Synthesis and Exploration of Abscisic Acid Receptor Agonists Against Dought Stress by Adding Constraint to a Tetrahydroquinoline-Based Lead Structure

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doi.org/10.1002/ejoc.202100415

Synthesis and Exploration of Abscisic Acid Receptor

Agonists Against Dought Stress by Adding Constraint to a

Tetrahydroquinoline-Based Lead Structure

Guido Bojack,^[a]Rachel Baltz,^[a]Jan Dittgen,^[a]Christian Fischer,^[a]Jörg Freigang,^[b]

Rahel Getachew,^[a]Erwin Grill,^[c]Hendrik Helmke,^[a]Sabine Hohmann,^[a]Gudrun Lange,^[a]

Stefan Lehr,^[a]Fabien Porée,^[a]Jana Schmidt,^[a]Dirk Schmutzler,^[a]Zhenyu Yang,^[c]and

Jens Frackenpohl*^[a]

New oxotetrahydroquinolinyl- and oxindolinyl sulfonamides

interacting with RCAR/(PYR/PYL) receptor proteins were identi-

fied as lead structures against drought stress in crops starting

from protein docking studies of a sulfonamide lead structure,

followed by in-depth SAR studies. Optimized five to six step

synthetic approaches via substituted amino oxo-tetrahydro-

quinolines and amino oxo-indolines as essential intermediates

gave access to the envisaged oxo-tetrahydroquinolinyl and

oxindolinyl sulfonamides. Whilst oxo-tetrahydroquinolinyl sulfo-

namides with additional carbon substituents or spiro-cycloalkyl

groups exhibited only low to moderate target affinities, the

corresponding spiro-oxindolinyl and oxo-tetrahydroquinolinyl

sulfonamides carrying optimized N-substituents revealed strong

interactions with RCAR/(PYR/PYL) receptor proteins in Arabidop-

sis thaliana. Remarkably, the in vitro activity observed for these

new compounds was on the same level as observed for the

naturally occurring plant hormone in line with strong efficacy

against drought stress in-vivo (canola and wheat as broad-acre

crops).

Introduction

technology,^[4,5]and exploring novel chemical active ingredients

inspired by naturally occurring plant hormones or signaling

pathways as the third approach. Abscisic acid (1, S-(+)-ABA), a

chiral sesquiterpenoid first discovered in the 1960’s,^[6–9]is one of

the most important plant hormones. It regulates not only

essential developmental signals in plants, e.g. seed maturation

or dormancy, but mediates – by regulating transpiration – how

plants adapt to different types of environmental abiotic stress

such as drought, heat or salinity.

Intriguingly, the foliar application of ABA to wheat grown

under near-field conditions improved water use efficiency

without significant growth trade-offs.^[10]As a consequence, new

synthetic small molecules that modify transpiration via the

same mechanism as natural phytohormone ABA can serve as

valuable tools for maximizing water use efficiency of crops

thereby safeguarding crop yield in anticipation of drought.

Investigations of ABA-mediated signaling have progressed

rapidly since the initial discovery of RCAR/(PYR/PYL) receptor

proteins as soluble ABA- receptors.^[11,12]It was shown via crystal

structural analysis that binding of ABA induced a conforma-

tional change in the highly conserved ABA receptor proteins

prompting an interaction with phosphoprotein phosphatases

2 C (PP2Cs) and thereby inhibiting their activity. Accordingly,

these findings have provided new insights into the structure

activity relationship (SAR) of ABA resulting in novel synthetic

analogues.

It is known that agricultural crops face significant losses due to

biotic stress such as pests, diseases, and weed damages, but

abiotic stress adversely affects crop production even further in

various parts of the world, decreasing average yields for most

of the crops severely.^[1]Moreover, the impact of abiotic stress is

expected to grow as a consequence of climate change. Among

the abiotic stress types affecting agricultural production,

drought stress is regarded as one of the major sources of crop

losses around the globe.^[2]Three major strategies have been

pursued so far to reduce the impact that drought has on crop

yield. These include firstly exploiting beneficial effects of crop

protection agents,^[3]secondly developing drought tolerant

crops through transgenic approaches or by using breeding

[a] Dr. G. Bojack, Dr. R. Baltz, Dr. J. Dittgen, C. Fischer, R. Getachew,

Dr. H. Helmke, S. Hohmann, Dr. G. Lange, Dr. S. Lehr, Dr. F. Porée,

Dr. J. Schmidt, D. Schmutzler, Dr. J. Frackenpohl

Research & Development, Weed Control, Division Crop Science

Bayer AG

Industriepark Höchst, 65926 Frankfurt am Main, Germany

E-mail: jens.frackenpohl@bayer.com

http://www.bayer.com

[b] Dr. J. Freigang

Research & Development, Research Technology, Division Crop Science,

Bayer AG

Alfred-Nobel-Straße 50, 40789 Monheim, Germany

[c] Prof. Dr. E. Grill, Dr. Z. Yang

Lehrstuhl für Botanik, Wissenschaftszentrum Weihenstephan,

Technische Universität München

Emil-Ramann-Straße 4, 85354 Freising, Germany

Whilst modifying the cyclohexenone moiety of ABA via

stabilization or further substitution has attracted considerable

synthetic interest due its potential to prevent formation of the

catabolite phaseic acid,^[13–21]a surprisingly limited number of

studies has been made so far to investigate open-chain

Supporting information for this article is available on the WWW under

https://doi.org/10.1002/ejoc.202100415

Part of the “Industrial Organic Synthesis” Special Collection.

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analogues of ABA or synthetic agonists. Recently, we have

analysis of the binding mode of sulfonamide-based 3^rdgen-

eration agonist cyanabactin 8 with PYR1 the beneficial impact

of p-cyano aniline moieties on the interaction of agonists with

ABA receptor proteins has also been observed.^[36]In good

correlation with X-ray analyses of cyano cyclopropyl analogues

of ABA (cf. 2a–c),^[24]this study confirmed our observations that

p-cyano aniline moieties (cf. phenylsulfonyl ethylenediamines

7a–b) are further effective isosteres of ABA’s cyclohexenone

headgroup. As is discussed further below the carbonyl group in

oxo-tetrahydroquinolines 5a–d also serves as a strong mimic of

ABA’s cyclohexenone carbonyl group. Thus, we have turned our

focus to the oxo-tetrahydroquinoline-based lead structure 5a

to complement our target-based high throughput screening

approach (HTS). Herein, we outline how we have identified new

lead structures interacting with RCAR/(PYR/PYL) receptor pro-

teins by carrying out an in-depth SAR starting from lead

structure 5a. In particular, we modified and transformed the

tetrahydroquinoline moiety at several positions hence empha-

sizing room for edging in further structural motifs into ABA-

receptor protein agonists.

shown that cyano-cyclopropyl groups in ABA analogues 2a–c

served as isosteres of the cyclohexanone moiety giving rise to

compounds with strong activity in vitro and in vivo.^[22]Synthetic

agonists of RCAR/(PYR/PYL) receptor proteins have also been

prepared by other groups,^[23–25]and the independent studies

have afforded three different generations of sulfonamide lead

structures. Likewise, naphthyl sulfonamides, such as 1^stgen-

eration lead structure pyrabactin 3a,^[26,27]structurally related

pyrimidine 3b, as well as five-membered analogs 4a–b,^[28]have

all exhibited good initial receptor affinity, albeit paired with

moderate beneficial effects in in vivo tests (Figure 1).

Besides, 2^ndgeneration lead structure quinabactin 5a, an

oxo-tetrahydroquinolinyl sulfonamide, and its close analogues

5b–d carrying different N-substituents, or analogues bearing

improved phenyl substituents (6a), a modified oxo-tetrahydro-

quinoline moiety (6c) have all shown enhanced in vitro activity

paired with significant in vivo efficacy.^[29–34]In our view these

observations foreshow that analogues of plant hormone ABA 1

with considerable structural changes can be identified. Recently,

3^rdgeneration sulfonamides that show promising receptor

binding, as well as significant in vivo efficacy in crops have been

identified as lead structures via a target-based high throughput Results and Discussion

screening (HTS) approach, e.g. phenylsulfonyl ethylenediamines

7a–b,^[35]and via target-based design, e.g. sulfonamide 8, both

carrying p-cyano aniline groups.^[36]In a thorough structural

Structure-based design and receptor docking studies. Ana-

lyzed from a synthetic chemist’s angle, various parts of the

tetrahydroquinoline lead structures 5a and 6b can be modified

to explore their impact on in vivo efficacy and receptor binding,

such as the sulfonamide bridge and several positions in the

lactam moiety. Several structural motifs could serve to sub-

stitute the sulfonamide moiety. For instance, carboxamide

spacers have been examined providing the first examples of

non-sulfonamide ABA receptor agonists Likewise, cyano- and

cyclo-propyl-substituted phenyl acetic acid amides and sub-

stituted triazolyl thioacetamides have shown good receptor

binding.^[35,37]

Additionally, we have shown that the sulfonamide group

could be shifted within the bridging moiety, e.g. in indoline

sulfonamides 9a–b,^[35]or could be replaced by a phosphinami-

date group (Figure 2, compound 10).^[38]Interestingly, the ring

contraction from a tetrahydroquinoline to an indoline moiety as

realized in ABA-agonists 9a–b was well tolerated regarding

target affinity. Particularly, this result prompted us to lay

emphasis on investigating the impact of tetrahydroquinoline

modifications on in vivo efficacy against drought stress in crops

and on target affinity. Based on our experience in agrochemical

active-ingredient research we have chosen oxo-tetrahydro-

quinolinyl sulfonamides 11a, 11b, 11g and 12c, as well as

spiro-cyclopropyl oxindolinyl sulfonamide 13a carrying steri-

cally more demanding or spirocyclic moieties as starting points

for our receptor docking studies and subsequent SAR inves-

tigations (Figure 2).

As a means to deduce the impact of modifying the oxo-

tetrahydroquinoline scaffold, we examined the crystal structure

of the complex between sulfonamide 6b (carrying a Chlorine-

atom instead of the methyl group in quinabactin 5a) and

AtRCAR11 and the related phosphatase AtHAB1 (Figure 3).

Figure 1. Abscisic acid, selected terpene analogues and sulfonamides

interacting with RCAR/(PYR/PYL) receptor proteins.

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The overall affinity is impelled further by a strong hydrophobic

effect with a significant contribution of the N-propyl moiety

and the Cl-atom in the 4-position of the benzyl group.^[35,36]

Upon having a closer look at the crystal structure, it became

clear that there is some space for introducing sterically more

demanding substituents than the unbranched N-propyl group

in 6b. Furthermore, small additional substituents of the

tetrahydroquinoline sp³-carbons should be tolerated as the

crystal structure analysis showed some limited space. We

started to investigate the impact of bulkier tetrahydroquinolinyl

N-substituents on receptor binding by docking target com-

pounds 11b and 11g into the crystal structure.^[39]In those cases

modelling investigations suggested that the hydrophobic 1,1’-

bi(cyclopropyl)-1-yl and bicyclo[1.1.1]cyclopent-1-yl moieties

replacing the N-propyl group should give rise to a significant

beneficial hydrophobic effect whilst all essential target inter-

actions are maintained (Figure 4). It could thus be assumed that

tetrahydroquinolinyl sulfonamides carrying substituted N-cyclo-

propyl and N-cyclobutyl groups would afford good target

affinities.

Exploring additional hydrophobic substituents at the tetra-

hydroquinoline sp³-carbons within our modelling studies

afforded less evident results. It could be deduced from these

investigations that the respective substituents had to be rather

small and should not exceed the size of methyl groups.

