Insights into the in Vitro and in Vivo SAR of Abscisic Acid – Exploring Unprecedented Variations of the Side Chain via Cross-Coupling-Mediated Syntheses

Article abstract of DOI:10.1002/ejoc.201701687

Novel analogues of the plant hormone abscisic acid (ABA) were designed and prepared to explore the impact that modifications of its terpenoid side chain have on receptor affinity and in vivo efficacy against drought stress in selected crops. Their efficient and versatile synthesis proceeded via Stille or Sonogashira couplings, shortening the synthetic route significantly. In line with molecular modelling and X-ray crystallography studies novel ABA-derivatives with small alkyl, cycloalkyl or haloalkyl substituents showed strong effects in vitro and in vivo against drought stress in crops, particularly canola and wheat.

Full text of DOI:10.1002/ejoc.201701687

DOI: 10.1002/ejoc.201701687
Full Paper
Plant Stress Efficacy
Insights into the in Vitro and in Vivo SAR of Abscisic Acid –
Exploring Unprecedented Variations of the Side Chain via
Cross-Coupling-Mediated Syntheses
Jens Frackenpohl,*[a] Erwin Grill,*[e] Guido Bojack,[a] Rachel Baltz,[c] Marco Busch,[a]
Jan Dittgen,[a] Jana Franke,[a] Jörg Freigang,[b] Susana Gonzalez,[a] Ines Heinemann,[a]
Hendrik Helmke,[a] Martin Hills,[a] Sabine Hohmann,[a] Pascal von Koskull-Döring,[a]
Jochen Kleemann,[a] Gudrun Lange,[a] Stefan Lehr,[a] Thomas Müller,[a] Elisabeth Peschel,[a]
Fabien Poree,[d] Dirk Schmutzler,[a] Arno Schulz,[a] and Lothar Willms,[a]Christian Wunschel
Abstract: Novel analogues of the plant hormone abscisic acid Sonogashira couplings, shortening the synthetic route signifi-
(ABA) were designed and prepared to explore the impact that cantly. In line with molecular modelling and X-ray crystallogra-
modifications of its terpenoid side chain have on receptor affin- phy studies novel ABA-derivatives with small alkyl, cycloalkyl or
ity and in vivo efficacy against drought stress in selected crops. haloalkyl substituents showed strong effects in vitro and in vivo
Their efficient and versatile synthesis proceeded via Stille or against drought stress in crops, particularly canola and wheat.
Introduction
mental stress such as drought or salinity stress. Studies on ab-
scisic acid (ABA) mediated signaling have progressed rapidly
since the discovery of RCAR/(PYR/PYL) receptor proteins as solu-
ble ABA-receptors.[5] These crucial findings have granted new
insights into the structure activity relationship of ABA. The ca-
tabolism of ABA has been well explored prior to identifying
the RCAR/(PYR/PYL) receptor proteins. Its principal pathway of
oxidation is through monooxygenase-mediated hydroxylation,
i.e. by the CYP707A-family, of the 8′-methyl group affording 8′-
hydroxy-abscisic acid which is in equilibrium with the cyclized
form phaseic acid.[6] Thus, various chemical modifications of the
cyclohexenone headgroup of ABA have been investigated
within recent years. It has been shown that metabolism-
resistant analogues altered at position C8′ were more stable
towards oxidation than ABA itself with 8′-ethynyl-ABA 2a,
8′-vinyl-ABA 2b and 8′-trifluoromethyl-ABA 2c exhibiting prom-
ising activity in vitro (Figure 1).[7]
Abiotic stress adversely affects crop production in various parts
of the world, decreasing average yields for most of the crops
significantly.[1] Among various abiotic stresses affecting agricul-
tural production, drought stress is considered to be the main
source of crop losses around the globe. There are various strate-
gies for reducing the impact that drought has on crop yield.
These include exploiting beneficial effects of crop protection
agents,[2] developing drought tolerant crops through transgenic
approaches or breeding,[3] but also exploring novel chemical
active ingredients inspired by naturally occurring plant hor-
mones. Abscisic acid [1, (S)-ABA], a chiral sesquiterpenoid first
discovered in the 1960s,[4] is an important plant hormone that
regulates developmental signals such as seed maturation or
dormancy and mediates the adaptation of plants to environ-
[a] Research & Development, Weed Control, Bayer AG, CropScience Division,
Industriepark Höchst
Geb. G836, 65926 Frankfurt am Main, 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
14 Impasse Pierre Baizet, 69263 Cedex 09, Lyon France
[d] Bayer SAS, Toxicology, Toxicology Research,
355, rue Dostoievski, CS 90153 Valbonne, 06906 Sophia-Antipolis Cedex,
France
Figure 1. The structure of (S)-abscisic acid (1) with formal atom numbering
and selected closely related analogues with modified headgroup.
[e] Lehrstuhl für Botanik, Wissenschaftszentrum Weihenstephan,
Technische Universität München
Furthermore, tetralone analogues of ABA have been pre-
pared,[8] as well as 3′-sulfanyl-ABA analogues,[9] ABA-analogues
with modified 7′-methyl group[10] or 3′-methyl- and 3′-halo-
alkyl-ABA.[11] Although varying the cyclohexenone moiety has
attracted significant synthetic interest, surprisingly few syn-
Emil-Ramann-Straße 4, 85354 Freising
E-mail: grill@wzw.tum.de
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201701687.
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thetic changes have been made regarding the (E,Z)-diene side 4 could then be transformed selectively into (E)-configured key
chain of ABA. 6-nor-ABA 2d, which lacks ABA's C6 methyl group, intermediate 5 via Pd(PPh3)2Cl2-mediated hydrostannylation.
has shown significantly reduced activity,[12] indicating the po-
Coupling of (E)-stannane 5 with a suitable freshly prepared
tential impact of side chain modifications on receptor binding
and in vivo efficacy. Herein, we outline results of our in-depth
evaluation of ABA derivatives with modified diene side chains,
and we discuss the structural basis of the effects of ABA deriva-
tives on drought stress observed within our in vivo-studies.
vinyl iodide 6a,b,e,q,r via an optimized Stille-coupling protocol
using Pd(MeCN)2Cl2 and CuI in N,N-DMF at slightly elevated
temperatures (40–50 °C) afforded protected ABA-analogues
7a,b,e,q,r.[16] Subsequent HCl-mediated deprotection yielded
ABA-desired analogues 8a,b,e,q,r. (Z)-Vinyl iodides 6a,b,e,q,r
were prepared starting from substituted alkynes within two
steps via a carboxylation and subsequent hydroiodination.[17] In
our hands, this methodology proved to be clearly superior to-
wards related approaches using hydrozirconation[18] and a Ne-
gishi-type cross-coupling of 9 to establish the (E,Z)-diene side
chain (e.g. 7q, yield < 10 %). Using (E)-stannane 5 as the key
intermediate thus allowed us to proceed without protecting the
tertiary alcohol group, while this proved to be mandatory for
preparing 7q via Zr-intermediate 9. As a result, ABA-analogues
8a,b,e,q,r could be prepared in 5 linear steps with overall yields
in the range of 30–40 %. Furthermore, upscaling of the Stille
coupling to a 30 mmol scale was feasible, allowing us to pre-
pare 8b,e,q,r in multigram quantities. Alkynol 4 also served as
key intermediate to prepare ABA-related enynes 10b,c,e,q,r via
Sonogashira coupling and subsequent acetal deprotection.
Both enantiomers of enyne 10c could be obtained via prep.
chiral HPLC, whereas the corresponding free enyne carboxylic
acid 10a was obtained via cleavage of the parent ester to avoid
formation of a butenolide.
Results and Discussion
Chemistry
Several syntheses of ABA have been reported,[13] however, not
all of them match the criteria crucial for efficiently preparing a
broad range of analogues for in vivo structure-activity relation-
ship (SAR) studies in greenhouse or field trials. Namely, low
number of overall steps, versatility, satisfying yield and selective
formation of the (E,Z)-diene side chain. Likewise, most synthe-
ses starting from commercially available 4-oxoisophorone 3
proceeded via hydride-mediated reduction of an intermediate
enyne system featuring i) additional steps due to re-oxidation
of the terminal alcohol moiety formed or incorporated, as well
as ii) insufficient (E)-selectivity as major drawbacks.[14] We thus
decided to explore a novel, more flexible approach using Stille
coupling as the key step. Protecting the less hindered carbonyl
group of 4-oxoisophorone 3 with 2,3-butanediol,[15] followed
by direct alkynylation using ethylenediamine lithium acetylide
complex afforded crystalline alkynol 4 in good yield (Scheme 1).
Scheme 2. Synthesis of ABA-analogues with R3
= haloalkyl. a) Ph3P=
CHC(O)CH3, Et2O, room temp., 20 h; b) CH3C(O)CH2CO2Et, tBuOK, Et2O, 0 °C
to room temp.; c) H3Mo12O48P, MoO3, CuSO4, H2O, air, toluene, reflux, 4 d; d)
2,3-butanediol, (H3CO)3CH, TsOH, dioxane; e) Li-acetylide ethylene diamine
complex, THF, room temp.; f) Bu3SnH, Pd(PPh3)2Cl2, THF, room temp.; g)
6c,q,r,u, Pd(MeCN)2Cl2, CuI, DMF, 50 °C; h) acetone, aq. HCl, room temp.; i)
Pd(PPh3)2Cl2, CuI, 6c,q,r,u, diisopropylamine, toluene, room temp.; j) acetone,
aq. HCl, room temp.
Scheme 1. Cross coupling mediated syntheses of ABA-analogues. a) 2,3-
butanediol, (H3CO)3CH, TsOH, dioxane; b) Li-acetylide ethylene diamine com-
plex, THF, room temp.; c) Bu3SnH, Pd(PPh3)2Cl2, THF, room temp.; d) 6a, b, e,
q, r, Pd(MeCN)2Cl2, CuI, DMF, 50 °C; e) acetone, aq. HCl, room temp.; f)
Et3SiOTf, 2,6-lutidine, CH2Cl2, 0 °C to room temp.; g) Cp2ZrHCl, CH2Cl2, room
temp.; h) 1. Pd(PPh3)2Cl2, DIBAL-H, ZnCl2, THF; i) TBAF, CH2Cl2; j) 1.
Pd(PPh3)2Cl2, CuI, diisopropylamine, toluene, room temp.; k) acetone, aq. HCl,
room temp.; l) chiral prep. HPLC.
With this robust synthetic route in hand we could prepare a
broad range of ABA-analogues with modified side chain (entries
3–33, Table 1). To further broaden our SAR-study we aimed to
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introduce these specific novel side chain variations into meta- 30a–c,t starting from dimethyl-benzoquinone or 2,6-dimethyl-
bolically stable ABA-analogues 2a–c. Having chosen C8′-CF3- phenol (Scheme 4).[21] Key intermediate 28 was formed in three
ABA 2c as a representative core structure, we decided to re- steps via acetal formation, cyclopropanation and subsequent
investigate and optimize the initial synthetic approaches to- lithium acetylide-mediated alkynylation in good yield, albeit as
wards 2c to provide a scalable synthesis (Scheme 2).[7e] With a mixture of diastereomers. 28 then served as starting point
particular emphasis on reducing the number of steps, we chose for Sonogashira and Stille coupling reactions affording desired
a direct allylic oxidation of 11 and its difluoromethyl analogue target compounds 31e and 30a–c,t. (Scheme 4). In accordance
21 to prepare haloalkyl-oxoisophorones 12 and 22.[19] Starting with results obtained for ABA-analogues 8a–r Pd(MeCN)2Cl2-
from a suitable fluorinated acetone cyclohexenones 11, 21 mediated Stille coupling proved again to be superior over
were prepared in two steps. MoO3/P-molybdate-mediated aero- hydride-based approaches via reduction of related alkynes 31
bic oxidation of 11, 21 using CuSO4 as an additive proved to and over other (E)-selective cross coupling reactions. As shown
be the best method in our hands affording 12, and 22 respec- for fluorinated ABA-derivatives 16c,q,r,u and 26c desired dienes
tively, in satisfying yields (65–75 %). In line with the transforma- 30a–c,t. and enyne 31e were obtained as mixtures of diastereo-
tions of 4-oxoisophorone 3 outlined in Scheme 1, alkynols 13 mers (80:20 ratio). With this additional set of double-modified
and 23, as well as stannanes 14 and 24 were then prepared ABA-analogues in hand, we had broadened the basis of our
as intermediates. Pd(MeCN)2Cl2-mediated Stille coupling with SAR-study further to assess the impact of side chain modifica-
subsequent acidic acetal cleavage afforded desired target com- tions at position C6 (R1).
pounds 16c,q,r,u and 26c, again in good yield. C8′-haloalkyl
ABA-derivatives 16c and 26c could thus be prepared in 8 steps
from triflouro or difluoro acetone in overall yields from 4–10 %
as mixtures of diastereomers. Typically, diastereomeric ratios
(dr) obtained were in the range of 88:12 (16c) to 62:38 (26c).
