thoroughly with di-
chloromethane, and the combined organic phases were then dried
with magnesium sulfate, filtered and concentrated under reduced
pressure. By final column chromatography purification of the result-
ing crude product (ethyl acetate/heptane gradient), it was possible
to obtain 1-[(2E)-1-cyclopentyl-1-hydroxy-3-(tributyl-stannyl)prop-2-
(4.11 g, 37.91 mmol, content 85 %, 80 mL). On completion of addi-
tion, the reaction solution was stirred at room temperature for 4 h,
then water was added and the mixture was concentrated under
reduced pressure. The remaining residue was admixed with water
and dichloromethane, and the aqueous phase was extracted re-
peatedly with dichloromethane. The combined organic phases were
dried with magnesium sulfate, filtered and concentrated under re-
duced pressure. By column chromatography purification of the
crude product obtained (ethyl acetate/heptane gradient), 1-(3-
hydroxy-4-methylpent-1-yn-3-yl)cyclopropanecarbonitrile 13a
en-1-yl]cyclopropanecarbonitrile 14c (1650 mg, 34 % of theoretical)
in the form of a colorless oil. H NMR (400 MHz, CDCl ): δ = 6.19 (d,
1
3
1
1
1
H), 6.13 (d, 1 H), 2.63–2.57 (m, 1 H), 1.88–1.80 (br. s, 1 H, OH),
.73–1.58 (m, 4 H), 1.56–1.47 (m, 8 H), 1.37–1.27 (m, 8 H), 1.26 (m,
H), 1.13 (m, 1 H), 1.05–0.98 (m, 2 H), 0.94–0.86 (m, 15 H) ppm.
(
2.59 g, 54 % of theoretical) was isolated as a colorless solid. 1
NMR (400 MHz, CDCl ): δ = 2.48 (s, 1 H), 2.41 (sept, 1 H), 2.19 (br. s,
H
3
1
H, OH), 1.34–1.28 (m, 3 H), 1.21 (m, 1 H), 1.12 (d, 3 H), 1.09 (d, 3
+
+
HRMS-ESI: calcd. for C24H44NOSn [M + H] 481.3137, found
81.3131.
2Z,4E)-6-(1-Cyanocyclopropyl)-3-ethyl-6-hydroxy-7-methyl-
octa-2,4-dienoic Acid (16c): Pent-2-ynecarboxylic acid (1500 mg,
5.2 mmol) was dissolved in concenctrated acetic acid (15 mL),
H) ppm. HRMS-ESI: calcd. for C H NO [M + H]+ 164.2164, found
+
1
0 14
4
1
64.2149.
(
1
-(1-Cyclopentyl-1-hydroxyprop-2-yn-1-yl)cyclopropanecarbo-
nitrile (13c): 1-(Cyclopentylcarbonyl)cyclopropanecarbonitrile
9.00 g, 55.14 mmol) was dissolved in abs. tetrahydrofuran (50 mL)
1
(
finely powdered sodium iodide (6876 mg, 45.8 mmol) was added
and the mixture was stirred at a temperature of 110 °C for 3 h. After
cooling to room temperature, methyl tert-butyl ether (MTBE) and
saturated sodium thiosulfate solution were added. The aqueous
phase was extracted repeatedly with MTBE, and the combined or-
ganic phases were dried with magnesium sulfate, filtered and con-
centrated under reduced pressure. By column chromatography pu-
rification of the resulting crude product (ethyl acetate/heptane gra-
dient), it was possible to obtain (2Z)-3-iodopent-2-enoic acid
in a round-bottom flask under argon and added dropwise to a solu-
tion, cooled to 0 °C, of a lithium acetylide-ethylenediamine complex
(
6.59 g, 71.68 mmol) in tetrahydrofuran (40 mL). On completion of
addition, the reaction solution was stirred at room temperature for
h, then water was added and the mixture was concentrated under
2
reduced pressure. The remaining residue was admixed with water
and CH Cl , and the aqueous phase was extracted repeatedly with
2
2
CH Cl . The combined organic phases were dried with MgSO , fil-
2
2
4
tered and concentrated under reduced pressure. By column chro-
matography purification of the crude product obtained (ethyl acet-
ate/heptane gradient), 1-(1-cyclopentyl-1-hydroxyprop-2-yn-1-yl)-
(
[