Likewise, the modelling studies gave borderline results for the

test whether the geminal methyl groups in compound 12c

would fit into the binding pocket. While the modelling software

still tolerated the two methyl groups, their distances to some of

the protein side chains appeared to be too short as the binding

pocket does not allow flexibility in this sub-pocket (Figure 5a).

The binding affinity of related substituted tetrahydroquinolinyl

sulfonamides might thus be reduced considerably, and we laid

particular emphasis on preparing compounds 12a–n to provide

experimental evidence. Reducing the ring size to a five

membered oxindoline ring led to a significantly improved

receptor fit. The oxindolinyl sulfonamide fitted well into the

pocket while preserving the essential interactions such as the

H-bond to the conserved water molecules (Figure 5b).

Figure 2. Synthetic ABA-receptor agonists with modified sulfonamide (pur-

ple), e.g. phosphinamidate 10, and target structures 11a, 11b, 11g, 12c and

13a of the work described herein, carrying a modified tetrahydroquinoline

moiety (dark blue=modifications in the 6-membered ring and light blue=

new N-substituents).

Figure 3. Essential Interactions of oxo-tetrahydroquinolinyl sulfonamide 6b

with AtRCAR11 and the related phosphatase AtHAB1 as determined via

crystal structure analysis. The H-bonds to glutamate 94 and to the essential

water molecule are indicated in dotted green lines. The contribution of

individual atoms is indicated by spheres and their size correlates with the

magnitude of their contribution.^[39]Green spheres show that these atoms

stabilize the interaction, whereas red spheres indicate destabilizing inter-

actions based on the HYDE scoring function.

Noteworthingly, the sulfonamide forms two essential hydrogen

bonds to the ABA receptor: Firstly, the carbonyl oxygen of the

oxo-tetrahydroquinoline moiety interacts with the conserved

water molecule. Secondly, the sulfonamide NH interacts with

the side chain of the conserved glutamate 94 moiety, whilst

only one of the sulfonyl oxygens interacts with the backbone

carbonyl group of histidine 60 via a bridging water molecule.

Figure 4. Putative binding mode of tetrahydroquinolinyl sulfonamides 11b

(Figure 4a, left) and 11g (Figure 4b, right). The contribution of individual

atoms is indicated by spheres and their size correlates with the magnitude

of their contribution.^[39]Green spheres show that these atoms stabilize the

interaction, whereas red spheres indicate destabilizing interactions.

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Figure 5. Superposition of the crystal structure of 6b with modelled 12c (5a)

and a corresponding dimethyl-substituted oxindolinyl sulfonamide (5b). The

distances between the geminal methyls are indicated, While the distances in

the case of 12c are quite short, the respective distances for the oxindoline

are larger suggesting a better fit into the protein.

Chemistry. Based upon our initial results regarding syn-

thetic approaches towards oxo-tetrahydroquinolinyl phosphina-

mi-dates^[38]four major synthetic pathways were developed and

applied to prepare oxo-tetrahydroquinolinyl sulfonamides carry-

ing the envisioned modifications. Particular emphasis has been

laid on upscaling potential, easy handling and on robust yield

of each step as we had to consider preparing multigram

quantities of the most active compounds for advanced bio-

logical testing. Firstly, oxo-tetrahydroquinoline which was easily

accessible via Friedel-Crafts alkylation could be converted in

three steps into key intermediate 19 via direct nitration, N-

alkylation with a primary sterically non-demanding alkyl halide

and subsequent Fe- or SnCl₂-mediated reduction of the nitro

group.^[31]Secondly, the required N-substituted 6-amino oxo-

tetrahydroquinolines 19 were prepared in four steps via (i) S_NAr

reaction of 6-ethyl (2E)-3-(2-fluoro-5-nitrophenyl)-acrylate 14

together with an appropriate amine to form intermediate 15,

preferably an amine with a larger bulky substituent, (ii)

hydroge-nation of the acrylic double bond of 15 using

Wilkinson’s catalyst to afford intermediate 16, (iii) NaH-

mediated cyclization and (iv) reduction of the nitro group

(Scheme 1).^[32]

In our hands, the hydrogenation using Wilkinson’s catalyst

afforded the desired products in higher yield (93–98%), i.e.

nearly quantitatively, than other conditions. Whilst the first

approach functioned well with sterically less demanding alkyl

halides (e.g. chloromethylcyclopropane or chloromethyl-cyclo-

butane), the second approach turned out to be particularly

suitable for sterically demanding amines or cycloalkyl amines.

Correspondingly, 1,1’-bi(cyclopropyl)-1-amine reacted easily

with fluorinated phenyl acrylate 14 to afford intermediate 19

giving access to envisioned sulfonamides 11d–e via final

coupling with a suitable substituted benzyl sulfonyl chloride

(Scheme 1). In order to prepare tetrahydroquinolinyl sulfona-

mides 12 with additional substituents at quaternary carbon

atom C4 in the oxotetrahydroquinoline moiety we chose a third

approach bearing slight modifications compared with the

synthetic routes outlined in Scheme 1. Nitro oxotetrahydroqui-

noline 20 was prepared via coupling of a suitable acrylate

carrying the desired substituents to form the quaternary atom

Scheme 1. Synthesis of tetrahydroquinolinyl sulfonamides 11a–z; a) R¹À NH₂,

°

N,N-DMF, EtN(i-Pr)₂, 50 C, 16 h; b) (Ph₃P)₃RhCl, abs. EtOH, H₂, r.t., 9 h; c) NaH,

°

abs. THF, 0 C – r.t.; d) SnCl₂À H₂O, abs. EtOH, 80 C, 5 h; or: Fe (powdered),

NH₄Cl, EtOH/H₂O (1:1); e) Et₃N, subst. aniline, THF, r.t., 2–6 h; f) Friedel-Crafts

°

alkylation with AlCl₃; g) conc. H₂SO₄, conc. HNO₃, À 20 C – r.t., 4 h; h) K₂CO₃,

1

°

R À I, N,N-DMF, r.t., 24 h; i) Et₃N, abs. THF, À 20 C – r.t., 1 h or pyridine, DMSO,

°

MeCN, 0 C – r.t., 3 h.

at C4 in target compounds 12a–c and 12j–n with aniline either

by using a TBTU-mediated or an acid chloride-based amide

formation, followed by Friedel-Crafts alkylation and subsequent

nitration. Thereafter, N-alkylation with a primary or secondary

sterically non-demanding alkyl iodide, followed by Fe- or SnCl₂-

mediated reduction of the nitro group in 21 afforded key

intermediate 22 which was then converted using a suitable

sulfonyl chloride into oxotetrahydro-quinolinyl sulfonamides

12a–c and 12j–n.

However, the synthetic route described in Scheme 2 turned

out to be unsuitable for preparing tetrahydroquinolinyl sulfona-

mides 12d–i with N-cyclopropyl substituents. Thus, we have

developed an adapted fourth route that proceeded via

amination in the first step, i.e. coupling of phenyl bromide with

the envisioned cyclopropylamine using Pd₂(dba)₃and

a

phosphorus-containing ligand (e.g. BINAP, t-BuXPhos).^[40]Alter-

natively, copper(II)salt-mediated coupling of a suitable cyclo-

propylamine with triphenylbismuth afforded the desired inter-

mediate 23 in good yield (Scheme 3).^[41]However, the Pd-

mediated amination proved to be more suitable for upscaling.

Thereafter, coupling with 3,3-dimethyl acrylate, subsequent

AlCl₃-mediated cyclization, followed by nitration and reduction

of the nitro group afforded amino N-cyclopropyl oxo-tetrahy-

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alkylation using a suitable Lewis acid to afford the desired N-

substituted dihydrooxindole 26 (Scheme 4). Alternatively, the

dihydro-oxindole 26 could be prepared from readily available

isatin by initially introducing the appropriate substituent R¹at

the nitrogen using a suitable halide and a carbonate base, for

example K₂CO₃or Cs₂CO₃, in a polar aprotic solvent, and

subsequently transforming the second carbonyl group into a

CH₂group by reaction with hydrazine hydrate at elevated

temperature following to a Wolff-Kishner protocol. Thereafter,

the desired spiro-cycloalkyl or spiro-heterocyclyl moiety was

introduced via double alkylation of a suitable dibromo alkane

or bis-sulfonylated triol except for the spiro-cyclopentenyl

group which was introduced in two steps, i.e. by two-fold

alkylation with allyl bromide and subsequent metathesis

reaction. Then, SnCl₂-mediated reduction of the NO₂-group and

final sulfonamide formation afforded desired target compounds

13a–p (Scheme 4).

A further option to prepare substituted dihydrooxindolyl-

amines 28 was offered by the reaction of p-acetylaminoaniline

Scheme 2. Synthesis of tetrahydroquinolinyl sulfonamides 12a–c & 12j–n;

a) aq. KOH; b) TBTU, DIPEA, aniline, CH₂Cl₂, r.t., 3 h; c) 1. SOCl₂, toluene, r.t. –

with

a suitably substituted bromoacetyl bromide already

carrying the desired cycloalkyl or heterocyclyl moiety and

ensuing Lewis acid-mediated cyclization (for example with

AlCl₃), followed by removal of the acetyl protective group with

HCl or DBU.

SAR-study. All target molecules that have been synthesized

to investigate the SAR of oxo-tetrahydroquinolinyl sulfonamides

11, 12 and oxo-spirocycloalkyl oxindolinyl sulfonamides 13

°

70 C, 4 h, 2. Et₃N, aniline, CH₂Cl₂; d) AlCl₃, CH₂Cl₂, 0 C – r.t., 2 h; e) conc.

1

°

HOAc, conc. HNO₃, 70 C, 2 h; f) Cs₂CO₃, R À I or R À OTf, dioxane, r.t, 24 h;

°

g) Zn, NH₄Cl, MeOH, H₂O; h) Et₃N, abs. THF, À 20 C – r.t, 1 h or pyridine,

°

DMSO, MeCN, 0 C – r.t., 3 h.

Scheme 3. Synthesis of substituted N-cyclopropyl tetrahydroquinolinyl sulfo-

namides 12d–i; a) Pd₂(dba)₃, BINAP, PhBr; b) BiPh₃, Cu(OAc)₂; c) 1. SOCl₂,

°

toluene, r.t. – 70 C, 4 h, 2. Et₃N, aniline, CH₂Cl₂; d) AlCl₃, CH₂Cl₂, 0 C – r.t., 2 h;

°

e) conc. HOAc, conc. HNO₃, 70 C, 2 h; f) SnCl₂À H₂O, abs. EtOH, 80 C, 5 h, or:

°

Fe (powdered), NH₄Cl, EtOH/H₂O (1:1); g) Et₃N, abs. THF, À 20 C – r.t., 1 h or

°

pyridine, DMSO, MeCN, 0 C – r.t., 3 h.

droquinoline 24 which was then sulfonylated to give target

compounds 12d–i.

The spiro-cycloalkyl and spiro-heterocyclyl oxindolinyl sulfo-

namides 13a–p could be prepared via an optimized 6-step

approach differing particularly in the first steps from the

reaction sequences outlined in Schemes 1–3 as more synthetic

options were available to prepare the key intermediate 28.^[42]

Firstly, we reacted an aniline correspondingly monosubsti-

tuted at the nitrogen by R¹with chloroacetyl chloride or bromo

acetyl bromide using a suitable base in a polar aprotic solvent.

