Structural exploration of cinnamate-based phosphonic acids as inhibitors of bacterial ureases

Article abstract of DOI:10.1016/j.ejmech.2018.09.074

The conjugated system of cinnamic acid, α-substituted with a phosphonoalkyl residue, was previously validated as a scaffold that provided one of the most potent organophosphorus inhibitors of bacterial urease. Following the idea of using Morita-Baylis-Hil

Full text of DOI:10.1016/j.ejmech.2018.09.074

Accepted Manuscript
Structural exploration of cinnamate-based phosphonic acids as inhibitors of bacterial
ureases
Vasilis Ntatsopoulos, Katarzyna Macegoniuk, Artur Mucha, Stamatia Vassiliou,
Łukasz Berlicki
PII:
S0223-5234(18)30859-6
10.1016/j.ejmech.2018.09.074
EJMECH 10785
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 August 2018
Revised Date: 27 September 2018
Accepted Date: 29 September 2018
Please cite this article as: V. Ntatsopoulos, K. Macegoniuk, A. Mucha, S. Vassiliou, Ł. Berlicki, Structural
exploration of cinnamate-based phosphonic acids as inhibitors of bacterial ureases, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.074.
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ACCEPTED MANUSCRIPT
Structural exploration of cinnamate-based phosphonic acids as inhibitors of bacterial
ureases
a
b
b
a,
Vasilis Ntatsopoulos , Katarzyna Macegoniuk , Artur Mucha , Stamatia Vassiliou *,
Łukasz Berlicki b,*
a
Laboratory of Organic Chemistry, Department of Chemistry, University of Athens,
Panepistimiopolis, Zografou, 15701 Athens, Greece
b
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of
Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
* Corresponding authors
SV. E-mail: svassiliou@chem.uoa.gr
LB. E-mail: lukasz.berlicki@pwr.edu.pl
Keywords: Morita-Baylis-Hillman bromides, palladium-catalyzed coupling, phosphonic
acids, enzyme inhibitors, structure-activity relationship.
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Highlights
-
-
-
Multistep synthesis of organophosphorus cinnamic acid-based compounds was
elaborated.
The most potent compounds showed slow-binding mechanism of inhibition and
submicromolar activity.
Molecular modelling indicated plausible mode of binding based on interaction with
metal ions and reaction with cysteine residue.
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Abstract
The conjugated system of cinnamic acid, α-substituted with a phosphonoalkyl residue, was
previously validated as a scaffold that provided one of the most potent organophosphorus
inhibitors of bacterial urease. Following the idea of using Morita-Baylis-Hillman adducts to
introduce the terminal phosphonic side chain functionality to the α,β-unsaturated system, we
currently report the synthesis and activity of an extended series of compounds. Cinnamates
modified with 3-phosphonopropyl and 4-phosphonobutyl side chains were obtained in a
convenient two-step procedure, which involved Pd-mediated transformations of the Morita-
Baylis-Hillman bromides as the key substrates. The introduction of a terminal alkenyl
fragment, which was achieved by Stille coupling with stannanes, was followed by a tandem
C-P bond formation/oxidation process. A submicromolar ligand of Sporosarcina pasteurii
urease (Ki = 0.509 µM) was identified among the active molecules. In addition, inhibitors of
Proteus mirabilis urease affected bacterial growth at the micromolar level. Based on the
structure-activity relationship and the mechanism of inhibition, we suggest a nontypical
mixed mode of action for the slow binding compounds. We presume that the molecular
distance between the phosphonic group and the backbone double bond allows a dual activity:
complexation of the acidic group with nickel ions and Michael addition of a cysteine forming
the active site lid.
Graphical abstract
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Introduction
Phosphonate and phosphinate groups are structural components/functionalities of invaluable
importance in the construction of enzyme inhibitors [1,2]. These molecular fragments often
serve to mimic the transition states of amide/ester bond hydrolysis; in addition, they are
responsible for the complexation of metal ions present in the active sites of corresponding
enzymes. A combination of both features offered by incorporated phosphonate/phosphinate
group(s) is particularly profitable for ligand affinity. Accordingly, the most widely studied
targets for such inhibitors are metallohydrolases, including metalloproteases, such as leucine
aminopeptidase, aminopeptidase N, angiotensin converting enzyme and matrix
metalloproteases, and other hydrolases, such as urease [3-5].
Urease is nickel-dependent enzyme that catalyzes the hydrolysis of urea to ammonia and
carbamate [6,7]. The active site of urease comprises two nickel ions complexed by four
histidine residues, carbamoylated lysine and an aspartate carboxylate [8]. The nucleophilic
hydroxyl group is activated by complexation with both nickel ions and reacts with a urea
molecule to produce the tetrahedral intermediate, which subsequently decomposes to
carbamate and ammonia [9]. Following the binding of the substrate, the active site is closed
with a movable loop. Although the size of the site is limited and its architecture is highly
conserved, the quaternary assembly of ureases is complex and can vary considerably. The
most typical bacterial enzyme comprises three units, each built by three subunit chains (α, β
and γ). However, in the case of Helicobacter pylori urease, a single unit is formed by two
chains (α and β), while the overall functional arrangement is a ball-shaped tetramer of trimers
with twelve active sites [10].
There are numerous compounds with known inhibitory activity against urease spanning
structurally diversified classes [5,11-13], which can be roughly divided into two groups
targeting (a) the catalytic nickel ions and (b) the cysteine residue placed on the movable loop
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covering the active site. The first group of ligands contains phosphordiamidates [14-16],
phosphonates [17], phosphinates [18-20], urea and thiourea derivatives [21,22], hydroxamic
acids [23], and heterocyclic compounds [13,24]. The second group contains Michael
acceptors [25], thiols [26], ebselen derivatives [27] and metal ions [28]. The ongoing interest
in the discovery and development of urease inhibitors is driven by their application for the
treatment of pathogenic states related to infections caused by urease-dependent
microorganisms [29,30]. Gastroduodenal peptic ulcers of H. pylori etiology are probably the
most widespread example of such a disease [31]. H. pylori urease releases large quantities of
ammonia, which gives rise to an increase in local pH and creates a microenvironment suitable
for survival and proliferation of the pathogen [32]. Ammonia is destructive to the stomach
mucosa, which consequently leads to devastating symptoms of the disease and the spreading
of bacteria. Thus, urease is the key virulence factor of H. pylori, and its inhibition is
considered a promising eradication strategy [33,34]. The second opportunity for the medical
application of urease inhibitors is the treatment of chronic infections of the urinary tract
colonized by Proteus species. Alkalization of urine related to the production of ammonia
causes the rapid formation of urinary stones, which is problematic, particularly for
catheterized patients [35,36]. The only drug available on the market contains
acetohydroxamic acid – a moderate urease inhibitor with numerous severe side effects
[37,38].