Likewise, haloalkyl-ABA related enynes 17c,q,r,u and 27c were
obtained via Sonogashira coupling and subsequent acetal de-
protection.
Scheme 4. Synthesis of C5′-C8′-cyclopropyl ABA-analogues. a) 2,3-butanediol,
(H3CO)3CH, pTsOH, dioxane; b) NaH, Me3SO+I–, DMF, room temp.; c) Li-acet-
ylide ethylene diamine complex, THF, room temp.; d) 2,3-butanediol,
PhI(OAc)2; e) Pd(PPh3)2Cl2, CuI, diisopropylamine, toluene, room temp.; f)
acetone, aq. HCl, room temp.; g) Bu3SnH, Pd(PPh3)2Cl2, THF, room temp.; h)
6a–c,t, Pd(MeCN)2Cl2, CuI, DMF, 50 °C; i) acetone, aq. HCl, room temp.
Scheme 3. Synthesis of ABA-analogues with cycloalkenyl and aryl moieties
stabilizing the terpenoid side chain. a) NIS, CH2Cl2; b) 1. Pd(MeCN)2Cl2, CuI,
In Vitro and in Vivo Structure–Activity Relationship Study
DMF, 50 °C; 2. aq. HCl, acetone; c) 1. Pd(PPh3)4, NaHCO3, dioxane, H2O, reflux;
2. acetone, aq. HCl, room temp.
A detailed analysis of the published structure of the ABA recep-
tor isoform RCAR12 and ABI1 in complex with ABA (3kdj), and
The utility of stannane 5 as a versatile key intermediate could the respective structure of RCAR11 in complex with ABA
be demonstrated further by preparing cyclized ABA-analogues (3kdi)[22a] indicated that there might be additional space in the
19 and 20a–b via Stille and Suzuki couplings. By converting 5 binding pocket into which the C6 methyl group of ABA is di-
into (E)-vinyl iodide 18 a Pd(PPh3)4-mediated Suzuki coupling rected (R1). Hence, we decided to explore this pocket by varying
could be used to prepare 20a without need for further protect- the size and the shape of the R1-substituents. Mainly hydro-
ing groups (Scheme 3). The free carboxylic acid moiety was phobic substituents were chosen since the protein pocket is
tolerated, and ABA-analogue 20a could be obtained in 58 % lined mostly with hydrophobic side chains suggesting that
yield (3 steps from 5) enabling us to evaluate the impact of a small hydrophobic moieties such as ethyl or cyclopropyl should
cyclohexenyl (19) or phenyl group (20a–b) as rigid replace- fit into the binding pocket whilst maintaining or even increas-
ments for the (Z)-configured double bond (Scheme 3). Addi- ing the affinity of the compounds to the target receptor. Like-
tionally, we applied our cross-coupling approach to a further wise, rigidified ABA-analogues 19 and 20a–b were prepared to
ABA analogue bearing a stabilized headgroup that exhibited explore the limits of this particular binding pocket. All ABA-
confirmed in vitro activity, namely C5′-C8′-cyclopropyl ABA.[20] analogues prepared were tested for target affinity, as well as for
Following the reaction sequences outlined in Scheme 1– beneficial effects in vivo under drought stress conditions upon
Scheme 2 we prepared C5′-C8′-cyclopropyl ABA analogues foliar application on plants. Wheat and canola were chosen as
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model plants for monocotyledonous and dicotyledonous spe-
cies, whereas in vitro tests were carried out at the ABA-receptor
system RCAR11 in Arabidopsis thaliana. All in vivo tests outlined
herein were carried out with several replicates using dose rates
based on our long-term experience in crop protection research.
The very high sequence identity between ABA receptors from A.
thaliana and canola was expected to lead to a good correlation
between in vitro binding at RCAR11 in A. thaliana and in vivo
effects measured in canola. However, subtle differences in ABA
binding sites between RCAR11 from A. thaliana and corre-
sponding ABA receptor isoforms in wheat may lead to different
in vivo efficacy in wheat. Based on our experiences regarding
herbicidal antidotes being used as Safeners[23] we also laid em-
phasis on exploring the impact of esters on in vivo efficacy.
Corresponding in vitro activities of esters were measured in the
presence of pig liver esterase to ensure consistent assessment
of structural changes at R1 and R3.
Table 1. SAR-results of selected diene-analogues of ABA.
Substituents[a]
In vitro activity
In vivo efficacy
against drought
stress
ABI1-
(AtRCAR11)[b]
Entry No.
R1
R2 R3
Activity
[%] 5 μ
pI50
Wheat[d] [250 g/ha][c]
Canola[d]
M
1
2
3
4
5
6
7
8
(S)-1 Me
(R)-1 Me
H
H
H
Me 100
Me 97
Me 99
7.1
6.8
5.8
+++
+++
+++
+++
+++
+++
+++
++++
+++
+++
++
++
++
0
++
++
+
++
0
0
0
+++
++++
++++
++++
++++
++++
0
++
0
0
++
8a
8b
8c
Et
Et
Et
Et Me 99[b]
Me Me 100[b]
nPr Me 95
5.9[b] ++++
6.1[b] +++
8d
8e
8f
8g
8h
8i
Et
5.7
++
nPr
nPr
nBu
Et Me 95[b]
H
H
5.8[b] +++
Me 97
Me 96
5.9
+++
+
+
9
5.6
In accordance with our initial expectations, in vitro and in
vivo efficacy correlated nicely, particularly in the case of in vitro
activity of A. thaliana and the in vivo efficacy in canola. In addi-
tion, there is an excellent agreement between in vitro activity
and the in vivo efficacies of acids and their respective esters
demonstrating the robustness of the test systems. Interestingly,
the stereoconfiguration at carbon C1′ did not have a significant
impact on in vivo efficacy as both enantiomers of ABA (Entries
1 and 2; Table 1) showed comparable effects. These findings
prompted us to use compounds 8, 16 and 26 as racemates
or diastereomeric mixtures in our SAR study. However, trans-
configured ABA (E,E)-1 (entry 34, Table 1) did not show benefi-
cial effects in canola and wheat emphasizing the importance of
an in-depth SAR of ABA-side chain variations.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
nBu Et Me 97[b]
5.6[b]
5.2
nHex
iPr
H
Me 83
+
8j
Et Me 96[b]
5.4[b] +++
8k
8l
sBu
H
Me 83
4.8
++
sBu Me Me 81[b]
4.7[b] +++
4.7[b] ++
n.d.[b] +++
8m
8n
8o
8p
8q
8r
sPent Et Me 80[b]
tBu
Et Me 42[b]
H
iPent
Me 10
n.d.
+
0
iPent Et Me 12[b]
CF3 Et Me 96[b]
CF3 Me Me 96[b]
n.d.[b]
5.9[b] ++++
5.9[b] ++++
5.7[b] +++
8s
8t
CF3
cPr
cPr
cBu
H
H
Me 98[b]
Me 100
6.6
6.2[b] ++++
5.9 +++
++++
8u
8v
8w
8x
8y
8z
16c
16d
16r
16u
26c
Et Me 100[b]
H
Me 100
cPent Et Me 41[b]
n.d.[b] ++
cHex Et Me 13[b]
Ph
Furyl
Et
n.d.[b]
n.d.[b]
4.8
+
+
0
Et Me 34[b]
Firstly, strong effects against drought stress were observed
for compounds with C6-ethyl moiety, i.e. 8a–d (entries 3–6,
Table 1), in line with good binding affinity to RCAR11. Interest-
ingly, esters and parent acid showed consistent effects in canola
and wheat. Derivatives with longer unbranched alkyl side
chains, such as n-propyl (8e–f, entries 7 and 8), n-butyl (8g–h,
entries 9, 10) and n-hexyl (8i, entry 11) showed a moderate
decline in in vitro activity due to increasing chain length,
whereas in vivo efficacy dropped significantly. Electron-with-
drawing groups at C6 were also well tolerated since ABA-ana-
logues with C6-CF3-group, i.e. 8q–s (entries 10–21, Table 1) also
showed strong effects in vivo with 8q exhibiting better efficacy,
both in wheat and canola, than ABA 1 (entry 1, Table 1). Whilst
small branched alkyl substituents (e.g. isopropyl 8j and sec-
butyl 8k–l) were still active in vitro and in vivo, substituents
with a quaternary carbon at C6 (e.g. tert-butyl 8n and isopentyl
8o,p) led to a loss in activity suggesting a clash within the
narrow pocket. Likewise, similar effects could be observed upon
H
Me 45
Me CF3 88[b]
5.3[b] +++
5.2[b] +++
5.4[b] +++
5.5[b] +++
4.9[b] ++
Et
nPr CF3 82[b]
++
++
++
++
CF3 Me CF3 86[b]
cPr
Et
Et CF3 95[b]
Me CHF2 80[b]
H
(E,E)-1 Me
Me 80
6.1
0
0
[a] cPr = cyclopropyl, sBu = but-2-yl, cPent = cyclopentyl, sPent = pent-2-yl,
iPent = 2-methylbut-2-yl, cHex = cyclohexyl. [b] Target affinity of esters was
measured after addition of pig liver esterase. [c] Standard application rate for
crop protection greenhouse trials. [d] A final expert assessment of efficacies
was made according to the following classification: i.e. “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);
please note: compounds 16 and 26 have been used as mixtures of dia-
stereomers (e.g. 16c with dr of 88:12 and 26c with dr of 62:38).
showed promising results, whereas related cyclopentyl and
introducing a phenyl or furyl group (8y, z, entries 27 and 28, cyclohexyl analogues only gave low activity, both in vitro and
Table 1). Hence, we introduced cycloalkyl substituents ranging in vivo. In contrast to conclusions from earlier studies,[24] we
from small cyclopropyl groups in 8t, 8u, 16u (entries 23, 24 and observed good receptor affinity as well as promising efficacy
against drought stress in vivo for ABA-derivatives with modified
32, Table 1) to a rather spacious cyclohexyl moiety in 8x (entry
16). C6-cyclopropyl ABA 8t showed the highest target affinity
observed among all analogues prepared within our SAR study
side chain substituents R1 at position C6.[25]
To further investigate the influence of substituents at posi-
in line with very strong effects against drought stress in vivo tion C6 on receptor binding, we determined the crystal struc-
which were superior to ABA 1. C6-cyclobutyl ABA 8v also ture of 8t in complex with RCAR11 and the phosphatase hab1.
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The superposition with published ABA complex structure evaluate the impact of the (E)-C4-C5-alkene linker, we have pre-
(3QN1)[22b] is shown in Figure 2 and demonstrates that intro- pared a set of corresponding enynes bearing modified substitu-
ducing a cyclopropyl group does not impact the principal bind- ents R1 at C6 (entries 1–22, Table 2).
ing mode significantly.