2100 mg, 58 % of theoretical) in the form of a colorless solid. 1-
(1E)-3-Hydroxy-4-methyl-1-(tributylstannyl)pent-1-en-3-yl]cyclo-
propane-carbonitrile 14a (390 mg, 0.86 mmol) and (2Z)-3-iodopent-
-enoic acid (194 mg, 0.86 mmol) were dissolved in N,N-dimethyl-
cyclopropanecarbonitrile 13c (2.55 g, 25 % of theoretical) was iso-
2
1
lated as a colorless solid. H NMR (400 MHz, CDCl ): δ = 2.65 (m, 1
3
formamide (4 mL) under argon in a flame-dried round-bottom flask,
dichloro[bis(acetonitrile)palladium(II)] (7 mg, 0.03 mmol) and cop-
per(I) iodide (131 mg, 0.80 mmol) were added and the mixture was
stirred at room temperature for 8 h. After the addition of aqueous
potassium fluoride solution, stirring of the reaction mixture contin-
ued at room temperature for 1 h. The aqueous phase was then
H), 2.47 (s, 1 H), 2.18 (br. s, 1 H, OH), 1.97–1.85 (m, 2 H), 1.73–1.59
(
1
m, 4 H), 1.45–1.39 (m, 1 H), 1.37–1.33 (m, 2 H), 1.30–1.26 (m, 2 H),
.22–1.19 (m, 1 H) ppm.
-[(1E)-3-Hydroxy-4-methyl-1-(tributylstannyl)pent-1-en-3-yl]-
cyclopropanecarbonitrile (14a): Tetrakis(triphenylphosphine)-
1
palladium(0) (198 mg, 0.17 mmol) was initially charged under argon repeatedly extracted thoroughly with dichloromethane, and the
in a flame-dried round-bottom flask, and abs. tetrahydrofuran combined organic phases were then dried with magnesium sulfate,
(
20 mL) and 1-(3-hydroxy-4-methylpent-1-yn-3-yl)cyclopropane-
filtered and concentrated under reduced pressure. By final column
carbonitrile 13a (560 mg, 3.41 mmol) were added. Stirring at room chromatography purification of the resulting crude product (ethyl
temperature for 5 min was followed by the addition of tributyltin acetate/heptane gradient), it was possible to obtain (2Z,4E)-6-(1-
hydride (1.10 mL, 4.12 mmol). The resulting reaction mixture was
stirred at room temperature for 1 h and then water was added. The
aqueous phase was repeatedly extracted thoroughly with CH Cl ,
cyanocyclopropyl)-3-ethyl-6-hydroxy-7-methylocta-2,4-dienoic acid
16c (83 mg, 37 % of theoretical) in the form of a colorless viscous
1
oil. H NMR (400 MHz, CDCl ): δ = 7.63 (d, 1 H), 6.28 (d, 1 H), 5.81
2
2
3
and the combined organic phases were then dried with magnesium
sulfate, filtered and concentrated under reduced pressure. By final
column chromatography purification of the resulting crude product
(s, 1 H), 2.97 (br. s, 1 H, OH), 2.89 (br. s, 1 H, OH), (s, 3 H), 2.43 (q, 2
H), 2.38 (sept, 1 H), 1.33 (m, 1 H) 1.21 (m, 1 H), 1.18 (t, 3 H), 1.09 (d,
3 H), 1.05 (m, 1 H), 0.97 (d, 3 H) ppm. 13C NMR (150 MHz, CDCl3):
δ = 170.5; 162.8; 157.4; 138.2; 125.8; 122.8; 115.9; 74.8; 36.6; 31.5;
(ethyl acetate/heptane gradient), it was possible to obtain 1-[(1E)-
Eur. J. Org. Chem. 2018, 1416–1425
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1422
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
+
2
1
2
7.6; 17.4; 16.5; 12.5; 10.3 ppm. LC-MS (ret.-time, min, [M /log p])
N,N-dimethylformamide (4 mL) under argon in a flame-dried round-
bottom flask, dichlorobis(acetonitrile)-palladium(II) (6 mg,
0.02 mmol) and copper(I) iodide (111 mg, 0.58 mmol) were added
and the mixture was stirred at room temperature for 12 h. After
the addition of aqueous potassium fluoride solution, stirring of the
reaction mixture continued at r.t. for 1 h. The aqueous phase was
then repeatedly extracted thoroughly with CH Cl , and the com-
.04 [263.33/2.15]. HRMS-ESI: calcd. for C15H23NO3+ [M + H]
64.1521, found 264,1558.