Our previous studies on inhibitors of bacterial ureases have led to the discovery of several
groups of phosphonate/phosphinate-based active compounds. Initially, aminophosphonates
and aminophosphinates [18,19,39], putative transition state analogues, were designed and
synthesized, and their activity was evaluated. Iterative optimization of these structures
provided inhibitors with submicromolar affinity to selected bacterial enzymes. Recently, we
developed a group of diverse organophosphorus compounds, the substituted cinnamic acid
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analogs, obtained conveniently by the addition of phosphorus nucleophiles to Morita-Baylis-
Hillman adducts (Fig. 1) [40]. The most active compound, (Z)-3-(4-methylphenyl)-2-
(phosphonomethyl)propenoic acid, exhibited Ki = 0.6 µM against Sporosarcina pasteurii
urease. Extensive structure-activity optimization of the novel framework allowed
identification of the promising points for potency improvement, namely, modification of
bulkiness and hydrophobicity of the aromatic ring as well as the separation distance between
the acidic groups (Fig. 1).
COOH
R
O
X
P
O OH
OH
OH
R = H, OH, CH3, CH2OH, CH2NH2
X = H, CH3, F, Cl, AcNH, CH3O
X
P
n
O OH
n = 1, 2
R = OH, X = CH3
Ki = 0.594 M, S. pasteurii urease
Figure 1. Structures of previously studied cinnamate-based phosphonic and phosphinic acids
obtained from the Morita-Baylis-Hillman acetates and evaluated as S. pasteurii urease
inhibitors (left panel). The extended compounds developed in the present study (right panel).
In the current study, we focused our attention on the further exploration of particular
structural features of cinnamic acid-based urease inhibitors substituted with a phosphonate
group. The key modifications involved the length of the α-substituent, thus the linkage
between the terminal phosphonic acid and cinnamate backbone, which was expected to affect
the mode of binding to the active site cleft. A dedicated synthetic approach was elaborated to
provide the target compounds, and their structure-activity relationship was analyzed for the
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inhibition of S. pasteurii and Proteus mirabilis ureases. For the latter microorganism, the
biological activity was supplemented by the results of cell proliferation studies.
Results and Discussion
Synthesis
The Morita-Baylis-Hillman (MBH) adducts, the starting materials for target phosphonic acids,
were synthesized in a three-step procedure (Scheme 1). First, benzaldehyde and its selected p-
substituted analogs (1a-e) were reacted with methyl acrylate under standard conditions of an
MBH reaction (in the presence of DABCO as the catalyst for an extended time) to provide the
corresponding allyl alcohols (2a-e) [40]. The MBH alcohols were further substituted with
bromine in 48% aqueous HBr in a regio- and stereoselective manner to give the
corresponding α-bromomethylcinnamates (3a-e) in excellent yield and in pure Z-form [41].
The introduction of the alkenyl moieties (either vinyl or allyl) to these compounds was
achieved by means of Pd-mediated Stille coupling between appropriate stannanes and
bromides [42]. Accordingly, methyl cinnamate α,β-unsaturated systems bearing the terminal
C=C at α-alkenyl residue 4a-e were obtained from tetravinylstannane in good to high yields.
The stereochemistry of products 4a-e was established by comparing 1H NMR parameters for
the olefinic and methylene protons with literature values and proved to be exclusively E
(stereoselectivity greater than 99/1 was observed in all cases) [43, 44]. However,
allyltributylstannane provided 5a-e in very good yields, and the E/Z ratio ranged from 86/14
to 100/0 in favor of E [45, 46]. The ratio of isomers was determined on the basis of the
1
integration of the isomeric olefinic protons and allylic methylene protons in the H NMR
spectrum. Because of the problematical separation of the two isomers by column
chromatography, 5a-e were further used as the diasteromeric mixture.
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OH
O
O
O
a
O
b
O
X
X
X
Br
2a-e
1a-e
3a-e
c
d
O
O
a: X = H
b: X = Me
c: X = F
O
O
d: X = Cl
e: X = OMe
X
X
4a-e
5a-e
Scheme 1. Preparation of the Morita-Baylis-Hillman bromides and Pd-mediated Stille
coupling with stannanes. Reagents and conditions: a) CH2=CHCO2Me, DABCO, rt, 3 d; b)
48% aq. HBr, cat. H2SO4, rt, 24 h; c) (CH2=CH2)4Sn, Pd(dba)2 (5 mol%), PPh3, LiCl, THF, rt,
24 h; d) CH2=CHCH2SnBu3, Pd(dba)2 (5 mol%), PPh3, LiCl, THF, rt, 24 h.
Direct phosphonic acid syntheses involving C-P bond formation using unactivated double
bond can be achieved by the low-yielding free-radical mediated addition of phosphorous acid
(H3PO3) to olefins [47]. This procedure has at least two shortcomings. First, polymerization is
unavoidable. Second, addition to the activated double bond present in the molecule cannot be
excluded. Therefore, the Pd-catalyzed tandem-phosphorus bond formation-oxidation process
developed by Montchamp [48] was tested as an alternative (Scheme 2). This approach is a
one-pot, one-step hydrophosphinylation-oxidation process using 50% aqueous H3PO2 as a
phosphorus source, Pd2dba3/xantphos as a catalyst and air as the oxidizing agent. The reaction
proceeded smoothly, providing E-phosphonic acids 7a-e in very good yields and retaining the
stereochemistry of their corresponding alkene precursors 4a-e. The same was observed for
alkenes 5a-e, which gave the corresponding phosphonic acids 8a-e, with an E/Z ratio ranging
from 84/16 to 100/0, which is similar to that of the starting materials. Alkaline hydrolysis of
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the carboxylic acid methyl esters followed by acidification provided the corresponding
products 9a-e and 10a-e in the form of free carboxylic acid in very good yields.
Scheme 2. Tandem hydrophosphinylation of unsaturated substrates and oxidation of
intermediates to phosphonic acids. Reagents and conditions: a) alkene 4a-e, Pd2dba3 (2
mol%), xantphos, DMF, 110 °C, air, 3-4 days; b) a) alkene 5a-e, Pd2dba3 (2 mol%), xantphos,
DMF, 110 °C, air, 3-4 days; c) NaOH 1.5 M, MeOH, rt, 15-20 days, then H+.
Biological Activity
The whole set of compounds differing by side chain lengths and phenyl substitutions,
including both acids and esters, was tested for inhibition of the model enzyme from S.
pasteurii (Table 1), and the comprehensive kinetic characterization including IC50s with and
without pre-incubation and Ki was performed. The state of the carboxylate group (free or
protected) was found to be most critical for the activity. In general, esters 7a-e and 8a-e
appeared to be much less potent than acids 9a-e and 10a-e. Only two esters showed inhibition
in the low micromolar range, e.g., methyl α-(3-phosphonopropyl)-p-methoxycinnamate 7e (Ki
≈ 6 µM) and methyl α-(4-phosphonobutyl)-p-methoxycinnamate 8d (Ki ≈ 5 µM), while the
majority of acids displayed Ki values below 3.4 µM.