We also determined a crystal structure with this class of li-
gands, namely with enyne 10s, in a complex with RCAR11 and
the phosphatase hab1. Accordingly, the crystal structure of 10s
(Figure 3) shows that the enyne binds in a very similar binding
mode than ABA. With the protein crystal structures of 8t and
10s in hand, further modelling studies were initiated to evalu-
ate the impact of stereocentres within the cyclohexenyl head-
group. In line with results observed within our in vivo screening
campaign it was shown that structural variations (i) of the diene
side chain or (ii) of key elements responsible for receptor bind-
ing (e.g. the cyclohexanone carbonyl group) had a stronger im-
pact on receptor binding and efficacy than changes in the ab-
solute configuration of stereocentres in the cyclohexanone moi-
ety (cf. also Table 1, entries 1, 2, 34).
Table 2. SAR-results of selected enyne-analogues of abscisic acid.
Figure 2. Crystal structure of 8t (green) in complex with RCAR11-hab1 and
superposition with ABA 1 (yellow) in complex structure (3QN1).
Furthermore, metabolically more stable derivatives 16c, 16d,
16r, 16u, 26c (entries 29–33) carrying a CF3- or CHF2-group at
C8′ showed comparable results, although on a slightly lower
level. Likewise, C5′-C8′-cyclopropyl analogues afforded moder-
ate target affinities in line with good effects in greenhouse ex-
periments when bearing small alkyl and cycloalkyl substituents
as in compounds 30a–c,t. (cf. Scheme 4). However, these results
were on a lower level than for parent compounds 8 (e.g. 30a:
pI50 5.1, wheat: +++, canola: ++; 30t: pI50 5.0, wheat: ++, canola:
+++; 31e: pI50 4.9, wheat: ++, canola: ++). Introducing addi-
tional substituents in the cyclohexenone moiety thus did not
improve in vivo efficacy. Interestingly, more rigid ABA-ana-
logues 19 and 20a,b showed only weak target affinity correlat-
ing with minor effects against drought stress in our in vivo tests.
These results emphasize our observation that small substituents
are tolerated at C6 suggesting a cavity in the ABA-receptor of
limited size. To broaden our SAR study further, as well as to
Substituents[a]
In vitro activity
ABI1-
In vivo efficacy
against drought stress
(AtRCAR11)[b]
Entry No.
R1
R2 R3
Activity
pI50
250 g/ha
[%] 5 μ
M
Wheat[c]
Canola[c]
1
2
10b
10c
Et
Et
Et Me
Me Me
100[b]
100[b]
100[b]
98[b]
92[b]
88[b]
97[b]
15[b]
100[b]
97[b]
100[b]
100[b]
42[b]
93[b]
86[b]
75[b]
93[b]
88[b]
85[b]
5.9[b]
6.1[b]
6.1[b]
5.9[b]
5.4[b]
5.3[b]
5.8[b]
n.d.[b]
6.2[b]
5.7[b]
6.3[b]
6.2[b]
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
++
0
3
4
(S)-10c Et
(R)-10c Et
Me Me
Me Me
5
6
7
10e
10h
10j
nPr
Et Me
nBu Et Me
iPr
Et Me
Et Me
Et Me
Me Me
8
9
10n
10q
10r
10s
10u
10w
17b
17c
17e
17j
17q
17u
27b
27c
27r
tBu
CF3
CF3
CF3
cPr
0
+++
++
+++
++++
++
++
++
+++
0
10
11
12
13
14
15
16
17
18
19
20
21
22
H
Me
Et Me
cPent Et Me
n.d.[b] ++
Et
Et
Et CF3
Me CF3
Et CF3
Et CF3
Et CF3
Et CF3
5.2[b]
5.4[b]
n.d.[b]
5.3[b]
5.4[b]
5.2[b]
4.9[b]
4.9[b]
5.7[b]
+
+++
+
++
++
+
nPr
iPr
CF3
cPr
Et
+++
++
++
+
+
+
+
+
Et CHF2 78[b]
Me CHF2 80[b]
Me CHF2 97[b]
Et
CF3
+
++
++
++
[a] cPr = cyclopropyl, cPent = cyclopentyl. [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: i.e.
“0” = no effect, “+” = slight beneficial effect, “++” = significant beneficial
effect, “+++” = strong beneficial effect against drought stress, “++++” = very
strong effect superior to internal standard ABA; please note: compounds 17
and 27 have been used as mixtures of diastereomers (dr 80:20).
A similar pattern of in vitro and in vivo activity could be
observed for compounds 10a–w, albeit on a slightly lower level.
In good accordance with results obtained for diene-analogues
with ethyl and cyclopropyl groups at C6 (Table 1) enynes 10b,
Figure 3. Crystal structure of 10s in complex with RCAR11-hab1.
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10c and 10u (entries 1, 2, 10, Table 2) showed the best activi-
ties, both in vitro and in vivo. Furthermore, unbranched alkyl
groups and the trifluoromethyl group were well tolerated ex-
hibiting good target affinity, as well as good effects in vivo
against drought stress. Derivatives with additional modifica-
tions at carbon C8′ gave weaker results, correlating with what
we had observed for related dienes with modified C8′-groups.
In line with results obtained for both enantiomers of ABA (en-
tries 1,2; Table 1) also both separated enantiomers of 10c
showed comparable effects in vivo against drought stress in
wheat and canola (entries 3, 4, Table 2). To validate our in vivo
screening results further, we have chosen selected ABA-ana-
logues for advanced green house trials (i.e. more replicates, ad-
ditional crops such as corn and barley, different corn varieties
and moderate stress levels). Accordingly, significant beneficial
effects against drought stress could also be observed in corn,
barley and wheat compared to untreated controls upon treat-
ment with 8b and 10b as representative examples (Figure 4).
Receptor Specificity
Selected ABA analogues were analyzed for their receptor speci-
ficity, i.e. 8t, 8v and 30a. Arabidopsis thaliana has a total of
14 ABA receptor isoforms (RCAR 1–14) and 9 isoforms of the
interacting phosphatase (e.g. HAB1 and ABI1) and is the plant
species in which the ABA receptor is extensively character-
ized.[26]
Figure 5. Ligand- and ABA receptor-specific stimulation of ABA signalling in
Arabidopsis cells. The ABA receptors RCAR1 to RCAR14 were ectopically ex-
pressed in protoplasts and the activation of the ABA-responsive luciferase
reporter expressed as fold induction.
Interestingly, ABA analogues 8t and 30a revealed varying
ABA receptor specificities, since 8t preferentially stimulated the
ABA response via RCAR11, whereas 30a also stimulated the ABA
response via RCAR1, RCAR5, and RCAR10. To further corro-
borate the ABA receptor specificity, we selected potent ABA
analogues with free acid moieties 8v and 30a, as well as nPr
ester 8d for in vitro analysis of ABA co-receptor regulation.[5a]
Inhibition of the protein phosphatase activity of ABA co-recep-
tors is strictly dependent on the presence of RCAR proteins and
ABA or ABA agonists.
Figure 4. Advanced drought stress trials with 10b and 8b in corn, barley and
wheat under moderate stress conditions, i.e. 40 % damage for corn, 48 %
damage for barley and 40 % for wheat, respectively.
Ectopic expression of RCARs in cell wall-free leaf cells, so-
called protoplasts, of A. thaliana stimulates ABA-responsive re-
All receptors were separately purified and analyzed for regu-
porter expression in an ABA-dependent manner.[5a] All ABA re- lation of PP2CA (ABI1) in the presence of 100 μ
M ABA analogues
ceptors were transiently expressed in protoplasts either in the and ABA (Figure 6). Free acids 8v and 30a emerged as potent
presence or absence of ABA and respective novel analogues 8t ABA agonists in these in vitro tests, whereas ester 8d was less
or 30a. Subsequently, the ABA response was recorded (Fig- active in line with our expectations as ester derivatives had
ure 5).
shown their full potential mainly in vivo.
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Experimental Section
In Vivo biology: 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-
tions. The test plants were treated at the early leaf stage (BBCH10
– BBCH13). To ensure uniform water supply before commencement
of 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), wettable gran-
ules (WG), suspension concentrates (SC) 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 follow-
ing conditions: “day” = 14 hours with illumination at 26 °C; “night” =
10 hours without illumination at 18 °C. The duration of the respec-
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
ended (by re-irrigating or transfer to a greenhouse with good
growth conditions) as soon as irreversible damage was observed on
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 % = 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 benefi-
cial effect, “++” = significant beneficial effect, “+++” = strong bene-
ficial effect against drought stress, “++++” = very strong effect su-
perior to internal standard ABA.
Figure 6. Ligand- and ABA receptor-specific inhibition of the protein phos-
phatase activity of PP2CA (ABI1). For the in vitro ABA receptor assay, 50 n
PP2CA and 100 n RCAR (RCAR1 to RCAR14) were incubated in the presence
of 100 μ ligand. The analyses were performed in triplicates.
M
M
M
Determination of IC50 values for all A. thaliana RCARs further
emphasized differences in selectivity between 8v and 30a at
low μ
RCAR5-, RCAR9-, and RCAR11-PP2CA holo-receptor complex
(IC50 values between 0.5–1.3 μ ), 30a regulated these protein
M
concentrations (Table 3). 8v sensitively regulated the
M
complexes approximately 4–20 times less efficiently. However,
30a was more effective in regulating RCAR4, RCAR6, RCAR8,
RCAR13, and RCAR14 respectively, by a factor of up to 15 com-
pared to 8v emphasizing its broad activity observed in proto-
plast tests (Figure 5).
Table 3. Receptor specificity of ABA agonists. The IC50-values of PP2CA inhibi-
tion are given in micromolar ligand concentration for distinct ABA receptors
RCAR1 to RCAR14 (+/– SE < 5 %).
RCAR
8v
30a
RCAR
8v
30a
IC50 [μM]
IC50 [μM]
IC50 [μM]
IC50 [μM]
1
2
3
4
5
6
7
45
25
8
9
50
0.9
> 100
1.3
4.7
3.8
4.0
> 100
20
11
6.5
6.1
> 100
> 100
> 100
0.5
> 100
> 100
> 100
> 100
10
10
19
10
11
12
13
14
> 100
> 100
> 100
Conclusions
In Vitro Biology: ABA signalling: Preparation and analysis of Arabid-
opsis protoplasts was performed as described in earlier studies.[27]
Briefly, protoplasts (105) were transfected with 5 μg DNA of reporter
construct (pRD29B::LUC), 3 μg of p35S::GUS plasmid as a control
We have developed a highly versatile and robust synthetic
route towards derivatives of abscisic acid, giving rise to a broad
variety of closely related analogues with particular focus on
structural changes at positions C6 and C8′. This synthetic ap- for internal normalization of expression, and 3 μg of RCAR effector
expression cassettes. The effector cassettes drive expression of
RCAR coding sequences under the control of the 35S promoter.[27]
All constructs used were verified by DNA sequence analysis. Total
amount of transfected DNA per assay remained constant by supple-
menting with DNA of the empty effector cassette. The protoplast
suspensions were incubated in the presence or absence of ABA and
ABA analogues immediate after transfection for 16–18 hours. Assays
were done in three replicates per data point. In vitro activity 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 RCARs and
proach enabled us to investigate in vivo activities of a large set
of ABA-analogues against drought stress. In summary, we have
identified six highly potent analogues of abscisic acid with im-
proved in vivo efficacy against drought stress in canola and
wheat. Results from in vitro measurements and crystal structure
analyses have confirmed a cavity in the ABA-receptor of limited
size tolerating small substituents. The best substituents at C6
were small lipophilic substituents like CF3, ethyl and cyclo-
propyl, while larger moieties appeared to be too large for the
binding pocket.