+
(
S)- and (R)-(2Z,4E)-6-(1-Cyanocyclopropyl)-3-ethyl-6-hydroxy-7-
methylocta-2,4-dienoic Acid [(S)-16c, (R)-16c]: Enantiomers of
2Z,4E)-6-(1-cyanocyclopropyl)-3-ethyl-6-hydroxy-7-methylocta-2,4-
dienoic acid 16c were also separated carefully via chiral prep. HPLC
to afford (S)-16c, retention time 19.607, [α] = –280 and (R)-16c,
ret. time 23.692, [α]D = +289 (chiral HPLC analysis: Chiralpak IG
(
2
2
bined organic phases were then dried with MgSO , filtered and
2
0
4
D
concentrated under reduced pressure. By final column chromatog-
raphy purification of the resulting crude product (ethyl acetate/
heptane gradient), it was possible to obtain ethyl (2E,4E)-6-(1-
cyanocyclopropyl)-6-cyclopentyl-6-hydroxy-3-(trifluoromethyl)hexa-
2
0
2
9
50/4.6 mm, flow rate 0.6 mL/min, eluent n-heptan/2-propanol
0:10).
Methyl (2E,4E)-6-(1-Cyanocyclopropyl)-6-hydroxy-7-methyl-3- 2,4-dienoate 16t (84 mg, 32 % of theoretical) in the form of a color-
1
(
tri-fluoromethyl)octa-2,4-dienoate (16e): Methyl 4,4,4-trifluoro-
less viscous oil. H NMR (400 MHz, CDCl ): δ = 7.49 (d, 1 H), 6.41 (d,
3
but-2-ynoate (500 mg, 3.01 mmol) was dissolved in concentrated 1 H), 6.31 (s, 1 H), 4.26 (q, 2 H), 2.69–2.63 (m, 1 H), 1.95–1.88 (m, 1
acetic acid (6 mL), finely powdered sodium iodide (1353 mg,
.03 mmol) was added and the mixture was stirred at a temperature
H), 1.68–1.60 (m, 4 H), 1.58 (br. s, 1 H, OH), 1.48–1.26 (m, 6 H) 1.19
(m, 1 H), 1.12 (m, 1 H), 1.03 (m, 1 H) ppm. HRMS-ESI: calcd. for
C H23NO F [M + H] 358.3672, found 358.3679.
18 3 3
9
+
+
of 110 °C for 4 h. After cooling to room temperature, methyl tert-
butyl ether (MTBE) and saturated sodium thiosulfate solution were
added. The aqueous phase was extracted repeatedly with MTBE,
and the combined organic phases were dried with magnesium sulf-
ate, filtered and concentrated under reduced pressure. By column
chromatography purification of the resulting crude product (ethyl
acetate/heptane gradient), it was possible to obtain methyl (2Z)-
Methyl (2Z)-6-(1-Cyanocyclopropyl)-3-ethyl-6-hydroxy-7-meth-
yloct-2-en-4-ynoate (17d): Methyl pent-2-ynoate (14.48 mmol)
was dissolved in concentrated acetic acid (15 mL), powdered so-
dium iodide (43.43 mmol) was added and the mixture was stirred
at a temperature of 110 °C for 3 h. After cooling to room temp.,
methyl tert-butyl ether (MTBE) and saturated sodium thiosulfate so-
lution were added. The aqueous phase was extracted repeatedly
with MTBE, and the combined organic phases were dried with mag-
nesium sulfate, filtered and concentrated under reduced pressure.