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Table 1. Inhibition of S. pasteurii and P. mirabilis ureases (ꢀꢁꢂꢃ, ꢄ ) with the inhibition of P.
ꢅ
mirabilis cell proliferation (ꢆꢁꢂꢃ), by methyl α-(3-phosphonopropyl)- and α-(4-
phosphonobutyl)cinnamates 7a-e and 8a-e, respectively, and the corresponding acids, 9a-e
ꢇꢈ
and 10a-e. ꢀꢁꢂꢃꢃ and ꢀꢁ , half maximal inhibitory concentration without pre-incubation and
ꢂꢃ
ꢇꢈ
with pre-incubation with inhibitors for 2 h, respectively; ꢆꢁ , lethal concentration, 50%,
ꢂꢃ
cells pre-incubated with inhibitors for 2 h. The most significant activities are highlighted in
bold.
Inhibition of S. pasteurii urease
Proteus mirabilis
ꢀꢁꢂꢃꢃ[µM]
449
ꢀꢁ [µM]
ꢇꢈ
ꢇꢈ
ꢇꢈ
No
Compound
ꢄ [µM]
ꢀꢁ [µM]
ꢆꢁ [µM]
ꢅ
ꢂꢃ
ꢂꢃ
ꢂꢃ
328
± 13
248
289
1592
± 133
1429
± 121
735
556
± 64
292
7a
± 22
265
± 12
227
7b
7c
7d
7e
8a
± 13
221
± 11
105
± 11
87.2
± 25
215
± 12
109
± 7.2
49.8
± 1.3
10.8*
± 0.46
54.0
± 1.3
± 3.3
42.5
± 65
521
± 17
89.6
± 7.5
236
± 5.6
35.3*
± 1.3
55.0
± 1.5
6.04
± 47
75.1
± 6.8
476
± 0.36
50.5
± 19
469
± 1.5
± 2.7
± 45
± 40
294
261
219
1552
659
8b
± 14
± 11
± 9.8
± 122
± 66
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164
130
116
630
1059
8c
8d
8e
± 5.8
± 4.5
± 5.0
± 62
± 144
58.3*
± 2.8
6.35*
4.71
27.4
638
± 0.23
± 0.18
± 2.1
± 79
24.5
22.6
21.4
162
160
± 1.1
± 0.99
± 0.84
± 15
± 22
135
± 16
139
9a
9b
9c
3.10*
± 0.12
30.0
0.743*
± 0.023
24.6
0.509
± 0.019
19.4
11.0
± 0.82
156
± 1.3
4.90
± 0.90
3.82
± 0.72
1.80
± 13
38.0
± 11
121
± 0.15
2.49
± 0.14
1.62
± 0.059
1.43
± 3.2
18.6
± 14
122
9d
9e
± 0.079
10.0
± 0.069
4.98
± 0.056
3.40
± 1.4
39.7
± 14
65.2
± 7.7
97.6
± 8.4
± 0.45
4.89
± 0.18
3.83
± 0.10
3.51
± 2.9
32.6
10a
± 0.16
± 0.12
± 0.13
± 2.6
9.24
8.53
7.21
52.6
49.8
10b
10c
± 0.39
± 0.28
± 0.22
± 4.6
± 4.2
9.98*
2.91*
2.58
14.1
192
± 0.36
± 0.11
± 0.11
± 1.1
± 23
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5.83
2.34
1.75
22.2
435
10d
10e
± 0.19
± 0.085
± 0.67
± 1.9
± 51
16.6
14.5
9.31
114
28.6
± 0.66
± 0.63
± 0.35
± 9.4
± 3.0
* slow binding inhibition.
To envisage the difference in complexation with the enzyme, we selected two fast-binding
analogs, namely, methyl α-(3-phosphonopropyl)-p-chlorocinnamate 7d (Ki = 42.5 µM) and α-
(3-phosphonopropyl)-p-chlorocinnamic acid 9d (Ki = 1.43 µM), and compared their
interaction modes by molecular modeling (Figure 2).
A
B
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Figure 2. Modeled binding mode of compounds 7d (A) and 9d (B) to S. pasteurii urease.
Inhibitors and enzyme amino acid residues are shown as sticks, while nickel ions as purple
spheres. Hydrogen bonds and interactions with metal ions are shown as thin green lines.
Both compounds fit well to the active site with the phosphonate group bound by two nickel
ions and a His222 residue. The key differences in binding involve a carboxyl moiety. In the
case of compound 9d, the carboxylate group formed two charge-assisted hydrogen bonds with
Arg339 and His323. Such interactions were not present for compound 7d where the methyl
carboxylate function was incorporated.
The high activity of acids 9a-e and 10a-e does not critically depend on the side chain length.
Both three- and four-carbon linkers between the phosphonic group and cinnamate backbone
seem to be optimal components, as they constitute equally potent molecules. Phenyl
substituents introduce disparate activities. In both series of compounds, p-methyl (9b and
10b) and p-methoxy analogs (9e and 10e) were less active (Ki = 3.5-19.4 µM) than the
halogen-substituted compounds (9c, 9d, 10c and 10d, Ki = 1.6-3.8 µM). This observation can
be explained by considering the change in the electron character of the ring substituent.
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Among the acids 9a-e and 10a-e, a single submicromolar ligand of S. pasteurii urease was
identified, namely, unsubstituted at aromatic ring α-(3-phosphonopropyl)cinnamic acid 9a (Ki
= 0.51 µM). This compound, as a few other potent molecules, displayed a slow-binding
mechanism of action (Table 2). Typically, the studied inhibitors were found to be fast-
binding, as they were characterized by comparable values of IC50 with and without
incubation. For the slow-binding compounds, pre-incubation significantly improved the
inhibitory activity. In order to further evaluate the slow-binding profile, koff and residence
time were measured (Table 2). Interestingly, compound 9a with the lowest Ki value (9a)
showed also the highest residence time (1466 s), thus, its potency exceeds previously
published fast-binding inhibitors [40]. While trying to recognize the mode of interaction of
acid 9a with urease at a molecular level, we performed molecular modeling and docking
studies (Fig. 3).·
Table 2. koff and residence time for slow-binding inhibitors of S. pateurii urease
Compound no
koff [s-1]
residence time [s]
0.00244 ± 0.00048
0.00206 ± 0.00044
0.000682 ±0.000042
0.00135 ± 0.00018
409
485
7e
8d
1466
740
9a
10c
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A
B
Figure 3. Modeled noncovalent (A) and covalent (B) binding mode of compound 9a to S.
pasteurii urease. Inhibitor and enzyme amino acid residues are shown as sticks, while nickel
ions as purple spheres. Hydrogen bonds and interactions with metal ions are shown as thin
green lines.
