Tetrahydroquinolinyl phosphinamidates and phosphonamidates enhancing tolerance towards drought stress in crops via interaction with ABA receptor proteins

Article abstract of DOI:10.1016/j.bmc.2020.115725

New phosphorous-containing lead structures against drought stress in crops interacting with RCAR/(PYR/PYL) receptor proteins were identified starting from in-depth SAR studies of related sulfonamide lead structures and protein docking studies. A converging 6-step synthesis via phosphinic chlorides and phosphono chloridates as key intermediates afforded envisaged tetrahydroquinolinyl phosphinamidates and phosphonamidates. Whilst tetrahydroquinolinyl phosphonamidates 13a,b exhibited low to moderate target affinities, the corresponding tetrahydroquinolinyl phosphinamidates 12a,b revealed confirmed strong affinities for RCAR/ (PYR/PYL) receptor proteins in Arabidopsis thaliana on the same level as essential plant hormone abscisic acid (ABA) combined with promising efficacy against drought stress in vivo (broad-acre crops wheat and canola).

Full text of DOI:10.1016/j.bmc.2020.115725

Bioorganic&MedicinalChemistry28(2020)115725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tetrahydroquinolinyl phosphinamidates and phosphonamidates enhancing
tolerance towards drought stress in crops via interaction with ABA receptor
proteins
⁎
Jens Frackenpohla, , Luka J.B. Deckera, Jan Dittgena, Jörg Freigangc, Pierre Génixb,
Hendrik Helmkea, Gudrun Langea, Peter Luemmena, Jana Schmidta, Dirk Schmutzlera,
Jean-Pierre Vorsb
a Research & Development, Weed Control - Bayer AG, CropScience Division, Industriepark Höchst, D-65926 Frankfurt am Main
b Research & Development, Disease Control - Bayer S.A.S., Crop Science Division, CRLD, 14 Impasse Pierre Baizet, 69263 Lyon, France
c Research & Development, Research Technology, Bayer AG, CropScience Division, Gebäude 6240, Alfred-Nobel-Straße 50, 40789 Monheim, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
New phosphorous-containing lead structures against drought stress in crops interacting with RCAR/(PYR/PYL)
receptor proteins were identified starting from in-depth SAR studies of related sulfonamide lead structures and
protein docking studies. A converging 6-step synthesis via phosphinic chlorides and phosphono chloridates as
key intermediates afforded envisaged tetrahydroquinolinyl phosphinamidates and phosphonamidates. Whilst
tetrahydroquinolinyl phosphonamidates 13a,b exhibited low to moderate target affinities, the corresponding
tetrahydroquinolinyl phosphinamidates 12a,b revealed confirmed strong affinities for RCAR/ (PYR/PYL) re-
ceptor proteins in Arabidopsis thaliana on the same level as essential plant hormone abscisic acid (ABA) com-
bined with promising efficacy against drought stress in vivo (broad-acre crops wheat and canola).
Phytochemistry
Drought stress
Phosphinamidates
Phosphonamidates
Abscisic acid receptor proteins
1. Introduction
of ABA to wheat grown under near-field conditions improved water use
efficiency without detectable growth trade-offs.10 Thus, new synthetic
Agricultural crops face losses due to biotic stress such as pests,
diseases, and weed damages, but abiotic stress adversely affects crop
production even further in various parts of the world, decreasing
average yields for most of the crops significantly.1 Its impact is expected
to grow as a consequence of climate change. Among various abiotic
stress types affecting agricultural production, drought stress is con-
sidered to be the main source of crop losses around the globe.2 So far,
there are three major strategies for reducing 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 breeding,4,5 but also exploring novel
chemical active ingredients inspired by naturally occurring plant hor-
mones or signaling pathways. Abscisic acid (1, S-(+)-ABA), a chiral
sesquiterpenoid first discovered in the 1960 s,6–9 is one of the most
important plant hormones regulating developmental signals in plants
such as seed maturation or dormancy. It also mediates the adaptation of
plants to environmental abiotic stress types such as drought, heat or
salinity stress by regulating transpiration. Interestingly, the application
small molecules that tune transpiration via the same mechanism as
phytohormone ABA in anticipation of drought may be valuable tools for
maximizing water use efficiency of crops thereby safeguarding crop
yield. Studies on ABA mediated signaling have progressed rapidly since
the discovery of RCAR/(PYR/PYL) receptor proteins as soluble ABA-
receptors.11,12 It was shown via crystal structural analysis that binding
of ABA to RCAR12 induced a conformational change in the highly
conserved ABA receptor proteins initiating an interaction with phos-
phoprotein phosphatases 2C (PP2Cs) thereby inhibiting their activity.
Hence, these findings have granted new insights into the structure ac-
tivity relationship (SAR) of ABA giving rise to novel synthetic analo-
gues.
Whilst modifying the cyclohexenone moiety of ABA via stabilization
or further substitution has attracted significant synthetic interest,13–20
surprisingly few approaches have been made to identify open-chain
analogues of ABA with promising target affinity. Recently, we have
shown that cyano-cyclopropyl groups in 2a-c served as suitable re-
placements of the cyclohexanone moiety leading to ABA analogues with
⁎ Corresponding author.
E-mail address: jens.frackenpohl@bayer.com (J. Frackenpohl).
https://doi.org/10.1016/j.bmc.2020.115725
Received 10 July 2020; Accepted 18 August 2020
Availableonline30August2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
Figure 1. Abscisic acid, selected terpene analogues and sulfonamides interacting with RCAR/(PYR/PYL) receptor proteins.
diamines 7a-b, and acylated indolinylmethyl sulfonamides 8a-b.34 It
has been shown that p-cyano aniline moieties are effective isosteres of
the carbonyl group in ABA’s cyclohexenone headgroup. The beneficial
impact of p-cyano aniline moieties on the interaction of agonists with
ABA receptor proteins has also been observed in a structural analysis of
the binding mode of sulfonamide-based 3rd generation agonist cyana-
bactin 9 with PYR1.35 In good correlation with X-ray analyses of cyano
cyclopropyl analogues of ABA (cf. 2a-c),23 this study showed that the 3-
cyclopropyl-4-cyanoaniline moiety mimics the oxygen of ABA’s cyclo-
hexenone engaging the key components required to stabilize the acti-
vated receptors. However, the structural diversity of lead structures 3–9
is rather limited as they all share a central sulfonamide group. Herein,
we outline how we identified new lead structures interacting with
RCAR/(PYR/PYL) receptor proteins in which the sulfonamide moiety
was replaced by phosphinamidates and phosphonamidates thus em-
phasizing opportunities to introduce further structural motifs into ABA-
related structures.
2. Results and discussion
2.1. Structure-based design and modeling studies
From a synthetic chemist’s perspective, various motifs could serve
as replacements for the sulfonamide moiety linking substituted aryl or
hetaryl groups in lead structures 3a, 5a, 8a, and cyanabactin 9, e.g.
carboxamides, substituted ketones or phosphonamidates. Carboxamide
spacers have already been investigated affording the first examples of
non-sulfonamide ABA receptor agonists: Cyano- and cyclopropyl-sub-
stituted phenyl acetic acid amides 10a-b and substituted triazolyl
thioacetamides 11a-b (with inverted acetamide units) have shown
strong receptor binding (Fig. 2).36,37 Interestingly, these promising hit
structures share p-cyano-substituted phenyl groups with an additional
group in the meta-position. This particular motif proved to be beneficial
for strong receptor affinity in sulfonamides 7a-b and 9. To complement
these approaches we have laid emphasis in our work outlined herein on
further sulfonamide replacements. Based on our experience in agro-
chemical research we have chosen phosphorous-containing groups, i.e.
phosphinamidates and phosphonamidates (cf. 12a, 13a, Fig. 2).
Phosphoryl and carbonyl groups can act as a hydrogen-bond ac-
ceptor, whilst modifying strongly the physicochemical properties.38
However, the number of bioactive molecules containing phosphinami-
date or phosphonamidate motifs is rather limited compared to related
sulfonamides.39–42 Some famous examples are naturally occurring
bialaphos and the potent herbicides phosphinotricine and glyphosate
showing the biological potential of these motifs.
Figure 2. Synthetic non-sulfonamide ABA-receptor agonists, e.g. phenyl acetic
acid amides, triazolyl thioacetamides, and target structures of the work de-
scribed herein, i.e. phosphinamidates and phosphinamidates.
strong activity in vitro and in vivo.21 Synthetic compounds interacting
with RCAR/(PYR/PYL) receptor proteins have also been prepared by
other groups.22–24 Likewise, substituted aryl sulfonamides, e. g. 1st
generation lead structure pyrabactin 3a,25,26 closely related pyrimidine
3b, as well as thiazoles and pyrazoles 4a-b,27 have all shown promising
initial receptor affinity and beneficial effects in in vivo tests (Fig. 1).
Furthermore, tetrahydroquinolinyl sulfonamides, e.g. 2nd generation
lead structure quinabactin 5a and its close analogues 5b-d with dif-
ferent N-substituents, with optimized phenyl substituents (6a) or ring
modifications (6b) have shown even improved in vitro activity.28–33 In
our view this indicates that ABA analogues with more profound struc-
tural changes can be identified. More recently, 3rd generation sulfo-
namide lead structures that exhibit good receptor binding and pro-
mising in vivo efficacy in wheat have been identified via target-based
high throughput screening (HTS), e.g. phenylsulfonyl ethylene-
2

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
In order to understand the impact of replacing the sulfur by a
phosphorous atom, we analyzed the crystal structure of the complex
between sulfonamide 6b (with a Cl-atom instead of a methyl group in
quinabactin 5a) and AtRCAR11 and the related phosphatase AtHAB1
(Figure 3).
