One-pot imination / Arbuzov reaction of 4-aminobutanal derivatives: Synthesis of 2-phosphorylpyrrolidines and evaluation of anticancer activity

Article abstract of DOI:10.1016/j.tet.2020.131369

A novel one-pot method for the preparation of N-substituted 2-phopshorylpyrrolidines from readily available 4,4-diethoxybutan-1-amine derivatives and P (III) acid chlorides is described. The presented method benefits from simple reaction and work-up proce

Full text of DOI:10.1016/j.tet.2020.131369

Journal Pre-proof
One-pot imination / Arbuzov reaction of 4-aminobutanal derivatives: Synthesis of 2-
phosphorylpyrrolidines and evaluation of anticancer activity
Andrey V. Smolobochkin, Rakhymzhan A. Turmanov, Almir S. Gazizov, Alexandra D.
Voloshina, Julia K. Voronina, Anastasiia S. Sapunova, Alexander R. Burilov, Michail
A. Pudovik
PII:
S0040-4020(20)30524-X
https://doi.org/10.1016/j.tet.2020.131369
TET 131369
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 4 May 2020
Revised Date: 23 June 2020
Accepted Date: 28 June 2020
Please cite this article as: Smolobochkin AV, Turmanov RA, Gazizov AS, Voloshina AD, Voronina
JK, Sapunova AS, Burilov AR, Pudovik MA, One-pot imination / Arbuzov reaction of 4-aminobutanal
derivatives: Synthesis of 2-phosphorylpyrrolidines and evaluation of anticancer activity, Tetrahedron
(2020), doi: https://doi.org/10.1016/j.tet.2020.131369.
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One-pot Imination / Arbuzov Reaction of 4-Aminobutanal Derivatives: Synthesis of 2-Phosphorylpyrrolidines
and Evaluation of Anticancer Activity.
Andrey V. Smolobochkin,a Rakhymzhan A. Turmanov,a,b Almir S. Gazizov,a,* Alexandra D. Voloshina,a Julia K.
Voronina,c,d Anastasiia S. Sapunovaa, Alexander R. Burilov,a Michail A. Pudovika
a Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences,
420088, Arbuzova str., 8, Kazan, Russian Federation
b Kazan National Research Technological University, 420015, 68 Karl Marx str., Kazan, Russian Federation
с
N.S. Kurnakov Institute of General and Inorganic Chemistry, RAS, 31 Leninsky Av., 119991 Moscow, Russian
Federation
d G.V. Plekhanov Russian University of Economics, 36 Stremyanny Per., Moscow 117997, Russian Federation
Abstract
A novel one-pot method for the preparation of N-substituted 2-phopshorylpyrrolidines from readily available
4,4-diethoxybutan-1-amine derivatives and P (III) acid chlorides is described. The presented method benefits
from simple reaction and work-up procedures, mild reaction conditions, avoids protecting group introduction-
removal stages and provides a straightforward access to target compounds. In vitro cytotoxicity studies
indicate that obtained 2-phopshorylpyrrolidines inhibit selectively M-Hela tumor cells growth. The activity of
some derivatives towards M-Hela and MCF-7 tumor cells is comparable to that of reference drug Tamoxifen. In
contrast to Tamoxifen and Doxorubicin tested compounds have no cytotoxic effects on normal cells.
Keywords: nitrogen heterocycles, phosphorylation, ureas, sulfonylamides, cytotoxicity
Introduction
Pyrrolidine scaffold is found in a diverse set of biologically active molecules, both natural1,2 (such as alkaloids
nicotine, hygrine or norhygrine) and synthetic.1–3 Among them, N-substituted pyrrolidine derivatives possessing
phosphoryl moiety are of a special interest. Being a bio-isosteric phosphorus analogues of a proline amino acid,
they exhibit a range of interesting biological properties. Thus, N-acyl-2-phosphorylpyrrolidines exhibit antiviral
activity;4,5 oligopeptides with N-acylphosphaproline fragment are of interest as inhibitors of various proteases6–
8 and activity-based probes for monitoring post-proline protease activity.9
Some of the N-acylphosphaproline derivatives are known to inhibit angiotensin-converting enzyme10,11 and
fibroblast activation protein (FAP) (Figure 1, A).12 Notably, FAP expression is seen on activated stromal
fibroblasts of more than 90% of all human carcinomas13 and targeting this enzyme is of interest in cancer
treatment.14 Additionally, our own work also indicates an anti-cancer potential of some N-sulfonyl-2-
phopshorylpyrrolidines (Figure 1, B).15 Considering this, we became interested in expanding the scope of
available N-substituted 2-phosphorylpyrrolidines and exploring their anti-tumor activity.
1