The reaction product was then cyclized via Friedel-Crafts

Scheme 4. Synthesis of spirocycloalkyl and spiroheterocyclyl oxindolinyl

sulfonamides 13a–p; a) K₂CO₃, R¹À I or R¹À OTf, N,N-DMF, r.t., 4 h;

°

b) N₂H₄À H₂O, 130 C; c) Et₃N, suitably substituted bromo acetyl bromide, abs.

°

CH₂Cl₂, r.t., 2 h; d) AlCl₃, CH₂Cl₂, 0 C – r.t, 2 h; e) suitable dibromo alkane or

°

bis-sulfonylated triol, NaH, N,N-DMF; f) conc. HOAc, conc. HNO₃, 70 C, 2 h;

°

g) SnCl₂À H₂O, abs. EtOH, 80 C, 5 h; h) pyridine, DMSO, MeCN, 0 C – r.t., 3 h;

i) Cs₂CO₃, R¹À I or R¹À OTf, dioxane, r.t., 24 h; j) aq. HCl or DBU, r.t., 2 h.

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were tested for target affinity in vitro, as well as for in vivo

particular, the 1,1’-bi(cyclopropyl)-1-yl and 1,1’-bi(cyclopropyl)-

2-yl groups at the N-R¹position in combination with 4-Me (11a,

11b, Table 1, entries 1, 2) and 4-Et-benzyl moieties (11c,

Table 1, entry 3) showed strong results with 11b being nearly

as potent in vitro as ABA 1 and clearly exceeding the target

activity of the initial sulfonamide lead structure 5a. Further-

more, N-[1,1’-bi(cyclopropyl)-1-yl]-substituted tetrahydroquino-

linyl sulfonamide 11b turned out to be active on the same level

as ABA 1 in our in vivo greenhouse trials, both in wheat and

canola. Interestingly, the 1,1’-bi(cyclopropyl) motif proved to be

a particularly promising alternative to the n-propyl group in 5a

as other N-cyclopropyl groups showed good target affinity

combined with moderate to good, but not outstanding in vivo

efficacy (11d, 11e, and 11f, Table 1, entries 4–6).

Expanding our approach by investigating substituted N-

cyclobutyl moieties afforded target compounds 11i–11o with

very strong in vitro activity. In particular, N-3-Me-cyclobut-1-yl

and N-3,3-F₂-cyclobut-1-yl groups proved to be highly beneficial

for target affinity showing pI₅₀values which were significantly

higher than those of standards ABA and 5a, e.g. 11i (entry 9,

efficacy under drought stress conditions upon foliar application

on plants. Wheat (triticum aestivum) and Canola (brassica napus)

were chosen as model plants for monocotyledonous and

dicotyledonous species, whereas in vitro tests were carried out

using the ABA-receptor RCAR11 in Arabidopsis thaliana. RCAR11

of Arabidopsis is a prototypic ABA receptor with abundant

structural information that provides a solid basis for SAR

analyses. Along with plant hormone ABA 1, we have tested

oxo-tetrahydroquinolinyl sulfonamide 5a, one of the lead

structures outlined in Figure 1, as reference in our in vitro and

in vivo tests (Table 1, entries 27, 28).

Promising in vitro activity could be observed for various

oxo-tetrahydroquinolinyl sulfonamides 11 carrying the substitu-

ents at the tetrahydroquinolinyl nitrogen that we had inves-

tigated within our docking studies described above, albeit only

selected combinations of substituents afforded higher target

affinities than ABA 1 (Table 1). Firstly, N-cyclopropyl moieties

bearing further substituents afforded promising binding affin-

ities in line with good in vivo efficacy against drought stress. In

Table 1. SAR-results of oxo-tetrahydroquinolinyl sulfonamides 11a–z.

Entry

No.

Substituents^[a]

R¹

In vitro activity ABI1-

(AtRCAR11)^[b]

Activity

In vivo efficacy vs. drought stress

R²

[250 g/ha]^[c]

pI₅₀

Wheat^[d]

Canola^[d]

[5 μM]

1

2

3

4

5

6

7

8

11a

11b

11c

11d

11e

11f

11g

11h

11i

1,1’-bi(cyclopropyl)-2-yl

1,1’-bi(cyclopropyl)-1-yl

1-Me-cycloprop-1-yl

1,2-Me₂-cycloprop-1-yl

Bicyclo[1.1.1]pent-1-yl

3-Me-cyclobut-1-yl

Spiro[3.3]hept-2-yl

2-Me-cyclobut-1-yl

3,3-F₂-cyclobut-1-yl

Oxetan-3-yl

Me

Et

Me

CN

Me

CF₃

Me

Cl

CN

Me

CN

Me

CN

Cl

Me

F

Et

Cl

Me

Et

Cl

+ + + +

+ + +

+ + + +

+ + +

+ + + +

+ + +

+ + + +

+ + +

6.3

6.9

5.4

6.4

5.9

5.3

6.9

7.1

8.0

7.7

4.7

5.6

5.0

7.8

7.5

7.4

5.9

6.0

5.8

6.2

5.9

6.4

8.2

4.7

n.d.

5.9

7.0

+ +

+ + +

+ +

+

+ + +

+

+ +

+ + +

+ +

+

+ +

+

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

11j

11k

11l

11m

11n

11o

11p

11q

11r

11s

11t

11u

11v

11w

11x

11y

11z

5a

+

+ + +

+ +

+

+ +

+ + +

+ +

+ + +

+ +

0

+ + +

+ +

+ + + +

+ + +

+ +

+

0

+ +

+ + +

Tetrahydropyran-4-yl

c-PrÀ CH₂-

c-BuÀ CH₂-

c-PentÀ CH₂-

c-HexÀ CH₂-

CF₃

Cl

Me

+

–

Adamantyl-CH₂-

n-Pr

0

+ + + +

+ +

+ + +

1

Abscisic acid

[a] c-Pr=cyclopropyl, c-Bu=cyclobutyl, n-Pr=n-propyl, c-Pent=cyclopentyl, c-Hexyl; [b] a final expert assessment of target affinity was made according to

the following classification: “-”=activity <30%, “+”=30%<activity <50%, “+ +”=50%<activity <70%, “+ + +”=70%<activity <90%„ “+ + + +”=

activity >90%; [c] standard application rate for crop protection greenhouse trials; [d] a final expert assessment of efficacies was made according to the

following classification: “0”=no effect, “+”=slight beneficial effect, “+ +”=significant beneficial effect, “+ + +”=strong beneficial effect against drought

stress, “+ + + +”=very strong effect superior to internal standard ABA (comparative visual assessment of greenmass).

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Table 1) and 11n, 11o (entries 14, 15, Table 1). However, their

sulfonamides proved to be highly sensitive towards changes in

the substitution pattern at position C-4. These observations

correlate well with the docking studies outlined above. Only

dimethyl-groups showed target affinities that were strong

high in vitro activity measured at AtRCAR11 only correlated with

good in vivo activity against drought stress in canola, but not in

wheat. In contrast, the oxetan-3-yl moiety in 11p afforded good

in vivo efficacy in wheat in line with strong target affinity

(entry 16, Table 1). Cyclopropyl- and cyclobutyl groups linked to

the tetrahydroquinolinyl nitrogen via a methylene spacer

afforded good target affinities paired with promising in vivo

efficacy against drought stress in both crops (11r–11w, entries

18–23, Table 1). Remarkably, compound 11s showed a better

efficacy than internal standard ABA 1. The p-fluorine substituent

in 11s proved to be particularly beneficial for in vivo efficacy

due to favorable physicochemical properties and presumably

enhanced stability towards metabolism compared with close

analogs 11r and 11t carrying alkyl substituents. Introducing

sterically more demanding cycloalkylmethylene groups led to a

substantial decline in both, target affinity and in vivo efficacy.

Likewise, cyclohexylmethylene- and adamantylmethylene-sub-

stituted tetrahydroquinolinyl sulfonamides 11y and 11z did not

show any beneficial in vivo effects.

Introducing substituents at carbon C-4 in the oxo-tetrahy-

dro-quinolinyl moiety led to a significantly reduced target

affinity compared with the strong in vitro activity outlined in

Table 1, i.e. adding a spiro-cyclobutyl group at position C-4 to

compound 11u affording related spiro-cyclobutyl tetrahydro-

quinolinone 12k with all other substituents remaining un-

changed led to a complete loss of activity in vitro and a

significantly reduced efficacy in vivo (cf. 11u, entry 21, Table 1 &

12k, entry 11, Table 2). In contrast to the strong efficacy of 11u

in wheat and canola, only minor beneficial effects could be

observed for 12k in wheat. Hence, oxo-tetrahydroquinolinyl

enough to measure pI₅₀values above 4.0, albeit on

a

significantly lower level than observed for oxo-tetrahydroquino-

linyl sulfonamides 11a–x without substituents at carbon C-4. All

dimethyl substituted analogs 12a–i, as well as spirocyclic oxo-

tetrahydroquinolinyl sulfonamides 12j–n exhibited weak to

moderate target affinities combined with no or only marginal

beneficial effects against drought stress in vivo (entries 1–14,

Table 2). Furthermore, the ring size of the spirocyclic moiety did

not have any significant impact on receptor binding or in vivo

efficacy.

Whilst oxo-tetrahydroquinolinyl sulfonamides 12a–n carry-

ing additional substituents at carbon C-4 showed only weak

target affinity and marginal in vivo efficacy, related spiro-

substituted oxo-indolinyl sulfonamides 13a–p afforded better

results, both in vitro and in vivo.

Remarkably, the ring contraction from tetrahydroquinolines

12 to indolines 13 led to improved target affinity in A. thaliana

paired with good overall in vivo results for the indolines

showing the best target interactions. Furthermore, consistent

in vivo activity against drought stress in wheat was observed for

all indolines 13a–p. Likewise, spiro-cyclopropyl substituted oxo-

indolinyl sulfonamides 13a–h showed consistent target affin-

ities with 13f and 13g (Table 3, entries 6 and 7) giving the best

results in line with good in vivo efficacy, both in canola and

wheat. Increasing the ring size of the spiro-cycloalkyl moiety to

form 4- and 5-membered rings was tolerated (entries 13i–n,

Table 3), whilst a further expansion to 6-membered moieties

Table 2. SAR-results of oxo-tetrahydroquinolinyl sulfonamides 12a–n.

Entry

No.

Substituents^[a]

R¹

In vitro activity

ABI1-(AtRCAR11)^[b]

Activity [5 μM]

In vivo efficacy vs. drought stress

R²

X

250 g/ha^[c]

pI₅₀

Wheat^[d]

Canola^[d]

1

2

3

4

5

6

7

8

12a

12b

12c

12d

12e

12f

12g

12h

12i

12j

12k

12l

12m

12n

CH₂À CN

CH₂CHF₂

cPr

Cl

Me

Cl

Me

–

-

–

+ + +

+

+ +

+

+ +

+

–

4.3

+

0

+

0

+ +

+

0

+

0

n.d.

4.1

4.3

4.0

n.d.

4.2

4.0

n.d.

0

cPr

+

0

+

0

1-MeÀ cPr

1-(MeOÀ CH₂)À cPr

1-MeÀ cPr

2-MeÀ cPr

CH₂À cPr

Br

CF₃

Me

CF₃

Cl

CF₃

Me

9

–

10

11

12

13

14

CH₂

(CH₂)₂

(CH₂)₃

[a] cPr=cyclopropyl, “–“=no bond between the gem-dimethyl groups [b] a final expert assessment of target affinity was made according to the following

classification: “n.d.”=not determined, “–”=activity<30%, “+”=30%<activity<50%, “+ +”=50%<activity<70%, “+ + +”=70%<activity<90%, “+

+ + +”=activity>90%; [c] standard application rate for crop protection greenhouse trials; [d] a final expert assessment of efficacies was made according to

the following classification: “0”=no effect, “+”=slight beneficial effect, “+ +”=significant beneficial effect, “+ + +”=strong beneficial effect against

drought stress, “+ + + +”=very strong effect superior to internal standard ABA (comparative visual assessment of greenmass).