The crystal structure shows that the sulfonamide forms two essential
hydrogen bonds to the ABA receptor. The sulfonamide amine interacts
directly with the side chain of the conserved glutamate 94, whilst only
one of the sulfonyl oxygens interacts with the backbone carbonyl group
of histidine 60 via a bridging water molecule. The overall affinity is
driven by a strong hydrophobic effect with a considerable contribution
of the N-propyl moiety and the chlorine atom in the 4-position of the
benzyl group.34,35 Based on recognizing that only one sulfonyl oxygen
is essential for receptor binding we selected prototypes of the envi-
sioned P-isosteres of sulfonamides, e.g. phosphinamidate 12a and
phosphonamidates 13a & 13 m (cf. Fig. 2, table 1), were docked into
the ABA binding site of the crystal structure of ABA-bound AtRCAR11
with related phosphatase AtHAB1. Resulting poses were then scored
using the HYDE scoring function (Fig. 3).43
Figure 3. Interaction of 6b with AtRCAR11 and the related phosphatase
AtHAB1 as determined in the crystal structure. The H-bonds to glutamate 94
and to the water are indicated in dotted green lines. The contribution of in-
dividual atoms is indicated by spheres.43 Green spheres show that these atoms
stabilize the interaction, while red spheres indicate that these atoms destabilize
the interaction. The size of the spheres correlates with the size of the con-
tribution of the corresponding atoms.
Analyzing the binding mode of the P-methyl group in phosphina-
midate 12a via docking studies (Fig. 4a) indicated that there should be
a very similar binding behaviour compared to sulfonamide 6b in the X-
ray structure (cf. Fig. 3). In particular, the H-bonds formed by the
sulfonamide moiety are very well mimicked by the phosphinamidate
group, and its methyl group fits nicely into a small hydrophobic pocket.
Figure 4. Modelled interaction of 12a (a), 13a (b) and 13 m (c) with AtRCAR11 and the related phosphatase AtHAB1. The contribution of individual atoms is
indicated by spheres.43 Green spheres show that these atoms stabilize the interaction, while red spheres indicate that these atoms destabilize the interaction. The size
of the spheres correlates with the size of the contribution of the corresponding atoms. Clashes in (c) are indicated by orange arrows. The crystal structure of 6b is
shown in mauve.
3

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
Scheme 1. Synthesis of tetrahydroquinolinyl phosphinamidates 12 and tetrahydroquinolinyl phosphonamidates 13; (a) Trimethyl or triethyl phosphite, benzyl
bromide, 100 °C, 10 h; (b) POCl3, 60 °C, 1.5 h; (c) Et3N, abs·THF, −20 °C – r.t, 1 h; (d) 1. N,N-DMF, 110 °C, 4 h, 2. aq. NaOH (10%), reflux, 2 h; (e) R1-NH2, N,N-DMF,
EtN(i-Pr)2, 50 °C, 16 h; (f) (Ph3P)3RhCl, abs. EtOH, H2, r.t., 9 h; g) NaH, abs·THF, 0° C – r.t; h) SnCl2-H2O, abs. EtOH, 80 °C, 5 h; alternatively: Fe (powdered), NH4Cl,
EtOH/H2O (1:1); (i) Et3N, substituted aniline, abs·THF, r.t., 2–6 h; (j) Friedel-Crafts alkylation with AlCl3; (k) conc. H2SO4, conc. HNO3, −20 °C – r.t., 4 h; (l) K2CO3,
R1-I, N,N-DMF, r.t., 24 h; (m) 1. POCl3, 60 °C, 1.5 h, 2. Et3N, abs·THF, −20 °C – r.t, 1 h.
The hydrophobic effect of small substituents in the para-position of
the benzyl moiety (i.e. F, Cl, Me) contributed within our docking studies
by roughly one pI50 unit to the overall binding affinity, while a benzyl
moiety carrying a p-cyano group decreased the affinity due to the de-
hydration penalty that has to be paid for a polar group located in a
hydrophobic pocket. These docking studies are in line with our ex-
perimental observations (cf. table 1). Substituents in the meta-position
induced some movements of the benzylic moiety in order to avoid steric
clashes which seem to weaken the binding. In addition, the N-Pr group
contributed by two orders of magnitude to the pI50 values, while the
smaller N-Me group stabilizes the binding only by one order of mag-
nitude. As a consequence, combining a N-Me group at R1 with a less
favourably substituted benzyl moiety resulted in a loss of receptor af-
finity. These findings are in line with the observed in vitro and in vivo
results outlined in table 1. When replacing the phosphinamidate’s me-
thyl group by a methoxy group, thus affording the related phospho-
namidate, there seemed to be still enough space, and no significant
movement of the respective phosphonamidate was required in the
docking study to avoid steric clashes (Figure 4b). The slightly lower
affinity may be due to the polar dehydration of the bridging oxygen, but
the same SAR applies. Larger hydrophobic substituents at position R1
such as N-CH2-c-Pr, N-1,1‘-(bicyclopropyl) or N-1-(spiro[3.3]hept-2-yl)
seemed to have similar hydrophobic effects as the N-propyl moiety.
However, increasing the phosphonamidate substituent at the P-centred
position R3 by introducing an ethoxy group (13 m, Figure 4c) showed in
our docking study that steric clashes with the amino acids proline 55,
arginine 79 and glutamate 94 require a significant movement of 13m in
order to fit into the binding pocket. Likewise, these phosphonamidates
(R3 = OEt) could not assume a pose without significant steric clashes,
while maintaining the H-bonds. This may explain why phosphonami-
dates 13k-13o neither showed binding to the ABA receptor nor in vivo
effects.
2.2. Chemistry
Based on our experiences with the synthesis of tetrahydroquinolinyl
sulfonamides30,31 two main synthetic approaches were used to prepare
tetrahydroquinolinyl amines. Firstly, dihydroquinolin-2(1H)-one which
was accessible via Friedel-Crafts acylation could be easily converted
into the corresponding intermediate 22 via direct nitration, N-alkyla-
tion with a primary sterically less demanding alkyl halide and Fe- or
SnCl2-mediated reduction of the nitro group. Secondly, the desired N-
substituted 6-amino 3,4-dihydroquinolin-2(1H)-ones 22 were prepared
in 4 steps via (i) SNAr reaction of 6- ethyl (2E)-3-(2-fluoro-5-ni-
trophenyl)acrylate 14 with a suitable amine, preferably an amine with
a larger substituent, (ii) hydrogenation of the acrylic double bond with
Wilkinson’s catalyst, (iii) lactam formation and (iv) subsequent reduc-
tion of the nitro group. The second approach proved to be particularly
suitable for sterically demanding amines or cycloalkyl amines, whilst
the first approach worked well with sterically less demanding alkyl
halides (e.g. n-propyl iodide or chloromethylcyclopropane). Likewise,
1,1′-bi(cyclopropyl)-1-amine reacted easily with fluorinated phenyl
acrylate 14 to afford intermediate 15 giving access to desired phos-
phonamides 13 h and 13n.
Substituted phosphinic chlorides and phosphono chloridate pre-
cursors could be prepared, for example, in a stepwise approach starting
with the reaction of a correspondingly substituted benzyl halide and a
substituted phosphorus compound such as trimethylphosphite, triethyl
phosphite, or diethylmethyl phosphonite using Arbuzov conditions in a
suitable polar-aprotic solvent (e.g. N,N-dimethylformamide or Et2O).
Alternatively, using a suitable base (e.g. sodium hydroxide) at elevated
temperature, diethyl methylphosphonite could be converted into the
corresponding acid intermediate 21 (cf. Scheme 1). In the next step, the
intermediates 19 and 21 obtained in this manner could be converted
into the corresponding phosphinic chloride and phosphono chloridate
4

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
Table 1
SAR-results of tetrahydroquinolinyl phosphinamidates 12 and phosphonamidates 13.
Substituents[a]
R1
In vitro activity - ABI1(AtRCAR)[b]
In vivo efficacy vs. drought stress
[250 g/ha][c]
Entry
No.
R2
R3
Activity
pI50
Wheat[c]
Canola[c]
[%] 5 μM
1
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
5a
n-Pr
4-Cl
4-Me
4-F
Me
+++
4.6
5.7
4.9
4.3
4.3
4.3
4.3
n.d.
n.d.
n.d.
n.d.
4.4
4.0
4.0
n.d.
n.d.
n.d.
4.2
4.0
4.0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
5.9
7.0
++
++
++
+
++
+++
+++
++
++
++
++
0
2
n-Pr
Me
++++
3
n-Pr
Me
++++
4
n-Pr
3-Me
3-F
Me
+++
5
n-Pr
Me
+++
+++
+
6
Me
4-Me
4-Cl
3-F
Me
+++
7
Me
Me
++
+
8
Me
Me
-
0
9
Me
3-Cl
4-CN
H
Me
-
0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Me
Me
+
0
+
n-Pr
Me
-
0
0
n-Pr
4-Cl
4-Me
4-F
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OEt
+++
++
+
++
++
+
n-Pr
++
n-Pr
++
+
n-Pr
4-CN
4-Me
4-Me
4-Me
4-Me
4-Me
4-Cl
3-F
+
0
+
Me
-
0
0
Et
+
0
+
CH2-c-Pr
++
++
++
+
++
+
1,1’-(Bicyclopropyl)
++
1-(Spiro[3.3]hept-2-yl)
++
+
Me
-
0
0
n-Pr
-
0
0
n-Pr
4-Me
4-Cl
4-Me
4-Me
4-Me
-
0
0
n-Pr
OEt
-
0
0
CH2-c-Pr
1,1’-(Bicyclopropyl)
n-Pr
OEt
-
0
0
OEt
-
0
0
Sulfonamide
++++
++++
++
+++
++
+++
1
Abscisic acid
ac-Pr = cyclopropyl, c-Bu = cyclobutyl, i-Pr = isopropyl; b a final expert assessment of target affinity was made according to the following classification: “-”: < 30%
, “+” = 30% < activity < 50%, “++” = 50% < activity < 70%, “+++” = 70% < activity < 90%, “++++” = activity > 90%; c standard application rate
d
for crop protection greenhouse trials; 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).
precursors (e.g. 20a,b, Scheme 1).44–46 Coupling of the substituted
phosphonyl chloride (20) and phosphinyl chloride precursors with the
appropriate oxotetra-hydroquinolinyl amines 22 with the aid of a sui-
table base (e.g. triethylamine, pyridine) under mild conditions in an
aprotic solvent (e.g. tetrahydrofuran, acetonitrile, or dichloromethane)
afforded the desired tetrahydroquinolinyl phosphinamidates and
-phosphonamidates 13a-n and 12a-k.47 The formation of PeN bonds
remains a challenge as is shown by low to moderate yields in the final
PeN coupling step of compounds 20 and 21 with amines 22 (Scheme
1). Furthermore, only a limited number of literature citations for the
formation of phosphinamidates and related phosphonamidates can be
found.48 Following the synthetic approaches outlined in scheme 1 we
have prepared target compounds covering structural variations at three
positions, firstly at the tetrahydroquinolinyl nitrogen (N-R1), secondly
in the P-benzyl moiety (R2) and finally at the central phosphorous atom
(P-R3).
thaliana.
