Ugi reaction on α-phosphorated ketimines for the synthesis of tetrasubstituted α-aminophosphonates and their applications as antiproliferative agents

Article abstract of DOI:10.3390/molecules26061654

An Ugi three-component reaction using preformed α-phosphorated N-tosyl ketimines with different isocyanides in the presence of a carboxylic acid affords tetrasubstituted α-aminophosphonates. Due to the high steric hindrance, the expected acylated amines u

Full text of DOI:10.3390/molecules26061654

molecules
Communication
Ugi Reaction on α-Phosphorated Ketimines for the Synthesis of
Tetrasubstituted α-Aminophosphonates and Their Applications
as Antiproliferative Agents
Adrián López-Francés , Xabier del Corte , Edorta Martínez de Marigorta , Francisco Palacios *
and Javier Vicario *
Departamento de Química Orgánica I, Centro de Investigación y Estudios Avanzados “Lucio Lascaray”,
Facultad de Farmacia, University of the Basque Country, UPV/EHU Paseo de la Universidad 7,
01006 Vitoria-Gasteiz, Spain; adrian.lopez@ehu.eus (A.L.-F.); xabier.delcorte@ehu.es (X.d.C.);
edorta.martinezdemarigorta@ehu.eus (E.M.d.M.)
* Correspondence: francisco.palacios@ehu.eus (F.P.); javier.vicario@ehu.eus (J.V.);
Tel.: +34-945-013-103 (F.P.); +34-945-013-087 (J.V.)
Abstract: An Ugi three-component reaction using preformed
different isocyanides in the presence of a carboxylic acid affords tetrasubstituted
Due to the high steric hindrance, the expected acylated amines undergo a spontaneous elimination of
the acyl group. The reaction is applicable to -aryl ketimines bearing a number of substituents and
several isocyanides. In addition, the densely substituted -aminophosphonate substrates showed
α
-phosphorated N-tosyl ketimines with
α
-aminophosphonates.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
α
α
Citation: López-Francés, A.; del
Corte, X.; Martínez de Marigorta, E.;
Palacios, F.; Vicario, J. Ugi Reaction
on α-Phosphorated Ketimines for the
Synthesis of Tetrasubstituted
α-Aminophosphonates and Their
Applications as Antiproliferative
Agents. Molecules 2021, 26, 1654.
https://doi.org/10.3390/
in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell line A549 (carcinomic
human alveolar basal epithelial cell).
Keywords: multicomponent synthesis; Ugi reaction; α-aminophosphonates; tetrasubstituted carbons;
antiproliferative effect
molecules26061654
1. Introduction
In the interdisciplinary research field of chemical biology and drug discovery, diversity-
oriented synthesis is an interesting model for the production of large chemical libraries of
small molecules, bearing multiple functional groups, in order to explore their influence
Academic Editors:
Ana Maria Gomez Neo and
Carlos Fernández Marcos
into the biological properties of those substrates [1–3]. At the heart of this concept, multi-
component reactions (MCRs) have become a mainstay of medicinal and organic chemistry
that allow the preparation of a broad spectrum of compounds with a reduced number of
synthetic steps [4,5]. In such synthetic procedures, three reactants or more are combined
Received: 25 February 2021
Accepted: 14 March 2021
Published: 16 March 2021
in the same pot to generate a new substrate, whose structure shows portions of all the
starting materials. The atom economy, efficiency, mild conditions and high convergence of
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
MCRs justify a central place in the toolbox of diversity-oriented synthesis [
numerous MCRs described along the last decades, the Ugi reaction [ ] has been verified
as one of the most important multicomponent processes for the synthesis of peptide-like
structures [ 10]. The Ugi reaction consists on a nucleophilic attack of an isonitrile to
an iminium ion , a salt composed of a carboxylic acid and an imine , which is often
generated in situ from a carbonyl derivative and an amine . Then, a second nucleophilic
attack of the carboxylate anion in the intermediate nitrilium species results in the forma-
. The reaction ends with an irreversible Mumm rearrangement of
-amido amide substrates in a very efficient manner (Scheme 1). Re-
6,7]. Among the
8
9,
6
5
4
3
1
2
7
Copyright:
© 2021 by the authors.
tion of acyl imidate
species , leading to
8
α
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
8
9
markably, the whole reaction is driven by the Mumm rearrangement since all other species
involved in the mechanism are in equilibrium. Due to its versatility, the Ugi reaction has
become increasingly practical in the synthesis of many active complex drugs and natural
products [11–13].
Molecules 2021, 26, 1654. https://doi.org/10.3390/molecules26061654
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1654
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Scheme 1. The accepted mechanism for an Ugi reaction.
On the other hand, the α-aminophosphonic acid framework enjoys significant atten-
tion in medicinal sciences, due to its unique ability to mimic the transition state of peptide
cleavage in an irreversible fashion, thus blocking very efficiently enzymes implicated in
proteolysis processes (Figure 1). For this reason,
and their phosphapeptides display an assorted biological activity, including anticancer
properties [14 18]. -Aminophosphonic acids can be considered as structural isosters of
-aminoacids, where the flat carboxylic acid group has been replaced by a phosphonic
acid group, and one of the most straightforward methods for the preparation of both com-
pounds, -aminoacids and -aminophosphonic acids, consists on the addition of carbon
nucleophiles to -iminoesters or -iminophosphonates, respectively [19 20]. While an Ugi
-iminoesters to afford -diamino acid derivatives is documented [21 22
-iminophosphonates as starting materials. In addition,
α-aminophosphonic acid derivatives
–
α
α
α
α
α
α
,
reaction using
α
α
,
α
,
]
no examples are described using
α
the use of ketones or ketimines as substrates in such reactions, in order to generate struc-
tures bearing tetrasubstituted carbons, entails additional obstacles, since the inherent steric
factors observed in these systems enhance the difficulty level in these synthetic method-
ologies [23]. In addition, the use of acyclic ketones typically requires preformation of the
imine intermediate in a separate step, and the yields of the Ugi are often modest [24–26].
Figure 1. Phosphonopeptides mimic the transition state for peptide cleavage.
In this context, during the course of our research on the addition of nucleophiles
to
α
-ketiminophosphonates, in the past, we achieved the synthesis of tetrasubtituted
27] using cyanide [28], organometallics [29] and nitromethane [30
α-aminophosphonates
[
]
as nucleophiles and, more recently, we have reported the first enantioselective Reformatsky
reaction using acyclic ketimines as substrates [31]. Continuing with our interest in the chem-
istry of organophosphorus compounds, we thought that α-ketiminophosphonates would
be excellent substrates in Ugi reactions for the generation of phosphorated peptide-like
structures bearing tetrasubstituted carbons. Due to the great occurrence of tetrasubstituted
carbons in natural products and drugs [32], the high affinity of
α-aminophosphonates to
proteolytic enzymes and the synthetic versatility of multicomponent reactions, a synthetic
protocol of an Ugi reaction using
organic and medicinal chemistry.
α-phosphorated ketimines would be of great value in

Molecules 2021, 26, 1654
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2. Results and Discussion
2.1. Chemistry
N-tosyl
α-ketiminophosphonates 10 can be synthesized by a formal oxidation of
trisubstituted aminophosphonates as reported in literature [28
,
31]. In our first experiment
we studied the Ugi reaction of N-tosyl ketimine 10a (R1 = Me, R2 = Ph) with phenyl
acetic acid 11 and cyclohexyl isocyanide 12a (R3 = Cy) under the typical reaction conditions
(Scheme 2). After stirring a mixture of the three compounds in CH2Cl2 at room temperature
for 1 h, NMR showed the complete disappearance of the starting materials and formation of
tetrasubstituted α-aminophosphonate 13a. Due to the insolubility of the starting materials,
the use of other environmentally friendly solvents led to the formation of substrate 13a in
lower yields and longer reaction times.
Scheme 2. Scope for the Ugi reaction of α-ketiminophosphonates 10.
With this result in hand, next we extended the Ugi protocol to different α-iminopho-
sphonates 10 and isocyanides 12 using phenylacetic acid 11 in CH2Cl2 (Scheme 2). First,
different isocyanides 12 were tested in the reaction using ketimine 10a (R1 = Me, R2 = Ph)
derived from dimethylphosphonate. The reactions proceed fast (1 h) and with good yields,
not only using cyclohexyl isocyanide 12a (R3 = Cy), but also with methyl isocyanoacetate
12b (R1 = CH2CO2Me) or benzyl isocyanide 12c (R3 = Bn) to afford
13b–c (Scheme 2).
α-aminophosphonates
Next, diethyl, dibenzyl and di-iso-propyl phosphonate substituted ketimines 10b–d
i
(R1 = Et, Bn, Pr,) were tested as electrophilic substrates with very good results but dif-
ferent reactivity. In the case of diethylphosphonates 13d
zylphosphonates 13g (R1 = Bn, R2 = Ph) the reactions proceed to full conversion after 6 h
and even longer reaction times of 14 h are needed for di-iso-propylphosphonates 13h
R1 = iPr, R2 = Ph) (Scheme 2). These differences in the reactivity related to the size of
–f
(R1 = Et, R2 = Ph), and diben-
–
j
(

Molecules 2021, 26, 1654
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the phosphonate substituents are in agreement with what has been observed in similar
reactions [28,30].
Then, the scope of the reaction was extended to the use of phosphorated ketimines
bearing substituted aromatic rings. Aromatic ketimines holding strong electron withdraw-
ing substituents such as a para-nitro group showed very good reactivity and aminophos-
phonate 13k was obtained in very good yield after 1 h at room temperature (Scheme 2).
The reaction is also fast using ketimines with halogenated aromatic groups. Several
halogen substituted aromatic ketimines were successfully used in the reaction, including
para-substituted aromatic rings containing bromine or chlorine to yield halogenated
α-
aminophosphonates in full conversion after 1 h (Scheme 2, 13l ). The reaction tolerates
–
m
also the presence of an ortho-fluor substituted aromatic ring in (Scheme 2, 13n) and even
the existence of a perfluorinated phenyl group (Scheme 2, 13o). Besides, when aromatic
ketimines substituted by electron donating groups were used as substrates, an increase in
the reaction times was observed. However,
full conversion after 14 h (Scheme 2).
α-aminophosphonates 13p–q were obtained in
Tetrasubstituted
1H, 31P, 19F and 13C NMR, IR spectra and high-resolution mass spectra (see Supplemen-
tary Materials for the detail). For example, 1H NMR spectrum
-aminophosphonate 13a
α-aminophosphonates 13 were characterized on the basis of their
α
presents the signals corresponding to the aliphatic cyclohexyl moiety with several chemical
shifts in the interval δH = 0.92–1.91 ppm for the five methylene groups and an additional
multiplet at δH = 3.77 ppm for the CH bonded to the nitrogen. The phosphonate moiety is
seen as two representative doublets at δH = 3.80 ppm (3JPH = 10.5 Hz) and δH = 3.99 ppm
(
3JPH = 10.7 Hz), typical for the diastereotopic methoxy groups at the phosphonate. The
presence of the tosyl group is evident from the chemical shift for its para-methyl substituent
at δH = 2.33 ppm, that appears as a singlet, and the two doublets at δH = 7.00 and 7.16 ppm
(3JHH = 8.3 Hz), corresponding to the four aromatic protons, that appear partially over-
lapped with the five protons of the phenyl substituent in the interval at δH = 6.99–7.25 ppm.
The sulfamide and amide NH protons appear as two doublets that interchange with D2O
at δH = 6.47 ppm (3JPH = 8.2 Hz) and δH = 6.76 ppm (3JHH = 6.4 Hz), respectively. Due
to the low interchange rate in such acidic protons, the signal corresponding to the NH of
the sulfamide moiety is coupled with the magnetically active phosphorus atom, while the
amide NH is coupled with the neighboring CH of the cyclohexyl group.
In addition, in the 13C NMR spectrum of
α-aminophosphonate 13a, the cyclohexyl
group can be detected by the chemical shift at δC = 49.8 ppm, corresponding to its methyne
group, bonded to the nitrogen atom and, due to the stereogenic center present in the
structure, the other five methylene carbons show five different signals at δC = 24.5, 24.6, 25.4,
32.1 and 32.3 ppm. Here, again, the two diastereotopic methoxy groups at the phosphonate
moiety are seen as two doublets at δH = 55.8 ppm (2JPC = 8.2 Hz) and δC = 55.2 ppm
(2JPC = 7.5 Hz). The most characteristic chemical shift of -aminophosphonate 13a in 13
α C
NMR is certainly the doublet corresponding to the quaternary carbon directly bonded
to the phosphonate that appears at δC = 68.5 ppm and presents a strong coupling with
the phosphorus atom (1JPC = 157.2 Hz). The presence of the tosyl group is here deduced
from the chemical shift corresponding to its para-methyl substituent at δC = 21.6 ppm and
the aromatic carbons with two signals at δC = 126.5 and 129.1 ppm for each of the two
couples of the equivalent CH carbons of the aromatic ring, as well as another two signals
for the two quaternary carbons at δC = 142.4 and 139.2 ppm, the latter seen as a doublet
due to the coupling with the phosphorus atom (4JPC = 1.6 Hz). In the aromatic region it
also appears the chemical shifts of the carbons corresponding to the phenyl ring, with
the signals corresponding to the two pairs of equivalent CH carbons at δC = 127.9 and
130.2 ppm, the second as a doublet coupled with the phosphorus atom (3JPC = 8.3 Hz). The
fifth aromatic CH appears at δC = 128.7 ppm and the quaternary carbon as a doublet at
δC = 131.9 (2JPC = 1.8 Hz). Surprisingly, the amide carbonyl group does not show coupling
with the phosphorus atom and the signal appears as a singlet at δC = 166.1 ppm.