Figure 1. Some N-substituted 2-phosphorylpyrrolidines with anti-tumor activity.
Synthetic routes to 2-phosphorylpyrrolidines have been reviewed16,17 and involve either the modification of
pre-formed pyrrolidine core18–21 or the intramolecular cyclization of phosphorylated butan-1-amine
derivatives.22–26 Recently, Ragulin et al27–29 has described the synthesis of (pyrrolidin-2-yl)phosphonates via
Kabachnik-Fields reaction of N-Cbz protected 4-aminobutanal with some hydrophosphoryl compounds. The
similar reaction was earlier reported by Yiotakis with coworkers.30 However, only a limited number of 2-
phosphorylpyrrolidines have been obtained and no systematic studies on this reaction have been undertaken.
Additionally, these reactions suffer from laborious multi-stage synthesis of starting compounds. Moreover,
they require a protection of amino group and provide N-(Fmoc/Cbz)-2-phosphorylpyrrolidines only. This
implies additional stages for introduction of protecting group, its removal upon completion of the reaction and
further modification of NH group to obtain target N-substituted compounds (Scheme 1, A).
Scheme 1. A) Amidoalkylation of hydrophosphoryl compounds by N-protected 4-aminobutanal; B) One-pot
synthesis of N-substituted 2-phosphorylpyrrolidines via intramolecular imination / Arbuzov reaction sequence
of 4,4-diethoxybutan-1-amine derivatives and P (III) acid chlorides.
Thus, we aimed at applying our own approach towards addressing these issues, particularly in conjunction with
our long-lasting efforts for exploring the application of 4,4-diethoxybutan-1-amine derivatives to 2-substituted
pyrrolidines synthesis. Our preliminary studies indicate31 that the reaction of some N-(4,4-diethoxybutyl)ureas
with chlorodiphenylphosphine affords diphenyl(N-carbamoylpyrrolidin-2-yl)phosphine oxides with reasonable
yields. The reaction presumably proceeds through in situ iminium ion formation and subsequent Arbuzov-type
phosphorylation. Herein, we describe a significant expansion of the scope of this reaction to other 4,4-
diethoxybutan-1-amine derivatives and application of this methodology to the synthesis of novel N-substituted
2

2-phosphorylpyrrolidines (Scheme 1, B). The proposed approach features advantages of easily available
reagents, simple one-pot procedure and mild reaction conditions. It avoids protecting group introduction-
removal stages and provides a straightforward entry to target compounds. Additionally, the synthesized
compounds were evaluated to establish their in vitro anti-tumor activity, providing encouraging preliminary
results in case of some derivatives.
Results and discussion
We started our studies by using chlorodiphenylphosphine and 1-(4,4-diethoxybutyl)-3-phenylurea 1a as the
model substrates. Our preliminary results indicated that N-(4,4-diethoxybutyl)ureas react smoothly with
chlorodiphenylphosphine at room temperature in dry chloroform in the presence of 1.1 equivalent of acetic
acid. Increasing the reaction temperature resulted in lower yield of pyrrolidine derivative (Table 1, entry 2).
Surprisingly, lowering the amount of acetic acid still afforded target pyrrolidine 3a in 68% yield (Table 1, entry
3). Notably, a yield was almost not affected by the acid amount. Moreover, the reaction proceeded without
any acid additive to afford compound 3a in even higher yield (Table 1, entry 4). However, the difference in
yields was only 8% (Table 1, entries 1 and 4). Taking this into account, all subsequent experiments were carried
out both with and without acid additive, and the best yield is reported for each case. Pleasingly, no tedious
workup procedure or column chromatography was required for product isolation and simple evaporation of
solvent and washing with diethyl ether was enough.
Table 1. Optimization study of the reaction of 1-(4,4-diethoxybutyl)-3-phenylurea 1a with
chlorodiphenylphosphine.
Entry AcOH (eq) Temperature Yield of 3a, %
1
2
3
4
1.1
1.1
0.5
0
r.t.
reflux
r.t.
69
53
68
77
r.t
With this in hand, we further explored the scope of the reaction by employing different N-(4,4-
diethoxybutyl)ureas with aryl- and alkyl substituents as shown in Scheme 2. Starting ureas were obtained
either via reaction of isocyanates or carbamoyl chlorides with 4,4-diethoxybutane-1-amine or by treatment of
appropriate amine with CDI followed by addition of 4,4-diethoxybutane-1-amine, as described previously.32
Ureas with both electron-donating and electron-withdrawing substituents at p-position of aryl moiety provided
target 2-phosphorylpyrrolidines 3b-e in moderate to high isolated yield. The highest yield (78%) was observed
in case of urea 1f possessing electron-withdrawing cyano group, whereas electron-donating methoxy group
lowered yield of product 3b to 47%. Ureas possessing halogen at the aromatic fragment, as well as
unsubstituted phenyl ring provided appropriate 2-phosphorylpyrrolidines in intermediate yields. Introducing
one more phenyl group at the nitrogen atom increased a yield of compound 3f a bit, however, this effect was
not pronounced. Urea 1g with alkyl substituent also behaved well, affording target pyrrolidine 3g in moderate
yield. Overall, ureas 1 tend to provide a bit higher yields (≈3-9%) of target pyrrolidines 3 in the presence of
acetic acid, except compounds 3e,g.
Employing more sterically demanding chlorobis(3,5-dimethylphenyl)phosphine as phosphoryl group source
also resulted in 2-phosphorylpyrrolidine 3h, however, in lower yield compared to a chlorodiphenylphosphine .
Unsymmetrical chloroethylphenylphosphine reacted similarly. Due to the presence of asymmetric tertiary
carbon and tetracoordinated phosphorus atoms pyrrolidine 3i was obtained as 1 : 3 diastereomeric mixture.
Multiple re-crystallizations from acetone afforded single diastereomer with 25% yield. Unfortunately, all the
3