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Table 3. SAR-results of oxo-indolinyl sulfonamides 13a–p.

Entry

No.

Substituents^[a]

R¹

In vitro activity

ABI1-(AtRCAR11)^[b]

Activity [5 μM]

In vivo efficacy vs. drought stress

R²

X

250 g/ha^[c]

pI₅₀

Wheat^[d]

Canola^[d]

1

2

3

4

5

6

7

8

13a

13b

13c

13d

13e

13f

13g

13h

13i

13j

13k

13l

13m

13n

13o

13p

Me

Et

n-Pr

i-Pr

Me

Et

CF₃

Me

CF₃

CN

Cl

CF₃

Me

CF₃

Cl

Me

Cl

Me

Cl

dir. bond

CH₂

(CH₂)₂

+ +

4.3

4.7

4.9

4.7

4.8

5.0

5.3

4.2

4.4

4.2

4.5

4.1

4.6

4.2

n.d.

+

0

+ + +

+ + + +

+ +

+

+ +

+

0

+

0

+

0

+ +

+

+ +

+

+ +

+ + +

+ +

0

9

10

11

12

13

14

15

16

Et

n-Pr

Allyl

Me

+ + +

+ +

–

À CH=CH-

(CH₂)₃

Cl

CH₂À OÀ CH₂

–

0

[a] i-Pr=iso-propyl, n-Pr=n-propyl, Allyl=À CH₂À CH=CH₂, “dir. bond“=X stands for a direct bond between the Me groups forming a spiro-cyclopropyl ring;

[b] a final expert assessment of target affinity was made according to the following classification: “n.d.”=not determined, “-”=activity<30%, “+”=30%<

activity<50%, “+ +”=50%<activity<70%, “+ + +”=70%<activity<90%„ “+ + + +”=activity>90%; [c] standard application rate for crop

protection greenhouse trials; [d] a final expert assessment of efficacies was made according to the following classification: “0”=no effect, “+”=slight

beneficial effect, “+ +”=significant beneficial effect, “+ + +”=strong beneficial effect against drought stress, “+ + + +”=very strong effect superior to

internal standard ABA (comparative visual assessment of greenmass).

such as spiro-cyclohexyl (13o, entry 15, Table 3) and spiro-

pyranyl (13p, entry 16, Table 3) led to a complete loss of in vitro

and in vivo efficacy. Thus, we have identified the limits of the

receptor cavity addressed by the spiro-cycloalkyl indolinyl

moiety, again in good accordance with our modelling studies

outlined above.

Interestingly, both saturated (13j–m, entries 10–13, Table 3)

and unsaturated (13n, entry 14, Table 3) 5-membered rings

were tolerated with 13m carrying a spiro-cyclopentyl group and

a

N-(n-propyl) moiety giving the highest target affinity.

Accordingly, example 13m exhibited strong initial in vivo

efficacy against drought stress in wheat. Likewise, spiro-cyclo-

butyl-substituted oxo-tetrahydroquinolinyl sulfonamides 12j

and 12k also showed in vivo activity only in wheat, albeit on a

lower level than 13m.

Figure 6. Advanced drought stress trials with 13m in wheat and barley

under moderate drought stress conditions, i.e. 44% damage for barley and

49% for wheat, respectively (application rate 25 g/ha).

In order to corroborate the results of our initial in vivo

screening campaign, we selected 13m with strong efficacy in

wheat and 11b with good efficacy in canola as prototypes for

advanced greenhouse trials (i.e. more replicates, different

application rates, e.g. 100 and 25 g/ha, additional crops such as

cotton and barley, different varieties and moderate stress

levels). Hence, significant beneficial effects against drought

stress could be observed in wheat upon treatment with 13m,

as the representative oxo-indolinyl sulfonamide, compared to

untreated controls (Figure 6). However, these promising effects

could not be seen in barley emphasizing the differences

between crops regarding responses towards drought stress and

chemical treatments.

Accordingly, the strong in vivo efficacy of some oxo-

tetrahydroquinolinyl sulfonamides in canola observed in our

initial screening (cf. Table 1) could also be validated in

advanced greenhouse trials as representative example 11b

afforded substantial beneficial effects under moderate drought

stress conditions in canola compared with the untreated control

(Figure 7).

To rationalize our SAR-results further we selected oxo-

tetrahydroquinolinyl sulfonamides 11a, 11b, 11j and 11r as

prototypes in thermographic experiments and to investigate

their capacity to activate the ABA response in cell wall-free leaf

cells, so-called protoplasts.^[11]Ectopic expression of the ABA-

receptors (AtRCAR11 from A. thaliana) and the corresponding

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showed consistent ABA agonistic effects, both in A. thaliana

and in wheat protoplasts, albeit on a lower level than ABA 1.

Remarkably, 11b and 11r nearly reached the activity level of

ABA 1 at higher concentrations. The results shown in Figure 8

are in good accordance with consistent target affinities and

in vivo efficacies shown in Table 1.

In line with the ABA-responsive gene expression experi-

ments outlined above the thermographic analysis of changes in

leaf temperature induced by ABA and oxo-tetrahydroquinolinyl

sulfonamides 11a, 11b, 11j and 11r showed consistent effects

with 11r giving the highest temperature increase among the

sulfonamides tested, matching the efficacy of ABA at low

concentration (10 μM) (Figure 9).

Figure 7. Advanced drought stress trials with 11b in canola under moderate

drought stress conditions, i.e. 48% (application rate 6.25 g/ha).

wheat ABA receptor TuRCAR11 stimulate ABA-responsive

reporter expression of protoplasts in the presence of ABA. In Conclusion

this analysis, Arabidopsis protoplasts were transfected with gene

expression cassettes for the ABA receptor, an ABA-responsive

luciferase reporter, and a constitutive reporter for normalization

were exposed towards the ligand for 18 hours (Figure 8). The

ABA responsive luciferase activity was expressed as fold

induction relative to the value without ligand administration.

Transfections without ABA receptor construct and with

mock ligand levels (-ABA, -RCAR) served as controls in the

experiments outlined in Figure 8. All four selected examples, i.e.

oxo-tetrahydroquinolinyl sulfonamides 11a, 11b, 11j and 11r,

In the SAR-study described herein, we have outlined how we

have analyzed, explored and optimized a lead structure that

had shown good initial in vitro activity by interacting with

RCAR/(PYR/PYL) receptor proteins in A. thaliana. Converging

and optimized 5–6 step syntheses via substituted amino oxo-

tetrahydroquinolines and amino oxindolines as essential inter-

mediates afforded the envisaged oxotetrahydroquinolinyl and

oxindolinyl sulfonamides. In conclusion, we have identified

several potent analogues of sulfonamide lead structure quina-

bactin with higher target affinity and, more importantly,

improved in vivo efficacy against drought stress in broad-acre

crops canola and wheat compared with the initial lead

structure. Corroborating results from in vitro measurements and

crystal structure analyses have confirmed a cavity in the ABA-

receptor of limited size tolerating sterically more demanding

substituents at the tetrahydroquinoline nitrogen bearing more

Figure 9. Changes in leaf temperature induced by ABA and by selected

sulfonamides. 9a) Thermogram of Arabidopsis rosettes, arranged on a single

tray, 1 day after spraying different concentrations of ABA and compounds

11a, 11b, 11j, and 11r (10, 30, and 100 μM). Each vertical row shows 4

plants serving as replicates. The hormone solution contained 0.05% DMSO

and 0.01% Tween20 in water. 0.1, 0.3, 1% DMSO combined with 0.01%

Tween20 served as a solvent control. The left bar gives the calibration of the

false color code. 9b) Increases in leaf temperature in response to ABA

agonists as shown on top and treated simultaneously (SE, n=5). Leaf

Figure 8. Regulation of ABA-responsive gene expression of oxo-tetrahydro-

quinolinyl sulfonamides 11a, 11b, 11j and 11r. Ectopic expression

°

temperature of control plants is 22.5�0.06 C.

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between 3 and 5 days; in the case of monocotyledonous crops, for

constraint. In particular, 1,1’-bi(cyclopropyl)-1-yl, bicyclo[1.1.1]-

pent-1-yl and cyclopropylmethylene substituents afforded the

most promising results. Remarkably, some of the new sulfona-

mides exceeded the in vitro activity of natural ligand ABA. By

reducing the ring size and by adding constraint via introducing

a spiro-cycloalkyl group we have further modified the initial

lead structure quinabactin. Likewise, we have identified two

spiro-cycloalkyl substituted oxindolinyl sulfonamides that

showed promising activity against drought stress in wheat in

different greenhouse trials. A good translation between in vitro

results and drought stress control in the model crop canola can

be observed for oxo-tetrahydroquinolinyl sulfonamides 11a–w.

High target affinities measured at AtRCAR11 from A. thaliana

correlate well with good in vivo efficacy in related dicotyledo-

nous crop canola. However, the RCAR11 homologues from

plants other than A. thaliana are functionally poorly character-

ized except that they positively regulate ABA responses.

Precisely deducing an in vitro-in vivo translation from receptor

example wheat, it varied between 6 and 10 days. The end of the

stress phase was followed by an approx. 5-7-day recovery phase,

during which the plants were once again kept under good growth

conditions in a greenhouse. In order to rule out any influence of

the effects observed by any fungicidal action of the test

compounds, it was additionally ensured that the tests proceeded

without fungal infection and without infection pressure. After the

recovery phase had ended, the intensities of damage were rated

visually in comparison to untreated, unstressed controls of the

same age (in the case of drought stress) or the same growth stage

(in the case of cold stress). The intensity of damage was first

recorded as a percentage (100%=plants have died, 0%=like

control plants). These values were then used to calculate the

efficacy of the test compounds (=percentage reduction in the

intensity of damage as a result of substance application), and a final

assessment of the respective efficacy was made, i.e. “0”=no effect,

“+”=slight beneficial effect, “+ +”=significant beneficial effect,

“+ + +”=strong beneficial effect against drought stress, “+ + +

+”=very strong effect superior to internal standard ABA.

In vitro biology. ABA signaling. – Preparation and analysis of

Arabidopsis protoplasts was performed as described in earlier

studies.^[43]Briefly, protoplasts (105) were transfected with 5 μg DNA

of reporter construct (pRD29B::LUC), 3 μg of p35S::GUS plasmid as a

control for internal normalization of expression, and 3 μg of RCAR

effector expression cassettes. The effector cassettes drive expres-

sion of RCAR coding sequences under the control of the 35S

promoter.^[43]All constructs used were verified by DNA sequence

analysis. Total amount of transfected DNA per assay remained

constant by supplementing 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 PP2Cs was performed as

described.^[11]For the determination of activity, the dephosphoryla-

tion 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) dimethyl sulphoxide

(DMSO) 0.5% (f.c.) and b) 5 μM (f.c.) abscisic acid (ABA). The assay

described here was generally conducted with substance concen-

trations of the appropriate chemical test substances in a concen-

tration range of 0.1 μM to 100 μM in a solution of DMSO and water.