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PP2Cs was performed as described.[5a] For the determination of ac-
tivity, 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 controls: a)
are reported. Further experimental details have been described in
the Supporting Information, as well as in references.[21,25]
8-Ethynyl-2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-en-8-
ol (4): In a round-bottom flask under argon, 2,2,6-trimethyl-1,4-
cyclohexanedione (15.40 g, 101.19 mmol) was dissolved in 2,3-
butanediol (90 mL), and abs. toluene (90 mL), and trimethyl ortho-
formate (33.21 mL, 303.56 mmol) and p-toluenesulfonic acid (1.22 g,
7.08 mmol) were added. The resulting reaction mixture was stirred
at 50 °C for 7 h. After cooling to room temperature, water and
toluene were added and the aqueous phase was extracted repeat-
edly with toluene. The combined organic phases were dried with
magnesium sulfate, filtered and concentrated under reduced pres-
sure. By column chromatography purification of the resulting crude
product (ethyl acetate/heptane gradient), 2,3,7,9,9-pentamethyl-1,4-
dioxaspiro[4.5]dec-6-en-8-one (20.01 g, 88 % of theoretical) was ob-
tained. In a round-bottom flask under argon, 2,3,7,9,9-pentamethyl-
1,4-dioxaspiro[4.5]dec-6-en-8-one (10.00 g, 44.58 mmol) was then
dissolved in tetrahydrofuran (50 mL) and added dropwise to a solu-
tion of a lithium acetylide/ethylenediamine complex (6.28 g,
57.96 mmol, 85 % pure) in tetrahydrofuran (70 mL). On completion
of addition, the reaction mixture 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
repeatedly with dichloromethane. The combined organic phases
were dried with magnesium sulfate, filtered and concentrated un-
der reduced pressure. By column chromatography purification of
the crude product obtained (ethyl acetate/heptane gradient), 8-
ethynyl-2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-en-8-ol
(10.02 g, 85 % of theoretical) was isolated as a colorless solid. 1H
NMR (400 MHz, CDCl3): δ = 5 ppm.41/5.39 (s, 1 H), 4.24–4.20/3.62–
3.54 (m, 2 H), 2.49 (s, 1 H), 2.08/2.01 (d, 1 H), 1.92/1.90 (s, 3 H), 1.88/
1.81 (d, 1 H), 1.26–1.21 (m, 3 H), 1.18–1.06 (m, 9 H) ppm. 13C NMR
(150 MHz, CDCl3): δ = 139.9/139.1; 125.5/125.3; 103.8/103.6; 84.4;
78.07; 77.90; 74.5/74.3; 73.9/73.7; 46.1/45.6; 39.1/38.9; 25.4;
dimethyl sulfoxide (DMSO) 0.5 % (f.c.) and b) 5 μM (f.c.) abscisic
acid (ABA). The assay described here was generally conducted with
substance concentrations of the appropriate chemical test substan-
ces in a concentration range of 0.1 μ
M to 100 μM in a solution of
DMSO and water. The substance solution thus obtained, if neces-
sary, 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 μL was introduced into each cavity of the microplate,
featuring the following composition:
(1) 5 μL 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 μL 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 dithio-
threitol (DTT)], b) 4 % by vol. of ABI1 dilution (protein stock solution
was diluted so as to give, after addition, a final concentration in the
assay of 0.15 μg ABI1/well), c) 4 % by vol. of RCAR11 dilution (en-
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
enzyme/well), d) 5 % by vol. of Tween20 (1 %), e) 47 % by vol. H2O
bi-dist.
(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
3.33 m MnCl2, d) 5 % by vol. of 25 m MUP, 5 % by vol. of Tween20
(1 %), 60 % by vol. of H2O bi-dist.
M
M
M
M
Enzyme buffer mix and substrate mix were made up 5 minutes prior
to the addition and warmed to a temperature of 35 °C. On comple-
tion of pipetting of all the solutions and on completion of mixing,
the plate was incubated at 35 °C for 20 min. Finally, a relative fluo-
rescence measurement was made at 35 °C with a BMG Labtech
“POLARstar Optima” microplate reader using a 340/10 nm excitation 22.5/21.9; 18.8/18.4; 17.0/16.8; 15.6/15.4. LC-MS (ret.-time, min,
filter and a 460 nm emission filter.
[M+/log p]) 1.47 [250.16/3.03]; 1.49 [250.16/3.10]; 1.51 [250.16/3.17].
HRMS-ESI: calcd. for C15H21O2 [M + H]+ – H2O 233.1542, found
+
Chemistry:
233.1543.
General: All reagent-grade solvents and chemicals were purchased
from standard commercial suppliers and used without further puri-
fication. All non-aqueous reactions were carried out under anhy-
drous 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]), with
typical gradients starting from an ethyl acetate/heptane ratio of
2,3,7,9,9-Pentamethyl-8-[(E)-2-(tributylstannyl)vinyl]-1,4-
dioxaspiro [4.5]dec-6-en-8-ol (5): Under argon, tetrakis(triphenyl-
phosphine) palladium(0) (231 mg, 0.20 mmol) was initially charged
in a round-bottom flask that had been dried by heating, and abs.
tetrahydrofuran (25 mL) and 8-ethynyl-2,3,7,9,9-pentamethyl-1,4-di-
oxaspiro[4.5]dec-6-en-8-ol 4 (1.0 g, 3.99 mmol) were added. Stirring
at room temperature for 5 min was followed by the addition of
tributyltin hydride (1.29 mL, 4.79 mmol). The resulting reaction mix-
ture was stirred at room temperature for 1 h and then water was
added. The aqueous phase was repeatedly extracted thoroughly
with dichloromethane, 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 2,3,7,9,9-pentamethyl-8-[(E)-2-(tributyl-
stannyl)vinyl]-1,4-dioxaspiro[4.5]dec-6-en-8-ol 5 (1.50 g, 66 % of
theoretical) in the form of a colorless oil. 1H NMR (400 MHz, CDCl3):
δ = 6.13 (d, 1 H), 5.93 (d, 1 H), 5.42 (s, 1 H), 4.22/3.63 (m, 2 H), 1.61
(s, 3 H), 1.59 (d, 1 H), 1.52 (d, 1 H), 1.49 (m, 6 H), 1.32–1.24 (m, 12
H), 1.09 (s, 3 H), 0.89 (m, 18 H). HRMS-ESI: calcd. for C27H50O3Sn+ [M
+ H]+ 543.2782, found 543.2771.
1
20:80, to a final ratio of 80:20. The H NMR, 13C NMR and 19F NMR
spectroscopy data reported for the chemical examples described
below (400 MHz for 1H NMR and 150 MHz for 13C NMR and 375 MH
z for 19F NMR, solvent: CDCl3, CD3OD or [D6]DMSO, internal stan-
dard: 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 are defined as follows: Me = CH3, Et = CH2CH3,
tHex = C(CH3)2CH(CH3)2, tBu = C(CH3)3, nBu = unbranched butyl,
nPr = unbranched propyl, cHex = cyclohexyl. In the case of dia-
stereomeric mixtures, either the significant signals for each of the
diastereomers or the characteristic signal of the main diastereomer
Ethyl (2Z)-3-[(E)-2-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-di-
oxaspiro [4.5]dec-6-en-8-yl)vinyl]hex-2-enoate (7e): Under
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1
argon, 2,3,7,9,9-pentamethyl-8-[(E)-2-(tributylstannyl)vinyl]-1,4-di- tained in the form of a colorless oil. H NMR (400 MHz, CDCl3): δ =
oxaspiro[4.5] dec-6-en-8-ol 5 (300 mg, 0.55 mmol) and ethyl (2Z)-3- 7.47 (d, 1 H), 6.32 (d, 1 H), 6.28/6.26 (s, 1 H), 5.59/5.39 (s, 1 H), 4.28–
iodohex-2-enoate 6c (156 mg, 0.55 mmol) in a round-bottom flask
that had been dried by heating were dissolved in N,N-dimethyl-
formamide (4 mL), dichlorobis(acetonitrile)palladium(II) (7 mg,
0.03 mmol) was added and the mixture was stirred at room temper-
ature for 3 h. After the addition of potassium fluoride solution, the
reaction mixture was stirred further at room temperature overnight,
and water was added. The aqueous phase was repeatedly extracted
thoroughly with CH2Cl2. The combined organic phases were then
dried with magnesium sulfate, filtered and concentrated under re-
duced pressure. Purification of the resulting crude product by col-
umn chromatography (ethyl acetate/heptane gradient) gave ethyl
(2Z)-3-[(E)-2-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]-
dec-6-en-8-yl)vinyl]hex-2-enoate 7e (140 mg, 41 % of theoretical) in
4.22/3.68–3.55 (m, 2 H), 3.79 (s, 3 H), 2.02 (d, 1 H), 1.83 (d, 1 H),
1.69/1.66 (s, 3 H), 1.65/1.60 (br. m, 1 H, OH), 1.25 (m, 3 H), 1.18 (m,
3 H), 1.10 (s, 3 H), 0.91 (s, 3 H).. LC-MS (ret.-time, min, [M+/log p])
diastereomer 1: 1.85 [404.18/4.16]; diastereomer 2: 1.87 [404.18/
4.25]. HRMS-ESI: calcd. for C20H27O5F3 [M + H]+ 405.1814, found
+
405.1819.
Ethyl (2Z)-3-[(E)-2-(1-Hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-
en-1-yl)vinyl]hex-2-enoate (8e): Ethyl (2Z)-3-[(E)-2-(8-hydroxy-
2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]-dec-6-en-8-yl)vinyl]hex-2-
enoate 7e (180 mg, 0.46 mmol) was dissolved in acetone (5 mL)
under argon in a round-bottom flask, and 3 drops of conc. hydro-
chloric acid were added. The resulting reaction solution was stirred
at room temperature for 30 min, and water was then added. After
removing acetone under reduced pressure, the aqueous phase was
extracted repeatedly with dichloromethane. The combined organic
phases were dried with magnesium sulfate, filtered and concen-
trated under reduced pressure. By final column chromatography
purification of the resulting crude product (ethyl acetate/heptane
gradient), ethyl (2Z)-3-[(E)-2-(1-hydroxy-2,6,6-trimethyl-4-oxocyclo-
hex-2-en-1-yl)vinyl]hex-2-enoate 8e (114 mg, 74 % of theoretical)
was obtained in the form of a colorless oil. 1H NMR (400 MHz,
CDCl3): δ = 7.77 (d, 1 H), 6.15 (d, 1 H), 6.05 (s, 1 H), 5.93 (s, 1 H),
4.19 (q, 2 H), 2.96 (br. s, 1 H, OH), 2.47 (d, 1 H), 2.31 (d, 1 H), 2.24 (t,
2 H), 1.92 (s, 3 H), 1.58 (m, 2 H), 1.27 (t, 3 H), 1.24 (s, 3 H), 1.12 (s, 3
H), 0.92 (t, 3 H) ppm. LC-MS (retention time, min [M+/log p]) 1.53
1
the form of a colorless oil. H NMR (400 MHz, CDCl3): δ = 7.68 (d, 1
H), 6.09 (d, 1 H), 5.97 (s, 1 H), 5.48 (s, 1 H), 4.19 (m, 3 H), 3.61 (m, 1
H), 2.29 (m, 1 H), 2.22 (m, 2 H), 2.02 (m, 1 H), 1.92 (m, 3 H), 1.67 (m,
2 H), 1.62 (m, 1 H), 1.28 (m, 6 H), 1.20–1.08 (m, 9 H), 0.91 (t, 3 H)
ppm. HRMS-ESI: calcd. for C23H36O5+ [M]+ 392.2562, found 392.2556.