Strong receptor affinity could be observed for phosphinamidates
carrying halogen or methyl substituents in the P-benzyl moiety com-
bined with an n-Pr substituent at the tetrahydroquinolinyl nitrogen,
albeit with lower pI50 values than ABA 1 as the internal standard
(Table 1). P-benzyl moieties carrying substituents in the 4-position of
the aryl unit afforded most promising binding affinities in line with
results from earlier studies in the field of tetrahydroquinolinyl sulfo-
namides. In particular, phosphinamidates carrying an unbranched n-Pr
substituent in the N-R1 moiety together with a 4-Cl-benzyl group (12a,
Table 1, entry 1), a 4-Me-benzyl group (12b, entry 2) and a 4-F-benzyl
moiety (12c, entry 3) as the P-substituent showed very good target
affinities together with promising efficacy against drought stress in vivo,
preferably in canola. Whilst introducing a small methyl substituent at
position R1 in combination with a 4-substituted P-benzyl group afforded
weaker effects, both in vitro and in vivo (12f, 12 g, 12j, cf. Table 1,
entries 6, 7, 10), a shift from 4- to 3-substituted P-benzyl moieties was
tolerated in N-(n-Pr)-tetrahydroquinolinyl phosphinamidates (12d,
12e, Table 1, entries 4,5). Remarkably, 3-F-benzyl substituted phos-
phinamidate 12e exhibited the strongest effects of all new compounds
against drought stress in vivo in wheat. Remarkably, these effects are on
the level of ABA 1 (Table 1, entry 28). Introducing two structural
variations compared with the strongest in vitro hit 12b, i.e. replacing
the n-Pr group by Me and changing the P-benzyl substitution from the
4- to 3-position or to an unsubstituted phenyl group, led to a complete
loss of activity, both in vitro and in vivo (12 h, 12i, 12j, 12 k, cf.
2.3. SAR study
All compounds that have been prepared to explore the SAR of tet-
rahydroquinolinyl phosphinamidates 12 and phosphon-amidates 13
were tested both, for target affinity in dedicated in vitro tests, as well as
for beneficial effects in vivo under drought stress conditions upon foliar
application on plants. Wheat and Canola were chosen as model plants
for monocotyledonous and dicotyledonous species, whereas in vitro
tests were carried out using the ABA-receptor RCAR11 in Arabidopsis
5

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
closely related phosphinamidate 12b (R1 = n-Pr, R2 = p-Me) showed a
comparable target affinity combined with better efficacy against
drought stress in canola. Phosphinamidate 12c and 12e also showed
stronger efficacy in vivo than sulfonamide 5a. Remarkably, phospho-
namidate 13g exhibited significant effects in vivo against drought stress,
both in wheat and canola, although its target affinity was only mod-
erate (pI50 4.2). The phosphinamidate and phosphonamidate moieties
in compounds 12 and 13 prepared in our SAR-study thus serve as a
suitable bioisosteric replacement of the sulfonamide moiety in tetra-
hydroquinolinyl sulfonamides (e.g. 5a, 6b).
To further validate our initial in vivo screening results, we selected
12e with strong efficacy in wheat and 13a with good efficacy in canola
as prototypes for advanced greenhouse trials (i.e. more replicates, dif-
ferent application rates, e.g. 100 and 25 g/ha, additional crops such as
corn and barley, different corn varieties and moderate stress levels).
Accordingly, significant beneficial effects against drought stress could
be observed in wheat, and particularly in barley compared to untreated
controls upon treatment with 12e as the representative phosphinami-
date (Fig. 5).
Figure 5. Advanced drought stress trials with 12e in wheat and barley under
moderate stress conditions, i.e. 45% damage for barley and 44% for wheat,
respectively.
Interestingly, the promising in vivo efficacy of several phosphona-
midates observed in our initial screening (cf. Table 1) could be con-
firmed as representative example 13a afforded significant beneficial
effects under moderate drought stress conditions in canola (Fig. 6).
3. Conclusions
We have identified new phosphorous-containing ABA-receptor
agonists showing activity against drought stress in field crops by in-
teracting with RCAR/(PYR/PYL) receptor proteins. These new sub-
stituted phosphinamidates and phosphonamidates were identified
starting from SAR studies of related sulfonamide lead structures and
protein docking studies. The docking studies showed that both phos-
phorous moieties could serve as bioisosteric replacements of the sul-
fonyl group in known sulfonamide ABA-receptor agonists. A converging
6-step synthesis proceeding via phosphinic chlorides and phosphono
chloridates as key intermediates has been developed to afford en-
visaged tetrahydroquinolinyl phosphinamidates 12 and phosphonami-
dates 13. Whilst phosphonamidates 13a-o exhibited low to moderate
target affinities, the corresponding phosphin-amidate 12b revealed
good affinities for RCAR/ (PYR/PYL) receptor proteins in Arabidopsis
thaliana on the same level as structurally related sulfonamide-based
lead structure quinabactin. Remarkably, 12b showed promising effi-
cacy against drought stress in vivo (broad-acre crops wheat and canola).
Furthermore, tetrahydroquinolinyl phosphonamidates (e.g. 13a)
proved to be active against drought stress in vivo. Our results demon-
strate that phosphinamidates 12 and phosphonamidates 13 are effec-
tive isosteres of the sulfonyl group in tetrahydroquinolinyl sulfonamide-
based ABA-receptor agonists, thus nicely complementing earlier work
in this field. These results should encourage other chemists involved in
life science research to use phosphorous-based chemistry in their work.
Figure 6. Advanced drought stress trials with 13a in canola under moderate
stress conditions, i.e. 48% damage.
Table 1, entries 8–11). Hence, phosphinamidates 12 proved to be
highly sensitive towards changes in the substitution pattern. These
observations are in line with the docking studies outlined above.
Likewise, phosphonamidates 13 also showed a high sensitivity to-
wards structural changes. Whilst N-(n-Pr)-tetrahydroquinolinyl phos-
phonamidates bearing a p-substituted P-benzyl moiety and a P-OMe
group (13a-13c, Table 1, entries 12–14) exhibited significant target
affinities, related analogues with smaller N-substituents in the tetra-
hydroquinolinyl moiety showed weaker or no target affinity and proved
to be inactive in vivo (cf. 13e, 13f, 13j, Table 1, entries 16, 17, 21).
Furthermore, phosphonamidates 13a-13c afforded weaker activities,
both in vitro and in vivo, than corresponding phosphinamidates 12a-
12c. We thus investigated the impact of N-substituents in the tetra-
hydroquinolinyl moiety of phosphonamidates further by carefully
modifying the n-Pr group and keeping the other substituents constant
(i.e. p-Me benzyl and OMe substituents on the phosphorous atom).
Based on promising results obtained in earlier studies of N-substituted
tetrahydroquinolinyl sulfonamides30,31 larger substituents such as cy-
clopropyl-methyl (13 g), 1,1′-(bicyclopropyl) (13 h), and 1-(spiro[3.3]
hept-2-yl) (13i) were synthesized affording moderate target affinities
and moderate effects in vivo with 13 g showing the best results. In
contrast to a change from N-(n-Pr) to N-Me activity in vitro and in vivo
was preserved upon introducing these particular N-substituents. How-
ever, they did not afford improved effects as shown in the related tet-
rahydroquinolinyl sulfonamides.30,31 A change of the P-benzyl sub-
stituent from the para- to the meta-position led to a loss of activity, both
in vitro and in vivo (13 k, Table 1, entry 22). Changing the alkoxy
substituent (R3) in the central phosphonamidate moiety from a
methoxy to an ethoxy group caused a significant decline in target af-
finity in line with a loss of in vivo efficacy as is shown by results ob-
tained for target compounds 13 l-13o (Table 1, entries 23–26).