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 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 pH 8, 500 mM NaCl, 3.33 mM MnCl₂,

40 mM dithiothreitol (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 (enzyme stock was diluted so as to give, on

proteins isolated from

a dicotyledonous model plant to

monocotyledonous crops such as wheat is thus a more

challenging task. As a result, differences could be observed for

several compounds regarding their in vivo efficacy in canola

and wheat. Introducing substituents such as fluorine and the

trifluoromethyl group that are known to show beneficial effects

in agrochemistry also provided good results in our SAR-study

emphasizing that additional factors such as stability towards

metabolism and physicochemical properties have to be consid-

ered when carrying out in vivo SAR-studies. Our results show

that it is worthwhile to carry out in-depth in vivo SAR studies of

initial agrochemical lead structures with emphasis on thorough

structural variations including branched and rigid moieties.

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

conditions. The test plants were treated at the early leaf stage

(BBCH10–BBCH13). To ensure uniform water supply before com-

mencement 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 granules (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 following conditions: “day”=14 hours

addition of the dilution to the enzyme buffer mix,

a final

°

with illumination at 26 C; “night”=10 hours without illumination at

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 mM Tris-HCl pH 8, b) 10% by

vol. of 500 mM NaCl, c) 10% by vol. of 3.33 mM MnCl₂, d) 5% by

vol. of 25 mM MUP, 5% by vol. of Tween20 (1%), 60% by vol. of

H₂O bi-dist. Enzyme buffer mix and substrate mix were made up

5 minutes prior to the addition and warmed to a temperature of

°

18 C. The duration of the respective stress phases was guided

mainly by the state of the untreated (=treated with blank

formulation but without test compound), stressed control plants

and thus varied from 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

°

35 C. On completion of pipetting of all the solutions and on

°

completion of mixing, the plate was incubated at 35 C for

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20 minutes. Finally, a relative fluorescence measurement was made

Ethyl 3-{2-[1,1’-bi(cyclopropyl)-2-ylamino]-5-nitrophenyl}propanoate

(650 mg, 2.04 mmol) was dissolved in abs. tetrahydrofuran (8 mL)

°

at 35 C with a BMG Labtech “POLARstar Optima” microplate reader

°

using a 340/10 nm excitation filter and a 460 nm emission filter.

and added dropwise to a suspension, cooled down to 0 C, of

sodium hydride (122 mg, 3.06 mmol, 60% suspension in oil) in abs.

tetrahydrofuran (5 mL) under argon. The resulting reaction mixture

Chemistry. General – All reagent-grade solvents and chemicals

were purchased from standard commercial suppliers and used

without further purification. All non-aqueous reactions were carried

out under anhydrous conditions using dry solvents. Reactions were

monitored by LC-MS or TLC carried out on 0.25 mm silica gel plates

(60F-254). TLC plates were visualized using UV light. Flash

chromatography was carried out using Biotage Isolera One systems

with pre-packed column cartridges (Biotage KP-Sil [40+M] or KP-Sil

[25+M]). The ¹H NMR, ¹³C NMR and ¹⁹F NMR spectroscopy data

which are reported for the chemical examples described below

(400 and 600 MHz for ¹H NMR and 150 MHz for ¹³C NMR and

375 MHz for ¹⁹F NMR, solvent: CDCl₃, CD₃OD or d₆-DMSO, internal

standard: tetramethylsilane δ=0.00 ppm), were obtained on a

Bruker instrument, and the signals listed have the meanings given

below: br=broad; s=singlet, d=doublet, t=triplet, dd=doublet

of doublets, ddd=doublet of a doublet of doublets, m=multiplet,

q=quartet, quint=quintet, sext=sextet, sept=septet, dq=dou-

blet of quartets, dt=doublet of triplets. The abbreviations used for

chemical groups are defined as follows: Me=CH₃, Et=CH₂CH₃, t-

Hex=C(CH₃)₂CH(CH₃)₂, t-Bu=C(CH₃)₃, n-Bu=unbranched butyl, n-

Pr=unbranched propyl, c-Hex=cyclohexyl, ArH=aromatic hydro-

gen, HetH=heteroaromatic hydrogen. In the case of diastereomeric

mixtures, either the significant signals for each of the diastereomers

or the characteristic signal of the main diastereomer is/are reported.

A selection of experimental procedures has been outlined below.

Further experimental details have been described in the suppl.

material, as well as in references,^[31,32]and.^[42]

°

was stirred at 0 C for 1 h, and then water was added cautiously,

followed by ethyl acetate after stirring for 5 min. The aqueous

phase was then extracted repeatedly with ethyl acetate. The

combined organic phases were dried over magnesium sulfate,

filtered and concentrated under reduced pressure. By column

chromatography purification of the crude product obtained (ethyl

acetate/heptane gradient), 1-[1,1’-bi(cyclopropyl)-2-yl]-6-nitro-3,4-

dihydroquinolin-2(1H)-one (480 mg, 86%) was isolated as a color-

1

less solid, H-NMR (400 MHz, CDCl₃δ, ppm) 8.18 (m, 1H), 8.05 (m,

1H), 7.41 (d, 1H), 2.91 (m, 2H), 2.68 (m, 2H), 2.57 (m, 1H), 1.08–0.97

(m, 2H), 0.90–0.83 (m, 1H), 0.69 (m, 1H), 0.58 (m, 1H), 0.48 (m, 1H),

0.32–0.21 (m, 2H). In the next step, 1-[1,1’-bi(cyclopropyl)-2-yl]-6-

nitro-3,4-dihydroquinolin-2(1H)-one (480 mg, 1.76 mmol) was

added together with tin(II) chloride dihydrate (1591 mg, 7.05 mmol)

to abs. ethanol (5 mL) and the mixture was stirred under argon at a

°

temperature of 80 C for 5 h. After cooling to room temperature,

the reaction mixture was poured into ice-water and then adjusted

to pH 12 using aqueous NaOH. The aqueous phase was then

extracted repeatedly with ethyl acetate. The combined organic

phases were dried over magnesium sulfate, filtered and concen-

trated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), 6-amino-1-[1,1’-bi(cyclopropyl)-2-yl]-3,4-dihydroquinolin-

2(1H)-one (410 mg, 58% of theory) was isolated as a highly viscous

1

foam, H-NMR (400 MHz, CDCl₃δ, ppm) 7.08 (d, 1H), 6.58 (m, 1H),

6.49 (m, 1H), 3.72–3.38 (br. s, 2H, NH), 2.69 (m, 2H), 2.56 (m, 2H),

2.48 (m, 1H), 1.05–0.96 (m, 1H), 0.92–0.79 (m, 2H), 0.69 (m, 1H), 0.51

(m, 1H), 0.40 (m, 1H), 0.33–0.17 (m, 2H). 6-Amino-1-[1,1’-bi-

N-{1-[1,1’-bi(cyclopropyl)-2-yl]-2-oxo-1,2,3,4-tetrahydroquinolin-

6-yl}-1-(4-methylphenyl)methanesulfonamide (11a): Ethyl (2E)-3-

(2-fluoro-5-nitrophenyl)acrylate (2000 mg, 8.36 mmol) and 1,1’-bi-

(cyclopropyl)-2-amine (739 mg, 7.60 mmol) were dissolved under

argon in abs. N,N-dimethylformamide (12 mL), and then N,N-

diisopropylethylamine (2.65 mL, 15.20 mmol) was added. The

(cyclopropyl)-2-yl]-3,4-dihydroquinolin-2(1H)-one

(41 mg,

0.08 mmol) was dissolved together with (4-methylphenyl)

methanesulfonyl chloride (19 mg, 0.09 mmol) in abs. acetonitrile

(5 mL) in a baked-out round-bottom flask under argon, then

pyridine (0.01 mL, 0.17 mmol) was added and the mixture was

stirred at room temperature for 8 h. The reaction mixture was then

concentrated under reduced pressure, the remaining residue was

admixed with dil. HCl and dichloromethane, and the aqueous phase

was extracted repeatedly with dichloromethane. The combined

organic phases were dried over magnesium sulfate, filtered and

concentrated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), N-{1-[1,1’-bi(cyclopropyl)-2-yl]-2-oxo-1,2,3,4-tetrahydro-

quinolin-6-yl}-1-(4-methylphenyl)methanesulfonamide 11a (11 mg,

32% of theory) was isolated as a colourless solid. ¹H-NMR (600 MHz,

CDCl₃δ, ppm) 7.23 (d, 2H), 7.20 (d, 2H), 7.18 (m, 1H), 7.00 (d, 1H),

6.95 (d, 1H), 6.18 (s, 1H, NH), 4.30 (s, 2H), 2.79–2.76 (m, 2H), 2.62–

2.59 (m, 2H), 2.52–2.49 (m, 1H), 2.36 (s, 3H), 1.08–0.99 (m, 2H), 0.97–

0.93 (m, 1H), 0.71–0.68 (m, 1H), 0.57–0.54 (m, 1H), 0.45–0.42 (m, 1H),

0.30–0.21 (m, 2H). ¹³C-NMR (150 MHz, CDCl₃δ, ppm) 169.7, 136.8,

136.1, 129.2, 128.6, 127.4, 125.9, 123.6, 118.3, 117.6, 114.9, 55.2,

30.3, 28.9, 23.2, 22.7, 19.1, 12.0, 8.9, 3.6, 2.2. LCMS (ESI, m/z): [M+

H]⁺411.3; HRMS (ESI, m/z): calcd. for C₂₃H₂₇N₂O₃S, 411.1742 [M+

H]⁺; found 411.1755.

°

resulting reaction mixture was stirred at a temperature of 50 C for

10 h and, after cooling to room temperature, water and ethyl

acetate were added. The aqueous phase was then extracted

repeatedly with ethyl acetate. The combined organic phases were

dried over magnesium sulfate, filtered and concentrated under

reduced pressure. By column chromatography purification of the

crude product obtained (ethyl acetate/heptane gradient), ethyl

(2E)-3-{2-[1,1’-bi(cyclopropyl)-2-ylamino]-5-nitrophenyl}-acrylate

(1730 mg, 65% of theory) was isolated as a colourless solid, ¹H-NMR

(400 MHz, CDCl₃δ, ppm) 8.27 (m, 1H), 8.17 (m, 1H), 7.68/7.60 (d,

1H), 7.07 (m, 1H), 6.47/6.43 (d, 1H), 5.18/5.04 (br. s, 1H, NH), 4.29 (q,

2H), 2.63/2.34 (m, 1H), 1.34 (t, 3H), 1.31–1.24 (m, 1H), 1.05–0.97 (m,

1H), 0.92–0.86 (m, 1H), 0.87–0.82 (m, 1H), 0.64–0.45 (m, 2H), 0.27–

0.23 (m, 1H), 0.21–0.15 (m, 1H). Ethyl (2E)-3-{2-[1,1’-bi(cyclopropyl)-

2-ylamino]-5-nitrophenyl}acrylate (1730 mg, 5.47 mmol) was then

dissolved in abs. ethanol (15 mL), and (Ph₃P)₃RhCl (400 mg,

0.43 mmol) was added. After stirring at room temperature for

5 min, hydrogen was introduced into the reaction solution with a

constant gas flow via a gas introduction apparatus for 9 h. The

progress of the reaction was monitored by LCMS. On completion of

conversion, the reaction solution was concentrated under reduced

pressure. By column chromatography purification of the crude

product obtained (ethyl acetate/heptane gradient), it was possible

to isolate ethyl 3-{2-[1,1’-bi(cyclopropyl)-2-ylamino]-5-nitrophenyl}

N-[1-(Bicyclo[1.1.1]pent-1-yl)-2-oxo-1,2,3,4-tetrahydroquinolin-6-

yl]-1-[4-(trifluoromethyl)phenyl]methanesulfonamide (11h): Ethyl

(2E)-3-(2-fluoro-5-nitrophenyl)acrylate (4000 mg, 16.7 mmol) and

bicyclo[1.1.1]pent-1-ylamine (1810 mg, 15.2 mmol) were dissolved

under argon in abs. N,N-dimethylformamide (30 mL), and then N,N-

diisopropylethylamine (5.0 mL, 30.4 mmol) was added. The result-

1

propanoate (650 mg, 37% of theory) as a colourless solid, H-NMR

(400 MHz, CDCl₃δ, ppm) 8.09 (m, 1H), 7.93 (m, 1H), 6.98 (m, 1H),

5.22 (br. m, 1H, NH), 4.15 (q, 2H), 2.78 (m, 2H), 2.66–2.47 (m, 2H),

2.31 (m, 1H), 1.27 (t, 3H), 1.02–0.87 (m, 2H), 0.72–0.67 (m, 1H), 0.60–

0.51 (m, 2H), 0.49–0.42 (m, 1H), 0.24–0.20 (m, 1H), 0.20–0.13 (m, 1H).