Ethyl (2E,4E)-5-(8-Hydroxy-2,3,7,9,9-pentamethyl-1,4-di-
oxaspiro[4.5]dec-6-en-8-yl)-3-(trifluoromethyl)penta-2,4-dieno-
ate (7q): Under argon, 2,3,7,9,9-pentamethyl-8-[(E)-2-(tributyl-
stannyl)vinyl]-1,4-dioxa-spiro[4.5] dec-6-en-8-ol 5 (300 mg,
0.55 mmol) and ethyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate 6q
(163 mg, 0.55 mmol) in a round-bottom flask that had been dried
by heating were dissolved in N,N-dimethylformamide (4 mL), di-
chlorobis(acetonitrile)palladium(II) (7 mg, 0.03 mmol) was added
and the mixture was stirred at room temperature for 3 h. After the
addition of potassium fluoride solution, the reaction mixture was
stirred further at r.t. overnight. The aqueous phase was then repeat-
edly extracted thoroughly with diethyl ether, and the combined
organic phases were then dried with MgSO4, filtered and concen-
trated under reduced pressure. By final column chromatography
purification of the resulting crude product (ethyl acetate/heptane
gradient), ethyl (2E,4E)-5-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-di-
oxaspiro[4.5]dec-6-en-8-yl)-3-(trifluoromethyl)penta-2,4-dienoate
7q (150 mg, 61 % of theoretical) was obtained in the form of a
[320.20/3.20]. HRMS-ESI: calcd. for C19H28O4 [M + H]+ – H2O
+
321.1990, found 321.1997.
Ethyl (2E,4E)-5-(1-Hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-
1-yl)-3-(trifluoromethyl)penta-2,4-dienoate (8q): Ethyl (2E,4E)-5-
(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-en-8-yl)-
3-(trifluoromethyl)penta-2,4-dienoate 7q (150 mg, 0.36 mmol) was
dissolved in acetone (5 mL) under argon in a round-bottom flask,
and 10 % hydrochloric acid was added. The resulting reaction solu-
tion was stirred at room temperature for 40 min, and water was
then added. After removing acetone under reduced pressure, the
aqueous phase was extracted repeatedly with dichloromethane.
The combined organic phases were dried with magnesium sulfate,
filtered and concentrated under reduced pressure. By column chro-
matography purification of the resulting crude product (ethyl acet-
ate/heptane gradient), ethyl (2E,4E)-5-(1-hydroxy-2,6,6-trimethyl-4-
oxocyclohex-2-en-1-yl)-3-(trifluoromethyl)-penta-2,4-dienoate 8d
(80 mg, 82 % of theoretical) was obtained in the form of a colorless
1
colorless oil. H NMR (400 MHz, CDCl3): δ = 7.47 (d, 1 H), 6.29 (d, 1
H), 6.25 (s, 1 H), 5.48 (s, 1 H), 4.26 (q, 2 H), 3.68 (m, 1 H), 3.59 (m, 1
H), 1.93 (d, 1 H), 1.83 (br. m, 1 H, OH), 1.77 (d, 1 H), 1.69 (s, 3 H),
1.32 (t, 3 H), 1.25 (m, 3 H), 1.18 (m, 3 H), 1.10 (s, 3 H), 0.91 (s, 3 H)
+
ppm. HRMS-ESI: calcd. for C21H29O5F3 [M]+ 418.1971, found
418.1980.
Methyl-(2E,4E)-5-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-di-
oxaspiro [4.5]dec-6-en-8-yl)-3-(trifluoromethyl)penta-2,4-di-
enoate (7r): Under argon, 2,3,7,9,9-pentamethyl-8-[(E)-2-(tributyl-
stannyl)vinyl]-1,4-dioxa-spiro[4.5] dec-6-en-8-ol 5 (15.0 g,
27.71 mmol) and methyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate 6r
(7.76 g, 27.71 mmol) in a round-bottom flask that had been dried
1
oil. H NMR (400 MHz, CDCl3): δ = 7.55 (d, 1 H), 6.37 (d, 1 H), 6.33
(s, 1 H), 5.97 (s, 1 H), 4.25 (q, 2 H), 2.47 (d, 1 H), 2.34 (d, 1 H), 1.92
(s, 3 H), 1.90 (br. s, 1 H, OH), 1.33 (t, 3 H), 1.11 (s, 3 H), 1.02 (s, 3 H)
ppm. LC-MS (ret. time, min, [M+/log p]) 1.46 [346.14/3.00]. HRMS-ESI:
calcd. for C17H19O3F3 [M + H]+ – H2O 329.1834, found 329.1830.
+
by heating were dissolved in N,N-dimethylformamide (100 mL), di- Methyl (2E,4E)-5-(1-Hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-
chlorobis(acetonitrile)palladium(II) (0.22 g, 0.83 mmol) and copper(I) en-1-yl)-3-(trifluoromethyl)penta-2,4-dienoate (8r): Methyl
iodide (4.22 g, 22.17 mmol) were added, and the mixture was stirred
at 50 °C for 5 h. After cooling to room temperature and subsequent
addition of potassium fluoride solution, the reaction mixture was
stirred further at room temperature overnight. The aqueous phase
was then repeatedly extracted thoroughly with ethyl acetate, 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), methyl (2E,4E)-5-(8-hydroxy-
2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-en-8-yl)-3-(trifluoro-
methyl)penta-2,4-dienoate 7r (6.72 g, 60 % of theoretical) was ob-
(2E,4E)-5-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-
en-8-yl)-3-(trifluoromethyl)penta-2,4-dienoate 7r (6.84 g,
16.91 mmol) was dissolved in acetone (15 mL) under argon in a
round-bottom flask, and 10 % hydrochloric acid was added (5 mL).
The resulting reaction solution was stirred at room temperature for
40 min, and water was then added. After removing acetone under
reduced pressure, the aqueous phase was extracted repeatedly with
dichloromethane. The combined organic phases were dried with
magnesium sulfate, filtered and concentrated under reduced pres-
sure. By column chromatography purification of the resulting crude
product (ethyl acetate/heptane gradient), methyl (2E,4E)-5-(1-
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hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)-3-(trifluoro- dried with magnesium sulfate, filtered and concentrated under re-
methyl)-penta-2,4-dienoate 8r (4.72 g, 84 % of theoretical) was ob-
tained in the form of a colorless oil. H NMR (400 MHz, CDCl3): δ =
duced pressure. By final column chromatography purification of the
crude product obtained (using an ethyl acetate/heptane gradient),
methyl (2E)-5-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-dioxaspiro-
[4.5]dec-6-en-8-yl)-3-(ethyl)pent-2-en-4-ynoate (860 mg, 78 % of
1
7.56 (d, 1 H), 6.39 (d, 1 H), 6.33 (s, 1 H), 5.97 (s, 1 H), 3.80 (s, 3 H),
2.47 (d, 1 H), 2.35 (d, 1 H), 1.92 (s, 3 H), 1.88 (br. s, 1 H, OH), 1.11 (s,
3 H), 1.02 (s, 3 H) ppm. LC-MS (ret. time, min, [M+/log p]) 1.47 theoretical) was isolated in the form of a colorless oil. 1H NMR
[332.12/2.67]. HRMS-ESI: calcd. for C16H19O4F3 [M + H]+ – H2O
332.1244, found 332.1235.
(400 MHz, CDCl3): δ = 5.99 (s, 1 H), 5.53/5.41 (s, 1 H), 4.23–4.15/
3.65–3.57 (m, 2 H), 3.72 (s, 3 H), 2.55–2.45 (br. s, 1 H, OH), 2.33–2.28
(m, 2 H), 2.08–2.02 (m, 1 H), 1.95–1.87 (m, 4 H), 1.26–1.13 (m, 15 H)
ppm. Methyl (2E)-5-(8-hydroxy-2,3,7,9,9-pentamethyl-1,4-diox-
aspiro[4.5]dec-6-en-8-yl)-3-(ethyl)pent-2-en-4-ynoate (750 mg,
2.07 mmol) was dissolved in acetone (4 mL) under argon in a round-
bottom flask, and 10 % hydrochloric acid (4 mL) was added. The
resulting reaction solution was stirred at room temp. for 45 min,
and water was then added. After removing acetone under reduced
pressure, the aqueous phase was extracted repeatedly with CH2Cl2.
The combined organic phases were dried with MgSO4, filtered and
concentrated under reduced pressure. By column chromatography
purification of the resulting crude product (ethyl acetate/heptane
gradient), methyl (2E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-
en-1-yl)-3-(ethyl)pent-2-en-4-ynoate 10c (470 mg, 78 % of theoreti-
+
Ethyl (2E)-5-(1-Hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-
yl)-3-(trifluoromethyl)pent-2-en-4-ynoate (10q): Copper(I) iodide
(46 mg, 0.24 mmol) and bis(triphenylphosphine)palladium(II)-
chloride (126 mg, 0.18 mmol) were initially charged under argon in
a round-bottom flask which had been dried by heating, and toluene
(9 mL) and ethyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate 6q (388 mg,
1.32 mmol) were added. Stirring at room temperature for 10 min
was followed by the dropwise addition of a solution of 8-ethynyl-
2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-en-8-ol 4 (300 mg,
1.19 mmol) in toluene (3 mL) and of diisopropylamine (0.34 mL,
2.39 mmol). The resulting reaction mixture was stirred at room tem-
perature for 3 h and then water was added. The aqueous phase was
extracted repeatedly with dichloromethane. The combined organic
phases were dried with magnesium sulfate, filtered and concen-
trated under reduced pressure. By final column chromatography
purification of the crude product obtained (using an ethyl acetate/
heptane gradient), ethyl (2E)-5-(8-hydroxy-2,3,7,9,9-pentamethyl-
1,4-dioxaspiro[4.5]dec-6-en-8-yl)-3-(tri-fluoromethyl)pent-2-en-4-
ynoate (300 mg, 57 % of theoretical) was isolated in the form of a
1
cal) was obtained in the form of a colorless oil. H NMR (400 MHz,
CDCl3): δ = 6.06 (s, 1 H), 5.87 (s, 1 H), 3.72 (s, 3 H), 2.91 (br. s, 1 H,
OH), 2.62 (br. d, 2 H), 2.44 (d, 1 H), 2.33 (q, 2 H), 2.16 (s, 3 H), 1.26
(s, 3 H), 1.16–1.13 (m, 6 H) ppm. 13C NMR (150 MHz, CDCl3): δ =
198.1; 165.4; 139.9; 137.6; 126.3; 124.2; 99.5; 85.9; 74.4; 51.5; 41.9;
31.5; 25.2; 19.6; 12.6 ppm. LC-MS (ret. time, min, [M+/log p]) 1.29
[290.15/2.44]. HRMS-ESI: calcd. for C17H22O4 [M + H]+ – H2O
+
1
colorless oil. H NMR (400 MHz, CDCl3): δ = 6.61 (s, 1 H), 5.56 (s, 1
290.1524, found 290.1519.
H), 4.22 (m, 3 H), 3.58 (m, 1 H), 2.21 (br. s, 1 H, OH), 1.99 (m, 1 H),
1.92 (m, 4 H), 1.31 (t, 3 H), 1.22–1.13 (m, 12 H) ppm. Ethyl (2E)-5-(8-
hydroxy-2,3,7,9,9-pentamethyl-1,4-dioxaspiro[4.5]dec-6-en-8-yl)-3-
(tri-fluoromethyl)pent-2-en-4-ynoate (200 mg, 0.48 mmol) was dis-
solved in acetone (5 mL) under argon in a round-bottom flask, and
10 % hydrochloric acid was added (3 mL). The resulting reaction
solution was stirred at room temperature for 45 min, and water was
then added. After removing acetone under reduced pressure, the
aqueous phase was extracted repeatedly with dichloromethane.