By column chromatography purification of the resulting crude prod-
uct (ethyl acetate/heptane gradient), it was possible to obtain
methyl (2Z)-3-iodopent-2-enoate (about 3.0 g, 72 % of theoretical)
in the form of a viscous oil. Copper(I) iodide (5 mg, 0.25 mmol) and
bis(triphenylphosphine)palladium(II) chloride (13 mg, 0.02 mmol)
were initially charged under argon in a flame-dried round-bottom
flask, and toluene (2 mL) and methyl (2Z)-3-iodopent-2-enoate
4
,4,4-trifluoro-3-iodobut-2-enoate (380 mg, 43 % of theoretical) in
the form of a viscous oil. 1-[(1E)-3-hydroxy-4-methyl-1-(tributyl-
stannyl)pent-1-en-3-yl]cyclopropanecarbonitrile 14a (300 mg,
0
.66 mmol) and methyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate
(
(
185 mg, 0.66 mmol) were dissolved in N,N-dimethylformamide
20 mL) under argon in a flame-dried round-bottom flask, tet-
rakis(triphenylphosphine)palladium(0) (76 mg, 0.07 mmol) and cop-
per(I) iodide (94 mg, 0.49 mmol) were added and the mixture was
stirred at room temperature for 16 h. After the addition of aqueous
potassium fluoride solution, stirring of the reaction mixture contin-
ued at room temperature for 1 h. The aqueous phase was then
repeatedly extracted thoroughly with dichloromethane, and the
combined organic phases were then dried with magnesium sulfate,
filtered and concentrated under reduced pressure. By final column
chromatography purification of the resulting crude product (ethyl
acetate/heptane gradient), it was possible to obtain methyl (2E,4E)-
(151 mg, 0.63 mmol) were added. Stirring at room temp. for 10 min
was followed by addition of a solution of 1-(3-hydroxy-4-methyl-
pent-1-yn-3-yl)cyclopropanecarbonitrile 13a (100 mg, 0.63 mmol)
(120 mg, 0.63 mmol) in toluene (3 mL) and of diisopropylamine
(0.26 mL, 1.88 mmol). The resulting reaction mixture was stirred at
room temp. for 3 h and then water was added. The aqueous phase
was extracted repeatedly with dichloromethane. The combined or-
ganic phases were dried with magnesium sulfate, filtered and con-
centrated under reduced pressure. By final column chromatography
purification of the crude product obtained (using an ethyl acetate/
heptane gradient), methyl (2Z)-6-(1-cyanocyclopropyl)-3-ethyl-6-
hydroxy-7-methyloct-2-en-4-ynoate 17d (149 mg, 78 % of theoreti-
6
2
-(1-cyanocyclopropyl)-6-hydroxy-7-methyl-3-(trifluoromethyl)octa-
,4-dienoate 16e (53 mg, 25 % of theoretical) in the form of a color-
1
less viscous oil. H NMR (400 MHz, CDCl ): δ = 7.44 (d, 1 H), 6.36 (d,
3
1
1
H), 6.32 (s, 1 H), 3.80 (s, 3 H), 2.42 (sept, 1 H), 1.63 (br. s, 1 H, OH),
.27 (m, 1 H) 1.22 (m, 1 H), 1.12 (m, 1 H), 1.05 (d, 3 H), 0.99 (m, 1
+
+
H), 0.97 (d, 3 H) ppm. HRMS-ESI: calcd. for C H NO F [M + H]
1
5
19
3 3
3
18.3043, found 318,3027.
1
cal) was isolated in the form of a colorless oil. H NMR (400 MHz,
CDCl ): δ = 6.02 (s, 1 H), 3.72 (s, 3 H), 2.63 (br. s, 1 H, OH), 2.28 (q,
3
Ethyl (2E,4E)-6-(1-Cyanocyclopropyl)-6-cyclopentyl-6-hydroxy-
2
1
H), 1.99 (m, 1 H), 1.88 (m, 1 H), 1.78 (m, 1 H), 1.50–1.41 (m, 4 H),
.34 (m, 1 H), 1.24 (m, 1 H), 1.15 (t, 3 H), 1.06 (t, 6 H) ppm. C NMR
3
-(trifluoromethyl)hexa-2,4-dienoate (16t): Ethyl 4,4,4-trifluoro-
1
3
but-2-ynoate (500 mg, 3.01 mmol) was dissolved in concentrated
acetic acid (6 mL), finely powdered sodium iodide (1353 mg,
(
150 MHz, CDCl ): δ = 165.3; 139.6; 124.1; 121.6; 96.4; 85.4; 63.4;
3
5
1.4; 37.8; 31.9; 18.3; 17.5; 13.9; 12.7; 11.1 ppm. LC-MS (ret. time,
9
.03 mmol) was added and the mixture was stirred at a temperature
+
+
min, [M /log p]) 2.84 [275.15/1.37]. HRMS-ESI: calcd. for C H NO
16
22
3
of 110 °C for 4 h. After cooling to r.t., methyl tert-butyl ether (MTBE)
and saturated sodium thiosulfate solution were added. The aqueous
phase was extracted repeatedly with MTBE, and the combined or-
+
[
M + H] 276.3431, found 276,3408.