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The observed inhibition kinetics suggest a mixed mechanism, where covalent and
noncovalent inhibition may occur. Noncovalent binding of compound 9a to S. pasteurii
urease (Figure 3A) was very similar to that modeled for compound 9d. Interactions of
inhibitor phosphonate with both nickel ions and His222 as well as hydrogen bonds between
carboxylate and Arg339 and His323 were found. However, the activated double bond was
positioned proximal to the reactive thiol group of Cys322; thus, we propose that noncovalent
binding is followed by a Michael addition to form a covalent yet reversible enzyme-ligand S-
adduct, and this result complicated the kinetics. The modeled structure of this 9a adduct
(Figure 3B) indicated that all the interactions found in the noncovalent complex are preserved,
and inhibitor conformation is similar.
All compounds were also assayed against P. mirabilis urease and achieved a significant level
ꢇꢈ
of potency, with ꢀꢁ values in the range of 11-1592 µM. The overall structure-activity
ꢂꢃ
relationship was well-reproduced in comparison to that of S. pasteurii urease inhibition.
Carboxylic acids were more potent than their corresponding esters, homologs were of
comparable activity to each other, and halogen-substituted phenyls gave rise to activity
ꢇꢈ
ꢇꢈ
improvement. 9a remained the most potent derivative, with ꢀꢁ = 11 µM (ꢀꢁ = 0.74 µM
ꢂꢃ
ꢂꢃ
for S. pasteurii urease). Interestingly, assayed compounds also showed significant
antimicrobial activity against P. mirabilis. However, the compounds exhibited a different
pattern of SAR in this case. Five compounds, ester 7d and acids 9e, 10a, 10b and 10e, were
ꢇꢈ
found to be moderately potent, with ꢆꢁ < 100 µM. The most structurally extended acids
ꢂꢃ
10b (α-(4-phosphonobutyl)-p-methylcinnamic acid) and 10e (α-(4-phosphonobutyl)-p-
ꢇꢈ
methoxycinnamic acid) displayed the highest activity: ꢆꢁ
≈ 50 µM and 29 µM,
ꢂꢃ
respectively. This result could be related to the lipophilicity and ability of compounds to cross
bacterial cell walls.
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Conclusions
Our previous studies on organophosphorus inhibitors of ureases indicated that ligands of high
affinity must fulfill challenging steric and functional restrictions to fit the specific active site
arrangement. Typically, among an extended set of homologs/analogs, only a single derivative
surpasses the other members in activity [40]. In this work, we identified optimized
organophosphorus scaffolds for a bacterial urease (Sporosarcina pasteurii). Several
compounds based on p-substituted 3-phosphonopropyl- and 4-phosphonobutylcinnamic acids
were found to be good inhibitors, with 9a being the most potent (Ki = 0.509 ± 0.019 µM).
Moreover, the most active compound showed slow-binding mechanism with residence time of
1466 s, what enhances its potency in comparison to previously published fast-binding
compounds based on similar scaffold [40]. To explain the slow-binding mechanism of
inhibition exhibited by this compound and other active structures, we also suggested a two-
step interaction/reaction mode. We proposed preliminary complexation of the nickel(II) ions
present in the active site with the phosphonic group, followed by -SH addition to the
unsaturated Michael acceptor, which was similar to an approach described elsewhere for other
activated multiple bonds [25]. The second step is slow, and, although covalent bond formation
is also reversible, its occurrence depends on the individual conformation of a ligand adopted
in the active site.
To identify therapeutic potential, the whole set of compounds was additionally tested for the
inhibition of urease from the pathogenic P. mirabilis strain. Following the high homology of
the two hydrolases, the phosphonic acids exhibited a similar structure-activity relationship
pattern. Antibacterial properties were further explored on the cell lines. The most lipophilic 4-
phosphonobutylcinnamic acids inhibited cell proliferation with IC50 at a micromolar level.
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Experimental
1. Chemistry – general
All used reagents were purchased at the highest quality from commercial suppliers, and were
used without further purification. Reactions were monitored by thin-layer chromatography
(TLC) carried out on 0.25 mm silica gel plates (silica gel 60F254) and components were
visualized by UV light absorbance. Purification of compounds by column chromatography
was carried out on silica gel (70-230 mesh) with the indicated solvents. 1H, 13C and 31P NMR
spectra were recorded on a 200 MHz Mercury spectrometer. 1H and 13C spectra are referenced
according to the residual peak of the solvent based on literature data. 31P NMR chemical shifts
are reported in ppm downfield from 85% H3PO4 (external standard). 13C and 31P NMR spectra
are fully proton decoupled. ESI mass spectral analyses were performed on a mass
spectrometer, using direct sample injection. Negative or positive ion ESI spectra were
acquired by adjusting the needle and cone voltages accordingly. HRMS spectra were
registered using a Brucker Maxis Impact QTOF mass spectrometer. To verify the purity of
new organophosphorus compounds, high-performance liquid chromatography (HPLC) was
used. The products were analyzed in a Gilson instrument (pump model 321) with a UV
detector (Gilson UV/VIS 151) equipped with a commercially prepacked reverse phase
analytical column (RPC18, 250 mm χ 4.6 mm, 5 µm Jupiter 300 Å, Phenomenex) and a
Widepore C18 guard column (Phenomenex). The mobile phase was a gradient between Α)
5% acetonitrile/water/0.1% TFA and B) 95% acetonitrile/water/0.1% TFA. The running
gradient was as follows: 0’ 5% B, 2’ 5% B; 17’ 100% B at a flow rate of 1 mL/min. Detection
was performed at 254 nm. The purity of the final compounds was confirmed to exceed 97%.
The spectroscopy and analytical data were obtained in the Laboratory of Organic Chemistry
of the University of Athens.
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2. Cross-coupling reaction of methyl (α-bromomethyl)cinnamate and tetravinylstannane,
4a-e
Pd(dba)2 (29 mg, 0.05 mmol, 5 mol%) was added to a stirred mixture of 3 (1 mmol),
tetravinylstannane (219 µL, 1.20 mmol), PPh3 (52 mg, 0.20 mmol) and LiCl (21 mg, 0.50
mmol) in dry THF (2 mL). The reaction mixture was degassed and stirred at room
temperature for 24 h under argon. After removal of the solvent the residue was purified by
column chromatography using petroleum ether (40-60 °C)/AcOEt, 9:1 as eluent.