Molecules 2021, 26, 1654
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The most relevant absorptions observed in IR spectrum correspond to the amide,
sulfamide and phosphonate moieties. The stretching vibration of amide and sulfamide NH
groups can be observed at
bands are observed at
=1678 and 1256 cm−1, correspond to the vibration of amide C=O
and phosphonate P=O bonds. Finally, the spectrum shows two characteristic absorptions
= 1333 and 1164 cm−1 that correspond to the asymmetric and symmetric stretching
ν
= 3426 and 3333 cm−1, respectively. In addition, two strong
ν
ν
vibration of the sulfonyl group.
Regarding the mechanism of the reaction, we theorized that compounds 13 might
be formed by a typical three-component Ugi reaction that leads to the formation of the
predicted phosphorated
acyl group, due to the high steric hindrance present in the intermediate 15
In fact, the same behavior has been observed in the acylcyanation reaction of N-tosyl
ketimines 10
R2 = Ar, PG = Ts) with pyruvonitrile [28]. In our attempts to detect the
acylated intermediate 15, different carboxylic acids were used in the reaction, but
α
-amido amide 15, followed by a spontaneous cleavage of the
(
Scheme 3).
(
α
-
aminophosphonate 13a was obtained in all cases, even when acetic, trifluoroacetic or
benzoic acid were used as reagents. Nevertheless, the reaction does not proceed in the
absence of a carboxylic acid, which at least indicates that the formation of iminium species
is crucial prior to the nucleophilic attack of isocyanide.
Scheme 3. Ugi reaction of N-tosyl ketimines 10 and 14, phenyl acetic acid 11 and isocyanides 12.
In order to check if the Mumm rearrangement was indeed taking place, next we used
N-trityl aldimine 14 (R2 = H, PG = CPh3) [33] as the electrophile substrate, in the presence
of phenylacetic acid 11 and cyclohexyl isocyanide 12a (R3 = Cy) (Scheme 3). Due to the
utilization of an aldimine derived electrophile in the reaction, a less hindered structure is
expected in the Ugi adduct, which may result in the isolation of species 15. However, in
this case, trisubstituted
α-aminophosphonate 16 was obtained in full conversion, where,
the formation of -amido amide 15 is followed by a spontaneous cleavage of the bulky
α
trityl protecting group (Scheme 3).
Although this last experiment supports an Ugi three-component mechanism of the
process, still we were skeptical about the real role of the carboxylic acid in the system. It
is true that, considering the accepted mechanism for the Ugi reaction, only through the
irreversible Mumm rearrangement all the equilibrium in the process can be displaced
to the final products. But yet, it might be vaguely possible that, in the case of our ke-
timines 10, a simple addition of isocyanide to iminium species could afford tetrasubstituted
α-aminophosphonate 13a after an irreversible hydrolysis of the nitrilium intermediate, due
to the presence of traces of water in the solvent. Then the key question to be addressed is:
is the third reactant of the multicomponent reaction a carboxylic acid or is it just water?
This matter could be resolved in view of the fact that the isolation of intermediate
15 was achieved when para-fluorophenyl or para-trifluoromethylphenyl substituted
α-
phosphorated ketimines 10l (R = CF3, F) were used as the electrophile unit in the Ugi
,m

Molecules 2021, 26, 1654
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reaction. Using phenylacetic acid 11 and cyclohexyl isocyanide 12a, phosphorated
α
-
amido amides 15a
,b
were obtained, without the elimination of the amide group (Scheme 4).
Although substrate 15b proved to be very stable, trifluoromethyl substituted α-amido
amide 15a underwent spontaneous hydrolysis of the amide under the air moisture to yield
tetrasubstituted α-aminophosphonate 13r.
Scheme 4. Ugi reaction of ketimines 10l,m.
NMR properties of phosphorated α-amido amides 15 were very similar to the parent
substrates 13 except for some significant differences. In the case of substrate 15b, the
presence of benzylamide group was evident in 13C NMR by the existence of the chemical
shifts for two carbonyl groups at δC = 176.4 and 165.3 ppm and a methylene carbon at
δC = 45.7 ppm (DEPT). Key features for this compound in 1H NMR spectrum are mainly
the two diastereotopic protons of the benzyl group that appear as doublets at δC = 3.92 and
4.16 ppm with a strong geminal coupling constant 2JHH = 17.1 Hz. It is also noteworthy
the presence of an atypical doublet for two equivalent aromatic protons at δC = 8.26 ppm
(
3JHH = 7.9 Hz) that corresponds either to the benzyl or the tosyl moiety that appears
especially deshielded, which is probably originated by the proximity of both aromatic rings
due to the steric crowding present in the structure.
In order to shed more light on this issue, we set up an additional experiment where
the three-component reaction was performed using of N-tosyl ketimine 10a, thioacetic
acid 17 and cyclohexyl isocyanide 12a in CDCl3. However, after 1h at room temperature a
complex mixture was observed in the reaction vessel. We hypothesized that the high steric
hindrance due to the presence of the tetrasubstituted carbon together with the higher Van
der Waals radius of the sulfur atom versus the oxygen (180 pm vs. 152 pm) could be the
reason of such different behavior.
For this reason, next we tried the Ugi reaction using a less sterically demanding
isocyanide such as methyl isocyanoacetate 12b (Scheme 5). In this case, formation of
thioamide 18 was observed in full conversion. The presence of a sulfur atom in the structure
confirms unambiguously the Ugi mechanism of our reaction through the formation of
iminium species 19 from
α-ketiminophosphonate 10a and thioacid 17, followed by a
nucleophilic attack of isocyanide 12b. Then, a second nucleophilic attack of thiocarboxylate
anion in the intermediate nitrilium species 20 results in the formation of acyl thioimidate 21
.
To complete the Ugi sequence, the acyl transfer from thioimidate 21 to the adjacent nitrogen
atom yields irreversibly phosphorated
α-amido amide 22 that, due to the high steric
hindrance owing to the presence of the tetrasubstituted carbon, undergoes a spontaneous
cleavage of the acyl group that affords finally tetrasubstituted α-aminophosphonate 18.

Molecules 2021, 26, 1654
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O
Ts
N
Ph
S
O
CDCl3 (MeO)2P
H
N
+
+
N
CO2Me
rt, 1h
Ts
CO2Me
(MeO)2P
O
Ph
C
N
H
HS
17
10a
12b
18
O
O
Ph
S
(MeO)2P
Ts
S
H
N
HN
CO2Me
Mumm
Ts
N
CO2Me
+
N
C
(MeO)2P
Ph
O
O
19
22
12b
rearrangement
O
O
Ph
S
O
(MeO)2P
Ph
N
CO2Me
(MeO)2P
Ts
S
N
Ts
H
N
N
CO2Me
H
O
20
21
Scheme 5. Ugi reaction of N-tosyl ketimine 10a, thioacetic acid 17 and methyl isocyanoacetate 12b.
Nevertheless, attempts to isolate compound 18 failed due to its decomposition during
the workup, but the identity of thioamide 22 was confirmed by NMR of the crude reaction.
31P NMR showed the disappearance of the starting imine (δP = 6.6 ppm) and the formation
1
of a major compound with a chemical shift at δP = 18.7 ppm. On the other hand, H
NMR showed two clear doublets at δH = 3.90 ppm (3JPH = 10.7 Hz) and δH = 3.79 ppm
(
3JPH = 10.8 Hz), typical for the diastereotopic methoxy groups at the phosphonate, that
suggest the formation of a stereogenic carbon close to the phosphorus atom and a broad
triplet that interchanges with D2O, at δH = 8.63 ppm (1JNH = 4.0 Hz), that may correspond
to the NH of thioamide group, where the proton is coupled with the quadrupolar nucleus
of 14N. More importantly, 13C NMR shows a doublet for the quaternary C-P (DEPT) at
δC = 58.6 ppm (1JPH = 167.2 Hz), and the characteristic chemical shift for the C=S group of
thioamides at δC = 199.2 ppm. A similar result was obtained using thiobenzoic acid instead
of thioacetic acid.
Additionally, the hydrolysis the phosphonate group to its phosphonic acid derivative
23 can be performed under mild conditions in chloroform by the treatment of 13b with
trimethylsilyl bromide at room temperature. The subsequent aqueous workup yields
α-aminophosphonic acid 23 in almost quantitative yield (Scheme 6).
Scheme 6. Hydrolysis of phosphonate ester 13b.
2.2. Biological Results
In vitro cytotoxicity of tetrasubstituted α-aminophosphonate derivatives 13, 15 and 23
was evaluated by testing their antiproliferative activities against A549 cell line (carcinomic
human alveolar basal epithelial cell). Cell counting kit (CCK-8) assay was used for the
evaluation of growth inhibition. Moreover, nonmalignant MRC5 lung fibroblasts were
tested for studying selective toxicity [34] and chemotherapeutic doxorubicin is used as
reference value. The cell proliferation inhibitory activity is shown as IC50 values (Table 1).