The combined organic phases were dried with magnesium sulfate,
filtered and concentrated under reduced pressure. By column chro-
matography purification of the resulting crude product (ethyl acet-
ate/heptane gradient), ethyl (2E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-
cyclohex-2-en-1-yl)-3-(trifluoromethyl)pent-2-en-4-ynoate 10q
(130 mg, 79 % of theoretical) was obtained in the form of a colorless
oil. 1H NMR (600 MHz, CDCl3): δ = 6.68 (s, 1 H), 5.90 (s, 1 H), 4.26
(q, 2 H), 2.87 (s, 1 H, OH), 2.58 (d, 1 H), 2.45 (d, 1 H), 2.16 (s, 3 H),
1.33 (t, 3 H), 1.25 (s, 3 H), 1.15 (s, 3 H) ppm. 13C NMR (150 MHz,
CDCl3): δ = 197.8; 162.8; 129.9; 126.9; 125.1; 121.6; 119.8; 102.4; 78.2;
75.2; 61.7; 24.9; 19.6; 14.1 ppm. LC-MS (ret. time, min, [M+/log p])
(R)- and (S)-Methyl (2Z)-5-(1-Hydroxy-2,6,6-trimethyl-4-oxo-
cyclohex-2-en-1-yl)-3-(ethyl)pent-2-en-4-ynoate [(R)-10c], [(S)-
10c]: Enantiomers of (2E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclo-
hex-2-en-1-yl)-3-(ethyl)pent-2-en-4-ynoate 10c were separated care-
fully via chiral prep. HPLC to afford (S)-10c, ret. time 19.550, [α]D20
=
–223.36 and (R)-10c, ret. time 24.03, [α]2D0 = +226.83 (chiral HPLC
analysis – Chiralpak IC, flow rate 0.6 mL/min, eluent n-heptan/iso-
propanol = 90:10).
(2E)-5-(1-Hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)-3-
(tri-fluoromethyl)pent-2-en-4-ynoic Acid (10s): In a round-bot-
tom flask, ethyl (2E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-
en-1-yl)-3-(trifluoromethyl)pent-2-en-4-ynoate 10q (130 mg,
0.38 mmol) was dissolved in a mixture of water and tetrahydrofuran
(1:1), and sodium hydroxide (38 mg, 0.94 mmol) was then added.
The resulting reaction mixture was stirred under reflux for 2 h and,
after cooling to room temperature, acidified with aqueous hydro-
chloric acid. The aqueous phase was repeatedly extracted
thoroughly with CH2Cl2, and the combined organic phases were
then dried with MgSO4, filtered and concentrated under reduced
pressure. By final column chromatography purification of the result-
ing crude product (ethyl acetate/heptane gradient), (2E)-5-(1-
hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)-3-(trifluoro-
methyl)pent-2-en-4-ynoic acid 10s (40 mg, 32 % of theoretical) was
obtained in the form of a colorless solid. 1H NMR (400 MHz, CDCl3):
δ = 10.14 (br. s, 1 H, OH), 6.70 (s, 1 H), 5.92 (s, 1 H), 2.61 (d, 1 H),
2.43 (d, 1 H), 2.30 (br. s, 1 H, OH), 2.16 (s, 3 H), 1.28 (s, 3 H), 1.13 (s, 3
H) ppm. HRMS-ESI: calcd. for C15H15O4F3+ [M + H]+ 317.0922, found
317.0930.
1.46 [344.12/3.00]. HRMS-ESI: calcd. for C17H20F3O4 [M + H]+
+
345.1314, found 345.1302.
Methyl (2Z)-5-(1-Hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-
yl)-3-(ethyl)pent-2-en-4-ynoate (10c): Copper(I) iodide (23 mg,
0.12 mmol) and bis(triphenylphosphine)palladium(II)chloride
(65 mg, 0.09 mmol) were initially charged under argon in a round-
bottom flask which had been dried by heating, and toluene (15 mL)
and methyl (2Z)-3-iodopent-2-enoate 6c (738 mg, 3.08 mmol) were
added. Stirring at room temperature for 10 min was followed by
the dropwise addition of a solution of 8-ethynyl-2,3,7,9,9-penta-
methyl-1,4-dioxaspiro[4.5]dec-6-en-8-ol 4 (770 mg, 3.08 mmol) in
toluene (5 mL) and of diisopropylamine (1.29 mL, 9.23 mmol). The
8-Ethynyl-2,3,7,9-tetramethyl-9-(trifluoromethyl)-1,4-di-
oxaspiro[4.5]-dec-6-en-8-ol (13): Acetylmethylenetriphenylphos-
resulting reaction mixture was stirred at room temperature for 3 h phorane (12.91 g, 40.57 mmol) was dissolved in a mixture of diethyl
and then water was added. The aqueous phase was extracted re-
peatedly with dichloromethane. The combined organic phases were
ether (30 mL) and dichloromethane (10 mL) and stirred for 5 min,
then 1,1,1-trifluoroacetone (5.00 g, 44.62 mmol) was added and the
Eur. J. Org. Chem. 2018, 1403–1415
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mixture was stirred at room temperature for 40 h. The precipitate
4 H), 1.910–1.87 (m, 1 H), 1.40 (s, 3 H), 1.25 (m, 3 H), 1.17 (m, 3 H).
formed was filtered off, the filter cake was washed with diethyl LC-MS (ret. time, min, [M+/log p]) 1.38 [304.13/2.90], 1.40 [304.13/
ether and the combined organic phases were concentrated cau-
tiously under slightly reduced pressure. The crude solution of (3Z)-
5,5,5-trifluoro-4-methylpent-3-en-2-one thus obtained was used
without further purification in the next reaction step and taken up
in toluene (25 mL). After the addition of ethyl acetoacetate (3.42 g,
26.29 mmol) and potassium tert-butoxide (0.88 g, 7.89 mmol), the
resulting reaction mixture was stirred under reflux conditions for
5 h. After cooling to r.t., water was added, the mixture was stirred
vigorously for 5 min and then the aqueous phase was extracted
repeatedly with CH2Cl2. The combined organic phases were dried
with MgSO4, 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
3,5-dimethyl-5-(trifluoromethyl)cyclohex-2-en-1-one (1.9 g, 38 % of
theoretical) in the form of a colorless oil. 3,5-Dimethyl-5-(trifluoro-
methyl)cyclohex-2-en-1-one (1.60 g, 8.33 mmol) was then dissolved
in toluene (60 mL), and molybdatophosphoric acid hydrate (30 mg,
0.02 mmol), copper(II) sulfate pentahydrate (4 mg, 0.02 mmol) and
molybdenum(VI) oxide (5 mg, 0.03 mmol) were added. The result-
ing reaction mixture was stirred with introduction of air under reflux
conditions for 4 d. After cooling to room temperature, water was
added, the mixture was stirred vigorously for 5 min and then the
aqueous phase was extracted repeatedly with CH2Cl2. The com-
bined organic phases were dried with MgSO4, filtered and concen-
trated under reduced pressure. By column chromatography purifica-
tion of the resulting crude product (ethyl acetate/heptane gradient),
it was possible to obtain 2,6-dimethyl-6-(trifluoromethyl)cyclohex-
2-ene-1,4-dione 12 (300 mg, 17 % of theoretical) in the form of a
3.00], 1.42 [304.13/3.07].
2,3,7,9-Tetramethyl-8-[(E)-2-(tributylstannyl)vinyl]-9-(trifluoro-
methyl)-1,4-dioxaspiro[4.5]dec-6-en-8-ol (14): Under argon,
tetrakis-(triphenylphosphine)palladium(0) (19 mg, 0.02 mmol) was
initially charged in a round-bottom flask that had been dried by
heating, and THF (5 mL) and 8-ethynyl-2,3,7,9-tetramethyl-9-
(trifluoromethyl)-1,4-di-oxaspiro[4.5]dec-6-en-8-ol 13 (100 mg,
0.33 mmol) were added. Stirring at room temperature for 5 min was
followed by the addition of tributyltin hydride (0.11 mL, 0.39 mmol).
The resulting reaction mixture was stirred at 55 °C for 1 h and, after
cooling to r.t., water was added. The aqueous phase was repeatedly
extracted thoroughly with CH2Cl2, and the combined organic pha-
ses were then dried with MgSO4, 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 2,3,7,9-tetramethyl-8-[(E)-2-(tributylstannyl)-
vinyl]-9-(trifluoro-methyl)-1,4-dioxaspiro[4.5]dec-6-en-8-ol 14
1
(160 mg, 82 % of theoretical) in the form of a colorless oil. H NMR
(400 MHz, CDCl3): δ = 6.33/6.31 (d, 1 H), 5.97/5.92 (d, 1 H), 5.51/5.42
(s, 1 H), 4.24/3.64 (m, 2 H), 2.46/2.35 (d, 1 H), 1.92 (br. s, 1 H, OH),
1.91/1.87 (d, 1 H), 1.64/1.62 (s, 3 H), 1.49 (m, 6 H), 1.32–1.26 (m, 9
H), 1.18 (m, 3 H), 0.89 (m, 18 H) ppm. HRMS-ESI: calcd. for
C27H48O3F3Sn+ [M + H]+ 597.2504, found 597.2518.
Ethyl (2Z,4E)-3-Cyclopropyl-5-[8-hydroxy-2,3,7,9-tetramethyl-9-
(tri-fluoromethyl)-1,4-dioxaspiro[4.5]dec-6-en-8-yl]penta-2,4-di-
enoate (15u): Under argon, 2,3,7,9-tetramethyl-8-[(E)-2-(tributyl-
stannyl)vinyl]-9-(trifluoromethyl)-1,4-dioxaspiro[4.5]dec-6-en-8-ol
14 (150 mg, 0.25 mmol) and ethyl (2Z)-3-cyclopropyl-3-iodoacrylate
6u (67 mg, 0.25 mmol) in a round-bottom flask that had been dried
by heating were dissolved in tetrahydrofuran (4 mL), dichlorobis-
(acetonitrile)palladium(II) (3 mg, 0.01 mmol) was added and the
mixture was stirred at room temperature for 3 h. After the addition
of potassium fluoride solution, the reaction mixture was stirred fur-
ther at room temperature overnight. The aqueous phase was then
repeatedly extracted thoroughly with diethyl ether, and the com-
bined organic phases were then dried with magnesium sulfate, fil-
tered and concentrated under reduced pressure. By final column
chromatography purification of the resulting crude product (ethyl
acetate/heptane gradient), ethyl (2Z,4E)-3-cyclopropyl-5-[8-hydroxy-
2,3,7,9-tetramethyl-9-(trifluoromethyl)-1,4-dioxaspiro[4.5]dec-6-en-
8-yl]penta-2,4-dienoate 15u (40 mg, 36 % of theoretical) was ob-
1
colorless oil. H NMR (400 MHz, CDCl3): δ = 6.69 (s, 1 H), 3.17 (d, 1
H), 2.80 (d, 1 H), 2.06 (s, 3 H), 1.48 (s, 3 H) ppm. 2,6-Dimethyl-6-
(trifluoromethyl)cyclohex-2-ene-1,4-dione 12 (520 mg, 2.52 mmol)
was dissolved in 2,3-butanediol (4 mL) under argon, and trimethyl
orthoformate (0.83 mL, 7.57 mmol) and p-toluenesulfonic acid
(30 mg, 0.18 mmol) were added. The resulting reaction mixture was
stirred at 50 °C for 6 h. After cooling to room temperature, water
and toluene were added and the aqueous phase was extracted
repeatedly with toluene. The combined organic phases were dried
with magnesium sulfate, filtered and concentrated under reduced
pressure. By column chromatography purification of the resulting
crude product (ethyl acetate/heptane gradient), 2,3,7,9-tetramethyl-
9-(trifluoromethyl)-1,4-dioxaspiro[4.5]dec-6-en-8-one (700 mg, 98 %
1
of theoretical) was obtained. H NMR (400 MHz, CDCl3): δ = 6.70 (s,
1
tained in the form of a colorless oil. H NMR (400 MHz, CDCl3): δ =
1 H), 4.35–4.28/3.76–3.67 (m, 2 H), 2.61 (d, 1 H), 2.43 (d, 1 H), 2.16
(s, 3 H), 1.34–1.26 (m, 6 H), 1.20 (m, 3 H). In a round-bottom flask
under argon, 2,3,7,9-tetramethyl-9-(trifluoromethyl)-1,4-di-
oxaspiro[4.5]-dec-6-en-8-one (700 mg, 2.52 mmol) was then dis-
solved in tetrahydrofuran (3 mL) and added dropwise to a solution
of a lithium acetylide/ethylenediamine complex (376 mg,
3.27 mmol, 80 % pure) in tetrahydrofuran (5 mL). On completion of
addition, the reaction mixture was stirred at r.t. for 4 h, then water
was added and the mixture was concentrated under reduced pres-
sure. The remaining residue was admixed with water and CH2Cl2,
and the aqueous phase was extracted repeatedly with CH2Cl2. The
combined organic phases were dried with magnesium sulfate, fil-
tered and concentrated under reduced pressure. By column chro-
matography purification of the crude product obtained (ethyl acet-
ate/heptane gradient), 8-ethynyl-2,3,7,9-tetramethyl-9-(trifluoro-
methyl)-1,4-dioxaspiro-[4.5]dec-6-en-8-ol 13 (550 mg, 68 % of theo-
7.80/7.78 (d, 1 H), 6.40/6.38 (d, 1 H), 5.62/5.60 (s, 1 H), 5.52/5.44 (s,
1 H), 4.28/3.63 (m, 2 H), 4.18 (q, 2 H), 2.52/2.41 (d, 1 H), 2.03/1.94
(d, 1 H), 2.00 (br. s, 1 H, OH), 1.72/1.69 (s, 3 H), 1.62/1.55 (m, 1 H),
1.40–1.34 (m, 3 H), 1.29–1.17 (m, 6 H), 0.92 (t, 3 H), 0.83 (m, 2 H),
0.58 (m, 2 H) ppm. HRMS-ESI: calcd. for C23H32O5F3 [M + H]+
+
445.2115, found 445.2109.