Methyl (E)-2-benzylidenepent-4-enoate, 4a [43, 44, 49]
1
Colorless oil, 0.31 g, yield 78%. Rf (PE/AcOEt, 9:1) = 0.67. H NMR (200 MHz, CDCl3) δ
7.82 (s, 1H), 7.41-7.31 (m, 5H), 6.11-5.92 (m, 1H), 5.15-5.06 (m, 2H), 3.81 (s, 3H), 3.29 (d, J
= 5.4 Hz, 2H). 13C NMR (50 MHz, CDCl3) δ 168.5, 140.5, 135.5, 135.3, 129.9, 129.2, 128.6,
128.4, 115.6, 52.1, 31.6.
Methyl (E)-2-(4-methylbenzylidene)pent-4-enoate, 4b [49]
1
Colorless oil, 0.36 g, yield 91%. Rf (PE/AcOEt, 9:1) = 0.59. H NMR (200 MHz, CDCl3) δ
7.81 (s, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 6.09-5.93 (m, 1H), 5.18-5.08
13
(m, 2H), 3.81 (s, 3H), 3.32 (d, J = 5.4 Hz, 2H), 2.36 (s, 3H). C NMR (50 MHz, CDCl3) δ
168.3, 140.4, 138.6, 135.4, 132.3, 129.2, 129.0, 128.8, 115.4, 51.7, 31.4, 21.1.
Methyl (E)-2-(4-fluorobenzylidene)pent-4-enoate, 4c
1
Colorless oil, 0.35 g, yield 85%. Rf (PE/AcOEt, 9:1) = 0.54. H NMR (200 MHz, CDCl3) δ
7.72 (s, 1H), 7.37-7.29 (m, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.02-5.86 (m, 1H), 5.10-4.99 (m,
2H), 3.76 (s, 3H), 3.22 (d, J = 5.3 Hz, 2H). 13C NMR (50 MHz, CDCl3) δ 168.1, 162.6 (d, 1JF-
C = 249.5 Hz), 139.2, 137.7, 135.2, 131.2 (d, 4JF-C = 3.4 Hz), 131.0 (d, 3JF-C = 8.3 Hz), 129.6,
20

ACCEPTED MANUSCRIPT
+
115.3 (d, 2JF-C = 21.4 Hz), 51.8, 31.3. MS (ESI) m/z calculated for C13H14FO2 [M+H]+ 221.1,
found 221.1. Elemental analysis: calculated (%) for C13H13FO2: C: 70.90 H: 5.95, found: C:
70.77 H: 5.86.
Methyl (E)-2-(4-chlorobenzylidene)pent-4-enoate, 4d [49]
1
Colorless oil, 0.38 g, yield 93%. Rf (PE/AcOEt, 9:1) = 0.60. H NMR (200 MHz, CDCl3) δ
7.72 (s, 1H), 7.31 (s, 4H), 6.06-5.87 (m, 1H), 5.13-5.01 (m, 2H), 3.79 (s, 3H), 3.23 (d, J=5.4
Hz, 2H). 13C NMR (50 MHz, CDCl3) δ 168.1, 139.1, 137.9, 135.1, 134.5, 133.6, 130.4, 128.6,
115.7, 52.0, 31.4.
Methyl (E)-2-(4-methoxybenzylidene)pent-4-enoate, 4e [49, 50]
1
Colorless oil, 0.31 g, yield 77%. Rf (PE/AcOEt, 9:1) = 0.43. H NMR (200 MHz, CDCl3) δ
7.76 (s, 1H), 7.37 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.10-5.91 (m, 1H), 5.15-5.05
13
(m, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.30 (d, J = 5.3 Hz, 2H). C NMR (50 MHz, CDCl3) δ
168.6, 159.9, 140.2, 135.4, 131.1, 127.7, 127.5, 115.4, 113.8, 55.1, 51.9, 31.5.
3. Cross-coupling reaction of methyl (α-bromomethyl)cinnamate and allyltributylstannane
5a-e
Pd(dba)2 (29 mg, 0.05 mmol, 5 mol%) was added to a stirred mixture of 3 (1 mmol),
allyltributylstannane (372 µL, 1.20 mmol), PPh3 (52 mg, 0.20 mmol) and LiCl (21 mg, 0.50
mmol) in dry THF (2 mL). The reaction mixture was degassed and stirred at room
temperature for 24 h under argon. After removal of the solvent the residue (containing mainly
the E isomer of the product) was purified by column chromatography using a gradient of
petroleum ether (40-60 °C) to petroleum ether/AcOEt, 9:1 as eluent.
21

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Methyl (E)-2-benzylidenehex-5-enoate, 5a [46, 51]
Colorless oil, 0.34 g, yield 80% (E/Z, 91:9). Rf (PE/AcOEt, 9:1) = 0.58. 1H NMR (200 MHz,
CDCl3) δ 7.69 (s, 1H), 7.39-7.32 (m, 5H), 5.96-5.75 (m, 1H), 5.10-4.96 (m, 2H), 3.82 (s, 3H),
13
2.68-2.60 (m, 2H), 2.36-2.25 (m, 2H). C NMR (50 MHz, CDCl3) δ 168.5, 139.3, 137.5,
135.4, 132.4, 129.1, 128.4, 128.3, 114.8, 51.8, 33.0, 26.7.
Methyl (E)-2-(4-methylbenzylidene)hex-5-enoate, 5b
Colorless oil, 0.37 g, yield 87% (E/Z, 89:11). Rf (PE/AcOEt, 9:1) = 0.66. 1H NMR (200 MHz,
CDCl3) δ 7.83 (s, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 6.13-5.93 (m, 1H),
13
5.26-5.12 (m, 2H), 3.96 (s, 3H), 2.85-2.77 (m, 2H), 2.52-2.42 (m, 5H). C NMR (50 MHz,
CDCl3) δ 168.8, 139.3, 138.5, 137.7, 132.6, 131.6, 129.3, 129.2, 114.8, 51.8, 33.1, 26.8, 21.2.
+
MS (ESI) m/z calculated for C15H19O2 [M+H]+ 231.3, found 231.3. Elemental analysis:
calculated (%) for C15H18O2: C: 78.23 H: 7.88, found: C: 78.10 H: 7.71.
Methyl (E)-2-(4-fluorobenzylidene)hex-5-enoate, 5c [46]
Colorless oil, 0.38 g, yield 88% (E/Z, 84:16). Rf (PE/AcOEt, 9:1) = 0.56. 1H NMR (200 MHz,
CDCl3) δ 7.62 (s, 1H), 7.36-7.29 (m, 2H), 7.05 (t, J = 8.7 Hz, 2H), 5.92-5.72 (m, 1H), 5.08-
4.94 (m, 2H), 3.79 (s, 3H), 2.64-2.56 (m, 2H), 2.33-2.25 (m, 2H). 13C NMR (50 MHz, CDCl3)
1
4
δ 168.5, 162.5 (d, JF-C = 249.3 Hz), 138.1, 137.5, 132.3, 131.5 (d, JF-C = 3.5 Hz), 131.0 (d,
3JF-C = 8.2 Hz), 115.5 (d, 2JF-C = 21.6 Hz), 115.0, 51.9, 33.0, 26.7.