Molecules 2021, 26, 1654
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Table 1. Antiproliferative activity of α-aminophosphonate derivatives 13, 15 and 23.
IC50 (µM)
IC50 (µM)
MRC5
Entry
Entry
Cmpd.
Cmpd.
A549
>50
MRC5
n.d.
A549
1
2
3
4
5
13a
13d
13g
13h
13k
6
7
13n
13p
13r
15a
23
>50
n.d.
>50
>50
>50
n.d.
>50
n.d.
14.56 ± 2.53
>50
16.136 ± 1.14
19.72 ± 3.70
>50
>50
8
>50
9
28.76 ± 3.20
>50
n.d.
10
In a preliminary study, we tested the cytotoxicity of simple phenyl substituted
aminophosphonates 13a as lead compounds. Although no grown inhibition activity
was observed for dimethyl and diethylphosphonates 13a (Table 1, Entries 1–2), diben-
zylphosphonate 13g showed some cytotoxicity against A549 cell line with an IC50 value of
16.46 1.49 M and, interestingly, very good selectivity was also obtained towards MRC5
nonmalignant cell line (Table 1, Entry 3). Besides, bulkier di-iso-propylphosphonate 13h
α-
–
j
,f
±
µ
,
derived from cyclohexyl isocyanide, presented an IC50 value of 19.72
Entry 4).
±
3.70 µM (Table 1,
Then we studied the introduction of substituents at the aromatic ring of tetrasubsti-
tuted aminophosphonates 13. Scarce cytotoxic effect was found for para-nitrophenyl substi-
tuted substrate 13k, bearing an electron poor aromatic group (Table 1, Entry 5). Although
the effect of the introduction of fluorine atoms in the structure of organic compounds is
rather difficult to predict, very often it leads to increased activities [35
next we tested the in vitro cytotoxicity of fluorine containing -aminophosphonates 13n
However, ortho-fluorophenyl and para-trifluoromethylphenyl substituted substrates 13n
presented IC50 values higher than 50 M (Table 1, Entries 6, 8). Interestingly, thioether
derived -aminophosphonate 13p, showed a considerable antiproliferative activity with an
IC50 value of 14.56 2.53 M and a very good selectivity towards MRC5 cell line (Table 1,
Entry 7). Phosphorated -amido amide 15a bearing a para-trifluomethylphenyl substituent
showed better toxicity than its parent compound 13r with an IC50 value of 28.76 3.20
–37]. For this reason,
α
.
,r
µ
α
±
µ
α
±
µM
and a good selectivity towards nonmalignant cells (Table 1, Entry 8 vs. Entry 9). Finally
phosphonic acid derivative 23 did not provide any toxicity against A549 cell line (Table 1,
Entry 10).
3. Materials and Methods
3.1. Chemistry
3.1.1. General Experimental Information
Solvents for extraction and chromatography were technical grade. All solvents used
in reactions were freshly distilled from appropriate drying agents before use. All other
reagents were recrystallized or distilled as necessary. All reactions were performed under
an atmosphere of dry nitrogen. Analytical TLC was performed with silica gel 60 F254
1
plates. Visualization was accomplished by UV light. H, 13C, 31P and 19F-NMR spectra
were recorded on a Varian Unity Plus (Varian Inc, NMR Systems, Palo Alto, CA, USA)
(at 300 MHz, 75 MHz, 120 MHz and 282 MHz respectively) and on a Bruker Avance 400
(Bruker BioSpin GmbH, Rheinstetten, Germany) (at 400 MHz for 1H, and 100 MHz for
13C). Chemical shifts (
for H and
δ
) are reported in ppm relative to residual CHCl3 (
δ
= 7.26 ppm
δ
= 77.16 ppm for 13C NMR) and using phosphoric acid (50%) as external
1
reference (δ = 0.0 ppm) for 31P NMR spectra. Coupling constants (J) are reported in Hertz.
Data for 1H NMR spectra are reported as follows: chemical shift, multiplicity, coupling
constant, integration. Multiplicity abbreviations are as follows: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet). 13C NMR peak assignments were supported by
distortionless enhanced polarization transfer (DEPT). High resolution mass spectra (HRMS)
were obtained by positive-ion electrospray ionization (ESI). Data are reported in the form

Molecules 2021, 26, 1654
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m/z (intensity relative to base = 100). Infrared spectra (IR) were taken in a Nicolet iS10
Thermo Scientific spectrometer (Thermo Scientific Inc., Waltham, Massachusetts, MA, USA)
as neat solids. Peaks are reported in cm−1
.
3.1.2. Compounds Purity Analysis
All synthesized compounds were analyzed by HPLC to determine their purity. The
analyses were performed on Agilent 1260 infinity HPLC system (Agilent, Santa Clara,
CA, USA) using a CHIRALPAK
Technologies, Illkirch Cedex, France) at room temperature. All the tested compounds were
dissolved in dichloromethane, and 5 L of the sample was loaded onto the column. Ethanol
®IA column (5
µm, 0.54 cm ø
×
25 cm, Daicel Chiral
µ
and heptane were used as the mobile phase, and the flow rate was set at 1.0 mL/min. The
maximal absorbance at the range of 190–400 nm was used as the detection wavelength. The
purity of all the tested
α-aminophosphonate derivatives 13, 15 and α-aminophosphonaic
acid 23 is >95%, which meets the purity requirement by the Journal.
3.1.3. Experimental Procedures and Characterization Data for Compounds 13, 15, 16
and 23
General Procedure for the Synthesis N-Tosyl α-Iminophosphonates 10
Following a literature procedure, [28
tuted N-tosyl -aminophosphonate (10 mmol) in CH2Cl2 (30 mL) was added trichloroiso-
cyanuric acid (6.97 g, 30 mmol). The resulting suspension was stirred at 0 ◦C until disap-
pearance of the starting N-tosyl
-aminophosphonate, as monitored by 31P NMR (14 to
48 h). The solid residue was eliminated by filtration to afford a clear solution of interme-
,31] to a solution of the corresponding tetrasubsti-
α
α
diate N-chloro α-aminophosphonate and then, poly(4-vinylpyridine) (3.0 g), previously
dried at 100 ◦C overnight, was added. The resulting suspension was stirred under reflux
overnight and the reaction was then filtered and concentrated under reduced pressure. The
resulting yellow oily crude was purified by crystallization from diethyl ether.
General Procedure for the Synthesis N-Trityl α-Iminophosphonate 14
Following a literature procedure, [33] N-bromosuccinimide (178 mg, 1 mmol) was
added on a solution of dimethyl ((tritylamino)methyl)phosphonate (457 mg, 1 mmol) in
CCl4 (3 mL). The mixture was stirred in quartz flask under UV light until the disappearance
of starting α
-aminophosphonate as monitored by 31P-NMR (δH 30.9 to 10.1 ppm). The re-
sulting suspension was filtered under inert atmosphere to afford a clear solution of dimethyl
(E)-((tritylimino)methyl)phosphonate that can be used without any further workup.
General Procedure for the Ugi Reaction of α-Phosphorated Ketimines 10 and 14
A mixture of α-iminophosphonate 10 or 14 (1 mmol), phenylacetic acid (11, 136 mg,
1 mmol) and isocyanide 12 (1.1 mmol) in ichloromethane (3 mL) was stirred at room
temperature until disappearance of the starting iminophosphonate 10 as monitored by 31P-
NMR. The reaction was concentrated under vacuum and the resulting crude residue was
purified by crystallization (Dichomethane/Hexanes 1:3), yielding
13 15 or 16. In some cases, a purification by column chromatography was necessary as
detailed for each compound.
α-aminophosphonates
,
Dimethyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate
13a). The general procedure was followed, using dimethyl (E)-(phenyl(tosylimino)methyl)
phosphonate (10a, 367 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and cyclo-
(
hexyl isocyanide (12a, 136
µ
L, 1.1 mmol) to afford 420 mg (85%) of 13a after 1 h as white
crystals after crystallization (Dichloromethane/Hexanes 1:3). M.p.: 206–208 ◦C. H-NMR
1
(400 MHz, CDCl3)
δ
7.25–7.15 (m, 3H, 3
×
CHAr), 7.16 (d, 3JHH = 8.3 Hz, 2H, 2 × CHAr),
7.05 (d, 3JHH = 8.5 Hz, 2H, 2
×
CHAr), 7.00 (d, 3JHH = 8.3 Hz, 2H, 2 × CHAr), 6.76 (d, 3JHH
=
6.4 Hz, 1H, NHCO), 6.47 (d, 3JPH = 8.2 Hz, 1H, NHTs), 3.99 (d, 3JPH = 10.7 Hz, 3H, OCH3),
3.80 (d, 3JPH = 10.5 Hz, 3H, OCH3), 3.77 (m, 1H, CHCy), 2.33 (s, 3H, CH3Ts), 1.91–1.44 (m,
4H, CH2Cy), 1.39–0.92 (m, 6H, 3
×
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
δ 166.1