Ethyl (2Z,4E)-3-Cyclopropyl-5-[1-hydroxy-2,6-dimethyl-4-oxo-6-
(tri-fluoromethyl)cyclohex-2-en-1-yl]penta-2,4-dienoate (16u):
Ethyl (2Z,4E)-3-cyclopropyl-5-[8-hydroxy-2,3,7,9-tetramethyl-9-(tri-
fluoromethyl)-1,4-dioxaspiro[4.5]dec-6-en-8-yl]penta-2,4-dienoate
15u (40 mg, 0.09 mmol) was dissolved in acetone (4 mL) under
argon in a round-bottom flask, and a few drops of concentrated
HCl were added. The resulting reaction solution was stirred at room
temperature for 4 h and then water was added. After removing
acetone under reduced pressure, the aqueous phase was extracted
repeatedly with CH2Cl2. The combined organic phases were dried
1
retical) was isolated as a colorless solid. H NMR (400 MHz, CDCl3):
δ = 5.50/5.48/5.40 (s, 1 H), 4.26–4.21/3.70–3.58 (m, 2 H), 2.63/2.60 with MgSO4, filtered and concentrated under reduced pressure. By
(s, 1 H), 2.53/2.32 (m, 1 H), 2.33/2.31 (br. s, 1 H, OH), 2.02–1.96 (m, column chromatography purification of the resulting crude product
Eur. J. Org. Chem. 2018, 1403–1415
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Full Paper
(ethyl acetate/heptane gradient), ethyl (2Z,4E)-3-cyclopropyl-5-[1-
(using an ethyl acetate/heptane gradient), ethyl (2E)-5-[8-hydroxy-
hydroxy-2,6-dimethyl-4-oxo-6-(trifluoro-methyl)cyclohex-2-en-1- 2,3,7,9-tetramethyl-9-(trifluoromethyl)-1,4-dioxa-spiro[4.5]dec-6-en-
yl]penta-2,4-dienoate 16u (15 mg, 45 % of theoretical) was ob-
tained in the form of a colorless oil. H NMR (400 MHz, CDCl3): δ =
8-yl]-3-(trifluoromethyl)pent-2-en-4-ynoate (110 mg, 52 % of theo-
1
retical) was isolated in the form of a colorless oil. 1H NMR (400 MHz,
7.88 (d, 1 H), 6.37 (d, 1 H), 6.01 (s, 1 H), 5.64 (s, 1 H), 4.17 (q, 2 H), CDCl3): δ = 6.65/6.63 (s, 1 H), 5.54/5.51/5.29 (s, 1 H), 4.28/3.96 (q, 2
2.92 (d, 1 H), 2.52 (d, 1 H), 2.39 (br. s, 1 H, OH), 1.98 (s, 3 H), 1.59
(m, 1 H), 1.30 (t, 3 H), 1.28 (s, 3 H), 0.88 (m, 2 H), 0.59 (m, 2 H)
H), 4.27/3.60 (m, 2 H), 2.62 (br. s, 1 H, OH), 2.47/2.34 (d, 1 H), 2.01/
1.99 (s, 3 H), 1.98/1.91 (d, 1 H), 1.42 (s, 3 H), 1.31 (t, 3 H), 1.28 (m, 3
H), 1.17 (m, 3 H) ppm. Ethyl (2E)-5-[8-hydroxy-2,3,7,9-tetramethyl-9-
(trifluormethyl)-1,4-dioxaspiro[4.5]dec-6-en-8-yl]-3-(trifluoro-
methyl)pent-2-en-4-ynoate (110 mg, 0.23 mmol) was dissolved in
acetone (5 mL) under argon in a round-bottom flask, and 5 drops of
concentrated hydrochloric acid were added. The resulting reaction
solution was stirred at room temperature for 4 h and then water
was added. After removing acetone under reduced pressure, the
aqueous phase was extracted repeatedly with dichloromethane.
The combined organic phases were dried with magnesium sulfate,
filtered and concentrated under reduced pressure. By column chro-
matography purification of the resulting crude product (ethyl acet-
ate/heptane gradient), ethyl (2E)-5-[1-hydroxy-2,6-dimethyl-4-oxo-6-
(trifluoromethyl)cyclohex-2-en-1-yl]-3-(trifluoromethyl)pent-2-en-4-
ynoate 17q (70 mg, 71 % of theoretical) was isolated in the form of
a colorless viscous oil. 1H NMR (400 MHz, CDCl3): δ = 6.72/6.71 (s,
1 H), 5.98/5.97 (s, 1 H), 4.28/3.93 (q, 2 H), 3.28 (br. s, 1 H, OH), 3.00
(d, 1 H), 2.66 (d, 1 H), 2.21/2.18 (s, 3 H), 1.49/1.37 (s, 3 H), 1.32/1.09
(t, 3 H) ppm. 13C NMR (150 MHz, CDCl3): δ = 194.9/193.6; 166.7/
166.6; 154.5; 135.0; 130.8/130.2; 127.8/126.7; 100.3/99.9; 85.6/84.3;
76.3; 74.2; 61.9/59.7; 41.4; 19.7/18.4; 14.2/14.1 ppm. 19F NMR
(376 MHz, CDCl3): δ = –67.09/–67.47 (s, CF3), –68.91 (s, CF3) ppm.
LC-MS (ret. time, min, [M+/log p]) 1.52 [398.10/3.27], HRMS-ESI:
ppm. HRMS-ESI: calcd. for C19H24O4F3 [M + H]+ 373.1547, found
+
373.1555.
Methyl (2Z,4E)-5-[1-Hydroxy-2,6-dimethyl-4-oxo-6-(trifluoro-
methyl)cyclohex-2-en-1-yl]-3-trifluormethyl-penta-2,4-dienoate
(16r): Target compound 16r (29 mg, 98 % of theoretical in the final
step) was prepared and isolated in the form of a colorless viscous
oil following the synthetic procedures described for 16u. 1H NMR
(400 MHz, CDCl3): δ = 7.63/7.52 (d, 1 H), 6.39 (s, 1 H), 6.30/6.25 (d,
1 H), 6.03 (s, 1 H), 3.81 (s, 3 H), 2.93 (d, 1 H), 2.50 (d, 1 H), 2.34 (br.
s, 1 H, OH), 1.95 (s, 3 H), 1.20 (s, 3 H) ppm. 13C NMR (150 MHz,
CDCl3): δ = 193.4; 164.7; 159.9; 137.1; 127.8, 126.7, 122.1, 121.4;
121.2; 78.2; 52.4, 42.9; 18.9; 18.6; 17.9; 16.8 ppm. LC-MS (ret. time,
+
min, [M+/log p]) 1.31 [386.1/2.97], HRMS-ESI: calcd. for C16H17O4F6
[M + H]+ 386.2862, found 386.2850.
Methyl (2Z,4E)-3-Ethyl-5-[1-hydroxy-2,6-dimethyl-4-oxo-6-(tri-
fluoro-methyl)cyclohex-2-en-1-yl]penta-2,4-dienoate (16c): Tar-
get compound 16c (147 mg, 79 % of theoretical in the final step)
was prepared and isolated in the form of a colorless viscous oil
following the synthetic procedures described for 16u. 1H NMR
(400 MHz, CDCl3): δ = 7.85 (d, 1 H), 6.01 (s, 1 H), 6.00 (d, 1 H), 5.79
(s, 1 H), 3.72 (s, 3 H), 2.91 (d, 1 H), 2.50 (d, 1 H), 2.43 (br. s, 1 H, OH),
2.40–2.36 (q, 2 H), 1.96 (s, 3 H), 1.27 (s, 3 H), 1.12 (t, 3 H) ppm. 13C
NMR (150 MHz, CDCl3): δ = 193.8; 166.6; 161.4; 154.3; 132.8, 128.5,
127.3, 117.6; 78.4; 51.3, 43.2; 27.2; 21.3; 18.8; 18.1; 17.5; 13.1 ppm.
calcd. for C17H15O3F6 [M + H]+ – H2O 381.2245, found 381.2241.
+
Ethyl (2E)-5-[1-Hydroxy-2,6-dimethyl-4-oxo-6-(trifluoro-
methyl)cyclo-hex-2-en-1-yl]-3-(ethyl)pent-2-en-4-ynoate (17b):
Target compound 17b (40 mg, 91 % of theoretical) was prepared
and isolated in the form of a colorless viscous oil following the
HRMS-ESI: calcd. for C17H21O4F3 [M + H]+ 346.3414, found
+
346.3422.
1
n-Propyl (2Z,4E)-3-Ethyl-5-[1-hydroxy-2,6-dimethyl-4-oxo-6-(tri-
fluoromethyl)cyclohex-2-en-1-yl]penta-2,4-dienoate (16d): Tar-
get compound 16d (128 mg, 83 % of theoretical) was prepared and
isolated in the form of a colorless viscous oil following the synthetic
synthetic procedures described for 17q. H NMR (400 MHz, CDCl3):
δ = 6.09/6.07 (s, 1 H), 5.94/5.92 (s, 1 H), 4.21–4.16 (q, 2 H), 3.67 (br.
s, 1 H, OH), 3.02/2.97 (d, 1 H), 2.70/2.62 (d, 1 H), 2.33 (q, 2 H), 2.21/
2.19 (s, 3 H), 1.50 (s, 3 H), 1.28 (t, 3 H), 1.15 (t, 3 H) ppm. 19F NMR
(376 MHz, CDCl3): δ = –67.14/–67.42 (s, CF3) ppm. LC-MS (ret. time,
1
procedures described for 16u. H NMR (400 MHz, CDCl3): δ = 7.86
min, [M+/log p]) 1.48 [358.14/3.16], HRMS-ESI: calcd. for C18H20O3F3
+
(d, 1 H), 6.43/6.00 (d, 1 H), 5.98 (s, 1 H), 5.80 (s, 1 H), 4.08 (q, 2 H),
2.90 (d, 1 H), 2.49 (d, 1 H), 2.41 (br. s, 1 H, OH), 2.40–2.36 (q, 2 H),
1.96 (s, 3 H), 1.71–1.65 (m, 2 H), 1.27 (s, 3 H), 1.13 (t, 3 H), 0.96 (t, 3
H) ppm. 13C NMR (150 MHz, CDCl3): δ = 193.8; 166.3; 161.5; 153.8;
132.6, 128.7, 127.3, 118.1; 78.4; 65.8; 50.6, 43.2; 27.2; 20.0; 18.8; 18.1;
13.1; 10.5 ppm. 19F NMR (376 MHz, CDCl3): δ = –67.07 (s, CF3) ppm.