4. Experimental
4.1. Biology
4.1.1. In vivo studies
Seeds of monocotyledonous and dicotyledonous crop plants were
laid out in sandy loam in wood-fiber pots, covered with soil and cul-
tivated in a greenhouse under good growth conditions. The test plants
were treated at the first leaf stage (BBCH10 – BBCH13). To ensure
uniform water supply before commencement of drought stress, the
potted plants were supplied with the maximum amount of water im-
mediately beforehand by dam irrigation and, after application, trans-
ferred into plastic inserts in order to prevent subsequent, excessively
rapid drying. The respective compounds, formulated in the form of
wettable powders (WP) or emulsion concentrates (EC), were sprayed
In addition to reference plant hormone ABA 1, we have tested tet-
rahydroquinolinyl sulfonamide 5a, one of the sulfonamide lead struc-
tures outlined in Fig. 1, in our in vitro and in vivo tests. Remarkably,
6

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
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 h with
illumination at 26 °C; “night” = 10 h without illumination at 18 °C. The
duration of the respective stress phases was guided mainly by the state
of the untreated (=treated with blank formulation but without test
compound), stressed control plants and thus varied from wheat to ca-
nola. It was ended (by re-irrigating or transfer to a greenhouse with
standard growth conditions) as soon as irreversible damage was ob-
served on the untreated, stressed control plants. In the case of dicoty-
ledonous crops, for example canola, the duration of the drought stress
phase varied between 3 and 5 days; in the case of monocotyledonous
crops, for example wheat, it varied between 6 and 10 days. The end of
the stress phase was followed by an approx. 5–7-day recovery phase,
during which the plants were once again kept under good growth
conditions in a greenhouse. In order to rule out any influence of the
effects observed by any fungicidal action of the test compounds, it was
additionally ensured that the tests proceeded without fungal infection
and without infection pressure. After the recovery phase had ended, the
intensities of damage were rated visually in comparison to untreated,
unstressed controls of the same age (in the case of drought stress). The
intensity of damage was first recorded as a percentage (100% = plants
have died, 0% = like control plants). These values were then used to
calculate the efficacy of the test compounds (=percentage reduction in
the intensity of damage as a result of substance application), and a final
assessment of the respective efficacy was made, i.e. “0” = no effect,
“+” = slight beneficial effect, “++” = significant beneficial effect,
“+++” = strong beneficial effect against drought stress, “++++”
= very strong beneficial effect superior to internal standard ABA.
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 pH8, 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 freshly made 5 min prior to
the addition and warmed to a temperature of 35 °C. On completion of
pipetting of all the solutions and on completion of mixing, the plate was
incubated at 35 °C for 20 min. Finally, a relative fluorescence mea-
surement was made at 35 °C with a BMG Labtech “POLARstar Optima”
microplate reader using a 340/10 nm excitation filter and a 460 nm
emission filter. An expert assessment of target affinity was made ac-
cording to the following classification: “-”: < 30%
, “+” =
30% < activity < 50%, “++” = 50% < activity < 70%, “+++”
= 70% < activity < 90%, “++++” = activity > 90%.
4.2. Chemistry
4.2.1. General
All reagent-grade solvents and chemicals were purchased from
standard commercial suppliers and used without further purification.
All non-aqueous reactions were carried out under anhydrous conditions
using dry solvents. Reactions were monitored by LC-MS or TLC carried
out on 0.25 mm silica gel plates (60F-254). TLC plates were visualized
using UV light. Flash chromatography was carried out using Biotage
Isolera One systems with pre-packed column cartridges (Biotage KP-Sil
[40 + M] or KP-Sil [25 + M]). The 1H NMR, 13C NMR and 19F NMR
spectroscopy data which are reported for the chemical examples de-
scribed below (400 MHz for 1H NMR and 150 MHz for 13C NMR and
375 MHz for 19F NMR, solvent: CDCl3, CD3OD or d6-DMSO, internal
standard: tetramethylsilane δ = 0.00 ppm), were obtained on a Bruker
instrument, and the signals listed have the meanings given below:
br = broad; s = singlet, d = doublet, t = triplet, dd = doublet of
doublets, ddd = doublet of a doublet of doublets, m = multiplet,
4.1.2. In vitro - AtABI1-(AtRCAR11)
The assay described hereinafter utilizes the inhibition of the phos-
phatase AtABI1 via the co-regulator RCAR11/PYR1 from Arabidopsis
thaliana. Expression and purification of RCARs and PP2Cs was per-
formed as described.11 A concise overview of the correlation between
PYR/PYL and RCAR numbering can be found in reference by Grill
et al.49 For the determination of activity, the dephosphorylation of 4-
methylumbelliferyl phosphate (MUP) was measured at 460 nm. The in
vitro assay was conducted in Greiner 96-well, 384-well or 1536-well PS
microplates, using two controls: a) dimethyl sulfoxide (DMSO) 0.5%
(f.c.) and b) 5 μM (f.c.) abscisic acid (ABA). For high throughput
screening 1 µg/ml ABI1 and 0,5 µg/ml RCAR11 were incubated with
100 µM MUP in reaction buffer (50 mM Tris/HCl pH 7.8, 50 mM NaCl,
0.3 mM MnCl2, 0.01% Tween, 0.01% BSA) at 32 °C for 80 min.
Fluorescence intensity was detected by a BMG Labtech Pherastar using
a 340 nm excitation filter and a 460 nm emission filter. For follow up
compound characterization the assay was conducted with substance
concentrations of the appropriate chemical test substances in a con-
centration range of 0.1 μM to 100 μM in a solution of DMSO and water.
The substance solution thus obtained, if necessary, was stirred with
esterase from porcine liver (EC 3.1.1.1) at room temperature for 3 h and
centrifuged at 4000 rpm for 30 min. A total volume of 45 μl was in-
troduced into each cavity of the microplate, having the following
composition: 1. 5 μl of substance solution, i.e. a) DMSO 5% or b) ab-
scisic 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 dithiothreitol (DTT)], b) 4% by vol. of AtABI1
or TaABI1 dilution (protein stock solution was diluted so as to give,
after addition, a final concentration in the assay of 0.15 μg ABI1/well),
c) 4% by vol. of AtRCAR11 dilution (enzyme stock was diluted so as to
give, on addition of the dilution to the enzyme buffer mix, a final
concentration in the assay of 0.30 μg enzyme/well), d) 5% by vol. of
q = quartet, quint = quintet, sext = sextet, sept = septet,
dq = doublet of quartets, dt = doublet of triplets. The abbreviations
used for chemical groups or dedicated hydrogen atoms are defined as
follows: Me = CH3, Et = CH2CH3, t-Hex = C(CH3)2CH(CH3)2, t-
Bu = C(CH3)3, n-Bu = unbranched butyl, n-Pr = unbranched propyl, c-
Hex = cyclohexyl, ArH = aromatic hydrogen, HetH = heteroaromatic
hydrogen. In the case of diastereomeric mixtures, either the significant
signals for each of the diastereomers or the characteristic signal of the
main diastereomer is/are reported. In the following sections, the yield
for the corresponding phosphinamidates 12a-k and phosphonamidates
13a-o is given as a combined yield over 3–4 steps as most of the
phosphorous chemistry was carried out with purification of the final
product only.
4.2.2. Preparation of phosphinamidates 12a-k
All cpds were prepared following the protocol outlined for the
synthesis of 12k.
4.2.2.1. P-(4-Chlorobenzyl)-P-methyl-N-(2-oxo-1-propyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12a). White solid; yield 11%;
1H NMR (400 MHz, CDCl3) δ 7.17–7.07 (m, 5H), 6.89 (m, 1H), 6.62 (m,
1H), 6.57 (br. s, 1H, NH), 3.86 (m, 2H), 3.35–3.23 (m, 2H), 2.83 (m,
2H), 2.62 (m, 2H), 1.68–1.59 (m, 2H), 1.52 (d, 3H), 0.95 (t, 3H). LCMS
(ESI, m/z): [M+H]+ 391.9. HRMS (ESI, m/z): calcd. for
C
20H24N2O2PCl, 390.8435 [M+H]+; found 390.8418.
4.2.2.2. P-(4-Methylbenzyl)-P-methyl-N-(2-oxo-1-propyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12b). White solid; yield 14%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.13 (m, 2H), 7.07 (m, 2H), 6.92 (m,
2H), 6.86 (m, 2H), 3.86 (m, 2H), 3.39–3.21 (m, 2H), 2.83 (m, 2H), 2.62
(m, 2H), 2.32 (s, 3H), 1.68–1.58 (m, 2H), 1.51 (d, 3H), 0.95 (t, 3H);
LCMS (ESI, m/z): [M]+ 370.4; HRMS (ESI, m/z): calcd. for
C
21H27N2O2P, 370.1810 [M+H]+; found 370.1819.
7

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
4.2.2.3. P-(4-Fluorobenzyl)-P-methyl-N-(2-oxo-1-propyl-1,2,3,4-
reduced pressure. By column chromatography purification of the crude
product obtained (ethyl acetate/heptane gradient), 6-nitro-3,4-
dihydroquinolin-2(1H)-one (20.0 g, 76% of theory) was isolated as a
colorless solid. 6-Nitro-3,4-dihydroquinolin-2(1H)-one (8.52 g,
44.38 mmol) was dissolved under argon in abs. N,N-dimethylformamide
(150 ml), the mixture was cooled to 0 °C and fine potassium carbonate
powder (7.40 g, 52.26 mmol) was added. After 15 min of stirring at a
temperature of 0 °C, n-propyl iodide (2 equiv, 88.771 mmol) was added.
The resulting reaction mixture was then stirred at room temperature for
24 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. Column
chromatography purification of the crude product obtained (ethyl
acetate/heptane gradient) gave 6-nitro-1-propyl-3,4-dihydroquinolin-
2(1H)-one (8.40 g, 87% of theory) as a colorless solid. In the next step,
6-nitro-1-propyl-3,4-dihydroquinolin-2(1H)-one (5.0 g, 24.27 mmol) was
dissolved in an ethanol/water mixture (ratio 1:1, 50 ml), and ammonium
chloride (12.96 g, 242.72 mmol) and iron powder (4.07 g, 72.82 mmol)
were added. The resulting reaction mixture was stirred at a temperature of
80 °C for 2 h and, after cooling to room temperature, concentrated. Ethyl
acetate and water were added to the residue and 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. Column chromatography purification of the crude
product obtained (ethyl acetate/heptane gradient) gave 6-amino-1-
tetrahydroquinolin-6-yl)phosphinamidate (12c). White solid; yield 19%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.15 (m, 2H), 7.02 (m, 2H), 6.92 (m,
2H), 6.87 (m, 2H), 4.54 (m, 1H), 3.86 (m, 2H), 3.35–3.23 (m, 2H), 2.84
(m, 2H), 2.62 (m, 2H), 1.68–1.59 (m, 2H), 1.52 (d, 3H), 0.95 (t, 3H);
LCMS (ESI, m/z): [M+H]+ 375.4; HRMS (ESI, m/z): calcd. for
C
20H24N2O2PF, 374.1559 [M+H]+; found 374.1549.