Molecules 2021, 26, 1654
10 of 19
(C=O), 142.5 (CquatTs), 139.2 (d, 4JPC = 1.6 Hz, CquatTs), 131.9 (d, 2JPC = 1.8 Hz, Cquat Ph),
130.2 (d, 3JPC = 8.3 Hz, 2
×
CHAr Ph), 129.1 (2
×
CHAr), 128.7 (CHAr), 127.9 (2 × CHAr),
1
2
126.5 (2
×
CHAr), 68.5 (d, JPC = 157.2 Hz, Cquat-P), 55.8 (d, JPC = 8.2 Hz, OCH3), 55.2
(d, 2JPC = 7.5 Hz, OCH3), 49.8 (CHCy), 32.3 (CH2Cy), 32.1 (CH2Cy), 25.4 (CH2Cy), 24.6
(CH2Cy), 24.5 (CH2Cy), 21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ
20.5 ppm. FTIR
(neat)
ν
: ν = 3426 (N-H st) 3333 (N-H st) 1678 (C=O st) 1256 (P=O st) 1333 (S=O st sym)
max
1164 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C23H32N2O6PS 495.1719,
Found 495.1718.
Methyl (2-(dimethoxyphosphoryl)-2-((4-methylphenyl)sulfonamido)-2-phenylacetyl)glycinate (13b).
The general procedure was applied starting from dimethyl (E)-(phenyl(tosylimino)methyl)-
phosphonate (10a, 367 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and methyl
2-isocyanoacetate (12b, 100 µL, 1.1 mmol) to afford 378 mg (78%) of 13b after 1 h as a white
solid after a chromatography column (Hexanes/AcOEt 1:1), followed by crystallization
◦
(Dichloromethane/Hexanes 1:3). M.p.: 130–132 C. 1H-NMR (400 MHz, CDCl3)
δ
7.37–7.30
3
(m, 2H, 2
7.11–6.95 (m, 4H, 4
CH2), 3.91 (d, 3JPH = 10.8 Hz, 3H, POCH3), 3.78 (d, 3JPH = 10.6 Hz, 3H, POCH3), 3.68 (s,
3H, COCH3), 2.33 (s, 3H, CH3Ts) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
169.2 (C=O),
×
CHAr), 7.23–7.16 (m, 3H, 3
×
CHAr), 7.15 (d, JHH = 5.5 Hz, 1H, NHCO),
×
CHAr), 6.64 (d, 3JPH = 6.8 Hz, 1H, NHTs), 4.00 (d, 3JHH = 5.5 Hz, 2H,
δ
167.8 (C=O), 142.7 (CquatTs), 139.0 (CquatTs), 131.5 (CquatPh), 130.2 (d, 3JPC = 7.8 Hz, 2
×
CHArPh), 129.1 (2
×
CHAr), 128.9 (CHAr), 128.0 (2
×
CHAr), 126.7 (2 × CHAr), 69.0 (d, 1JPC
= 155.3 Hz, Cquat-P), 55.6 (d, 2JPC = 8.0 Hz, POCH3), 55.4 (d, 2JPC = 7.5 Hz, POCH3), 52.5
(COCH3), 42.2 (CH2), 21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ 18.9 ppm. FTIR
(neat)
ν
: ν = 3437 (N-H st) 3323 (N-H st) 1675 (C=O st) 1266 (P=O st) 1338 (S=O st sym)
max
1165 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M+H]+ calcd for C20H26N2O8PS 485.1147,
Found 485.1149.
Dimethyl (2-(benzylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate (13c).
The general procedure was applied starting from dimethyl (E)-(phenyl(tosylimino)methyl)
phosphonate (10a, 367 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and (iso-
cyanomethyl) benzene (12c, 134 µL, 1.1 mmol) to afford 402 mg (80%) of 13c after 1 h as
white crystals after a chromatography column (Hexanes/AcOEt 7:3), followed by crystal-
lization (Dichloromethane/Hexanes 1:3). M.p.: 150–152 ◦C. 1H-NMR (400 MHz, CDCl3)
δ
7.32–7.09 (m, 11H, 11
NHCO), 6.74 (d, 3JPH = 6.6 Hz, 1H, NHTs), 4.51 (dd, 2JHH = 14.9 Hz, 3JHH = 6.2 Hz, 1H,
ACHB), 4.35 (dd, 2JHH = 14.9 Hz, 3JHH = 5.7 Hz, 1H, CHAC B), 3.91 (d, 3JPH = 10.8 Hz
3H, OCH3), 3.67 (d, 3JPH = 10.6 Hz, 3H, OCH3), 2.34 (s, 3H, CH3Ts) ppm. 13C-NMR {1H}
(101 MHz, CDCl3) 167.2 (C=O), 142.7 (CquatTs), 139.2 (CquatTs), 137.2 (CquatPh), 132.0
×
CHAr), 7.08–6.99 (m, 3H, 3
×
CHAr), 6.97 (d, 3JHH = 6.3 Hz, 1H,
C
H
H
,
δ
3
(CquatPh), 130.1 (d, JPC = 8.1 Hz, 2
×
CHArPh), 129.1 (2
×
CHAr), 128.8 (CHAr), 128.7
(
2 × CHAr), 128.1 (2 CHAr), 127.8 (2
×
×
CHAr), 127.7 (CHAr), 126.6 (2
×
CHAr), 68.8 (d,
1JPC = 156.0 Hz, Cquat-P), 55.6 (d, JPC = 8.0 Hz, OCH3), 55.1 (d, JPC = 7.6 Hz, OCH3),
2
2
44.7 (CH2), 21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ 19.3 ppm. FTIR (neat)
ν
max: ν = 3428 (N-H st) 3341 (N-H st) 1675 (C=O st) 1255 (P=O st) 1332 (S=O st sym) 1160
(S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H28N2O6PS 503.1406,
Found 503.1411.
Diethyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate
(
13d). The general procedure was applied starting from diethyl (E)-(phenyl(tosylimino)methyl)
phosphonate (10b, 395 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and cyclohexyl
isocyanide (12a, 136 L, 1.1 mmol) to afford 434 mg (83%) of 13d after 6 h as white
crystals after a chromatography column (Hexanes/AcOEt 8:2), followed by crystallization
µ
(Dichloromethane/Hexanes 1:3). M.p.: 122–124 C. 1H NMR (400 MHz, CDCl3)
(m, 5H, 5 CHAr), 7.05–6.95 (m, 4H, 4
CHAr), 6.73 (d, 3JHH = 6.4 Hz, 1H, NHCO), 6.54
(d, 3JPH = 8.1 Hz, 1H, NHTs), 4.47–4.28 (m, 2H, OCH2), 4.27–4.06 (m, 2H, OCH2), 3.76 (m,
1H, CHCy), 2.33 (s, 3H, CH3Ts), 1.91–1.47 (m, 6H, 3
CH2Cy), 1.40 (t, 3JHH = 7.0 Hz, 3H,
H3CH2), 1.27 (t, 3JHH = 7.0 Hz, 3H, CH3CH2) 1.30–0.94 (m, 4H, 2 CH2Cy) ppm. 13C
δ 7.28–7.10
◦
×
×
×
C
×

Molecules 2021, 26, 1654
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NMR {1H} (101 MHz, CDCl3)
130.4 (d, 3JPC = 7.8 Hz, 2
δ
166.4 (C=O), 142.4 (CquatTs), 139.3 (CquatTs), 132.1 (CquatPh),
×
CHArPh), 129.0 (2
×
CHAr), 128.6 (CHAr), 127.8 (2
×
CHAr),
1
2
126.6 (2
×
CHAr), 68.6 (d, JPC = 156.2 Hz, Cquat-P), 65.5 (d, JPC = 8.3 Hz, OCH2), 65.0
(d, 2JPC = 7.6 Hz, OCH2), 49.7 (CHCy), 32.2 (CH2Cy), 32.2 (CH2Cy), 25.4 (CH2Cy), 24.6
(CH2Cy), 24.5 (CH2Cy), 21.5 (CH3Ts), 16.6 (d, 3JPC = 5.7 Hz, H3CH2), 16.4 (d, 3JPC = 5.8
Hz, 17.1 ppm. FTIR (neat)
H3CH2) ppm. 31P-NMR (121 MHz, CDCl3)
C
C
δ
ν
:
ν
= 34−321
max
(N-H st) 3340 (N-H st) 1672 (C=O st) 1253 (P=O st) 1338 (S=O st sym) 1165 (S=O st as) cm
.
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C25H36N2O6PS 523.2032, Found 523.2034.
Methyl (2-(diethoxyphosphoryl)-2-((4-methylphenyl)sulfonamido)-2-phenylacetyl)glycinate (13e).
The general procedure was applied starting from diethyl (E)-(phenyl(tosylimino)methyl) phos-
phonate (10b, 395 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and methyl
2-isocyanoacetate (12b, 100
µ
L, 1.1 mmol) to afford 453 mg (88%) of 13e after 6 h◦as
white crystals after crystallization (Dichloromethane/Hexanes 1:3). M.p.: 147–149 C.
1H-NMR (400 MHz, CDCl3)
+ NHCO), 7.06–6.92 (m, 4H, 4
δ
7.36–7.29 (m, 2H, 2
×
CHAr), 7.21–7.11 (m, 4H, 3
×
CHAr
3
×
CHAr), 6.61 (d, JPH3 = 6.9 Hz, 1H, NHTs), 4.34–4.19
5
(m, 2H, OCH2), 4.18–4.04 (m, 2H, OCH2), 3.96 (dd, JHH = 5.5 Hz, JPH = 2.7 Hz, 2H,
3
NCH2), 3.65 (s, 3H, OCH3), 2.29 (s, 3H, CH3Ts), 1.30 (t, JHH = 6.9 Hz, 3H, CH3CH2),
3
1.19 (t, JHH = 7.0 Hz, 3H, CH3CH2) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
δ 169.2
4
(C=O), 167.9 (C=O), 142.6 (CquatTs), 139.09 (d, JPC = 1.4 Hz, CquatTs), 131.6 (CquatPh),
3
130.4 (d, JPC = 7.6 Hz
,
2 × CHArPh), 129.0 (2
×
CHAr), 128.7 (CHAr), 127.8 (2
×
CHAr),
1
2
126.7 (2 × CHAr), 69.1 (d, JPC = 153.9 Hz, Cquat-P), 65.4 (d, JPC = 8.0 Hz, OCH2), 65.1
(d, 2JPC = 7.5 Hz, OCH2), 52.4 (OCH3), 42.2 (NCH2), 21.5 (CH3Ts), 16.5 (d, 3JPC = 5.7 Hz,
C
H3CH2), 16.4 (d, 3JPC = 5.6 Hz H3CH2) ppm. 31P-NMR (121 MHz, CDCl3)
, C δ 16.7 ppm.
FTIR (neat)
ν
: ν = 3425 (N-H st) 3331 (N-H st) 1678 (C=O st) 1256 (P=O st) 1332 (S=O
max
st sym) 1165 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C22H30N2O8PS
513.1460, Found 513.1462.
Diethyl (2-(benzylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate (13f).
The general procedure was applied starting from diethyl (E)-(phenyl(tosylimino)methyl) phos-
phonate (10b, 395 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and (isocyanomethyl)
benzene (12c, 134
µL, 1.1 mmol) to afford 411 mg (78%) of 13f after 6 h as white crystals
◦
1
after crystallization (Dichloromethane/Hexanes 1:3). M.p.: 140–142 C. H-NMR (400
MHz, CDCl3)
δ
7.34–7.14 (m, 10H, 10
×
CHAr), 7.11–6.97 (m, 5H, 4
×
CHAr + NHCO),
6.69 (d, 3JPH = 5.7 Hz, 1H, NHTs), 4.52 (dd, 2JHH = 14.9, 3JHH = 6.2 Hz, 1H, NCHACHB),
4.44–4.22 (m, 3H, NCHACHB + OCH2), 4.17–3.95 (m, 2H, OCH2), 2.35 (s, 3H, CH3Ts), 1.35
(t, 3JHH = 7.0 Hz, 3H, CH3CH2), 1.17 (t, 3JHH = 7.0 Hz, 3H, CH3CH2) ppm. 13C-NMR {1H}
(101 MHz, CDCl3)
δ 167.5 (C=O), 142.5 (CquatTs), 139.3 (CquatTs), 137.2 (CquatPh), 132.1
(CquatPh), 130.2 (d, 3JPC = 7.7 Hz, 2 × CHArPh), 129.1 (2 × CHAr), 128.7 (2 × CHAr), 128.6
(CHAr), 128.0 (2
×
CHAr), 127.8 (2
×
CHAr), 127.7 (CHAr), 126.6 (2
×
CHAr), 68.9 (d, 1JPC
2
2
= 155.0 Hz, Cquat-P), 65.5 (d, JPC = 8.2 Hz, OCH2), 65.0 (d, JPC = 7.5 Hz, OCH2), 44.7
(NCH2), 21.6 (CH3Ts), 16.5 (d, 3JPC = 5.7 Hz,
C
H3CH2), 16.3 d, 3JPC = 5.7 Hz,
C
H3CH2)
: ν = 3433 (N-H st) 3342
max
ppm. 31P-NMR (121 MHz, CDCl3)
δ
16.9 ppm. FTIR (neat)
ν
(N-H st) 1679 (C=O st) 1257 (P=O st) 1330 (S=O st sym) 1162 (S=O st as) cm−1. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C26H32N2O6PS 531.1719, Found 531.1728.
Dibenzyl (2-(benzylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate (13g).
The general procedure was applied starting from dibenzyl (E)-(phenyl(tosylimino)methyl) phos-
phonate (10c, 520 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and cyclohexyl
isocyanide (12a, 136 µL, 1.1 mmol) to afford 583 mg (90%) of 13g after 6 h as white crys-
tals after a chromatography column (Hexanes/AcOEt 8:2), followed by crystallization
◦
(Ether/Pentane 1:3). M.p.: 80–82 C. 1H-NMR (400 MHz, CDCl3)
δ
7.44–7.25 (m, 10H, 10
×
×
CHAr), 7.24–7.13 (m, 5H, 5
= 8.1 Hz, 2H, 2
CHAr), 6.82 (d, 3JHH = 6.6 Hz, 1H, NHCO), 6.44 (d, 3JPH = 8.0 Hz, 1H,
NHTs), 5.31–5.09 (m, 2H, OCH2), 5.06–4.91 (m, 2H, OCH2), 3.77–3.63 (m, 1H, CHCy), 2.33
(s, 3H, CH3Ts), 1.80–0.81 (m, 10H, 5
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
×
CHAr), 7.04 (t, 3JHH = 7.7 Hz, 2H, 2
CHAr), 6.96 (d, 3JHH
×
×
δ