°

ing reaction mixture was stirred at a temperature of 50 C for 10 h

and, after cooling to room temperature, water and ethyl acetate

were added. The aqueous phase was then extracted repeatedly

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with ethyl acetate. The combined organic phases were dried over

451.3; HRMS (ESI, m/z): calcd. for C₂₂H₂₂N₂O₃F₃S, 451.1303 [M+H]⁺;

found 451.1313.

magnesium sulfate, filtered and concentrated under reduced

pressure. By column chromatography purification of the crude

product obtained (ethyl acetate/heptane gradient), ethyl (2E)-3-[5-

N-[1-(Cyclopropylmethyl)-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl]-

1-(4-ethylphenyl)methane sulfonamide (11t): 3,4-Dihydroquinolin-

2(1H)-one (770 mg, 3.83 mmol) was added to conc. acetic acid

(5 ml), and fuming nitric acid (0.21 ml, 5.06 mmol) was then added

carefully. The resulting reaction mixture was stirred at room

temperature for 2 h and then diluted with ice-water. The aqueous

phase was then repeatedly extracted with ethyl acetate. The

combined organic phases were dried over magnesium sulfate,

filtered and concentrated under reduced pressure. By column

chromatography purification of the crude product obtained (ethyl

acetate/heptane gradient), 6-nitro-3,4-dihydroquinolin-2(1H)-one

(500 mg, 68% of theory) was isolated as a colorless solid. 6-Nitro-

3,4-dihydroquinolin-2(1H)-one (500 mg, 2.60 mmol) was dissolved

under argon in abs. N,N-dimethylformamide and admixed with fine

potassium carbonate powder (1.08 mg, 7.81 mmol). After stirring at

room temperature for 5 min, chloromethylcyclopropane (306 mg,

3.38 mmol) and potassium iodide (6 mg, 0.04 mmol) were added.

nitro-2-(bicyclo[1.1.1]pent-1-ylamino)phenyl]acrylate

68% of theory) was isolated as a colorless solid. Ethyl (2E)-3-[5-

nitro-2-(bicyclo[1.1.1]pent-1-ylamino)phenyl]acrylate (3120 mg,

(3120 mg,

10.3 mmol) was then dissolved in abs. ethanol (200 mL), and

(Ph₃P)₃RhCl (477 mg, 0.52 mmol) was added. After stirring at room

temperature for 5 min, hydrogen was introduced into the reaction

solution with a constant gas flow via a gas introduction apparatus

for 10 h. The progress of the reaction was monitored by LCMS. On

completion of conversion, the reaction solution was concentrated

under reduced pressure. By column chromatography purification of

the crude product obtained (ethyl acetate/heptane gradient), it was

possible to isolate ethyl 3-[5-nitro-2-(bicyclo[1.1.1]pent-1-ylamino)

phenyl]propanoate (2740 mg, 88% of theory) as a colorless solid.

Ethyl 3-[5-nitro-2-(bicyclo[1.1.1]pent-1-ylamino)phenyl]propanoate

(2740 mg, 9.0 mmol) was dissolved in abs. tetrahydrofuran (100 mL)

and added dropwise over a period of 30 minutes to a suspension,

°

The resulting reaction mixture was stirred at 120 C for 2 h and,

°

cooled down to 0 C, of sodium hydride (540 mg, 13.5 mmol, 60%

suspension in oil) in abs. tetrahydrofuran (50 mL) under argon. The

after cooling to room temperature, water and ethyl acetate were

added. The aqueous phase was then repeatedly extracted with

ethyl acetate. The combined organic phases were dried over

magnesium sulfate, filtered and concentrated under reduced

pressure. By column chromatography purification of the crude

product obtained (ethyl acetate/heptane gradient), 1-

(cyclopropylmethyl)-6-nitro-3,4-dihydroquinolin-2(1H)-one (600 mg,

94% of theory) was isolated as a colourless solid. ¹H-NMR (400 MHz,

CDCl₃δ, ppm) 8.17 (dd, 1H), 8.08 (d, 1H), 7.22 (d, 1H), 3.91 (d, 2H),

3.04 (m, 2H), 2.73 (m, 2H), 1.12 (m, 1H), 0.55 (m, 2H), 0.45 (m, 2H). In

the next step, 1-(cyclopropylmethyl)-6-nitro-3,4-dihydroquinolin-

2(1H)-one (600 mg, 2.44 mmol) was added together with tin(II)

chloride dihydrate (2.19 g, 9.75 mmol) to abs. ethanol and the

°

resulting reaction mixture was stirred at 0 C for 1 h, and then water

was added cautiously, followed by ethyl acetate after stirring for

5 min. The aqueous phase was then extracted repeatedly with ethyl

acetate. The combined organic phases were dried over magnesium

sulfate, filtered and concentrated under reduced pressure. By

column chromatography purification of the crude product obtained

(ethyl acetate/heptane gradient), 6-nitro-1-(bicyclo[1.1.1]pent-1-yl)-

3,4-dihydroquinolin-2(1H)-one (1520 mg, 65%) was isolated as a

colorless solid. In the next step, 6-nitro-1-(bicyclo[1.1.1]pent-1-yl)-

3,4-dihydroquinolin-2(1H)-one (2130 mg, 8.25 mmol) was added

together with ammonium chloride (4410 mg, 82.5 mmol) and iron

powder (1380 mg, 24.7 mmol) to abs. ethanol (150 mL) and water

(75 mL), and the mixture was stirred under argon at a temperature

°

mixture was stirred under argon at a temperature of 80 C for 5 h.

After cooling to room temperature, the reaction mixture was

poured into ice-water and then adjusted to pH 12 with aqueous

NaOH. The aqueous phase was then repeatedly extracted with ethyl

acetate. The combined organic phases were dried over magnesium

sulfate, filtered and concentrated under reduced pressure. By

column chromatography purification of the crude product obtained

(ethyl acetate/heptane gradient), 6-amino-1-(cyclopropylmethyl)-

3,4-dihydroquinolin-2(1H)-one (481 mg, 91% of theory) was iso-

lated as a colourless solid. ¹H-NMR (400 MHz, CDCl₃δ, ppm) 6.94 (d,

1H), 6.58 (dd, 1H), 6.53 (d, 1H), 3.83 (d, 3H), 2.81 (m, 2H), 2.61 (m,

2H), 1.12 (m, 1H), 0.47 (m, 2H), 0.39 (m, 2H). 6-Amino-1-

°

of 80 C for 1 h. After cooling to room temperature, the reaction

mixture was filtered through Celite and washed through thoroughly

with methanol and concentrated under reduced pressure. The

residue was taken up with dichloromethane and water and

extracted thoroughly. The aqueous phase was re-extracted repeat-

edly with dichloromethane. The combined organic phases were

dried over magnesium sulfate, filtered and concentrated under

reduced pressure. By column chromatography purification of the

crude product obtained (ethyl acetate/heptane gradient), 6-amino-

1-(bicyclo[1.1.1]pent-1-yl)-3,4-dihydroquinolin-2(1H)-one (1210 mg,

64% of theory) was isolated as a highly viscous foam. 6-Amino-1-

cyclopropylmethyl-3,4-dihydroquinolin-2(1H)-one

(90 mg,

(bicyclo[1.1.1]pent-1-yl)-3,4-dihydroquinolin-2(1H)-one

(60 mg,

0.42 mmol) was dissolved together with (4-ethylphenyl)

methanesulfonyl chloride (100 mg, 0.46 mmol) in abs. acetonitrile

(5 mL) in a baked-out round-bottom flask under argon, then

pyridine (0.07 mL, 0.83 mmol) was added and the mixture was

stirred at room temperature for 9 h. The reaction mixture was then

concentrated under reduced pressure, the remaining residue was

admixed with dil. HCl and dichloromethane, and the aqueous phase

was extracted repeatedly with dichloromethane. The combined

organic phases were dried over magnesium sulfate, filtered and

concentrated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), N-[1-(cyclopropylmethyl)-2-oxo-1,2,3,4-tetrahydroquino-

lin-6-yl]-1-(4-ethylphenyl)methanesulfonamide 11t (114 mg, 69% of

0.26 mmol) was dissolved together with (4-trifluoromethyl-phenyl)

methanesulfonyl chloride (65 mg, 0.29 mmol) in abs. acetonitrile

(5 mL) in a baked-out round-bottom flask under argon, then

pyridine (0.04 mL, 0.47 mmol) was added and the mixture was

stirred at room temperature for 8 h. The reaction mixture was then

concentrated under reduced pressure, the remaining residue was

admixed with dil. HCl and dichloromethane, and the aqueous phase

was extracted repeatedly with dichloromethane. The combined

organic phases were dried over magnesium sulfate, filtered and

concentrated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), N-[1-(bicyclo[1.1.1]pent-1-yl)-2-oxo-1,2,3,4-tetrahydroqui-

nolin-6-yl]-1-[4-(trifluoromethyl)phenyl]methanesulfonamide 11h

1

theory) was isolated as a colorless solid. H-NMR (600 MHz, CDCl₃δ,

1

(56 mg, 47% of theory) was isolated as a colorless solid. H-NMR

ppm) 7.24 (d, 2H), 7.22 (d, 2H), 7.08 (d, 1H), 6.98 (m, 2H), 6.24 (s, 1H,

NH), 4.30 (s, 2H), 3.86 (d, 2H), 2.89 (m, 2H), 2.68–2.64 (m, 4H), 1.25 (t,

3H), 1.15–1.09 (m, 1H), 0.53–0.50 (m, 2H), 0.45–0.41 (m, 2H). ¹³C-

NMR (150 MHz, CDCl₃δ, ppm) 169.9, 145.3, 137.4, 131.4, 130.8,

128.4, 127.9, 125.7, 121.0, 120.1, 115.9, 57.4, 45.9, 31.6, 28.6, 25.7,

(600 MHz, CDCl₃δ, ppm) 7.63 (d, 2H), 7.46 (d, 2H), 7.22 (d, 1H), 6.97

(dd, 1H), 6.94 (d, 1H), 6.50 (br. s, 1H, NH), 4.39 (s, 2H), 2.80–2.76 (m,

2H), 2.53 (m, 2H), 2.41 (s, 6H). ¹³C-NMR (150 MHz, CDCl₃δ, ppm)

171.9, 137.6, 137.7, 132.6, 131.9, 131.6, 131.4, 130.5, 125.8, 120.6,

120.2, 119.1, 57.3, 54.2, 33.5, 25.8, 25.4. LCMS (ESI, m/z): [M+H]⁺

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15.4, 9.8, 4.2. LCMS (ESI, m/z): [M+H]⁺399.0; HRMS (ESI, m/z):

theory) was isolated as a colorless solid. 6’-Nitro-1’H-spiro

calcd. for C₂₂H₂₇N₂O₃S, 399.1742 [M+H]⁺; found 399.1742.