4.2.2.4. P-(3-Methylbenzyl)-P-methyl-N-(2-oxo-1-propyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12d). White solid; yield 9%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.19 (m, 1H), 7.09–6.96 (m, 2H),
6.94–6.81 (m, 5H), 3.89 (m, 2H), 3.44–3.27 (m, 2H), 2.87 (m, 2H), 2.65
(m, 2H), 1.72–1.60 (m, 2H), 1.56 (d, 3H), 0.98 (t, 3H); LCMS (ESI, m/z):
[M]+ 370.4; HRMS (ESI, m/z): calcd. for C21H27N2O2P, 370.1810 [M
+H]+; found 370.1802.
4.2.2.5. P-(3-Fluorobenzyl)-P-methyl-N-(2-oxo-1-propyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12e). White solid; yield 8%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.33 (m, 1H), 7.01–6.95 (m, 2H),
6.93–6.88 (m, 3H), 6.87 (m, 2H), 3.89 (m, 2H), 3.44–3.27 (m, 2H), 2.87
(m, 2H), 2.65 (m, 2H), 1.72–1.60 (m, 2H), 1.56 (d, 3H), 0.98 (t, 3H);
LCMS (ESI, m/z): [M+H]+ 375.4; HRMS (ESI, m/z): calcd. for
C
20H24N2O2PF, 374.1559 [M+H]+; found 374.1545.
4.2.2.6. P-(4-Methylbenzyl)-P-methyl-N-(2-oxo-1-methyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12f). White solid; yield 17%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.13 (m, 2H), 7.09–7.03 (m, 3H),
6.98 (m, 1H), 6.79 (m, 1H), 6.58–6.52 (m, 1H), 3.39–3.23 (m, 2H), 3.29
(s, 3H), 2.86–2.77 (m, 2H), 2.63–2.57 (m, 2H), 1.52 (d, 3H); LCMS (ESI,
m/z): [M+H]+ 353.3.
propyl-3,4-dihydroquinolin-2(1H)-one (4.0 g, 94% of theory) as
a
colorless solid. 1H NMR (400 MHz, d6-DSMO δ, ppm) 6.79 (d, 1H), 6.45
(m, 1H), 6.42 (m, 1H), 4.85 (br. s, 2H, NH2), 3.75 (m, 2H), 2.68 (m, 2H),
2.43 (m, 2H), 1.52 (m, 2H), 0.85 (t, 3H). Methyldiethyl phosphite (1
equiv, 8.07 mmol) and benzyl bromide (1.54 ml, 8.07 mmol) were added
to a multi-necked flask which had been dried by heating and then stirred
together under continuous nitrogen flow at a temperature of 100 °C for
10 h. After complete conversion, without further purification, distilled
POCl3 (1 equiv) was added to the resulting crude product and the mixture
was stirred under argon at a temperature of 60 °C for 1.5 h. After complete
conversion, the benzyl(methyl)phosphine chloride obtained was, without
further purification, directly reacted in the next step. Under argon, 6-
amino-1-propyl-3,4-dihydroquinolin-2(1H)-one (1 equiv., 4.13 mmol) was
dissolved together with benzyl(methyl)phosphine chloride (1 equiv.,
4.13 mmol) in abs. tetrahydrofuran (10 ml) in a round-bottom flask
which had been dried by heating, the mixture was cooled to a temperature
of −20 °C, then triethylamine (1.2 ml, 8.26 mmol) was added and the
mixture was stirred at room temperature for 2 h. The reaction mixture was
then concentrated under reduced pressure, dil. HCl 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 concentrated under
reduced pressure. Column chromatography purification of the crude
product obtained (ethyl acetate/heptane gradient) gave P-benzyl-P-
methyl-N-(2-oxo-1-propyl-1,2,3,4-tetrahydroquinolin-6-yl)phosphinamide
12 k (103 mg, 7% of theory over three steps) as a colorless solid.1H-NMR
(400 MHz, CDCl3 δ, ppm) 7.37–7.26 (m, 6H), 6.83 (m, 1H), 6.61 (m, 1H),
6.45 (br. s, 1H, NH), 3.75 (m, 2H), 3.35–3.30 (m, 3H), 2.81 (m, 2H), 2.62
(m, 2H), 1.52 (m, 2H), 1.46 (m, 3H), 0.85 (t, 3H); LCMS (ESI, m/z): [M]+
356.4; HRMS (ESI, m/z): calcd. for C20H25N2O2P, 356.1654 [M+H]+;
found 356.1642.
4.2.2.7. P-(4-Chlorbenzyl)-P-methyl-N-(2-oxo-1-methyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12 g). White solid; yield 8%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.28 (m, 2H), 7.12 (m, 2H), 6.92 (m,
2H), 6.86 (m, 1H), 4.54 (m, 1H), 3.35–3.26 (m, 2H), 3.32 (s, 3H), 2.86
(m, 2H), 2.64 (m, 2H), 1.52 (d, 3H); LCMS (ESI, m/z): [M+H]+ 363.8.
4.2.2.8. P-(3-Fluorbenzyl)-P-methyl-N-(2-oxo-1-methyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12 h). White solid; yield 12%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.28 (m, 1H), 6.99–6.93 (m, 4H),
6.90–6.82 (m, 2H), 4.54 (m, 1H), 3.42–3.24 (m, 2H), 3.32 (s, 3H), 2.86
(m, 2H), 2.63 (m, 2H), 1.53 (d, 3H); LCMS (ESI, m/z): [M+H]+ 347.3.
4.2.2.9. P-(3-Chlorbenzyl)-P-methyl-N-(2-oxo-1-methyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12i). White solid; yield 11%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.33 (m, 1H), 7.18–7.06 (m, 4H),
6.92 (m, 2H), 6.86 (m, 1H), 4.54 (m, 1H), 3.35–3.26 (m, 2H), 3.32 (s,
3H), 2.86 (m, 2H), 2.64 (m, 2H), 1.52 (d, 3H); LCMS (ESI, m/z): [M]+
362.8. HRMS (ESI, m/z): calcd. for C18H20N2O2PCl, 362.0951 [M
+H]+; found 362.0966.
4.2.2.10. P-(4-Cyanobenzyl)-P-methyl-N-(2-oxo-1-methyl-1,2,3,4-
tetrahydroquinolin-6-yl)phosphinamidate (12j). White solid; yield 14%;
1H NMR (400 MHz, CDCl3 δ, ppm) 7.61 (m, 2H), 7.32 (m, 2H), 6.94 (m,
2H), 6.85 (m, 1H), 4.56 (m, 1H), 3.42–3.25 (m, 2H), 3.32 (s, 3H), 2.87
(m, 2H), 2.64 (m, 2H), 1.52 (d, 3H); LCMS (ESI, m/z): [M+H]+ 354.3.
4.2.2.11. P-Benzyl-P-methyl-N-(2-oxo-1-propyl-1,2,3,4-tetra-hydroquinolin-
6-yl)phosphinamidate 12 k. 3,4-Dihydroquinolin-2(1H)-one (20.0 g,
136.05 mmol) was added to conc. sulfuric acid (200 ml) and cooled to
−20 °C, and fuming nitric acid (4 ml, 95.24 mmol) was then added
dropwise carefully over a period of 30 min. The resulting reaction mixture
was stirred at −20 °C for 2 h and at room temperature for a further 2 h
and then carefully 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
4.2.3. Preparation of phosphonamidates 13a-o
All cpds were prepared following the protocols outlined for the
syntheses of 13 g, 13 h, 13i, 13 l, 13n, 13o.
4.2.3.1. Methyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(4-chlorobenzyl)phosphonamidate 13a.. White solid; yield 7%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.47 (m, 2H), 7.34 (m, 2H), 6.90 (m,
1H), 6.81 (m, 1H), 6.69 (m, 1H), 4.93 (br. s, 1H, NH), 3.87 (m, 2H),
8

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3.73/3.68 (d, 3H), 3.27/3.02 (d, 2H), 2.83–2.78 (m, 2H), 2.64–2.58 (m,
2H), 1.70–1.61 (m, 2H), 0.98 (t, 3H); LCMS (ESI, m/z): [M+H]+ 407.8;
HRMS (ESI, m/z): calcd. for C20H24N2O3PCl, 406.8429 [M]+; found
406.8444.
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 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 isolated as a
colorless solid. 1H NMR (400 MHz, CDCl3 δ, 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). Trimethyl phosphite (1 equiv, 8.07 mmol) and
4-methylbenzyl bromide (1 equiv, 8.07 mmol) were added to a multi-
necked flask which had been dried by heating and then stirred together
under continuous nitrogen flow at a temperature of 100 °C for 10 h. After
complete conversion, without further purification, distilled POCl3 (1
equiv) was added to the resulting crude product and the mixture was
stirred under argon at a temperature of 60 °C for 1.5 h. After complete
conversion, the methyl (4-methylbenzyl)phosphono-chloridate obtained
was, without further purification, directly reacted in the next step. In a
round-bottom flask which had been dried by heating, under argon, 6-
amino-1-cyclopropylmethyl-3,4-dihydroquinolin-2(1H)-one (960 mg,
4.57 mmol) was dissolved in abs. tetrahydrofuran (2 ml) and slowly
added dropwise under argon to a solution, cooled to −20 °C, of methyl (4-
methylbenzyl)-phosphonochloridate (1000 mg, 4.57 mmol) in abs.
tetrahydrofuran (10 ml) in a round-bottom flask which had been dried
beforehand by heating. The resulting reaction mixture was stirred at
−20 °C for 10 min, triethylamine (1.27 ml, 9.15 mmol) was then added
and the mixture was subsequently stirred at room temperature for 2 h. The
reaction mixture was then filtered, the filter cake was washed with
tetrahydrofuran and the filtrate was concentrated under reduced pressure.