Molecules 2021, 26, 1654
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166.1 (C=O), 142.5 (CquatTs), 139.2 (CquatTs), 135.9 (d, 3JPC = 5.2 Hz, CquatBn), 135.7 (d, 3JPC
= 6.0 Hz, CquatBn), 131.9 (CquatPh), 129.1 (2
×
CHAr), 128.8–128.5 (m, 9
×
CHAr), 128.3 (2
×
CHAr), 128.2 (2
×
CHAr), 127.9 (2
×
CHAr), 126.6 (2
×
CHAr), 70.6 (d, 2JPC = 8.0 Hz, OCH2),
70.1 (d, 2JPC = 7.5 Hz, OCH2), 68.9 (d, 1JPC = 156.4 Hz, Cquat-P), 49.8 (CHCy), 32.1 (CH2Cy),
32.0 (CH2Cy), 25.4 (CH2Cy), 24.5 (CH2Cy), 24.5 (CH2Cy), 21.6 (CH3Ts) ppm. 31P-NMR
(121 MHz, CDCl3)
δ
(ppm): 17.7 ppm. FTIR (neat)
ν
: ν = 3417 (N-H st) 3324 (N-H st)
max
1677 (C=O st) 1259 (P=O st) 1336 (S=O st sym) 1162 (S=O st as) cm−1. HRMS (ESI-TOF)
m/z: [M + H]+ calcd for C35H40N2O6PS 647.2346, Found 647.2348.
Di-iso-propyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate
(
13h). The general procedure was applied starting from di-iso-propyl (E)-(phenyl(tosylimi-
no)methyl) phosphonate (10d, 423 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol)
and cyclohexyl isocyanide (12a, 136 L, 1.1 mmol) to afford 474 mg (86%) of 13h after
µ
14 h as white crystals after a chromatography column (Hexanes/AcOEt 8:2), followed by
crystallization (Dichloromethane/Hexanes 1:3). M.p.: 141–143 ◦C. 1H-NMR (400 MHz,
CDCl3)
CHAr), 6.63 (d, 3JHH = 7.3 Hz, 1H, NHCO), 6.58 (d, 3JPH = 8.0 Hz, 1H, NHTs), 4.88 (m,
1H, CH3C ), 4.76 (m, 1H, CH3C ), 3.77 (m, 1H, CHCy), 2.32 (s, 3H, CH3Ts), 1.93–1.44 (m,
6H, 3
CH2Cy), 1.37 (d, 3JHH = 6.1 Hz, 3H, CH3CH), 1.36 (d, 3JHH = 6.2 Hz, 3H, CH3CH),
1.24 (d, JHH = 6.1 Hz, 3H, CH3CH), 1.15 (d, JHH = 6.2 Hz, 3H, CH3CH), 1.40–0.97 (m,
4H, 2 166.4 (C=O), 142.3 (CquatTs),
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
139.4 (CquatTs), 132.4 (CquatPh), 130.3 (d, 3JPC = 7.7 Hz, 2
CHAr), 129.0 (2 CHAr), 128.4
CHAr), 73.9 (d, 2JPC = 7.8 Hz, CH3 H), 73.8 (d, 2JPC
CH2Cy),
H3CH), 24.2 (2
H3CH), 21.5 (CH3Ts) ppm
δ
7.32–7.27 (m, 2H, 2
×
CHAr), 7.21–7.09 (m, 3H, 3
×
CHAr), 7.05–6.93 (m, 4H, 4
×
H
H
×
3
3
×
δ
×
×
(CHAr), 127.7 (2
= 8.2 Hz, CH3
×
CHAr), 126.7 (2
×
C
1
C
H), 68.8 (d, JPC = 156.0 Hz, Cquat-P), 49.6 (CHCy), 32.2 (2
×
25.5 (CH2Cy), 24.5 (d, 3JPC = 7.2 Hz,
C
H3CH), 24.3 (d, 3JPC = 6.5 Hz,
C
×
CH2Cy), 23.9 (d, 3JPc = 5.5 Hz, H3CH), 23.5 (d, 2JPc = 5.8 Hz,
C
C
.
31P-NMR (121 MHz, CDCl3)
δ
15.8 ppm. FTIR (neat)
ν
: ν = 3424 (N-H st) 3345 (N-H st)
max
1680 (C=O st) 1258 (P=O st) 1335 (S=O st sym) 1165 (S=O st as) cm−1. HRMS (ESI-TOF)
m/z: [M + H]+ calcd for C27H40N2O6PS 551.2345, Found 551.2348.
Methyl (2-(di-iso-propoxyphosphoryl)-2-((4-methylphenyl)sulfonamido)-2-phenylacetyl)glycinate (13i).
The general procedure was applied starting from diisopropyl (E)-(phenyl(tosylimino)-
methyl) phosphonate (10d, 423 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol)
and methyl 2-isocyanoacetate (12b, 100 µL, 1.1 mmol) to afford 335 mg (62%) of 13i after
14 h as white crystals after a chromatography column (Hexanes/AcOEt 6:4), followed by
crystallization (Dichloromethane/Hexanes 1:3). M.p.: 146–148 ◦C. 1H-NMR (400 MHz,
CDCl3)
4H, 4
(hept, 3JHH = 6.3 Hz, 1H, C
δ
7.44–7.35 (m, 2H, 2
×
CHAr), 7.25–7.14 (m, 4H, 3
×
CHAr + NHCO), 7.08–6.98 (m,
CH3), 4.74
×
CHAr), 6.43 (d, 3JPH = 7.8 Hz, 1H, NHTs), 4.82 (hept, 3JHH = 6.0, 1H, C
H
H
CH3), 4.03 (d, 3JHH = 5.2 Hz, 2H, CH2), 3.72 (s, 3H, OCH3), 2.33
(s, 3H, CH3Ts), 1.34 (d, 3JHH = 6.0 Hz, 3H, CH3CH), 1.31 (d, 3JHH = 6.3 Hz, 3H, CH3CH),
1.23 (d, 3JHH = 6.3 Hz, 3H, CH3CH), 1.11 (d, 3JHH = 6.0 Hz, 3H, CH3CH) ppm. 13C-NMR
{1H} (101 MHz, CDCl3) 169.3 (C=O), 167.9 (d, 2JPC = 1.3 Hz, C=O), 142.6 (CquatTs), 139.0 (d,
δ
4JPC = 1.3 Hz, CquatTs), 131.8 (d, 2JPC = 1.1 Hz, CquatPh), 130.3 (d, 3JPC = 7.5 Hz, 2
129.1 (2 CHAr), 128.6 (CHAr), 127.7 (2 CHAr), 126.8 (2
CHAr), 74.4 (d, 2JPC = 7.9 Hz,
CH), 74.1 (d, 2JPC = 8.0 Hz, CH), 69.5 (d, 1JPC = 152.4 Hz, Cquat-P), 52.5 (OCH3), 42.2 (CH2),
×
CHAr),
×
×
×
3
3
24.3 (d, JPC = 5.0 Hz,
CH3CH), 24.2 (d, JPC = 4.5 Hz, C ,
H3CH), 23.8 (d, 3JPC = 5.7 Hz
CH3CH), 23.4 (d, 3JPC = 6.0 Hz, CH3CH), 21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ
15.1 ppm. FTIR (neat)
ν
: ν = 3425 (N-H st) 3334 (N-H st) 1674 (C=O st) 1254 (P=O
max
st) 1330 (S=O st sym) 1163 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C24H34N2O8PS 541.1773, Found 541.1778.
Di-iso-propyl (2-(benzylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-phenylethyl)phosphonate
(
13j). The general procedure was applied starting from di-iso-propyl (E)-(phenyl(tosylimin-
o)methyl) phosphonate (10d, 423 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol)
and (isocyanomethyl) benzene (12c, 134 L, 1.1 mmol) to afford 464 mg (83%) of 13j after
14 h as white crystals after a chromatography column (Hexanes/AcOEt 8:2), followed by
µ

Molecules 2021, 26, 1654
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crystallization (Dichloromethane/Hexanes 1:3). M.p.: 100–102 ◦C. 1H-NMR (400 MHz,
CDCl3)
5H, 5
(hept, 3JHH = 6.1 Hz, 1H, CH), 4.55 (dd, 2JHH = 14.9, 3JHH = 6.3 Hz, 1H, C
2JHH = 14.9, 3JHH = 5.3 Hz, 1H, CHACHB), 2.33 (s, 3H, CH3Ts), 1.33 (d, 3JHH = 6.1 Hz, 3H,
H3CH), 1.30 (d, 3JHH = 6.2 Hz, 3H, CH3CH), 1.14 (d, 3JHH = 6.1 Hz, 3H, CH3CH), 1.06 (d,
3JHH = 6.2 Hz, 3H, CH3CH) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
167.5 (C=O), 142.5
(CquatTs), 139.3 (d, 4JPC = 1.3 Hz, CquatTs), 137.2 (CquatPh), 132.4 (CquatPh), 130.2 (d, 3JPC
= 7.6 Hz, 2 CHAr), 129.0 (2 CHAr), 128.7 (2 CHAr), 128.5 (CHAr), 127.8 (2 CHAr),
127.8 (2 CHAr), 127.7 (CHAr), 126.8 (2
CHAr), 74.1 (d, 2JPC = 8.0 Hz, 2
1JPC = 154.6 Hz, Cquat-P), 44.6 (CH2), 24.3 (d, 2JPC = 3.6 Hz
Hz,
H3CH), 23.8 (d, 2JPc = 5.8 Hz, C 3CH), 23.5 (d, 2JPC = 5.7 Hz
δ
7.39–7.31 (m, 2H, 2
CHAr), 6.59 (d, 3JPH = 7.4 Hz, 1H, NHTs), 4.85 (hept, 3JHH = 6.2 Hz, 1H, CH), 4.64
ACHB), 4.33 (dd,
×
CHAr), 7.30–7.13 (m, 8H, 7
×
CHAr + NHCO), 7.06–6.98 (m,
×
H
C
δ
×
×
×
×
×
×
×
CH), 69.1 (d,
2
,
C
H3CH), 24.1 (d, JPC = 3.0
C
H
,
C
H3CH), 21.6 (CH3Ts)
: ν = 3423 (N-H st) 3336
max
ppm. 31P-NMR (121 MHz, CDCl3)
δ
15.4 ppm. FTIR (neat)
ν
(N-H st) 1678 (C=O st) 1256 (P=O st) 1331 (S=O st sym) 1162 (S=O st as) cm−1. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C28H36N2O6PS 559.2032, Found 559.2038.
Dimethyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-1-(4-nitrophenyl)-2-oxoeth yl)ph-
osphonate (13k). The general procedure was applied starting from dimethyl (E)-((4-nitrophe-
nyl)(tosylimino)methyl)phosphonate (10e, 412 mg, 1 mmol), phenylacetic acid (11, 136 mg,
1 mmol) and cyclohexyl isocyanide (12a, 136 µL, 1mmol) to afford 418 mg (78%) of 13k
after 1 h as white crystals after a chromatography column (Hexanes/AcOEt 7:3), followed
by crystallization (Dichloromethane/Hexanes 1:3). M.p.: 100–102 ◦C. 1H-NMR (400 MHz,
CDCl3)
= 8.7 Hz, 2H, 2
δ
7.88–7.84 (m, 2H, 2
×
CHAr), 7.52 (d, 3JHH = 8.7 Hz, 2H, 2
×
CHAr), 7.22 (d, 3JHH
×
CHAr), 7.08–7.01 (m, 2H, 2
×
CHAr), 6.79 (d, 3JHH = 8.2 Hz, 1H, NHCO),
3
3
3
6.77 (d, JPH = 8.1 Hz, 1H, NHTos), 3.96 (d, JPH = 10.9 Hz, 3H, OCH3), 3.76 (d, JPH
=
10.7 Hz, 3H, OCH3), 3.76 (m, 1H, CHCy), 2.37 (s, 3H, CH3Ts), 1.93–1.51 (m, 4H, 2
×
CH2Cy),
1.41–0.97 (m, 6H, 3
×
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
δ
165.0 (C=O), 147.5
3
(Cquat-NO2), 143.6 (Cquat Ts), 139.7 (Cquat Ts), 138.8 (CquatAr), 130.9 (d, JPC = 7.6 Hz, 2
×
CHAr), 129.3 (2
×
CHAr), 126.5 (2
×
CHAr), 122.8 (2
×
CHAr), 67.8 (d, 1JPC = 153.9 Hz,
Cquat-P), 56.0 (d, 2JPC = 8.0 Hz, OCH3), 55.6 (d, 2JPC = 7.6 Hz, OCH3), 50.0 (CHCy), 32.3
(CH2Cy), 32.2 (CH2Cy), 25.4 (CH2Cy), 24.6 (CH2Cy), 24.5 (CH2Cy), 21.6 (CH3Ts) ppm.
31P-NMR (121 MHz, CDCl3)
δ
18.8 ppm. FTIR (neat)
ν
: ν = 3422 (N-H st) 3346 (N-H st)
max
1677 (C=O st) 1263 (P=O st) 1348 (S=O st sym) 1165 (S=O st as) cm−1. HRMS (ESI-TOF)
m/z: [M + H]+ calcd for C23H31N3O8PS 540.1569, Found 540.1571.
Dimethyl (1-(4-bromophenyl)-2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxoethyl)p-
hosphonate (13l). The general procedure was applied starting from dimethyl (E)-((4-bromop-
henyl)(tosylimino)methyl)phosphonate (10f, 446 mg, 1 mmol), phenylacetic acid (11
136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136 L, 1 mmol) to afford 495 mg (87%)
of 13l after 1 h as white crystals after crystallization (Dichloromethane/Hexanes 1:3). M.p.:
,
µ
◦
1
3
150–152 C. H-NMR (400 MHz, CDCl3)
δ
7.16 (d, JHH = 8.3 Hz, 2
×
CHAr), 7.09 (m,
seen as s, 4H, 4
×
CHAr), 7.04 (d, 3JHH = 8.3 Hz, 2H, 2
×
CHAr), 6.77 (d, 3JHH = 7.4 Hz,
1H, NHCO), 6.60 (d, 3JPH = 8.2 Hz, 1H, NHTs), 3.96 (d, 3JPH = 10.8 Hz, 3H, OCH3), 3.77
3
(d, JPH = 10.6 Hz, 3H, OCH3), 3.74 (m, 1H, CHCy), 2.36 (s, 3H, CH3Ts), 1.91–1.48 (m,
CH2Cy), 1.41–0.95 (m, 6H, 3
165.7 (C=O), 143.0 (CquatTs), 138.9 (CquatTs), 131.8 (d, JPC = 8.1 Hz, 2
4H, 2
δ
×
×
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
3
×
CHAr), 131.1
(CquatAr), 130.9 (2
×
CHAr), 129.2 (2
×
CHAr), 126.5 (2
×
CHAr), 123.4 (Cquat-Br), 67.9 (d,
1JPC = 155.9 Hz, Cquat-P), 55.9 (d, JPC = 8.2 Hz, OCH3), 55.4 (d, JPC = 7.5 Hz, OCH3),
2
2
49.8 (CHCy), 32.3 (CH2Cy), 32.2 (CH2Cy), 25.4 (CH2Cy), 24.6 (CH2Cy), 24.5 (CH2Cy), 21.6
(CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ
19.2 ppm. FTIR (neat)
ν
: ν = 3421 (N-H st)
max
3320 (N-H st) 1670 (C=O st) 1250 (P=O st) 1343 (S=O st sym) 1165 (S=O st as) cm−1. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C23H31BrN2O6PS 573.0824, Found 573.0830.
Dimethyl (1-(4-chlorophenyl)-2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxoethyl)p-
hosphonate (13m). The general procedure was applied starting from dimethyl (E)-((4-
chlorophenyl)(tosylimino)methyl) phosphonate (10g, 402 mg, 1 mmol), phenylacetic acid