Methyl (E)-2-(4-chlorobenzylidene)hex-5-enoate, 5d [46, 51, 52]
Colorless oil, 0.37 g, yield 86% (E/Z, 86:14). Rf (PE/AcOEt, 9:1) = 0.65. 1H NMR (200 MHz,
CDCl3) δ 7.60 (s, 1H), 7.33 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 5.87-5.71 (m, 1H),
22

ACCEPTED MANUSCRIPT
13
5.07-4.94 (m, 2H), 3.79 (s, 3H), 2.62-2.54 (m, 2H), 2.32-2.21 (m, 2H). C NMR (50 MHz,
CDCl3) δ 168.3, 137.9, 137.4, 134.2, 133.9, 133.1, 130.4, 128.6, 115.0, 52.0, 33.0, 26.7.
Methyl (E)-2-(4-methoxybenzylidene)hex-5-enoate, 5e [46, 51]
1
Colorless oil, 0.36 g, yield 83%. Rf (PE/AcOEt, 9:1) = 0.56. H NMR (200 MHz, CDCl3) δ
7.65 (s, 1H), 7.36 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 5.99-5.79 (m, 1H), 5.13-4.98
(m, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 2.71-2.63 (m, 2H), 2.38-2.27 (m, 2H). 13C NMR (50 MHz,
CDCl3) δ 168.9, 159.7, 139.0, 137.7, 131.0, 130.2, 127.9, 114.8, 113.9, 55.2, 51.8, 32.9, 26.7.
4. Synthesis of phosphonic acids via tandem C-P bond formation/oxidation reaction, 7a-e
and 8a-e
To a solution of 50% aqueous H3PO2 (0.528 g, 4 mmol, 2 eq.) in DMF (4 mL) was added
alkene 4a-e or 5a-e (2 mmol, 1 eq.), followed by Pd2dba3 (0.0184 g, 0.020 mmol, 2 mol% Pd)
and xantphos (0.0254 g, 0.044 mmol) at room temperature. The reaction was heated at 110 °C
under air (septum with a needle) until complete oxidation of the intermediate H-phosphinic
acid (according to 31P NMR monitoring of a sample taken from the reaction mixture),
typically 3 to 4 days. The mixture was then concentrated in vacuo. The residue was diluted
with EtOAc and washed with 2 M HCl. The aqueous phase was extracted with EtOAc (twice)
and the combined organic fractions were washed with brine, dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatography using
CHCl3/MeOH/AcOH, 7:1:0.5 as eluent.
Methyl (E)-3-phenyl-2-(3-phosphonopropyl)propenoate, 7a
1
White solid, 0.12 g, yield 93%. Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.30. H NMR (200
MHz, CD3COOD) δ 7.69 (s, 1H), 7.52-7.11 (m, 5H), 3.80 (s, 3H), 2.73-2.42 (m, 2H), 1.94-
23

ACCEPTED MANUSCRIPT
13
1.49 (m, 4H). C NMR (50 MHz, CD3COOD) δ 170.2, 140.8, 136.4, 133.4, 130.2, 129.8,
129.5, 52.7, 30.4, 29.6, 29.2, 28.9 (d, JP-C = 139.6 Hz), 23.7. 31P NMR (81 MHz, CD3COOD)
δ 38.89. HRMS m/z calculated for C13H16O5P- [M-H]- 283.0741, found 283.0730. Elemental
analysis: calculated (%) for C13H17O5P: C: 54.93 H: 6.03, found: C: 54.99 H: 6.10
Methyl (E)-3-(4-methylphenyl)-2-(3-phosphonopropyl)propenoate, 7b
1
White solid, 0.13 g, yield 84%. Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.27. H NMR (200
MHz, CD3COOD) δ 7.66 (s, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 3.79 (s,
13
3H), 2.68-2.49 (m, 2H), 2.32 (s, 3H), 1.88-1.61 (m, 4H). C NMR (50 MHz, CD3COOD) δ
170.3, 140.9, 139.7, 133.4, 132.4, 130.4, 130.2, 52.7, 30.4, 29.6, 29.2, 28.9 (d, JP-C = 136.2
Hz), 23.6, 21.2. 31P NMR (81 MHz, CD3COOD) δ 38.35. HRMS m/z calculated for
C14H18O5P- [M-H]- 297.0897, found 297.0895. Elemental analysis: calculated (%) for
C14H19O5P: C: 56.37 H: 6.42, found: C: 56.54 H: 6.53.
Methyl (E)-3-(4-fluorophenyl)-2-(3-phosphonopropyl)propenoate, 7c
1
White solid, 0.12 g, yield 93%. Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.27. H NMR (200
MHz, CD3COOD) δ 7.66 (s, 1H), 7.45-7.39 (m, 2H), 7.13 (t, J = 8.0 Hz, 2H), 3.80 (s, 3H),
2.69-2.45 (m, 2H), 1.86-1.59 (m, 4H). 13C NMR (50 MHz, CD3COOD) δ 170.0, 163.6 (d, 1JF-
C = 247.9 Hz), 139.7, 139.3, 133.2, 132.5 (d, 4JF-C = 3.1 Hz), 116.4 (d, 2JF-C = 21.6 Hz), 52.7,
31
30.4, 29.4, 29.1, 28.8 (d, JP-C = 135.4 Hz), 23.6. P NMR (81 MHz, CD3COOD) δ 38.33.
HRMS m/z calculated for C13H15FO5P- [M-H]- 301.0647, found 301.0642. Elemental analysis:
calculated (%) for C13H16FO5P: C: 51.66 H: 5.34, found: C: 51.48 H: 5.42.
Methyl (E)-3-(4-chlorophenyl)-2-(3-phosphonopropyl)propenoate, 7d
24

ACCEPTED MANUSCRIPT
1
White solid, 0.11 g, yield 92%. Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.35. H NMR (200
MHz, CD3COOD) δ 7.64 (s, 1H), 7.38 (s, 4H), 3.80 (s, 3H), 2.65-2.44 (m, 2H), 1.89-1.55 (m,
4H). 13C NMR (50 MHz, CD3COOD) δ 169.8, 139.1, 135.1, 134.9, 134.1, 131.7, 129.6, 52.7,
31
30.4, 29.5, 29.1, 28.7 (d, JP-C = 138.0 Hz), 23.6. P NMR (81 MHz, CD3COOD) δ 38.04.
HRMS m/z calculated for C13H15ClO5P- [M-H]- 317.0351, found 317.0342. Elemental
analysis: calculated (%) for C13H16ClO5P: C: 48.99 H: 5.06, found: C: 49.21 H: 5.12.