By column chromatography purification of the crude product obtained
(ethyl acetate/heptane gradient), methyl N-[1-(cyclopropylmethyl)-2-oxo-
1,2,3,4-tetrahydroquinolin-6-yl]-P-(4-methylbenzyl)-phos-phonamidate
13 g (209 mg, 10% of theory) was isolated as a colorless solid. 1H NMR
(400 MHz, CDCl3 δ, ppm) 7.09–7.04 (m, 4H), 7.02 (m, 1H), 6.83 (m, 1H),
6.73 (m, 1H), 5.01 (br. s, 1H, NH), 3.84 (d, 2H), 3.76 / 3.53 (d, 3H), 3.25/
3.00 (d, 2H), 2.87–2.82 (m, 2H), 2.65–2.61 (m, 2H), 2.32/2.30 (s, 3H),
1.13 (m, 1H), 0.53–0.48 (m, 2H), 0.45–0.41 (m, 2H); 13C NMR (150 MHz,
CDCl3 δ, ppm) 170.3, 141.4, 138.9, 134.3, 133.8, 130.0, 129.4, 129.2,
128.4, 128.2, 123.8, 117.0, 116.1, 53.7, 48.2, 32.9, 28.8, 25.2, 23.6, 21.5,
7.6, 7.2; LCMS (ESI, m/z): [M]+ 398.2; HRMS (ESI, m/z): calcd. for
4.2.3.2. Methyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(4-methylbenzyl)phosphonamidate 13b.. White solid; yield 12%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.07–7.03 (m, 4H), 6.88 (m, 1H), 6.80
(m, 1H), 6.72 (m, 1H), 4.90 (br. s, 1H, NH), 3.87 (m, 2H), 3.75/3.69 (d,
3H), 3.26/3.05 (d, 2H), 2.83–2.78 (m, 2H), 2.65–2.60 (m, 2H), 2.32 (s,
3H), 1.71–1.63 (m, 2H), 0.96 (t, 3H); LCMS (ESI, m/z): [M+H]+ 387.4.
4.2.3.3. Methyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(4-fluorobenzyl)phosphonamidate 13c.. White solid; yield 9%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.15 (m, 2H), 7.02 (m, 2H), 6.92 (m,
2H), 6.80 (m, 1H), 4.94 (br. s, 1H, NH), 3.87 (m, 2H), 3.71/3.65 (d,
3H), 3.24/3.04 (d, 2H), 2.83–2.78 (m, 2H), 2.65–2.60 (m, 2H),
1.71–1.63 (m, 2H), 0.96 (t, 3H); LCMS (ESI, m/z): [M+H]+ 391.3.
4.2.3.4. Methyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(4-cyanobenzyl)phosphonamidate 13d.. White solid; yield 11%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.63 (m, 2H), 7.33 (m, 2H), 6.93 (m,
2H), 6.85 (m, 1H), 4.99 (br. s, 1H, NH), 3.87 (m, 2H), 3.73/3.68 (d,
3H), 3.27/3.02 (d, 2H), 2.83–2.78 (m, 2H), 2.64–2.58 (m, 2H),
1.70–1.61 (m, 2H), 0.98 (t, 3H); LCMS (ESI, m/z): [M]+ 397.4.
4.2.3.5. Methyl N-[1-(methyl)-2-oxo-1,2,3,4-tetrahydroquino-lin-6-yl]-P-
(4-methylbenzyl)phosphonamidate 13e.. White solid; yield 15%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.08–7.03 (m, 4H), 6.87–6.79 (m,
2H), 6.72 (m, 1H), 5.14 (br. s, 1H, NH), 3.73/3.52 (d, 3H), 3.33 (s, 3H),
3.27/3.02 (d, 2H), 2.85–2.82 (m, 2H), 2.65–2.62 (m, 2H), 2.30 (s, 3H);
LCMS (ESI, m/z): [M+H]+ 359.4.
4.2.3.6. Methyl N-[1-(ethyl)-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl]-P-(4-
methylbenzyl)phosphonamidate 13f.. White solid; yield 6%; 1H NMR
(400 MHz, CDCl3 δ, ppm) 7.12–7.00 (m, 4H), 6.89 (m, 1H), 6.81 (m,
1H), 6.70 (m, 1H), 5.08 (br. s, 1H, NH), 3.97–3.92 (m, 2H), 3.75 (d,
3H), 3.43/3.23 (d, 2H), 2.83–2.78 (m, 2H), 2.63–2.58 (m, 2H), 2.30 (s,
3H), 1.26–1.22 (t, 3H). LCMS (ESI, m/z): [M]+ 373.2. HRMS (ESI, m/z):
calcd. for C20H25N2O3P, 372.3979 [M]+; found 372.3967.
4.2.3.7. Methyl N-[1-(cyclopropylmethyl)-2-oxo-1,2,3,4-tetrahydroquinolin-
6-yl]-P-(4-methylbenzyl)phosphonamidate
13g.. 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. The resulting
reaction mixture was stirred at 120 °C for 2 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-3,4-dihydroquinolin-2(1H)-one (600 mg, 94% of theory) was
isolated as a colorless solid. 1H NMR (400 MHz, CDCl3 δ, ppm) 8.17
(dd, 1H), 8.08 (d, 1H), 7.22 (d, 1H), 3.91 (d, 2H), 3.04 (m, 2H), 2.73 (m,
C
22H27N2O3P, 398.4351 [M]+; found 398.4338.
4.2.3.8. Methyl N-{1-[1,1′-bi(cyclopropyl)-1-yl]-2-oxo-1,2,3,4-tetrahydro
quinolin-6-yl}-P-(4-methylphenyl)-phosphon-amidate 13 h.. Ethyl (2E)-3-
(2-fluoro-5-nitrophenyl)acrylate (1000 mg, 4.18 mmol) and 1,1′-bi
(cyclopropyl)-1-amine (508 mg, 3.80 mmol) were dissolved under
argon in abs. N,N-dimethylformamide (10 ml), and then N,N-
diisopropylethylamine (1.32 ml, 7.60 mmol) was added. The resulting
reaction mixture was stirred at a temperature of 50 °C for a total of 16 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. Column
chromatography purification of the crude product obtained (ethyl
acetate/heptane gradient) gave ethyl (2E)-3-{2-[1,1′-bi(cyclopropyl)-
1-ylamino]-5-nitrophenyl}acrylate (570 mg, 43% of theory) as a
colorless solid, 1H NMR (400 MHz, CDCl3 δ, ppm) 8.27 (d, 1H), 8.16
(m, 1H), 7.63 (d, 1H), 7.18 (d, 1H), 6.46 (d, 1H), 5.19 (br. s, 1H, NH),
4.29 (q, 2H), 1.35 (t, 3H), 1.33–1.27 (m, 1H), 0.78 (m, 4H), 0.49 (m,
2H), 0.18 (m, 2H). Ethyl (2E)-3-{2-[1,1′-bi(cyclopropyl)-1-ylamino]-5-
nitrophenyl}acrylate (570 mg, 1.80 mmol) was then dissolved in abs.
9

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
ethanol (10 ml), and (Ph3P)3RhCl (167 mg, 0.18 mmol) was added.
After stirring at room temperature for 5 min, hydrogen was introduced
methylphenyl)phosphonamidate 13 h (243 mg, 11% of theory) as a
colorless solid.1H-NMR (400 MHz, CDCl3 δ, ppm) 7.28–7.22 (m, 2H),
7.13 (m, 1H), 7.10–7.04 (m, 2H), 6.81 (m, 1H), 6.68 (m, 1H), 5.11 (br.
s, 1H, NH), 3.72/3.52 (d, 3H), 3.30–2.90 (m, 2H), 3.25/3.00 (d, 2H),
2.82–2.53 (m, 2H), 2.32/2.27 (s, 3H), 1.47 (m, 1H), 1.09 (m, 1H), 0.99
(m, 1H), 0.78–0.70 (m, 1H), 0.59–0.42 (m, 4H), 0.27 (m, 1H). LCMS
(ESI, m/z): [M]+ 424.5; HRMS (ESI, m/z): calcd. for C24H29N2O3P,
424.1916 [M]+; found 424.1929.
into the reaction solution with
a constant gas flow via a gas
introduction apparatus for about 9 h. The progress of the reaction
was monitored by LC-MS. On completion of conversion, the reaction
solution was concentrated under reduced pressure. Column
chromatography purification of the crude product obtained (ethyl
acetate/heptane gradient) gave ethyl 3-{2-[1,1′-bi(cyclopropyl)-1-
ylamino]-5-nitrophenyl}propanoate (200 mg, 35% of theory) as a
colorless solid, 1H NMR (400 MHz, CDCl3 δ, ppm) 8.07 (m, 1H), 7.94
(d, 1H), 7.11 (d, 1H), 5.40 (br. s, 1H, NH), 4.18 (q, 2H), 2.77 (m, 2H),
2.64 (m, 2H), 1.30–1.24 (m, 4H), 0.76 (m, 4H), 0.46 (m, 2H), 0.17 (m,
4.2.3.9. Methyl N-[2-oxo-1-(spiro[3.3]hept-2-yl)-1,2,3,4-tetrahydroquinolin-
6-yl]-P-(4-methylphenyl)phosphonamidate 13i.. Ethyl (2E)-3-(2-fluoro-5-
nitrophenyl)acrylate (1000 mg, 4.18 mmol) and spiro[3.3]hept-2-
ylamine (561 mg, 3.80 mmol) were dissolved under argon in abs. N,N-
dimethylformamide, and then N,N-diisopropylethylamine (1.32 ml,
7.60 mmol) was added. The 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. Column chromatography purification of the crude
product obtained (ethyl acetate/heptane gradient) gave ethyl (2E)-3-[5-
nitro-2-(spiro[3.3]hept-2-ylamino)phenyl]acrylate (500 mg, 36% of
theory) as a colorless solid, 1H NMR (400 MHz, CDCl3 δ, ppm) 8.26 (d,
1H), 8.11 (m, 1H), 7.67 (d, 1H), 6.51 (m, 1H), 6.47 (d, 1H), 4.82 (br. m,
1H, NH), 4.29 (q, 2H), 3.88 (m, 1H), 2.60 (m, 2H), 2.12 (m, 2H), 2.01 (m,
2H), 1.95–1.85 (m, 4H), 1.37 (t, 3H). Ethyl (2E)-3-[5-nitro-2-(spiro[3.3]
hept-2-ylamino)phenyl]acrylate (500 mg, 1.51 mmol) was then dissolved
in abs. ethanol (8 ml), and (Ph3P)3RhCl (70 mg) 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. Column chromatography purification of the crude product
obtained (ethyl acetate/heptane gradient) gave ethyl 3-[5-nitro-2-(spiro
[3.3]hept-2-ylamino)phenyl]propanoate (490 mg, 97% of theory) as a
colorless solid, 1H NMR (400 MHz, CDCl3 δ, ppm) 8.04 (dd, 1H), 7.94 (d,
1H), 6.43 (d, 1H), 5.11 (br. m, 1H, NH), 4.28 (q, 2H), 3.87 (m, 1H), 2.81
(m, 2H), 2.67 (m, 2H), 2.58 (m, 2H), 2.11 (m, 2H), 2.00 (m, 2H),
1.92–1.85 (m, 4H), 1.28 (t, 3H). Ethyl 3-[5-nitro-2-(spiro[3.3]hept-2-
ylamino)phenyl]propanoate (490 mg, 1.47 mmol) was dissolved in abs.