Molecules 2021, 26, 1654
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(
11, 136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136
µL, 1 mmol) to afford 422 mg
(80%) of 13m after 1 h as white crystals after a chromatography column (Hexanes/AcOEt
7:3), followed by crystallization (Dichloromethane/Hexanes 1:3). M.p.: 165–167 ◦C. 1H-
NMR (400 MHz, CDCl3)
δ
7.19–7.12 (m, 4H, 4
×
CHAr), 7.04 (d, 3JHH = 8.2 Hz, 2H, 2
×
3
3
CHAr), 6.95 (d, JHH = 8.2 Hz, 2H, 2
×
CHAr), 6.76 (d, JHH = 7.2 Hz, 1H, NHCO), 6.58
(d, 3JPH = 8.1 Hz, 1H, NHTs), 3.97 (d, 3JPH = 10.8 Hz, 3H, OCH3), 3.78 (d, 3JPH = 10.6 Hz,
3H, OCH3), 3.75 (m, 1H, CHCy), 2.36 (s, 3H, CH3Ts), 1.91–0.95 (m, 10H, 5
×
CH2Cy) ppm.
165.7 (C=O), 142.9 (CquatTs), 139.0 (CquatTs), 135.0
CHAr), 130,6 (Cquat-Cl), 129.1 (2 CHAr), 128.0 (2
13C-NMR {1H} (101 MHz, CDCl3)
δ
(CquatAr), 131.5 (d, 3JPC = 7.7 Hz, 2
×
×
×
CHAr), 126.5 (2
×
CHAr), 67.8 (d, 1JPC = 155.9 Hz, Cquat-P), 55.9 (d, 2JPC = 8.2 Hz, OCH3),
55.4 (d, 2JPC = 7.5 Hz, OCH3), 49.8 (CHCy), 32.3 (CH2Cy), 32.2 (CH2Cy), 25.4 (CH2Cy), 24.6
(CH2Cy), 24.5 (CH2Cy), 21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ
(ppm): 19.2 ppm.
FTIR (neat)
ν
: ν = 3417 (N-H st) 3319 (N-H st) 1670 (C=O st) 1258 (P=O st) 1353 (S=O st
max
sym) 1160 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C23H31ClN2O6PS
529.1331, Found 529.1335.
Dimethyl (2-(cyclohexylamino)-1-(2-fluorophenyl)-1-((4-methylphenyl)sulfonamido)-2-oxoethyl)p-
hosphonate (13n). The general procedure was applied starting from dimethyl (E)-((2-fluorop-
henyl)(tosylimino)methyl) phosphonate (10h, 385 mg, 1 mmol), phenylacetic acid (11, 136
mg, 1 mmol) and cyclohexyl isocyanide (12a, 136
13n after 1 h as white crystals after crystallization (Dichloromethane/Hexanes 1:3). M.p.:
203–205 ◦C. 1H-NMR (400 MHz, CDCl3) 7.88 (t, 3JFH = 7.3 Hz, 1H, CHAr), 7.22 (m, 1H,
CHAr), 7.18–7.09 (m, 3H, 3 CHAr), 7.03–6.93 (m, 3H, 3 CHAr), 6.41 (m, 1H, NHCO), 5.98
(d, 3JPH = 8.2 Hz, 1H, NHTs), 4.03 (d, 3JPH = 10.7 Hz, 3H, OCH3), 3.87 (d, 3JPH = 10.5 Hz, 3H,
OCH3), 3.75 (m, 1H, CHCy), 2.32 (s, 3H, CH3Ts), 1.88–1.44 (m, 4H, 2 CH2Cy), 1.38–0.78
(m, 6H, 3 165.3 (C=O), 160.6 (dd,
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
1JFC = 252.7, 3JPC = 13.1 Hz, CF). 142.7 (CquatTs), 138.4 (d, CquatTs), 134.2 (d, 3JFC = 5.2 Hz,
CHAr), 131.5 (d, 3JFC = 9.1 Hz, CHAr), 129.0 (2 CHAr), 123.9 (d, 4JFC
CHAr), 126.6 (2
= 3.4 Hz, CHAr), 119.9 (dd, 2JFC = 10.8, 2JPC = 4.6 Hz, CquatAr), 115.7 (d, 2JFC = 22.5 Hz,
µL, 1 mmol) to afford 370 mg (73%) of
δ
×
×
×
×
δ
×
×
1
2
2
CHAr), 64.6 (d, JPC = 158.9 Hz, Cquat-P), 56.2 (d, JPC = 8.5 Hz, OCH3), 55.6 (d, JPC
7.7 Hz, OCH3), 50.0 (CHCy), 32.2 (2 CH2Cy), 25.4 (CH2Cy), 24.7 (CH2Cy), 24.6 (CH2Cy),
21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3) 17.8 ppm. 19F-NMR (282 MHz, CDCl3)
=
×
δ
δ
−
105.5 ppm. FTIR (neat)
ν
: ν = 3417 (N-H st) 3319 (N-H st) 1672 (C=O st) 1268 (P=O
max
st) 1352 (S=O st sym) 1165 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C23H31FN2O6PS 513.1624, Found 513.1630.
Dimethyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-(perfluorophenyl)et-
hyl)phosphonate (13o). The general procedure was applied starting from dimethyl (E)-
((perfluorophenyl)(tosylimino)methyl) phosphonate (10i, 457 mg, 1 mmol), phenylacetic
acid (11, 136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136
µL, 1 mmol) to afford 514 mg
(88%) of 13o after 1 h as white crystals after crystallization (Dichloromethane/Hexanes
◦
1
1:3). M.p. (Dichloromethane/Hexanes) = 189–191 C. H-NMR (400 MHz, CDCl3)
δ
3
3
7.31 (d, JHH = 8.1 Hz, 2H, 2
×
CHAr), 7.11 (d, JHH = 8.1 Hz, 2H, 2
×
CHAr), 6.92 (d,
3JHH = 7.3 Hz, 1H, NHCO), 6.40 (br s, 1H, NHTs), 4.01 (d, 3JPH = 10.9 Hz, 3H, OCH3), 3.82
(d, 3JPH = 10.7 Hz, 3H, OCH3), 3.79–3.69 (m, 1H, CHCy), 2.36 (s, 3H, CH3Ts), 1.96–0.94 (m,
10H, 5
×
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
δ
164.0 (C=O), 146.4 (m, 2
×
CF),
144.0 (CquatTs), 142.1 (m, 2
×
CF), 137.8 (CquatTs), 137.5 (m, CF), 129.2 (2
×
CHAr), 126.3 (2
×
CHAr), 108.9 (m, CquatAr), 61.2 (d, 1JPC = 159.7 Hz, Cquat-P), 56.4 (d, 2JPC = 8.6 Hz, OCH3),
55.9 (d, 2JPC = 8.1 Hz, OCH3), 50.3 (CHCy), 32.3 (CH2Cy), 31.9 (CH2Cy), 25.4 (CH2Cy),
24.7 (CH2Cy), 24.6 (CH2Cy), 21.5 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ
15.9 ppm.
19F-NMR (282 MHz, CDCl3) δ −129.4,
−
151.7,
−
162.3 ppm. FTIR (neat)
ν
:
ν
= 34−117
max
(N-H st) 3316 (N-H st) 1677 (C=O st) 1268 (P=O st) 1330 (S=O st sym) 1165 (S=O st as) cm
.
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C23H27F5N2O6PS 585.1249, Found 584.1166.
Dimethyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-(4-((trichlorometh-
yl)thio)phenyl)ethyl)phosphonate (13p). The general procedure was applied starting from