Ethyl (2Z)-6-(1-Cyanocyclopropyl)-3-cyclopropyl-6-hydroxy-7-
methyloct-2-en-4-ynoate (17i): Ethyl 3-cyclopropylprop-2-ynoate
(2.00 g, 14.48 mmol) was dissolved in concentrated acetic acid
(15 mL), finely powdered sodium iodide (6.51 g, 43.43 mmol) was
added and the mixture was stirred at a temperature of 110 °C for
3 h. After cooling to room temperature, methyl tert-butyl ether
ganic phases were dried with MgSO , filtered and concentrated.
4
under reduced pressure. By column chromatography purification of
the resulting crude product (ethyl acetate/heptane gradient), it was
possible to obtain ethyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate
(380 mg, 45 % of theoretical) in the form of a viscous oil. 1-[(2E)-1-
Cyclopentyl-1-hydroxy-3-(tributylstannyl)prop-2-en-1-yl]cycloprop- (MTBE) and saturated sodium thiosulfate solution were added. The
anecarbonitrile 14c (350 mg, 0.73 mmol) and ethyl (2Z)-4,4,4- aqueous phase was extracted repeatedly with MTBE, and the com-
trifluoro-3-iodobut-2-enoate (214 mg, 0.73 mmol) were dissolved in bined organic phases were dried with magnesium sulfate, filtered
Eur. J. Org. Chem. 2018, 1416–1425
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1423
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
+
+
and concentrated under reduced pressure. By column chromatogra- H) ppm. HRMS-ESI: calcd. for C H NOSn [M + H] 468.3038, found
23 43
phy purification of the resulting crude product (ethyl acetate/hept-
ane gradient), it was possible to obtain ethyl (2Z)-3-cyclopropyl-3-
iodoacrylate (3.01 g, 74 % of theoretical) in the form of a viscous
oil. Copper(I) iodide (47 mg, 0.25 mmol) and bis(triphenylphos-
phine)palladium(II) chloride (129 mg, 0.18 mmol) were initially
charged under argon in a flame-dried round-bottom flask, and tolu-
ene (6 mL) and ethyl (2Z)-3-cyclopropyl-3-iodoacrylate (326 mg,
468.3029.
Methyl (2E,4E)-6-(1-Cyanocyclobutyl)-6-hydroxy-7-methyl-3-
trifluoromethyl)octa-2,4-dienoate (21e): Methyl 4,4,4-trifluoro-
but-2-ynoate (500 mg, 3.01 mmol) was dissolved in concentrated
acetic acid (6 mL), finely powdered sodium iodide (1353 mg,
(
9.03 mmol) was added and the mixture was stirred at a temperature
of 110 °C for 4 h. After cooling to room temp., MTBE and saturated
sodium thiosulfate solution were added. The aqueous phase was
extracted repeatedly with MTBE, and the combined organic phases
were dried with magnesium sulfate, filtered and concentrated un-
der reduced pressure. By column chromatography purification of
the resulting crude product (ethyl acetate/heptane gradient), it was
possible to obtain methyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate
1.23 mmol) were added. Stirring at room temperature for 10 min
was followed by the dropwise addition of a solution of 1-(3-
hydroxy-4-methylpent-1-yn-3-yl)cyclopropanecarbonitrile 13a
(200 mg, 1.23 mmol) in toluene (9 mL) and of diisopropylamine
(0.34 mL, 2.45 mmol). The resulting reaction mixture was stirred at
room temp. for 3 h and then water was added. The aqueous phase
was extracted repeatedly with CH Cl . The combined organic pha-
2
2
(380 mg, 43 % of theoretical) in the form of a viscous oil. 1-[(1E)-3-
ses were dried with MgSO , filtered and concentrated under re-
4
Hydroxy-4-methyl-1-(tributylstannyl)pent-1-en-3-yl]cyclobutane-
carbonitrile 20a (300 mg, 0.64 mmol) and methyl (2Z)-4,4,4-tri-
fluoro-3-iodobut-2-enoate (179 mg, 0.64 mmol) were dissolved in
N,N-dimethylformamide (5 mL) under argon in a flame-dried round-
bottom flask, dichlorobis(acetonitrile)palladium(II) (5 mg,
duced pressure. By final column chromatography purification of the
crude product obtained (using an ethyl acetate/heptane gradient),
ethyl (2Z)-6-(1-cyanocyclopropyl)-3-cyclopropyl-6-hydroxy-7-meth-
yloct-2-en-4-ynoate 17i (190 mg, 47 % of theoretical) was isolated
1
in the form of a colorless oil. H NMR (400 MHz, CDCl ): δ = 6.17 (s,
3
0.02 mmol) and copper(I) iodide (98 mg, 0.51 mmol) were added
1
H), 4.17 (q, 2 H), 2.46 (sept, 1 H), 2.01 (br. s, 1 H, OH), 1.68 (m, 1
H), 1.47 (m, 1 H), 1.39 (m, 1 H), 1.30 (m, 1 H), 1.28 (t, 3 H), 1.21 (m,
H), 1.12 (d, 3 H), 1.09 (d, 3 H), 0.87 (m, 4 H) ppm. HRMS-ESI: calcd.