tetrahydrofuran (8 ml) and, under argon, added dropwise to a suspension,
cooled to 0 °C, of sodium hydride (88 mg, 2.21 mmol, 60% suspension in
oil) in abs. tetrahydrofuran (5 ml). 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. Column chromatography purification of the crude
product obtained (ethyl acetate/heptane gradient) gave 6-nitro-1-(spiro
2H).
Ethyl
3-{2-[1,1′-bi(cyclopropyl)-1-ylamino]-5-nitrophenyl}
propanoate (200 mg, 0.63 mmol) was dissolved in abs.
tetrahydrofuran (8 ml) and, under argon, added dropwise to a
suspension, cooled to 0 °C, of sodium hydride (38 mg, 0.94 mmol,
60% suspension in oil) in abs. tetrahydrofuran (5 ml). 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. Column chromatography
purification of the crude product obtained (ethyl acetate/heptane
gradient)
gave
1-[1,1′-bi(cyclopropyl)-1-yl]-6-nitro-3,4-dihydro
quinolin-2(1H)-one (90 mg, 53%) as a colorless solid, 1H NMR
(400 MHz, CDCl3 δ, ppm) 8.16 (m, 1H), 8.04 (m, 1H), 7.53 (d, 1H),
2.93 (m, 2H), 2.78–2.58 (m, 2H), 1.44 (m, 1H), 1.23 (m, 1H), 1.03 (m,
1H), 0.91–0.82 (m, 2H), 0.60–0.45 (m, 3H), 0.28 (m, 1H). In the next
step, 1-[1,1′-bi(cyclopropyl)-1-yl]-6-nitro-3,4-dihydroquinolin-2(1H)-
one (90 mg, 0.33 mmol) was, together with tin(II) chloride dihydrate
(298 mg, 1.32 mmol), added to abs. ethanol (5 ml) 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 with aqueous NaOH. 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. Column chromatography purification of the crude
product obtained (ethyl acetate/heptane gradient) gave 6-amino-1-
[1,1′-bi(cyclopropyl)-1-yl]-3,4-dihydroquinolin-2(1H)-one (70 mg,
87% of theory) as a highly viscous foam, 1H NMR (400 MHz, CDCl3
δ, ppm) 7.18 (m, 1H), 6.58 (m, 1H), 6.48 (d, 1H), 2.78 (m, 2H), 2.59
(m, 2H), 1.47 (m, 1H), 1.08 (m, 1H), 0.98 (m, 1H), 0.90–0.81 (m, 2H),
0.60–0.43 (m, 3H), 0.28 (m, 1H). Trimethyl phosphite (1 equiv,
8.07 mmol) and 4-methylbenzyl bromide (1 equiv, 8.07 mmol) were
added to a multi-necked flask which had been dried by heating and
then stirred together under continuous nitrogen flow at a temperature
of 100 °C for 10 h. After complete conversion, without further
purification, distilled POCl3 (1 equiv) was added to the resulting
crude product and the mixture was stirred under argon at
a
temperature of 60 °C for 1.5 h. After complete conversion, the methyl
(4-methylbenzyl)phosphonochloridate obtained was, without further
purification, directly reacted in the next step. In a round-bottom flask
which had been dried by heating, under argon, 6-amino-1-[1,1′-bi
[3.3]hept-2-yl)-3,4-dihydroquinolin-2(1H)-one (180 mg, 43%) as a
colorless solid, 1H NMR (400 MHz, CDCl3 δ, ppm) 8.11–8.07 (m, 2H),
6.84 (d, 1H), 4.29 (m, 1H), 2.92 (m, 2H), 2.74 (m, 2H), 2.61 (m, 2H), 2.12
(m, 2H), 2.07 (m, 2H), 1.94–1.83 (m, 4H). In the next step, 6-nitro-1-(spiro
[3.3]hept-2-yl)-3,4-dihydroquinolin-2(1H)-one (180 mg, 2.44 mmol) was,
together with tin(II) chloride dihydrate (567 mg, 2.52 mmol), added to
abs. ethanol 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 with aqueous NaOH. 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. Column chromatography
purification of the crude product obtained (ethyl acetate/heptane
(cyclopropyl)-1-yl]-3,4-dihydro-quinolin-2(1H)-one
(1108
mg,
4.57 mmol) was dissolved in abs. tetrahydrofuran (2 ml) and slowly
added dropwise under argon to a solution, cooled to −20 °C, of methyl
(4-methylbenzyl)phosphonochloridate (1000 mg, 4.57 mmol) in abs.
tetrahydrofuran (10 ml) in a round-bottom flask which had been dried
beforehand by heating. The resulting reaction mixture was stirred at
−20 °C for 10 min, triethylamine (1.27 ml, 9.15 mmol) was then added
and the mixture was subsequently stirred at room temperature for 2 h.
The reaction mixture was then filtered, the filter cake was washed with
tetrahydrofuran and the filtrate was concentrated under reduced
pressure. Column chromatography purification of the crude product
obtained (ethyl acetate/heptane gradient), gave methyl N-{1-[1,1′-bi
(cyclopropyl)-1-yl]-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl}-P-(4-
gradient)
gave
6-amino-1-(spiro[3.3]hept-2-yl)-3,4-dihydroquinolin-
2(1H)-one (151 mg, 94% of theory) as a highly viscous foam, 1H NMR
(400 MHz, CDCl3 δ, ppm) 6.59–6.53 (m, 3H), 4.32–3.80 (br. s, 2H, NH2),
4.21 (m, 1H), 2.72 (m, 2H), 2.67 (m, 2H), 2.48 (m, 2H), 2.12–2.06 (m,
10

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
4H), 1.93–1.80 (m, 4H). Trimethyl phosphite (1 equiv, 8.07 mmol) and 4-
methylbenzyl bromide (1 equiv, 8.07 mmol) were added to a multi-necked
flask which have been dried by heating and then stirred together under
continuous nitrogen flow at a temperature of 100 °C for 10 h. After
complete conversion, a partial amount of the resulting crude product
(170 mg) was, without further purification, taken up in aqueous NaOH
(10% strength, 5 ml), and the mixture was stirred under reflux conditions
for 2 h. After cooling to room temperature, dil. hydrochloric acid was
added carefully, followed by thorough extraction with dichloromethane
and water. The combined organic phases were dried over magnesium
sulfate, filtered and concentrated under reduced pressure. Without further
purification, the resulting crude product (109 mg, 0.54 mmol) was initially
charged into abs. N,N-dimethylformamide (2 ml) and cooled to 0 °C. After
5 min of stirring at 0 °C, oxalyl chloride (0.12 ml, 1.63 mmol) was added
and the reaction mixture obtained was stirred at room temperature for one
and a half hours and then concentrated under reduced pressure. The
resulting methyl (4-methylbenzyl)-phosphonochloridate was taken up in
abs. dichloromethane (5 ml) and cooled to a temperature of −20 °C, and a
solution of 6-amino-1-(spiro[3.3]hept-2-yl)-3,4-dihydro-quinolin-2(1H)-
one (167 mg, 0.65 mmol) mmol) in abs. dichloromethane (2 ml) was
added. This was followed by the dropwise addition of N-
ethyldiisopropylamine (0.17 ml, 1.19 mmol). The resulting reaction
mixture was stirred at −20 °C for 10 min and then at room temperature
for 2 h. The reaction mixture was then filtered, the filter cake was washed
with dichloromethane and the filtrate was concentrated under reduced
pressure. Column chromatography purification of the crude product
obtained (ethyl acetate/heptane gradient) gave methyl N-[2-oxo-1-(spiro
[3.3]hept-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]-P-(4-methylphenyl)
phosphonamidate 13i (10 mg, 4% of theory) as a highly viscous foam. 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.15–7.11 (m, 2H), 7.09–7.05 (m, 2H),
6.99 (m, 1H), 6.83 (m, 1H), 6.68–6.65 (m, 1H), 5.15 (br. s, 1H, NH),
4.24–4.19 (m, 1H), 3.65/3.43 (m, 3H), 3.14/3.03 (d, 2H), 2.73–2.69 (m,
2H), 2.67–2.63 (m, 2H), 2.52–2.46 (m, 2H), 2.32 (s, 3H), 2.15–2.03 (m,
4H), 1.85–1.70 (m, 4H). LCMS (ESI, m/z): [M]+ 438.5
phosphonochloridate (1030 mg, 4.43 mmol) in abs. tetrahydrofuran
(8 ml) in a round-bottom flask which had been dried beforehand by
heating. The resulting reaction mixture was stirred at −20 °C for
10 min, triethylamine (1.23 ml, 8.86 mmol) was then added and the
mixture was subsequently stirred at room temperature for 2 h. The
reaction mixture was then filtered, the filter cake was washed with
tetrahydrofuran and the filtrate was concentrated under reduced
pressure. Column chromatography purification of the crude product
obtained (ethyl acetate/heptane gradient) gave ethyl N-[1-(n-
propylmethyl)-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl]-P-(4-
methylbenzyl)phosphonamidate 13 l (224 mg, 12% of theory) as a
colourless solid. 1H NMR (600 MHz, CDCl3 δ, ppm) 7.16–03 (m, 4H),
6.87 (m, 1H), 6.81 (m, 1H), 6.72 (m, 1H), 5.00 (br. m, 1H, NH),
4.24–4.17 (m, 1H), 4.08–4.00 (m, 1H), 3.90–3.84 (m, 2H), 3.27/3.02
(d, 2H), 2.83–2.78 (m, 2H), 2.63–2.59 (m, 2H), 2.30 (s, 3H), 1.71–1.63
(m, 2H), 1.33 (t, 3H), 0.96 (t, 3H); LCMS (ESI, m/z): [M]+ 400.2; logp
2.44.