Molecules 2021, 26, 1654
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dimethyl (E)-((tosylimino)(4-((trichloromethyl)thio)phenyl)methyl) phosphonate (10j),
phenylacetic acid (11, 136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136 µL, 1 mmol)
to afford 580 mg (92%) of 13p after 14 h as white crystals after a chromatography column
(Hexanes/AcOEt 7:3), followed by crystallization (Dichloromethane/Hexanes 1:3). M.p.:
148–150 ◦C. 1H-NMR (400 MHz, CDCl3)
δ
7.49 (d, 3JHH = 8.4 Hz, 2H, 2
×
CHAr), 7.42 (d,
CHAr), 7.06 (d, 3JHH = 8.1 Hz,
CHAr), 6.75 (d, 3JHH = 6.8 Hz, 1H, NHCO), 6.58 (d, 3JPH = 8.2 Hz, 1H, NHTs), 3.96
(d, 3JPH = 10.8 Hz, 3H, OCH3), 3.77 (d, 3JPH = 10.6 Hz, 3H, OCH3), 3.71 (m, 1H, CHCy),
2.33 (s, 3H, CH3Ts), 1.91–1.45 (m, 4H, 2 CH2Cy), 1.39–0.94 (m, 6H, 3 CH2Cy) ppm.
13C-NMR {1H} (101 MHz, CDCl3)
165.4 (C=O), 143.1 (CquatTs), 139.1 (CquatTs), 136.4 (2
CHAr), 136.2 (CquatAr), 131.2 (CquatAr) 130.8 (d, 3JPC = 7.9 Hz, 2
CHAr), 129.3 (2
3JHH = 8.4 Hz, 2H, 2
2H, 2
×
CHAr), 7.24 (d, 3JHH = 8.1 Hz, 2H, 2
×
×
×
×
δ
×
×
×
CHAr), 126.5 (2
×
CHAr), 98.2 (CCl3), 68.0 (d, 1JPC = 155.9 Hz, Cquat-P), 55.9 (d, 2JPC = 8.1
Hz, OCH3), 55.4 (d, 2JPC = 7.5 Hz, OCH3), 49.8 (CHCy), 32.2 (CH2Cy), 32.0 (CH2Cy), 25.4
(CH2Cy), 24.5 (CH2Cy), 24.4 (CH2Cy), 21.6 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ
(ppm) 19.0 ppm. FTIR (neat)
ν
: ν = 3412 (N-H st) 3337 (N-H st) 1677 (C=O st) 1265
max
(P=O st) 1353 (S=O st sym) 1160 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C24H31Cl3N2O6PS2 643.0427, Found 643.0430.
Dimethyl (1-(3-chloro-4-methoxyphenyl)-2-(cyclohexylamino)-1-((4-methylphenyl)-sulfonamido)-
2-oxoethyl)-phosphonate (13q). The general procedure was followed, using dimethyl (E)-((3-
chloro-4-methoxyphenyl)(tosylimino)-methyl) phosphonate (10k, 431 mg, 1 mmol), pheny-
lacetic acid (11, 136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136 µL, 1 mmol) to afford
418 mg (75%) of 13q after 14 h as white crystals after crystallization (Dichloromethane/Hexanes
1:3). M.p.: 115–117 ◦C. 1H-NMR (400 MHz, CDCl3)
= 4.4 Hz, 1H, NHCO), 7.15 (d, 3JHH = 8.0 Hz, 2H, 2
CHAr), 6.78–6.29 (m, 2H, 2
= 10.7 Hz, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.83 (d, 3JPH = 10.4 Hz, 3H, OCH3), 3.79 (m,
1H, CHCy), 2.34 (s, 3H, CH3Ts), 1.87–1.02 (m, 10H, 5
CH2Cy) ppm. 13C-NMR {1H}
(101 MHz, CDCl3) 165.9 (C=O), 155.2 (Cquat), 143.2 (Cquat Ts), 138.7 (Cquat Ts), 132.0 (d,
δ
7.45 (m, 1H, CHAr), 7.34 (d, 3JHH
×
CHAr), 7.02 (d, 3JHH = 8.0 Hz, 2H,
2
×
×
CHAr), 6.53 (d, 3JPH = 7.5 Hz, 1H, NHTs), 4.04 (d, 3JPH
×
δ
3JPC = 10.9 Hz, CHAr), 131.0 (d, 3JPC = 6.7 Hz, CHAr), 129.2 (2
×
CHAr Ts), 126.4 (2
×
CHAr
Ts), 124.3 (Cquat), 121.9 (Cquat), 110.2 (CHAr), 67.5 (d, 1JPC = 157.8 Hz, Cquat-P), 56.3 (OCH3),
56.2 (d, 2JPC = 7.4 Hz, OCH3), 55.4 (d, 2JPC = 7.5 Hz, OCH3), 49.8 (CHCy), 32.3 (CH2Cy),
32.2 (CH2Cy), 25.4 (CH2Cy), 24.6 (CH2Cy), 24.5 (CH2Cy), 21.6 (CH3 Ts) ppm. 31P-NMR
(121 MHz, CDCl3)
δ
19.1 ppm. FTIR (neat)
ν
: ν = 3434 (N-H st) 3323 (N-H st) 1677 (C=O
max
st) 1258 (P=O st) 1334 (S=O st sym) 1160 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+
calcd for C24H33ClN2O7PS 559.1436, Found 559.1437.
Dimethyl (2-(cyclohexylamino)-2-oxo-1-(2-phenyl-N-tosylacetamido)-1-(4-(trifluoromethyl)p-
henyl)ethyl)phosphonate (15a). The general procedure was applied starting from dimethyl
(E)-((tosylimino)(4-(trifluoromethyl)phenyl)methyl) phosphonate (10l, 435 mg, 1 mmol),
phenylacetic acid (11, 136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136 µL, 1 mmol)
to afford 579 mg (85%) of 15a after 1 h as white crystals after a chromatography column
(Hexanes/AcOEt 7:3), followed by crystallization (Dichloromethane/Hexanes 1:3). M.p.:
◦
84–86 C. 1H-NMR (400 MHz, CDCl3)
δ
8.20 (d, 3JHH = 8.0 Hz, 2H, 2
×
CHAr), 7.78 (d, 3JHH
= 8.3 Hz, 2H, 2
CHAr), 7.28–7.17 (m, 5H, 5
1H, C
ACHB), 3.98 (d, 2JHH = 17.1 Hz, 1H, CHAC
3.80–3.68 (m, 1H, CHCy), 3.50 (d, 3JPH = 11.5 Hz, 3H, OCH3), 2.44 (s, 3H, CH3Ts), 1.98–0.90
(m, 10H, 6 176.6 (C=O), 164.7 (d,
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
2JPC = 5.2 Hz, C=O), 144.8 (CquatTs), 138.5 Cquat), 138.4 (CquatTs), 133.9 (CquatCF3), 133.6
×
CHAr), 7.57 (d, 3JHH = 8.3 Hz, 2H, 2
CHAr), 5.95 (br s, 1H, NHCO), 4.20 (d, 2JHH = 17.1 Hz,
B), 3.84 (d, 3JPH = 11.2 Hz, 3H, OCH3),
×
CHAr), 7.36 (d, 3JHH = 8.0 Hz, 2H,
2
×
×
H
H
×
δ
(CquatAr), 130.2 (2
CHAr), 128.0 (2
×
CHAr), 129.9 (2
×
CHAr), 129.2 (d, 3JPC = 5.3 Hz, 2
×
CHAr), 128.4 (2
×
×
CHAr), 127.3 (d, 4JFC = 3.8 Hz, 2
×
CHAr), 125.1 (CHAr), 123.9 (q, 1JFC
=
272.3 Hz, CF3), 79.4 (d, 1JPC = 147.5 Hz, Cquat-P), 55.4 (d, 2JPC = 7.3 Hz, OCH3), 55.1 (d, 2JPC
= 7.7 Hz, OCH3), 49.2 (CHCy), 45.7 (CH2Bn), 32.4 (CH2Cy), 31.9 (CH2Cy), 25.5 (CH2Cy),
24.6 (CH2Cy), 24.4 (CH2Cy), 21.8 (CH3Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ (ppm):