Methyl (E)-3-(4-methoxyphenyl)-2-(3-phosphonopropyl)propenoate, 7e
1
White solid, 0.11 g, yield 80%. Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.30. H NMR (200
MHz, CD3COOD) δ 7.64 (s, 1H), 7.38 (d, J = 7.6 Hz, 2H), 6.95 (d, J = 7.6 Hz, 2H), 3.80 (s,
13
6H), 2.68-2.50 (m, 2H), 1.88-1.60 (m, 4H). C NMR (50 MHz, CD3COOD) δ 170.5, 161.0,
140.6, 132.3, 130.8, 128.6, 114.9, 55.5, 52.6, 30.4, 29.6, 29.2, 28.9 (d, JP-C = 137.5 Hz), 23.6.
31P NMR (81 MHz, CD3COOD) δ 38.19. HRMS m/z calculated for C14H18O6P- [M-H]-
313.0846, found 313.0840. Elemental analysis: calculated (%) for C14H19O6P: C: 53.50 H:
6.09, found: C: 53.28 H: 6.16.
Methyl (E)-3-phenyl-2-(4-phosphonobutyl)propenoate, 8a
1
White solid, 0.12 g, yield 95% (E/Z, 91:9). Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.27. H
NMR (200 MHz, CD3COOD) δ 7.67 (s, 1H), 7.47-7.26 (m, 5H), 3.79 (s, 3H), 2.62-2.41 (m,
2H), 1.80-1.48 (m, 6H). 13C NMR (50 MHz, CD3COOD) δ 170.2, 140.1, 136.4, 133.8, 130.1,
129.4, 129.0, 52.6, 31.3, 31.0, 30.4, 28.6 (d, JP-C = 134.1 Hz), 27.9, 24.0. 31P NMR (81 MHz,
CD3COOD) δ 39.14. HRMS m/z calculated for C14H18O5P- [M-H]- 297.0897, found 297.0888.
Elemental analysis: calculated (%) for C14H19O5P: C: 56.37 H: 6.42, found: C: 56.48 H:
6.52.
25

ACCEPTED MANUSCRIPT
Methyl (E)-3-(4-methylphenyl)-2-(4-phosphonobutyl)propenoate, 8b
1
White solid, 0.094 g, yield 70% (E/Z, 90:10). Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.28. H
NMR (200 MHz, CD3COOD) δ 7.63 (s, 1H), 7.28 (d, J = 7.4 Hz, 2H), 7.20 (d, J = 7.4 Hz,
13
2H), 3.78 (s, 3H), 2.61-2.43 (m, 2H), 2.32 (s, 3H), 1.76-1.51 (m, 6H). C NMR (50 MHz,
CD3COOD) δ 170.4, 140.2, 139.7, 133.5, 132.8, 130.3, 130.1, 52.7, 31.3, 30.9, 30.4, 28.6 (d,
31
JP-C = 136.2 Hz), 27.9, 24.0, 21.3. P NMR (81 MHz, CD3COOD) δ 39.09. HRMS m/z
calculated for C15H20O5P- [M-H]- 311.1054, found 311.1050. Elemental analysis: calculated
(%) for C15H21O5P: C: 57.69 H: 6.78, found: C: 57.51 H: 6.87.
Methyl (E)-3-(4-fluorophenyl)-2-(4-phosphonobutyl)propenoate, 8c
1
White solid, 0.081 g, yield 76% (E/Z, 89:11). Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.24. H
NMR (200 MHz, CD3COOD) δ 7.63 (s, 1H), 7.47-7.30 (m, 2H), 7.13 (t, J = 7.8 Hz, 2H), 3.79
13
(s, 3H), 2.60-2.37 (m, 2H), 1.83-1.43 (m, 6H). C NMR (50 MHz, CD3COOD) δ 170.1,
1
4
3
163.6 (d, JF-C = 248.0 Hz), 138.9, 133.7, 132.7 (d, JF-C = 3.0 Hz), 130.8 (d, JF-C = 8.6 Hz),
2
31
116.4 (d, JF-C = 22.3 Hz), 52.7, 31.2, 30.8, 30.4, 28.5 (d, JP-C = 136.1 Hz), 27.8, 23.9. P
NMR (81 MHz, CD3COOD) δ 39.14. HRMS m/z calculated for C14H17FO5P- [M-H]-
315.0803, found 315.0803. Elemental analysis: calculated (%) for C14H18FO5P: C: 53.17 H:
5.74, found: C: 53.29 H: 5.80.
Methyl (E)-3-(4-chlorophenyl)-2-(4-phosphonobutyl)propenoate, 8d
1
White solid, 0.091 g, yield 86% (E/Z, 86:14). Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.27. H
NMR (200 MHz, CD3COOD) δ 7.61 (s, 1H), 7.38 (s, 4H), 3.80 (s, 3H), 2.56-2.40 (m, 2H),
1.75-1.52 (m, 6H). 13C NMR (50 MHz, CD3COOD) δ 169.9, 139.0, 135.1, 134.5, 131.6,
130.4, 129.5, 52.7, 31.1, 30.8, 30.4, 28.5 (d, JP-C = 138.8 Hz), 27.9, 23.9.
26

ACCEPTED MANUSCRIPT
31P NMR (81 MHz, CD3COOD) δ 39.01. HRMS m/z calculated for C14H17ClO5P- [M-H]-
331.0508, found 331.0500. Elemental analysis: calculated (%) for C14H18ClO5P: C: 50.54 H:
5.45, found: C: 50.67 H: 5.50.
Methyl (E)-3-(4-methoxyphenyl)-2-(4-phosphonobutyl)propenoate, 8e
1
White solid, 0.082 g, yield 82%. Rf (CHCl3/MeOH/AcOH, 7:1:0.5) = 0.28. H NMR (200
MHz, CD3COOD) δ 7.60 (s, 1H), 7.43-7.25 (m, 2H), 7.03-6.83 (m, 2H), 3.79 (s, 6H), 2.66-
13
2.40 (m, 2H), 1.79-1.50 (m, 6H). C NMR (50 MHz, CD3COOD) δ 170.6, 161.0, 140.2,
132.1, 131.4, 128.8, 114.9, 55.6, 52.6, 31.2, 30.8, 30.4, 28.6 (d, JP-C = 132.7 Hz), 27.9, 24.0.
31P NMR (81 MHz, CD3COOD) δ 39.21. HRMS m/z calculated for C15H20O6P- [M-H]-
327.1003, found 327.0998. Elemental analysis: calculated (%) for C15H21O6P: C: 54.88 H:
6.45, found: C: 54.99 H: 6.40.