4.2.3.13. Ethyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(4-chlorobenzyl)phosphonamidate 13 m.. White solid; yield 9%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.47 (m, 2H), 7.34 (m, 2H), 6.90 (m,
1H), 6.81 (m, 1H), 6.70 (m, 1H), 5.02 (br. m, 1H, NH), 4.24–4.17 (m,
1H), 4.08–4.00 (m, 1H), 3.90–3.84 (m, 2H), 3.27/3.02 (d, 2H),
2.83–2.78 (m, 2H), 2.63–2.59 (m, 2H), 2.30 (s, 3H), 1.71–1.63 (m,
2H), 1.33 (t, 3H), 0.96 (t, 3H); LCMS (ESI, m/z): [M]+ 420.9
4.2.3.14. Ethyl N-[1-(cyclopropylmethyl)-2-oxo-1,2,3,4-tetrahydroquinolin-6-
yl]-P-(4-methylbenzyl)phosphonamidate 13n.. Triethyl phosphite (1 equiv,
8.07 mmol) and 4-methylbenzyl bromide (1 equiv, 8.07 mmol) were
added to a multi-necked flask which had been dried by heating and then
stirred together under continuous nitrogen flow at a temperature of 100 °C
for 10 h. After complete conversion, without further purification, distilled
POCl3 (1 equiv) was added to the resulting crude product and the mixture
was stirred under argon at a temperature of 60 °C for 1.5 h. After complete
conversion, the ethyl (4-methylbenzyl)-phosphonochloridate obtained
was, without further purification, directly reacted in the next step. In a
round-bottom flask which had been dried by heating, under argon, 6-
amino-1-cyclopropylmethyl-3,4-dihydroquinolin-2(1H)-one (890 mg,
4.11 mmol) was dissolved in abs. tetrahydrofuran (2 ml) and slowly
added dropwise under argon to a solution, cooled to −20 °C, of ethyl (4-
methylbenzyl)phosphonochloridate (957 mg, 4.11 mmol) in abs.
tetrahydrofuran (8 ml) in a round-bottom flask which had been dried
beforehand by heating. The resulting reaction mixture was stirred at
−20 °C for 10 min, triethylamine (1.15 ml, 8.23 mmol) was then added
and the mixture was subsequently stirred at room temperature for 2 h. The
reaction mixture was then filtered, the filter cake was washed with
tetrahydrofuran and the filtrate was concentrated under reduced pressure.
By column chromatography purification of the crude product obtained
(ethyl acetate/heptane gradient), ethyl N-[1-(cyclopropylmethyl)-2-oxo-
1,2,3,4-tetrahydroquinolin-6-yl]-P-(4-methylbenzyl)phosphonamidate
13n (236 mg, 9% of theory) was isolated as a colorless solid. 1H NMR
(400 MHz, CDCl3 δ, ppm) 7.17–7.03 (m, 4H), 7.00 (m, 1H), 6.83 (m, 1H),
6.72 (m, 1H), 5.03 (br. m, 1H, NH), 4.24–4.19 (m, 1H), 4.12–4.01 (m,
1H), 3.87 (m, 2H), 3.27/3.02 (d, 2H), 2.87–2.82 (m, 2H), 2.65–2.61 (m,
2H), 2.32/2.30 (s, 3H), 1.33 (t, 3H), 1.13 (m, 1H), 0.53–0.48 (m, 2H),
0.45–0.39 (m, 2H).; LCMS (ESI, m/z): [M]+ 412.2; logp 2.59.
4.2.3.10. Methyl N-[1-methyl-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-P-
(4-chlorobenzyl)phosphonamidate 13j.. White solid; yield 5%; 1H NMR
(400 MHz, CDCl3 δ, ppm) 7.47 (m, 2H), 7.34 (m, 2H), 6.90 (m, 1H),
6.81 (m, 1H), 6.69 (m, 1H), 4.93 (br. s, 1H, NH), 3.87 (m, 2H), 3.73/
3.68 (d, 3H), 3.29 (s, 3H), 3.25/3.04 (d, 2H), 2.83–2.78 (m, 2H),
2.64–2.58 (m, 2H); LCMS (ESI, m/z): [M+H]+ 379.7.
4.2.3.11. Methyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(3-chlorobenzyl)phosphonamidate 13 k.. White solid; yield 7%; 1H
NMR (400 MHz, CDCl3 δ, ppm) 7.32 (m, 1H), 7.06–6.95 (m, 2H),
6.91–6.81 (m, 2H), 6.69 (m, 1H), 4.98 (br. s, 1H, NH), 3.87 (m, 2H),
3.73/3.68 (d, 3H), 3.27/3.02 (d, 2H), 2.83–2.78 (m, 2H), 2.64–2.58 (m,
2H), 1.70–1.61 (m, 2H), 0.98 (t, 3H); LCMS (ESI, m/z): [M+H]+ 407.8.
4.2.3.12. Ethyl N-[1-(n-propyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-6-yl]-
P-(4-methylbenzyl)phosphonamidate 13 l.. Triethyl phosphite (2.00 g,
12.04 mmol) and 4-methylbenzyl bromide (15.65 mmol) were added to
a microwave vessel which had been dried by heating and then stirred
together in the microwave under nitrogen at a temperature of 140 °C for
1 h. After complete conversion, the resulting crude product was purified
by column chromatography (heptane/ethyl acetate gradient), distilled
POCl3 (4.43 mmol) was then added to a partial amount of the resulting
purified intermediate (4.43 mmol) and the mixture was stirred under
argon at a temperature of 60 °C for 1.5 h. After complete conversion,
the reaction mixture was concentrated carefully and the ethyl (4-
methylbenzyl)phosphonochloridate obtained was, without further
purification, directly reacted in the next step. In a round-bottom flask
which had been dried by heating, under argon, 6-amino-1-propyl-3,4-
dihydroquinolin-2(1H)-one (904 mg, 4.43 mmol) was dissolved in abs.
tetrahydrofuran (2 ml) and slowly added dropwise under argon to a
4.2.3.15. Ethyl
N-{1-[1,1′-bi(cyclopropyl)-1-yl]-2-oxo-1,2,3,4-tetrahydro
quinolin-6-yl}-P-(4-methylphenyl)phosphonamidate 13o. Triethyl phosphite
(1 equiv, 8.07 mmol) and 4-methylbenzyl bromide (1 equiv, 8.07 mmol)
were added to a multi-necked flask which had been dried by heating and
then stirred together under continuous nitrogen flow at a temperature of
100 °C for 10 h. After complete conversion, without further purification,
distilled POCl3 (1 equiv) was added to the resulting crude product and the
mixture was stirred under argon at a temperature of 60 °C for 1.5 h. After
complete conversion, the ethyl (4-methylbenzyl)-phosphonochloridate
solution,
cooled
to
−20
°C,
of
ethyl
(4-ethylbenzyl)
11

J. Frackenpohl, et al.
Bioorganic&MedicinalChemistry28(2020)115725
obtained was, without further purification, directly reacted in the next
step. In a round-bottom flask which had been dried by heating, under
argon, 6-amino-1-[1,1′-bi(cyclopropyl)-1-yl]-3,4-dihydroquino-lin-2(1H)-
one (1042 mg, 4.29 mmol) was dissolved in abs. tetrahydrofuran (2 ml)
and slowly added dropwise under argon to a solution, cooled to −20 °C, of
ethyl (4-methylbenzyl)phosphonochloridate (1000 mg, 4.29 mmol) in abs.
tetrahydrofuran (10 ml) in a round-bottom flask which had been dried
beforehand by heating. The resulting reaction mixture was stirred at
−20 °C for 10 min, triethylamine (1.19 ml, 8.54 mmol) was then added
and the mixture was subsequently stirred at room temperature for 2 h. The
reaction mixture was then filtered, the filter cake was washed with
tetrahydrofuran and the filtrate was concentrated under reduced pressure.
Column chromatography purification of the crude product obtained (ethyl
acetate/heptane gradient) gave ethyl N-{1-[1,1′-bi(cyclopropyl)-1-yl]-2-
oxo-1,2,3,4-tetrahydro-quinolin-6-yl}-P-(4-methyl-phenyl)
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115725.
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Acknowledgements
We would like to thank Matthias Jank, Susanne Ries, Gudrun Fey,
Peter Zöllner and Martin Annau for valuable analytical support. We
would like to thank Sanjay Bhavsar for preparing some of the target
compounds. Furthermore, we would like to thank David Barber for
fruitful discussions during the preparation of the manuscript.
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