Molecules 2021, 26, 1654
16 of 19
22.1 ppm. 19F-NMR (282 MHz, CDCl3) δ −63.1.ppm. FTIR (neat)
ν
:
ν
= 3430 (N-H st)
max
1676 (C=O st) 1248 (P=O st) 1330 (S=O st sym) 1160 (S=O st as) cm−1.HRMS (ESI-TOF) m/z:
[M + H]+ calcd for C32H36F3N2O7PS 680.1933, Found 680.1934.
Dimethyl (2-(cyclohexylamino)-1-((4-methylphenyl)sulfonamido)-2-oxo-1-(4-(trifluoromethyl)p-
henyl)ethyl)phosphonate (13r). Exposure of 15a under air moisture for 48 h yields 13r in
quantitative yield as white crystals after crystallization (Dichloromethane/Hexanes 1:3).
◦
M.p.: 88–90 C. 1H-NMR (400 MHz, CDCl3)
δ
7.36 (d, 3JHH = 7.8 Hz, 2H, 2
×
CHAr), 7.22 (d,
3JHH = 7.8 Hz, 2H, 2
×
CHAr), 7.11 (d, 3JHH = 8.4 Hz, 2H, 2
×
CHAr), 6.98 (d, 3JHH = 8.4 Hz,
2H, 2
(d, 3JPH = 10.8 Hz, 3H, OCH3), 3.80 (m, 1H, CHCy), 3.78 (d, 3JPH = 10.6 Hz, 3H, OCH3), 2.32
(s, 3H, CH3Tos), 1.91–0.94 (m, 10H, 5
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
165.4 (C=O), 143.0 (CquatTs), 138.8 (CquatTs), 135.8 (Cquat), 130.6 (q seen as d, 3JFC = 7.9
×
CHAr), 6.84 (d, 3JHH = 7.8 Hz, 1H, NHCO), 6.70 (d, 3JPH = 8.1 Hz, 1H, NHTs), 3.99
×
δ
Hz, 2
×
CHAr), 130.7 (d, 2JPC = 32.8 Hz, CquatCF3), 129.2 (2
×
CHAr), 126.3 (2
×
CHAr),
124.6 (d, 4JFC = 3.7 Hz, 2
×
CHAr), 123.7 (q, 1JFC = 272.5 Hz, CF3), 68.0 (d, 1JPC = 155.2 Hz,
Cquat-P), 55.8 (d, 2JPC = 8.1 Hz, OCH3), 55.5 (d, 2JPC = 7.6 Hz, OCH3), 49.9 (CHCy), 32.2
(CH2Cy), 32.1 (CH2Cy), 25.3 (CH2Cy), 24.6 (CH2Cy), 24.6 (CH2Cy), 21.4 (CH3Tos) ppm.
31P-NMR (121 MHz, CDCl3)
δ
(ppm): 20.0 ppm. 19F-NMR (282 MHz, CDCl3) δ −63.5.ppm.
FTIR (neat)
ν
: ν = 3430 (N-H st) 3332 (N-H st) 1677 (C=O st) 1259 (P=O st) 1326 (S=O st
max
sym) 1165 (S=O st as) cm−1.HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H30F3N2O6PS
562.1514, Found 562.1519.
Dimethyl (2-(cyclohexylamino)-1-(4-fluorophenyl)-2-oxo-1-(2-phenyl-N-tosylacetamido)eth-
yl)phosphonate (15b). The general procedure was followed, using dimethyl (E)-(4-fluorophenyl
phenyl(tosylimino)methyl) phosphonate (10m, 385 mg, 1 mmol), phenylacetic acid (11
136 mg, 1 mmol) and cyclohexyl isocyanide (12a, 136 L, 1 mmol) to afford 536 mg (85%)
of 15b after 14 h as white crystals after column chromatography (Hexanes/AcOEt 8:2),
,
µ
◦
1
followed by crystallization (Dichloromethane/Hexanes 1:3). M.p.: 90–92 C. H-NMR
(400 MHz, CDCl3)
δ
1H-NMR (400 MHz, CDCl3)
δ
8.26 (d, 3JHH = 7.9 Hz, 2H, 2
CHAr), 7.28–7.18 (m, 5H, 5
×
CHAr),
CHAr),
7.05 (m, 2H, 2
×
CHAr), 7.37 (d, 3JHH = 8.1 Hz, 2H, 2
×
×
7.03 (dd, seen as t, 3JHH = 8.5 Hz, 3JHH = 8.5 Hz, 2H, 2
×
CHAr), 5.93 (broad s, 1H, NH),
4.17 (d, 2JHH = 17.1 Hz, 1H, CH2), 3.92 (d, 2JHH = 17.1 Hz, 1H, CH2), 3.82 (d, 3JPH = 11.2 Hz,
3H, OCH3), 3.73 (m, 1H, CHCy), 3.50 (d, 3JPH = 11.5 Hz, 3H, OCH3), 2.44 (s, 3H, CH3Ts),
1.85–0.94 (m, 10H, 5
(d, 2JPC = 4.8 Hz, C=O), 162.4 (d, 1JFC = 248.9 Hz, CAr-F), 144.6 (Cquat), 138.6 (Cquat), 133.8
(Cquat), 131.9 (m, Cquat + 2 CHAr), 130.2 (2 CHAr), 129.8 (2 CHAr), 128.4 (2 CHAr),
×
CH2Cy) ppm. 13C-NMR {1H} (101 MHz, CDCl3)
δ 176.4 (C=O), 165.3
×
×
×
×
128.0 (2
×
CHAr), 127.2 (CHAr), 115.2 (d, 2JFC = 21.6 Hz, 2
×
CHAr), 78.9 (d, 1JPC = 148.5 Hz,
Cquat-P), 55.2 (d, 3JPc = 7.2 Hz, CH3O), 54.9 (d, 3JPC = 7.7 Hz, CH3O), 48.9 (CH Cy), 45.7
(CH2 Bn), 32.4 (CH2 Cy), 31.9 (CH2 Cy), 25.6 (CH2 Cy), 24.5 (CH2 Cy), 24.4 (CH2 Cy), 21.8
(CH3 Ts) ppm. 31P-NMR (121 MHz, CDCl3)
δ δ
21.8 ppm. 19F NMR (282 MHz, CDCl3):
−
113.9 ppm. FTIR (neat)
ν
: ν = 3426 (N-H st) 1677 (C=O st) 1263 (P=O st) 1348 (S=O st
max
sym) 1156 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C31H36FN2O7PS
631.2043, Found 631.2040.
Dimethyl (2-(cyclohexylamino)-2-oxo-1-(2-phenylacetamido)ethyl)phosphonate (16). The gen-
eral procedure was followed, using dimethyl (E)-dimethyl (E)-(phenyl(tritylimino)meth-
yl)phosphonate (14, 455 mg, 1 mmol), phenylacetic acid (11, 136 mg, 1 mmol) and cyclo-
hexyl isocyanide (12a, 136
µL, 1 mmol) to afford 536 mg (65%) of 16 after 14 h as white
crystals after column chromatography (Hexanes/AcOEt 8:2), followed by crystallization
◦
(Dichloromethane/Hexanes 1:3). M.p.: 121–122 C. 1H-NMR (300 MHz, CDCl3)
(400 MHz, CDCl3) 7.43–7.20 (m, 5H, 5
CHAr), 5.25 (d, 2JPH = 20.6 Hz, 1H, CH-P), 4.91
(br s, 2H, 2 NH), 3.73 (br s, 3H, OCH3), 3.70 (br s, 3H, OCH3), 3.63 (m, 1H, CHCy), 3.58
(s, 2H, CH2), 1.84–1.65 (m, 4H, 2 CH2Cy), 1.36–1.13 (m, 6H, 3
CH2Cy) ppm. 13C-NMR
{1H} (101 MHz, CD3OD) 174.3 (C=O), 172.0 (d, 2JPC = 5.3 Hz, C=O), 135.3 (Cquat), 129.0
(2 CHAr), 128.3 (2
CHAr), 126.7 (CHAr), 53.5 (d, 3JPc = 6.8 Hz, CH3O), 53.3 (d, 3JPc
6.4 Hz, CH3O), 50.5 (d, 1JPC = 148.0 Hz, Cquat-P), 49.0 (CH Cy), 42.0 (CH2 Bn), 32.2 (CH2
δ
1H-NMR
δ
×
×
×
×
δ
×
×
=

Molecules 2021, 26, 1654
17 of 19
Cy), 32.1 (CH2 Cy), 25.3 (CH2 Cy), 24.7 (CH2 Cy), 24.6 (CH2 Cy) ppm. 31P-NMR (121 MHz,
CDCl3)
δ
21.0 ppm. FTIR (neat)
ν
: ν = 3436 (N-H st) 3334 (N-H st) 1674 (C=O st) 1265
max
(P=O st) 1342 (S=O st sym) 1160 (S=O st as) cm−1.HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C18H27N2O5P, 383.1736, Found 383.1741.
Procedure for the Hydrolysis of Phosphonate Ester 13b
A solution of α-aminophosphonate 13b, (242 mg, 0.5 mmol) and bromotrimethylsilane
(765 mg, 2.5 mmol) was stirred at room temperature in chloroform (3 mL) for 24 h until
disappearance of the starting materials as monitored by 31P-NMR. Water (3 mL) was then
added and the volatiles were distilled off at reduced pressure. The crude residue was
crystallized from a mixture Dichloromethane/methanol (95:5) to afford 267 mg (99%) of
23 as a white solid. M.p.: 204–206 ◦C. 1H-NMR (400 MHz, D2O)
δ
7.39 (d, 3JHH = 7.3 Hz,
CHAr), 7.20–7.14 (m, 2H, 3H, 3 CHAr),
ACHB), 3.72 (d, 2JHH = 17.9 Hz
2H, 2 × CHAr), 7.25 (d, 3JHH = 8.5 Hz, 2H, 2
×
×
7.12–7.05 (m, 2H, 2
×
CHAr), 3.98 (d, 2JHH = 17.9 Hz, 1H, C
H
,
1H, CHACHB), 3.60 (s, 3H, OCH3), 2.20 (s, 3H, CH3Ts) ppm. 13C-NMR {1H} (101 MHz,
D2O)
171.7 (C=O), 170.7 (C=O), 133.1 (d, 2JPC = 4.3 Hz, Cquat), 130.2 (d, 3JPC = 5.8 Hz,
CHAr), 129.4 (2 CHAr), 128.1 (CHAr), 127.4 (2 CHAr), 126.3 (2 CHAr), 69.9 (d,
1JPC = 128.5 Hz, Cquat-P), 52.8 (OCH3), 42.0 (CH2), 20.6 (CH3Ts) ppm. 31P-NMR (121 MHz,
δ
2
×
×
×
×
D2O)
δ
13.1 ppm. FTIR (neat)
ν
: ν = 3447 (N-H st) 3367 (N-H st) 1674 (C=O st) 1290
max
(P=O st) 1357 (S=O st sym) 1172 (S=O st as) cm−1. HRMS (ESI-TOF) m/z: [M + H]+ for
C18H21N2O8PS 457.0834, Found 457.0831.
3.2. Biology
3.2.1. Materials
Reagents and solvents were used as purchased without further purification. All
stock solutions of the investigated compounds were prepared by dissolving the powered
materials in appropriate amounts of Dimethylsulfoxide (DMSO). The final concentration
◦
of DMSO never exceeded 5% (v/v) in reactions. The stock solution was stored at 5 C until
it was used.
3.2.2. Cell Culture
Human epithelial lung carcinoma cells (A549) (ATCC® CCL-185
™, ATCC - Manassas,
VA, USA) were grown in Kaighn’s Modification of Ham’s F-12 Medium (ATCC® 30-2004
™
™
,
,
ATCC - Manassas, VA, United States) and lung fibroblast cells (MRC5) (ATCC® CCL-171
ATCC - Manassas, VA, USA) were grown in Eagle’s Minimum Essential Medium (EMEM,
ATCC® 30-2003
™
, ATCC - Manassas, VA, USA). Both were supplemented with 10% of
fetal bovine serum (FBS) (Sigma-Aldrich, Spain) and with 1% of NORMOCIN solution
(Thermo Fisher, Waltham, Massachusetts (MA), United States). Cells were incubated at
◦
37 C and 5% CO2 atmosphere, and were split every 3–4 days to maintain monolayer
coverage. For cytotoxicity experiments, A549 cells were seeded in 96-well plates at a
density of 2.5–3 × 103 cells per well and incubated overnight to achieve 70% of confluence
at the time of exposition to the cytotoxic compound.
3.2.3. Cytotoxicity Assays
Cells were exposed to different concentrations of the cytotoxic compounds and were
incubated for 48 h. Then, 10 µL of cell counting kit-8 was added into each well for
additional two hours incubation at 37 ◦C. The absorbance of each well was determined
by an Automatic Elisa Reader System (Thermo Scientific Multiskan FC Automatic Elisa
Reader System, Thermo Scientific, Shangai, China) at 450 nm wavelength.
4. Conclusions
In conclusion, we report an efficient Ugi methodology using ketimines for the prepa-
ration of tetrasubstituted
α-aminophosphonates holding a variety of substituents. Despite
the difficulty often observed for the utilization of ketones or ketimines in Ugi reactions,

Molecules 2021, 26, 1654
18 of 19
α
-phosphorated ketimines react under mild conditions to give the Ugi adducts after the
spontaneous cleavage of the amide moiety. Clear evidences of the Ugi mechanism are
provided, using thioacids. Moreover, obtained -aminophosphonate derivatives 13g 13h,
α
,
13p and 15a showed in vitro cytotoxicity inhibiting the growth of human tumor cell line
A549 (carcinomic human alveolar basal epithelial cell), and a high selectivity toward MRC5
nonmalignant lung fibroblasts. As far as we know this is the first example of much hindered
tetrasubstituted α-aminophosphonates showing antiproliferative activity.
1
Supplementary Materials: H, 13C, 19F and 31P-NMR copies of compounds 13, 15, 16, 23.
Author Contributions: Conceptualization, A.L.-F., X.d.C., E.M.d.M., F.P. and J.V.; methodology,
A.L.-F. and X.d.C.; software, A.L.-F. and X.d.C.; validation, E.M.d.M. and J.V.; formal analysis, A.L.-F.
and X.d.C.; investigation, A.L.-F. and X.d.C.; resources, E.M.d.M., F.P. and J.V.; data curation, A.L.-F.
and X.d.C.; writing—original draft preparation, J.V.; writing—review and editing, A.L.-F., X.d.C.,
E.M.d.M., F.P. and J.V.; visualization, E.M.d.M., F.P. and J.V.; supervision, E.M.d.M. and J.V.; project
administration, E.M.d.M. and J.V.; funding acquisition, E.M.d.M., F.P. and J.V. All authors have read
and agreed to the published version of the manuscript.
Funding: Financial support by Ministerio de Economía, Industria y Competividad (MINECO, CTQ-
2015-67871R) and Gobierno Vasco (GV, IT 992-16) is gratefully acknowledged. X.d.C. and A.L.-F.
thank the Basque Country Government for a predoctoral grant.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the supplementary
materials file or on request from the corresponding author (1H, 13C, 19F and 31P-NMR and HRMS
spectra and cytotoxicity essays).
Acknowledgments: The authors thank for technical and human support provided by SGIker
(UPV/EHU/ERDF, EU).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available.
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