[cyclobutyl-1,4’-quinolin]-2’(3’H)-one (100 mg, 1.0 equiv.) was dis-

solved under argon in abs. dioxane (2 mL) and admixed with fine

cesium carbonate powder (400 mg, 3.0 equiv.). After stirring at

room temperature for 5 min, cyclobutylmethyl bromide (110 mg,

2.0 equiv.) and potassium iodide (35 mg, 0.1 equiv.) were added at

room temperature. The resulting reaction mixture was stirred at

N-[1’-(Cyclopropylmethyl)-2’-oxo-2’,3’-dihydro-1’H-spiro[cyclobu-

tane-1,4’-quinolin]-6’-yl]-1-(4-trifluoromethylphenyl)

methanesulfon-amide (12j): Triethyl phosphonoacetate (3.32 g,

1.0 equiv) was dissolved in abs. tetrahydrofuran and added to a

°

suspension, cooled down to 0 C, of sodium hydride (0.58 g,

1.02 equiv, 60% dispersion) in abs. tetrahydrofuran (5 mL). The

°

150 C under microwave conditions for 1 h and, after cooling to

room temperature, water and ethyl acetate were added. The

aqueous phase was then repeatedly extracted with ethyl acetate.

The combined organic phases were dried over magnesium sulfate,

filtered and concentrated under reduced pressure. By column

chromatography purification of the crude product obtained (ethyl

acetate/heptane gradient), 1-(cyclopropylmethyl)-6’-nitro-1’H-spiro

[cyclobutyl-1,4’-quinolin]-2’(3’H)-one (70 mg, 60% of theory) was

isolated as a colorless solid. In the next step, 1-(cyclopropylmethyl)-

°

resulting reaction mixture was stirred at a temperature of 0 C for

10 minutes and then admixed with a solution of cyclobutanone

(1.0 g, 1.0 equiv) in abs. tetrahydrofuran (5 mL), and the mixture

was stirred at room temperature for a further 4 h. After the cautious

addition of water, the reaction mixture was concentrated under

reduced pressure and admixed with dichloromethane. The aqueous

phase was then repeatedly extracted with dichloromethane. The

combined organic phases were dried over magnesium sulfate,

filtered and concentrated under reduced pressure. By column

chromatography purification of the crude product obtained (ethyl

acetate/heptane gradient), ethyl cyclobutylideneacetate (1.5 g, 75%

of theory) was isolated. Ethyl cyclobutylideneacetate (1.0 g,

1.0 equiv) was dissolved in methanol and admixed with a 1 M

solution of KOH in aq. methanol. The resulting reaction mixture was

stirred at room temperature for 16 h, then neutralized with dil. HCl,

admixed with water, concentrated under reduced pressure and

then admixed with dichloromethane. The aqueous phase was then

repeatedly extracted with dichloromethane. The combined organic

phases were dried over magnesium sulfate, filtered and concen-

trated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), cyclobutylideneacetic acid (0.40 g, 51% of theory) was

isolated. Aniline (0.26 g, 1 equiv.) was dissolved in dichloromethane

6’-nitro-1’H-spiro[cyclobutyl-1,4’-quinolin]-2’(3’H)-one

(50 g,

1 equiv.) was added together with zinc dust (55 mg, 5 equiv.) and

ammonium chloride (90 mg, 10 equiv.) to methanol/water (5:1)

°

and the mixture was stirred under argon at a temperature of 70 C

for 2 h. After cooling to room temperature, the reaction mixture

was poured onto ice-water and then adjusted to pH 12 with 6 N

NaOH. The aqueous phase was then repeatedly extracted with ethyl

acetate. The combined organic phases were dried over magnesium

sulfate, filtered and concentrated under reduced pressure. By

column chromatography purification of the crude product obtained

(ethyl acetate/heptane gradient), 6‘-amino-1-(cyclopropylmethyl)-

1’H-spiro[cyclobutyl-1,4’-quinolin]-2’(3’H)-one (35 mg, 70% of

theory) was isolated as

a

colorless solid. 6‘-Amino-1-

(cyclopropylmethyl)-1’H-spiro[cyclobutyl-1,4’-quinolin]-2’(3’H)-one

(1.0 equiv.) was dissolved together with 4-trifluoro-meth-

ylbenzylsulfonyl chloride (1.1 equiv) in abs. dichloromethane (5 mL)

in a baked-out round-bottom flask under argon, then pyridine

(0.15 mL, 5 equiv.) was added and the mixture was stirred at room

temperature for 1 h. The reaction mixture was then concentrated

under reduced pressure, the remaining residue was admixed with

dil. HCl and dichloromethane, and the aqueous phase was

extracted repeatedly with dichloromethane. The combined organic

phases were dried over magnesium sulfate, filtered and concen-

trated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), N-[1’-(cyclopropylmethyl)-2’-oxo-2’,3’-dihydro-1’H-spiro

[cyclo-butane-1,4’-quinolin]-6’-yl]-1-(4-trifluoromethylphenyl)

°

(5 mL) and cooled down to a temperature of 0 C, and diisopropy-

lethylamine (1.98 mL, 4.0 equiv.), cyclopentylideneacetic acid

(0.30 g, 1.0 equiv.) and N,N,N’,N’-tetramethyl-O-(benzotriazol-1-yl)

uronium tetrafluoroborate (0.97 g, 1.1 equiv.) were added. The

resulting reaction mixture was stirred at room temperature for 3 h,

and water and dichloromethane were then added. The aqueous

phase was then repeatedly extracted with dichloromethane. The

combined organic phases were dried over magnesium sulfate,

filtered and concentrated under reduced pressure. By column

chromatography purification of the crude product obtained (ethyl

acetate/heptane gradient), 2-cyclobutylidene-N-phenylacetamide

(0.27 g, 54% of theory) was isolated. In the next step, aluminum

trichloride (0.42 g, 3.0 equiv.) was initially charged in abs. dichloro-

ethane (5 mL) under argon in a baked-out round-bottom flask and

then, while cooling with ice, a solution of 2-cyclobutylidene-N-

phenylacetamide (0.20 g, 1.0 equiv.) in abs. dichloroethane (5 mL)

was added. The resulting reaction mixture was stirred at room

temperature for a further 4 h and then added cautiously to ice-

water. After adding aqueous HCl and dichloromethane, the

aqueous phase was extracted repeatedly with dichloromethane.

The combined organic phases were dried over magnesium sulfate,

filtered and concentrated cautiously under reduced pressure. By

column chromatography purification of the crude product obtained

(ethyl acetate/heptane gradient), 1’H-spiro[cyclobutyl-1,4’-quinolin]-

2’(3’H)-one was isolated as a colorless solid. 1’H-Spiro[cyclobutyl-

1,4’-quinolin]-2’(3’H)-one (0.2 g, 1 equiv.) was added to conc. acetic

methanesulfon-amide 12j (88 mg, 43% of theory) was isolated as a

1

colorless solid. H-NMR (600 MHz, CDCl₃δ, ppm) 7.65 (d, 2H), 7.49

(d, 2H), 7.22 (d, 1H), 7.11 (d, 1H), 7.06 (m, 1H), 6.30 (br. s, 1H, NH),

4.41 (s, 2H), 3.87 (m, 2H), 2.77 (s, 2H), 2.28–2.22 (m, 2H), 2.14–2.07

(m, 3H), 2.01–1.94 (m, 1H), 1.12–1.07 (m, 1H), 0.49–0.46 (m, 2H), 0.39

(m, 2H). ¹³C-NMR (150 MHz, CDCl₃δ, ppm) 169.1, 136.4, 135.6, 132.7,

131.4, 131.2, 125.8, 120.1, 117.8, 116.6, 57.4, 45.6, 43.4, 39.8, 31.2,

14.9, 9.7, 4.0. LCMS (ESI, m/z): [M+H]⁺479.5; HRMS (ESI, m/z):

calcd. for C₂₄H₂₆N₂O₃F₃S, 479.1616 [M+H]⁺; found 479.1635

N-[1’-(Cyclopropylmethyl)-2’-oxo-2’,3’-dihydro-1’H-spiro[cyclo-

hexane-1,4’-quinolin]-6’-yl]-1-(4-methylphenyl)

methanesulfonamide (12n): Triethyl phosphonoacetate (232.0 g,

1.0 equiv) was dissolved in abs. tetrahydrofuran (200 mL) and

°

added to a suspension, cooled down to 0 C, of sodium hydride

°

acid (1.5 mL) and then cautiously admixed at 0 C with fuming nitric

acid (0.5 mL). The resulting reaction mixture was then stirred at

(42.0 g, 1.02 equiv, 60% dispersion) in abs. tetrahydrofuran

(200 mL). The resulting reaction mixture was stirred at a temper-

°

90 C for 2 h and, after cooling to room temperature, cautiously

ature of 0 C for 10 minutes and then admixed with a solution of

diluted with ice-water. The aqueous phase was then repeatedly

extracted with ethyl acetate. The combined organic phases were

dried over magnesium sulfate, filtered and concentrated under

reduced pressure. By column chromatography purification of the

crude product obtained (ethyl acetate/heptane gradient), 6’-nitro-

1’H-spiro[cyclobutyl-1,4’-quinolin]-2’(3’H)-one (100 mg, 78% of

cyclohexanone (100.0 g, 1.0 equiv) in abs. tetrahydrofuran (300 mL),

and the mixture was stirred at room temperature for a further 4 h.

After the cautious addition of water, the reaction mixture was

concentrated under reduced pressure and admixed with dichloro-

methane. The aqueous phase was then repeatedly extracted with

dichloromethane. The combined organic phases were dried over

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magnesium sulfate, filtered and concentrated under reduced

spiro-[cyclopentyl-1,4’-quinolin]-2’(3’H)-one (700 mg, 58% of

theory) was isolated as a colorless solid. In the next step, 1-

(cyclopropylmethyl)-6’-nitro-1’H-spiro[cyclohexyl-1,4’-quinolin]-

2’(3’H)-one (900 mg, 1.0 equiv.) together with zinc powder (931 mg,

5.0 eq.) and ammonium chloride (759 mg, 5.0 eq.) were added to

pressure. By column chromatography purification of the crude

product obtained (ethyl acetate/heptane gradient), ethyl cyclo-

hexylideneacetate (105.0 g) was isolated. Ethyl cyclopentylidenea-

cetate (105.0 g, 1.0 equiv) was dissolved in methanol and admixed

with a 1 M solution of KOH in aq. methanol (1000 mL). The resulting

reaction mixture was stirred at room temperature for 16 h, then

neutralized cautiously with dil. HCl, admixed with water, concen-

trated under reduced pressure and then admixed with dichloro-

methane. The aqueous phase was then repeatedly extracted with

dichloromethane. The combined organic phases were dried over

magnesium sulfate, filtered and concentrated under reduced

pressure. By column chromatography purification of the crude

product obtained (ethyl acetate/heptane gradient), cyclohexylide-

neacetic acid (70.0 g, 80% of theory) was isolated. Aniline (41.5 g,

1.0 eq.) was dissolved in dichloromethane (500 mL) and cooled

°

abs. methanol at a temperature of 0 C and the mixture was stirred

under argon at room temperature for 1 h. The reaction mixture was

then poured onto ice-water and subsequently adjusted to pH 12

with 6 N NaOH. The aqueous phase was then repeatedly extracted

with ethyl acetate. The combined organic phases were dried over

magnesium sulfate, filtered and concentrated under reduced

pressure. By column chromatography purification of the crude

product obtained (ethyl acetate/heptane gradient), 6‘-amino-1-

(cyclopropylmethyl)-1’H-spiro[cyclohexyl-1,4’-quinolin]-2’(3’H)-one

(750 mg, 92% of theory) was isolated as a colorless solid. 6‘-Amino-

1-(cyclopropylmethyl)-1’H-spiro[cyclohexyl-1,4’-quinolin]-2’(3’H)-one

(70 mg, 1.0 equiv.) was dissolved together with 4-meth-

ylbenzylsulfonyl chloride (70 mg, 1.1 equiv) in abs. dichloromethane

(5 mL) under argon in a baked-out round-bottom flask under argon,

then pyridine (0.1 mL, 5.0 equiv.) was added and the mixture was

stirred at room temperature for 1 h. The reaction mixture was then

concentrated under reduced pressure, the remaining residue was

admixed with dil. HCl and dichloromethane, and the aqueous phase

was extracted repeatedly with dichloromethane. The combined

organic phases were dried over magnesium sulfate, filtered and

concentrated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient), N-[1’-(cyclopropylmethyl)-2’-oxo-2’,3’-dihydro-1’H-spiro