and the mixture was stirred at room temp. for 12 h. After adding
KF solution, stirring of the reaction mixture continued at room tem-
perature for 1 h. The aqueous phase was then repeatedly extracted
with diethyl ether, and the combined organic phases were then
dried with magnesium sulfate, filtered and concentrated under re-
duced pressure. By final column chromatography purification of the
resulting crude product (ethyl acetate/heptane gradient), it was
possible to obtain methyl (2E,4E)-6-(1-cyanocyclobutyl)-6-hydroxy-
1
+
+
for C H NO [M + H] 302.3803, found 302.3798.
18
24
3
1
-(3-Hydroxy-4-methylpent-1-yn-3-yl)cyclobutanecarbonitrile
(19a): 1-Isobutyrylcyclobutanecarbonitrile (9.00 g, 60.0 mmol) was
dissolved in tetrahydrofuran (50 mL) in a round-bottom flask under
argon and added dropwise to a solution, cooled to 0 °C, of a lithium
acetylide-ethylenediamine complex (7.92 g, 77.0 mmol, content
7-methyl-3-(trifluoromethyl)octa-2,4-dienoate 21e (46 mg, 21 % of
1
9
0 %) in tetrahydrofuran (20 mL). On completion of addition, the
theoretical) in the form of a colorless oil. H NMR (400 MHz, CDCl ):
3
reaction solution was stirred at room temp. for 2 h, then water was δ = 7.53 (d, 1 H), 6.31 (s, 1 H), 6.30 (d, 1 H), 3.80 (s, 3 H), 2.65 (m, 1
added and the mixture was concentrated under reduced pressure.
The remaining residue was admixed with water and dichloro-
methane, and the aqueous phase was extracted repeatedly with
dichloromethane. The combined organic phases were dried with
H), 2.40 (m, 1 H), 2.29 (m, 2 H), 2.11 (m, 1 H), 1.80 (m, 1 H), 1.73 (br.
s, 1 H, OH), 1.30 (m, 1 H), 0.94 (d, 3 H), 0.91 (d, 3 H) ppm. HRMS-
+
+
ESI: calcd. for C H NO F [M + H] 331.3303, found 331.3295.
16
20
3 3
Methyl (2Z)-6-(1-Cyanocyclobutyl)-6-hydroxy-7-methyl-3-tri-
fluoro-methyloct-2-en-4-ynoate (22e): Methyl 4,4,4-trifluorobut-
MgSO , filtered and concentrated under reduced pressure. By chro-
4
matography purification of the crude product obtained (ethyl acet-
ate/heptane gradient), 1-(3-hydroxy-4-methylpent-1-yn-3-yl)cyclo-
2-ynoate (500 mg, 3.01 mmol) was dissolved in concentrated acetic
acid (6 mL), finely powdered sodium iodide (1353 mg, 9.03 mmol)
was added and the mixture was stirred at a temperature of 110 °C
for 4 h. After cooling to room temperature, MTBE and saturated
sodium thiosulfate solution were added. The aqueous phase was
extracted repeatedly with MTBE, and the combined organic phases
were dried with magnesium sulfate, filtered and concentrated un-
der reduced pressure. By column chromatography purification of
butanecarbonitrile 19a (2.50 g, 24 % of theoretical) was isolated as
1
a colorless waxy solid. H NMR (400 MHz, CDCl ): δ = 2.70 (m, 2 H),
3
2
.64 (s, 1 H), 2.36 (m, 2 H), 2.24 (m, 2 H), 2.12 (br. s, 1 H, OH), 1.08
+
+
(d, 3 H), 0.98 (d, 3 H) ppm. HRMS-ESI: calcd. for C H NO [M + H]
11 16
1
78.2430, found 178.2404.