attempts to obtain crystals suitable for x-ray analysis failed, so we were not able to determine exact
configurations of chiral centers.
We also involved chloro(diethylamino)phenylphosphine into a reaction with urea 1a with the aim of obtaining
appropriate amidate derivative. However, no desired product was observed in this case. The reaction resulted
in previously described33,34 bispyrrole 4 formation. We attribute this to a greater basicity of aminophosphines
compared to diarylphosphines and will discuss it in more details below.
EtO OEt
O
O
P
AcOH, CHCl3,
r.t., 24 h
X
X
P
2
+
Y
N
Cl
Y
R
O
N
N
H
R NH
3
H
1
P(O)Ph2
P(O)Ph2
P(O)Ph2
N
O
N
O
3b
47%
N
O
NH
NH
3c
HN
3a
55%
Ph
69%
MeO
Br
P(O)Ph2
P(O)Ph2
P(O)Ph2
N
O
N O
3d
52%
3e
78%a
N
O
NH
N
NH
Ph
Ph
3f
NC
F
73%
P(O)Ph2
O
P
Et
O
3h
47%
N
O
Ph
N
P
O
N
NH
3g
45%a
HN
Ph
O
3i
25%
HN
Ph
H
N
N
N
Ph
O
Ph
4
O
N
H
isolated yield is given;
a reaction was carried out without acid additive
Scheme 2. Reaction scope with N-(4,4-diethoxybutyl)ureas 1 and P (III) acid chlorides
Next, we tried N-(4,4-diethoxybutyl)-4-methylbenzenesulfonamide 5a in the reaction with
a
chlorodiphenylphosphine in the presence of acetic acid (Scheme 3). Albeit the desired 2-phosphorylpyrrolidine
7a was obtained, the yield was rather low. Subsequent studies revealed that the best yield is achieved when a
reaction is carried out without acetic acid additive and 1-tosyl-2-ethoxypyrrolidine 6a is taken instead of
sulfonamide 5a. It should be emphasized that both N-(4,4-diethoxybutyl)sulfonamides 5 and 2-
ethoxypyrrolidines 6 can easily be obtained in one-step and one-pot manner from 4,4-diethoxybutan-1-amine
and appropriate sulfonyl chloride35 (see Supporting information for additional details on compounds 5a and 6a
synthesis). Thus, no additional stages are introduced by this replacement.
Ph2P-Cl (2a)
AcOH, CHCl3,
rt, 24 h
Ph2P-Cl (2a)
OEt
OEt
CHCl3,
Ph
O
rt, 24 h
P
EtO
Ts
Ph
N
N
67%
HN
35%
Ts
Ts
5a
6a
7a
4