[cyclohexane-1,4’-quinolin]-6’-yl]-1-(4-methylphenyl)methan-sulfo-

namide 12n (60 mg, 48% of theory) was isolated as a colourless

°

down to a temperature of 0 C, and diisopropylethylamine (354 mL,

4.0 equiv.), cyclohexylideneacetic acid (70.0 g, 1.0 equiv.) and

N,N,N’,N’-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluorobo-

rate (180 g, 1.1 equiv.) were added. The resulting reaction mixture

was stirred at room temperature for 3 h, and water and dichloro-

methane were then added. The aqueous phase was then repeatedly

extracted with dichloromethane. The combined organic phases

were dried over magnesium sulfate, filtered and concentrated

under reduced pressure. By column chromatography purification of

the crude product obtained (ethyl acetate/heptane gradient), 2-

cyclohexylidene-N-phenylacetamide (80.0 g, 74% of theory) was

isolated. In the next step, aluminum trichloride (93.2 g, 3.0 equiv.)

was initially charged in abs. dichloromethane (400 mL) under argon

in a baked-out round-bottom flask and then, while cooling with ice,

1

a

solution of 2-cyclohexylidene-N-phenylacetamide (50.0 g,

solid. H-NMR (400 MHz, CDCl₃δ, ppm) 7.66 (d, 2H), 7.48 (d, 2H),

1.0 equiv.) in abs. dichloromethane (100 mL) was added. The

resulting reaction mixture was stirred at room temperature for a

further 4 h and then added cautiously to ice-water. After adding

aqueous HCl and dichloromethane, the aqueous phase was

extracted repeatedly with dichloromethane. The combined organic

phases were dried over magnesium sulfate, filtered and concen-

trated cautiously under reduced pressure. By column chromatog-

raphy purification of the crude product obtained (ethyl acetate/

heptane gradient), 1’H-spiro[cyclohexyl-1,4’-quinolin]-2’(3’H)-one

(12.0 g, 24% of theory) was isolated as a colourless solid. 1’H-Spiro

[cyclohexyl-1,4’-quinolin]-2’(3’H)-one (12.0 g, 55.80 mmol, 1 eq.) was

added to conc. acetic acid (100 mL) and then cautiously admixed

with fuming nitric acid (20 mL). The resulting reaction mixture was

7.14–7.08 (m, 2H), 7.05 (m, 1H), 6.13 (s, 1H, NH), 4.39 (s, 2H), 3.39 (m,

2H), 2.67 (s, 2H), 1.78 (m, 1H), 1.71–1.54 (m, 8H), 1.29 (m,1H), 1.11

(m, 1H), 0.50 (m, 2H), 0.43 (m, 2H). LCMS (ESI, m/z): [M+H]⁺453.4;

HRMS (ESI, m/z): calcd. for C₂₆H₃₃N₂O₃S, 453.2212 [M+H]⁺; found

453.2224.

N-(1’-Methyl-2’-oxo-1’,2’-dihydrospiro[cyclobutane-1,3’-indol]-5’-

yl)-1-(4-trifluoromethylphenyl)methanesulfonamide (13i): In

a

round-bottom flask which had been dried by heating, and under

argon, 1-methyl-1,3-dihydro-2H-indol-2-one (1.00 g, 7 mmol) and

1,3-dibromopropane (2.06 g, 10 mmol) were dissolved in abs. N,N-

dimethylformamide, and the mixture was stirred at room temper-

°

ature for 5 min. The reaction solution was then cooled to 0 C, and

sodium hydride (0.82 g, 20 mmol, 60% strength dispersion) was

then added a little at a time. The resulting reaction mixture was

stirred for about 2 h, methanol (4 ml) was then added and after a

further 5 min sat. ammonium chloride solution (15 ml) and water

(200 ml) were added. The aqueous phase was extracted intensively

with ethyl acetate. The combined organic phases were dried over

magnesium sulfate, filtered and concentrated under reduced

pressure. Purification of the resulting crude product by column

chromatography (gradient ethyl acetate/heptane) gave 1’-meth-

ylspiro[cyclobutane-1,3’-indol]-2’(1’H)-one (360 mg, 29% of theory).

¹H-NMR (400 MHz, CDCl₃δ, ppm) 7.52 (d, 1H), 7.27 (m, 1H), 7.09 (m,

1H), 6.77 (d, 1H), 3.20 (s, 3H), 2.67 (m, 2H), 2.33 (m, 4H). 1’-

°

stirred at 70 C for 1 h and, after cooling to room temperature,

cautiously diluted with ice-water. The aqueous phase was then

repeatedly extracted with ethyl acetate. The combined organic

phases were dried over magnesium sulfate, filtered and concen-

trated under reduced pressure. By column chromatography

purification of the crude product obtained (ethyl acetate/heptane

gradient),

(2500 mg, 17% of theory) was isolated as a colourless solid. 6’-

Nitro-1’H-spiro[cyclohexyl-1,4’-quinolin]-2’(3’H)-one (1000 mg,

6’-nitro-1’H-spiro[cyclohexyl-1,4’-quinolin]-2’(3’H)-one

3.84 mmol, 1.0 equiv.) was dissolved under argon in a mixture of

abs. dioxane and N,N-dimethylformamide (5:1, 12 mL) and admixed

with fine cesium carbonate powder (3800 mg, 3.0 eq.). After stirring

at room temperature for 5 min, cyclopropylmethyl bromide

(1400 mg, 2.0 equiv.) and potassium iodide (64 mg, 0.1 equiv.) were

added at room temperature. The resulting reaction mixture was

Methylspiro[cyclobutane-1,3’-indol]-2’(1’H)-one

(360 mg,

1.92 mmol) was added to conc. acetic acid (5 ml), and fuming nitric

acid (0.21 ml, 5.06 mmol) was then added carefully. The resulting

reaction mixture was stirred at room temperature for 2 h and then

diluted with ice-water. The aqueous phase was then repeatedly

extracted with ethyl acetate. The combined organic phases were

dried over magnesium sulfate, filtered and concentrated under

reduced pressure. Purification of the resulting crude product by

column chromatography (gradient ethyl acetate/heptane) gave 1’-

°

stirred at 150 C under microwave conditions for 1 h and, after

cooling to room temperature, water and ethyl acetate were added.

The aqueous phase was then repeatedly extracted with ethyl

acetate. The combined organic phases were dried over magnesium

sulfate, filtered and concentrated under reduced pressure. By

column chromatography purification of the crude product obtained

(ethyl acetate/heptane gradient), 1-(cyclopropylmethyl)-6’-nitro-1’H-

methyl-5’-nitrospiro[cyclobutane-1,3’-indol]-2’(1’H)-one

(380 mg,

85% of theory) as a colorless solid.¹H-NMR (400 MHz, CDCl₃δ, ppm)

Eur. J. Org. Chem. 2021, 3442–3457

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3455

Full Papers

doi.org/10.1002/ejoc.202100415

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°

and stirred under argon at a temperature of 80 C for 5 h. After

cooling to room temperature, the reaction mixture was poured into

ice-water and then adjusted to pH 12 using aqueous NaOH. The

aqueous phase was then repeatedly extracted with ethyl acetate.

The combined organic phases were dried over magnesium sulfate,

filtered and concentrated under reduced pressure. Purification of

the resulting crude product by column chromatography (gradient

ethyl acetate/heptane) gave 5’-amino-1’-methylspiro[cyclobutane-

1,3’-indol]-2’(1’H)-one (230 mg, 66% of theory) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃δ, ppm) 6.98 (d, 1H), 6.62 (m, 2H), 3.14 (s,

3H), 2.64 (m, 2H), 2.38–2.20 (m, 4H). Under argon, 5’-amino-1’-

methylspiro[cyclobutane-1,3’-indol]-2’(1’H)-one

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0.79 mmol) and (4-trifluoromethylphenyl)methanesulfonyl chloride

(156 mg, 0.76 mmol) were dissolved in abs. acetonitrile (5 ml) in a

round-bottom flask which had been dried by heating, pyridine

(0.11 ml, 1.38 mmol) and dimethyl sulfoxide (0.03 ml, 0.42 mmol)

were then added and the mixture was stirred at room temperature

for 6 h. The reaction mixture was then concentrated under reduced

pressure, water, dil. hydrochloric acid and dichloromethane were

added to the residue that remained and the aqueous phase was

extracted repeatedly with dichloromethane. The combined organic

phases were dried over magnesium sulfate, filtered and concen-

trated under reduced pressure. Purification of the resulting crude

product by column chromatography (gradient ethyl acetate/

heptane) gave N-(1’-methyl-2’-oxo-1’,2’-dihydrospiro[cyclo-butane-

1,3’-indol]-5’-yl)-1-(4-trifluoromethylphenyl-)methanesulfonamide

1

13i (118 mg, 46% of theory) as a colorless solid. H-NMR (600 MHz,

CDCl₃δ, ppm) 7.64 (d, 2H), 7.47 (d, 2H), 7.36 (d, 1H), 7.05 (d, 1H),

6.74 (d, 1H), 6.46 (br. s, 1H, NH), 4.38 (s, 2H), 3.19 (s, 3H), 2.68–2.64

(m, 2H),1.42–2.35 (m, 1H), 2.33–2.28 (m, 2H), 2.23–2.18 (m, 1H). ¹³C-

NMR (150 MHz, CDCl₃δ, ppm) 179.9, 141.2, 135.8, 132.6, 131.6,

131.3, 131.1, 126.5–120.5 (q, CF₃), 125.9, 121.9, 117.6, 108.1, 57.1,

48.3, 31.2, 26.3, 16.7. LCMS (ESI, m/z): [M+H]⁺425.2; HRMS (ESI,

m/z): calcd. for C₂₀H₁₉F₃N₂O₃S, 425.1147 [M+H]⁺; found 425.1147.

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Conflict of Interest

The authors declare no conflict of interest.

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Manuscript received: April 5, 2021

Revised manuscript received: May 19, 2021

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3457

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Synthesis and Exploration of Abscisic Acid Receptor Agonists Against Dought Stress by Adding Constraint to a Tetrahydroquinoline-Based Lead Structure

DOI: 10.1002/ejoc.202100415

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