5. Alkaline hydrolysis of carboxylate methyl esters, 9a-e and 10a-e
To a stirred solution of a methyl ester (1 mmol) in MeOH (3.3 mL), 4 M NaOH (1.25 mL, 5
mmol) was added and the reaction mixture was stirred at room temperature till total
consumption of the starting material (the reaction progress was monitored by TLC), typically
15-20 days. Then, the volatiles were evaporated in vacuo and the residue was acidified with 6
M HCl to pH 1. The aqueous phase was extracted with AcOEt, dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatography using
CHCl3/MeOH/AcOH, 7:2:1 as eluent.
(E)-3-Phenyl-2-(3-phosphonopropyl)propenoic acid, 9a
White solid, 0.051 g, yield 48%. Rf (CHCl3/MeOH/AcOH, 7:2:1) = 0.31. 1H NMR (200 MHz,
13
CD3COOD) δ 7.80 (s, 1H), 7.49-7.30 (m, 5H), 2.67-2.50 (m, 2H), 1.90-1.63 (m, 4H). C
27

ACCEPTED MANUSCRIPT
NMR (50 MHz, CD3COOD) δ 173.8, 142.3, 136.2, 132.8, 130.4, 129.8, 129.6, 30.5, 29.3,
31
29.0, 28.9 (d, JP-C = 133.7 Hz), 23.6. P NMR (81 MHz, CD3COOD) δ 39.01. HRMS m/z
calculated for C12H14O5P- [M-H]- 269.0584, found 269.0583. Elemental analysis: calculated
(%) for C12H15O5P: C: 53.34 H: 5.60, found: C: 53.45 H: 5.50.
(E)-3-(4-Methylphenyl)-2-(3-phosphonopropyl)propenoic acid, 9b
White solid, 0.049 g, yield 42%. Rf (CHCl3/MeOH/AcOH, 7:2:1) = 0.41. 1H NMR (200 MHz,
CD3COOD) δ 7.76 (s, 1H), 7.33 (d, J = 7.5 Hz, 2H) , 7.22 (d, J = 7.5 Hz, 2H), 2.67-2.51 (m,
2H), 2.33 (s, 3H), 1.92-1.65 (m, 4H). 13C NMR (50 MHz, CD3COOD) δ 174.2, 142.3, 140.2,
31
133.4, 131.8, 130.6, 130.3, 30.4, 29.4, 29.0, 28.9 (d, JP-C = 136.7 Hz), 23.6, 21.2. P NMR
(81 MHz, CD3COOD) δ 39.25. HRMS m/z calculated for C13H16O5P- [M-H]- 283.0741, found
283.0744. Elemental analysis: calculated (%) for C13H17O5P: C: 54.93 H: 6.03, found: C:
55.09 H: 6.10.
(E)-3-(4-Fluorophenyl)-2-(3-phosphonopropyl)propenoic acid, 9c
White solid, 0.059 g, yield 56%. Rf (CHCl3/MeOH/AcOH, 7:2:1) = 0.35. 1H NMR (200 MHz,
CD3COOD) δ 7.76 (s, 1H), 7.53-7.38 (m, 2H), 7.14 (t, J = 8.2 Hz, 2H), 2.67-2.47 (m, 2H),
13
1
1.90-1.63 (m, 4H). C NMR (50 MHz, CD3COOD) δ 173.7, 163.8 (d, JF-C = 248.4 Hz),
2
141.1, 140.7, 132.7, 132.5, 116.5 (d, JF-C = 21.8 Hz), 30.4, 29.2, 28.9, 28.8 (d, JP-C = 135.2
31
Hz), 23.5. P NMR (81 MHz, CD3COOD) δ 38.65. HRMS m/z calculated for C12H13FO5P-
[M-H]- 287.0490, found 287.0493. Elemental analysis: calculated (%) for C12H14FO5P: C:
50.01 H: 4.90, found: C: 50.19 H: 4.84.
(E)-3-(4-Chlorophenyl)-2-(3-phosphonopropyl)propenoic acid, 9d
28

ACCEPTED MANUSCRIPT
White solid, 0.062 g, yield 62%. Rf (CHCl3/MeOH/AcOH, 7:2:1) = 0.35. 1H NMR (200 MHz,
CD3COOD) δ 7.74 (s, 1H), 7.41 (s, 4H), 2.67-2.48 (m, 2H), 1.89-1.58 (m, 4H). 13C NMR (50
MHz, CD3COOD) δ 173.4, 140.8, 135.4, 134.8, 133.6, 131.9, 129.7, 30.4, 29.2, 28.9, 28.8 (d,
31
JP-C = 135.5 Hz), 23.5. P NMR (81 MHz, CD3COOD) δ 38.68. HRMS m/z calculated for
C12H13ClO5P- [M-H]- 303.0195, found 303.0192. Elemental analysis: calculated (%) for
C12H14ClO5P: C: 47.31 H: 4.63, found: C: 47.43 H: 4.70.
(E)-3-(4-Methoxyphenyl)-2-(3-phosphonopropyl)propenoic acid, 9e
White solid, 0.035 g, yield 34%. Rf (CHCl3/MeOH/AcOH, 7:2:1) = 0.47. 1H NMR (200 MHz,
CD3COOD) δ 7.75 (s, 1H), 7.42 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 7.9 Hz, 2H), 3.81 (s, 3H),
2.71-2.54 (m, 2H), 1.87-1.64 (m, 4H). 13C NMR (50 MHz, CD3COOD) δ 173.6, 161.4, 139.4,
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132.6, 130.2, 128.5, 115.0, 55.6, 32.7, 30.3, 29.2, 29.1 (d, JP-C = 136.0 Hz), 23.6. P NMR
(81 MHz, CD3COOD) δ 38.68. HRMS m/z calculated for C13H16O6P- [M-H]- 299.0690, found
299.0693. Elemental analysis: calculated (%) for C13H17O5P: C: 52.00 H: 5.71, found: C:
52.21 H: 5.67.
(E)-3-Phenyl-2-(4-phosphonobutyl)propenoic acid, 10a
White solid, 0.049 g, yield 51% (E/Z, 91:9). Rf (CHCl3/MeOH/AcOH, 7:2:1) = 0.40. 1H NMR
(200 MHz, CD3COOD) δ 7.77 (s, 1H), 7.40 (s, 5H), 2.66-2.39 (m, 2H), 1.80-1.49 (m, 6H).
13C NMR (50 MHz, CD3COOD) δ 174.0, 141.8, 136.4, 133.4, 130.3, 129.5, 129.2, 31.2, 30.9,
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30.5, 28.6 (d, JP-C = 137.0 Hz), 27.7, 24.0. P NMR (81 MHz, CD3COOD) δ 39.84. HRMS
m/z calculated for C13H16O5P- [M-H]- 283.0741, found 283.0740. Elemental analysis:
calculated (%) for C13H17O5P: C: 54.93 H: 6.03, found: C: 54.80 H: 6.07.
(E)-3-(4-Methylphenyl)-2-(4-phosphonobutyl)propenoic acid, 10b
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