1
-[(1E)-3-Hydroxy-4-methyl-1-(tributylstannyl)pent-1-en-3-yl]-
cyclobutanecarbonitrile (20a): Tetrakis(triphenylphosphine)palla- the resulting crude product (ethyl acetate/heptane gradient), it was
dium(0) (261 mg, 0.23 mmol) was initially charged under argon in
a flame-dried round-bottom flask, and tetrahydrofuran (20 mL) and
possible to obtain methyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate
(380 mg, 43 % of theoretical) in the form of a viscous oil. Copper(I)
iodide (6 mg, 0.03 mmol) and bis(triphenylphosphine)palladium(II)
1
-(3-hydroxy-4-methylpent-1-yn-3-yl)cyclobutanecarbonitrile
(1000 mg, 5.64 mmol) were added. Stirring at room temp. for 5 min chloride (18 mg, 0.03 mmol) were initially charged under argon in
was followed by the addition of tributyltin hydride (1.82 mL,
.77 mmol). The resulting reaction mixture was stirred at room
temp. for 1 h and then water was added. The aqueous phase was
repeatedly extracted with dichloromethane, and the combined or-
a flame-dried round-bottom flask, and toluene (6 mL) and methyl
(2Z)-4,4,4-trifluoro-3-iodobut-2-enoate (237 mg, 0.85 mmol) were
added. Stirring at room temperature for 10 min was followed by
the dropwise addition of a solution of 1-(3-hydroxy-4-methylpent-1-
6
ganic phases were then dried with magnesium sulfate, filtered and yn-3-yl)cyclobutanecarbonitrile 19a (150 mg, 0.85 mmol) in toluene
concentrated under reduced pressure. By final column chromatog- (7 mL) and of diisopropylamine (0.36 mL, 2.54 mmol). The resulting
raphy purification of the resulting crude product (ethyl acetate/
heptane gradient), it was possible to obtain 1-[(1E)-3-hydroxy-4-
reaction mixture was stirred at room temp. for 3 h and then water
was added. The aqueous phase was extracted repeatedly with di-
chloromethane. The combined organic phases were dried with
methyl-1-(tributylstannyl)-pent-1-en-3-yl]cyclobutane-carbonitrile
1
2
0a (1.40 g, 53 % of theoretical) in the form of a colorless oil. H
MgSO , filtered and concentrated under reduced pressure. By final
4
NMR (400 MHz, CDCl ): δ = 6.23 (d, 1 H), 5.98 (d, 1 H), 2.78 (m, 1 column chromatography of the crude product obtained (using an
3
H), 2.30 (m, 1 H), 2.22 (m, 3 H), 2.02 (m, 1 H), 1.77 (m, 1 H), 1.67 (br.
s, 1 H, OH), 1.53–1.45 (m, 6 H), 1.35–1.27 (m, 6 H), 0.96–0.85 (m, 21
ethyl acetate/heptane gradient), methyl (2Z)-6-(1-cyanocyclobutyl)-
6-hydroxy-7-methyl-3-trifluoromethyloct-2-en-4-ynoate 22e
Eur. J. Org. Chem. 2018, 1416–1425
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1424
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
(
147 mg, 53 % of theoretical) was isolated in the form of a colorless
D. D. Woolard, D. O. Wilson, D. C. Leep, G. D. Venburg, WO2016/187370,
2016; c) G. Wang, R. Hopkins, D. C. Leep, D, D. Woolard, G. D. Venburg,
WO2016/187370, 2016.
1
oil. H NMR (400 MHz, CDCl ): δ = 6.71 (s, 1 H), 3.83 (s, 3 H), 2.79–
2
1
3
.68 (m, 2 H), 2.47 (br. s, 1 H, OH), 2.41 (m, 4 H), 2.32 (m, 2 H),
.94 (m, 1 H), 1.13 (d, 3 H), 1.03 (d, 3 H) ppm. HRMS-ESI: calcd. for
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Received: December 21, 2017
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