LC-MS (ret. time, min, [M+/log p]) 1.56 [374.3/3.28], HRMS-ESI: calcd.
[M + H]+ – H2O 340.0426, found 340.0429.
Acknowledgments
We would like to thank Matthias Jank, Susanne Ries, Gudrun
Fey, Peter Zöllner and Martin Annau for valuable analytical sup-
port and we are grateful to Christian Kornbauer for technical
assistance. Furthermore, we would like to thank Stephen Lindell
and David Barber for fruitful discussions during the preparation
of the manuscript.
for C19H26O4F3 [M + H]+ 375.1783, found 375.1779.
+
Ethyl (2E)-5-[1-Hydroxy-2,6-dimethyl-4-oxo-6-(trifluoro-
methyl)cyclo-hex-2-en-1-yl]-3-(trifluoromethyl)pent-2-en-4-yno-
ate (17q): Copper(I) iodide (16 mg, 0.09 mmol) and bis(triphenyl-
phosphine)palladium(II)chloride (45 mg, 0.06 mmol) were initially
charged under argon in a flame-dried round-bottom flask, and
toluene (3 mL) and ethyl (2Z)-4,4,4-trifluoro-3-iodobut-2-enoate
(126 mg, 0.43 mmol) were added. Stirring at room temperature for
10 min was followed by the dropwise addition of a solution
of 8-ethynyl-2,3,7,9-tetramethyl-9-(trifluoromethyl)-1,4-di-
oxaspiro[4.5]dec-6-en-8-ol 13 (130 mg, 0.43 mmol) in toluene (1 mL)
and of diisopropylamine (0.12 mL, 0.85 mmol). The resulting reac-
tion mixture was stirred at room temperature for 3 h and then water
was added. The aqueous phase was extracted repeatedly with
CH2Cl2. The combined organic phases were dried with magnesium
sulfate, filtered and concentrated under reduced pressure. By final
column chromatography purification of the crude product obtained
Keywords: Phytochemistry · Terpenoids · Drought stress ·
Plant hormone · Stille coupling
[1] a) S. Y. Salehi-Lisar, H. Bakhshayeshan-Agdam in Drought Stress Tolerance
in Plants, Vol 1; Physiology and Biochemistry (Eds.: M. A. Hossain, S. H.
Wani, S. Bhattacharjee, D. J. Burritt, L.-S. P. Tran), Springer Verlag, Berlin,
2016, pp. 1–16.
[2] a) D. S. Battisti, R. L. Naylor, Science 2009, 323, 240.
[3] a) H. Campos, M. Cooper, J. E. Habben, G. O. Edmeades, J. R. Schussler,
Field Crops Res. 2004, 90, 19; b) K. S. Nemali, C. Bonin, F. G. Dohlemann,
M. Stephens, W. R. Reeves, D. E. Nelson, P. Castiglioni, J. E. Whitsel, B.
Eur. J. Org. Chem. 2018, 1403–1415
www.eurjoc.org
1414
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Sammons, R. A. Silady, D. Anstrom, R. E. Sharp, O. R. Patharkar, D. Clay,
M. Coffin, M. A. Nemeth, M. E. Leibman, M. Luethy, M. Lawson, Plant Cell
Environ. 2015, 38, 1866.
[14] a) J. R. Hanson, C. Uyanik, J. Chem. Res. Synop. 2003, 2003, 426; b) X.
Han, J. Fan, H. Lu, X. Li, H. Li, D. Yang, Y. Zhang, Y. Xiao, Z. Qin, Bioorg.
Med. Chem. 2015, 23, 6210.
[4] a) F. T. Addicott, J. L. Lyon, Annu. Rev. Plant Physiol. 1969, 20, 139; b) B. V.
Milborrow, Annu. Rev. Plant Physiol. 1974, 25, 259; c) S. R. Cutler, P. L.
Rodriguez, R. R. Finkelstein, S. R. Abrams, Annu. Rev. Plant Biol. 2010, 61,
651; d) E. Nambara, A. Marion-Poll, Annu. Rev. Plant Biol. 2005, 56, 165.
[5] a) Y. Ma, I. Szostkiewicz, A. Korte, D. Moes, Y. Yang, A. Christmann, E. Grill,
Science 2009, 324, 1064; b) S.-Y. Park, P. Fung, P. Fung, N. Nishimura, D. R.
Jensen, H. Fujii, Y. Zhao, S. Lumba, J. Santiago, A. Rodriguez, T.-F. F. Chow,
S. E. Alfred, D. Bonetta, R. Finkelstein, N. J. Provart, D. Desveaux, P. L.
Rodriguez, P. McCourt, J.-K. Zhu, J. I. Schroeder, B. F. Volkman, S. R. Cutler,
Science 2009, 324, 1068.
[15] a) T. R. Smith, A. J. Clark, G. J. Clarkson, P. C. Taylor, A. Marsh, Org. Biomol.
Chem. 2006, 4, 4186; b) A careful screening of different acetal protecting
groups has been carried out proving 2,3-butanediol-based protecting
group being the most suitable with superior stability compared with 1,2-
ethanediol or 1,3-propanediol.
[16] T. Kajikawa, K. Aoki, R. S. Singh, T. Iwashita, T. Kusumoto, H. A. Frank, H.
Hashimoto, S. Katsumura, Org. Biomol. Chem. 2009, 7, 3723.
[17] J. Chen, G.-Q. Lin, Z.-M. Wang, H.-Q. Liu, Synlett 2002, 8, 1265.
[18] Y. Donde, M. Holobowski, M. F. Posner, R. M. Burk, M. E. Garst, U. S. 2006/
0111430, 2006.
[19] a) T. K. M. Shing, Y.-Y. Yeung, P. L. Su, Org. Lett. 2006, 8, 3149; b) E. F.
Murphy, M. Schneider, T. Mallat, A. Baiker, Synthesis 2001, 547; c) A.
Hanyu, Y. Sakurai, S. Fujibayashi, S. Sakaguchi, Y. Ishii, Tetrahedron Lett.
1997, 38, 5659.
[6] a) K. Ueno, Y. Todoroki, Curr. Med. Chem. 2010, 17, 3231; b) A. J. Cutler,
J. E. Krochko, Trends Plant Sci. 1999, 4, 472; c) R. Zhou, A. J. Cutler, S. J.
Ambrose, M. M. Galka, K. M. Nelson, T. M. Squires, M. K. Loewen, A. S.
Jadhav, A. R. S. Ross, D. C. Taylor, S. R. Abrams, Plant Physiol. 2004, 134,
361; d) J. J. Balsevich, A. J. Cutler, N. Lamb, L. J. Friesen, E. U. Kurz, M. R.
Perras, S. R. Abrams, Plant Physiol. 1994, 106, 135.
[7] a) P. A. Rose, A. J. Cutler, N. M. Irvine, A. C. Shaw, T. M. Squires, M. K.
Loewen, S. R. Abrams, Bioorg. Med. Chem. Lett. 1997, 7, 2543; b) S. R.
Abrams et al. WO1997/23441, 1997; c) A. J. Cutler, P. A. Rose, T. M.
Squires, M. K. Loewen, A. C. Shaw, J. W. Quail, J. E. Krochko, S. R. Abrams,
Biochemistry 2000, 39, 13614; d) Y. Todoroki, N. Hirai, K. Koshimizu, Phyto-
chemistry 1995, 38, 561; e) B. T. Kim, Y. K. Min, T. Asami, N. K. Park, I. H.
Jeong, K. Y. Cho, S. Yoshida, Bioorg. Med. Chem. Lett. 1995, 5, 275.
[8] J. M. Nyangulu, K. M. Nelson, P. A. Rose, Y. Gai, M. Loewen, B. Lougheed,
J. W. Quail, A. J. Cutler, S. A. Abrams, Org. Biomol. Chem. 2006, 4, 1400.
[9] J. Takeuchi, M. Okamoto, T. Akiyama, T. Muto, S. Yajima, M. Sue, M. Seo,
Y. Kanno, T. Kamo, A. Endo, E. Nambara, N. Hirai, T. Ohnishi, S. R. Cutler,
Y. Todoroki, Nat. Chem. Biol. 2014, 10, 477.
[20] a) Y. Todoroki, M. Sawada, M. Matsumoto, S. Tsukada, K. Ueno, M. Isaka,
M. Owaki, N. Hirai, Bioorg. Med. Chem. 2004, 12, 363; b) Y. Todoroki, S.-I.
Nakano, N. Hirai, H. Ohigashi, Tetrahedron 1996, 52, 8081.
[21] J. Frackenpohl, T. Müller, P. v. Koskull-Döring, I. Heinemann, C. H. Ro-
singer, I. Häuser-Hahn, M. J. Hills, WO2012/139892, 2012.
[22] a) P. Yin, H. Fan, Q. Hao, X. Yuan, D. Wu, Y. Pang, C. Yan, W. Li, J. Wang,
N. Yan. Nat. Struct. Mol. Biol. 2009, 16, 1230–1236; b) F. Dupeux, R. Antoni,
K. Betz, J. Santiago, M. Gonzalez-Guzman, L. Rodriguez, S. Rubio, S. Y.
Park, S. R. Cutler, P. L. Rodriguez, J. A. Márquez, Plant Physiol. 2011, 156,
106.
[23] a) H. Kraehmer, B. Laber, C. H. Rosinger, A. Schulz, Plant Physiol. 2014,
166, 1119; b) H. Ahrens, G. Lange, T. Müller, C. H. Rosinger, L. Willms, A.
van Almsick, Angew. Chem. Int. Ed. 2013, 52, 9388–9398; Angew. Chem.
2013, 125, 9558.
[24] a) S. C. Chen, J. M. MacTaggart Agric. Biol. Chem. 1986, 50, 1097; b) K.
Yamashita, T. Watanabe, M. Watanabe, T. Oritani, Agric. Biol. Chem. 1982,
46, 3069.
[25] a) J. Frackenpohl, T. Müller, P. v. Koskull-Döring, I. Heinemann, C. H. Ro-
singer, I. Häuser-Hahn, M. J. Hills, WO2012/139890, 2012; b) J. Fracken-
pohl, T. Müller, P. v. Koskull-Döring, I. Heinemann, C. H. Rosinger, I. Häu-
ser-Hahn, M. J. Hills, WO 2012139891, 2012.
[10] G. T. Wang, D. Heiman, G. D. Venburg, J. H. Lustig, M. A. Surpin, R. E.
Fritts, F. P. Silverman, D. D. Woolard, WO2016/168535, 2016.
[11] a) G. T. Wang, D. Heiman, G. D. Venburg, E. Nagano, M. A. Surpin, J. H.
Lustig, WO2016/007587, 2016; b) F. P. Silverman, G. Wang, K. A. Falco,
D. D. Woolard, D. O. Wilson, D. C. Leep, G. D. venburg, WO2016/187370,
2016.
[12] a) J. Takeuchi, T. Ohnishi, M. Okamoto, Y. Todoroki, Bioorg. Med. Chem.
Lett. 2015, 25, 3507; b) K. Ueno, Y. Araki, N. Hirai, S. Saito, M. Mizutani,
K. Sakata, Y. Todoroki, Bioorg. Med. Chem. 2005, 13, 3359.
[26] S. Fuchs, S. V. Tischer, C. Wunschel, A. Christmann, E. Grill, Proc. Natl.
Acad. Sci. USA 2014, 111, 5741.
[27] D. Moes, A. Himmelbach, A. Korte, G. Haberer, E. Grill, Plant J. 2008, 54,
806–819.
[13] a) M. G. Constantino, P. Losco, E. E. Castellano, J. Org. Chem. 1989, 54,
681; b) M. G. Constantino, P. M. Donate, N. Petragnani, J. Org. Chem.
1986, 51, 253; c) J. Cornforth, J. E. Hawes, R. Mallaby, Aust. J. Chem. 1992,
45, 179.
Received: December 4, 2017
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