Scheme 3. Reaction of sulfonylamide 5a and 2-ethoxypyrrolidine 6a with Ph2PCl
Further, we explored the scope of 1-sulfonyl-2-ethoxypyrrolidines 6 (Scheme 4). Yields of target 1-sulfonyl-2-
phopshorylpyrrolidines varied somewhat. In contrast to ureas 1, in case of compounds 6 higher yields were
achieved without acid additive. The exceptions are compounds 7h-j. The difference in all cases was not big,
though (≈5-10%). No direct correlation between substituents at a sulfur atom and a yield of desired compound
was observed. Thus, both aromatic substituents, such as p-methylphenyl and p-chlorophenyl, and aliphatic
ones (methyl, ethyl) provided pyrrolidines 7a,c and 7f,g with similar yields (63-67%). A presence of
polyaromatic and heteroaromatic substituents lowered yields of compounds 7e and 7i to about 50%. Just as in
case of ureas 1, sterically hindered bis(3,5-dimethylphenyl)chlorophosphine resulted in lower yield of 2-
phosphorylpyrrolidine 7j compared to 7b (58 vs 86%). The lowest yields were observed with 3-chloropropyl
and phenyl substituents (pyrrolidines 7h and 7d). Considering a significantly higher solubility of these
compounds, we attribute this to losses during purification rather than effects of substituents.
Scheme 4. Reaction scope with 1-sulfonyl-2-ethoxypyrrolidines 6 and P (III) acid chlorides
The structure of compounds 3a,b,h, and 7a,d,g was confirmed by x-ray analysis (see Supporting information for
additional details). The diphenylphosphoryl-pyrrolidine fragment in crystals of studied compounds has the
same geometry, only slightly differing in the orientation of phenyl rings (Figure 2). It is interesting to note that
the non-rigid five-membered cycle retains its conformation in all 6 studied crystals, and only in 7a it is
disordered at two positions.
5

Figure 2. The molecular structure of compounds 3a and 7d in crystals. Ellipsoids are shown with 50%
probability.
A plausible reaction mechanism in the presence of acetic acid includes the initial interaction of
chlorodiphenylphosphine with acetic acid to produce acetoxy derivative A (Scheme 5). Simultaneous acid-
promoted cyclization of N-(4,4-diethoxybutyl)urea results in intermediate 2-ethoxypyrrolidine 6 through
oxonium ion B. Notably, this cyclization is a reversible process in acidic media, according to our previous
studies.35 The easy ring opening in 2-ethoxypyrrolidine 6 may be attributed to the presence of electron-
donating ethoxy group, which stabilizes cationic intermediate B. This is further supported by acid-mediated
ring opening of N-carbamoyl- and N-sulfonyl-2-arylpyrrolidines,36–38 where aryl substituent also stabilizes
intermediate open-chain carbocation. Subsequent protonation of ethoxy group of compound 6 and elimination
of ethanol molecule yields iminium ion C. Taking into account the possibility of backward addition of ethanol to
iminium ion,39,40 this step should also be considered as reversible. Further reaction of iminium ion C with
acetoxy derivative A leads to target 2-phosphorylpyrrolidines 3,7. In fact, this is conventional two-stage
Arbuzov reaction. The first stage is a reversible addition of P-nucleophile A to cationic carbon atom leading to
quasi-phosphonium salt D.41–43 The second stage is an irreversible rearrangement of intermediate D to target 2-
phosphorylpyrrolidines 3,7 driven by stable P=O double bond formation.
Alternatively, N-(4,4-diethoxybutyl)urea may react with chlorodiphenylphosphine resulting in α-chloroether F.
Although this process is not described for phosphorus acid chlorides, similar reaction of acyl chlorides is well
known and used for α-haloether synthesis.44,45 Thus formed α-chloroether is highly reactive species and
undergo easily intramolecular cyclization to give 2-ethoxypyrrolidine 6. Next reaction stages are same for both
pathways, except that phosphineous acid ethyl ester serves as P-nucleophile.
In case of (diethylamino)phenylchlorophosphine the intermediate acetoxy/alkoxy derivative A/E exhibits
enhanced basicity due to mutual influence of phosphorus and nitrogen atoms.46 This leads to its protonation
and prevents its reaction with iminium ion C. Thus, a deprotonation / self-condensation of pyrrolinium cation C
according to previously described mechanism33 becomes a major reaction pathway, which affords bispyrrole
derivative 4 (see Supporting information for plausible mechanism of bispyrrole 4 formation).
6

R = Ac or Et
Cl
Ph
Ph OAc
P
AcOH
- HCl
P
Ph
A,E
Ph
2a
Ph
A
R
P
Ph
O
OEt
H
H
OEt
OEt
OEt
X
- EtOH
- H
- EtOH
H
N
X
N
6
N
X
NH
C
1
B
X
Ph2PCl
- HCl
OEt
Ph
O
Ph
O
Ph
Ph
Cl
R
P
Ph
P
Ph
NH
P
X
F
N
OEt
E
N
X
X
- ROEt
R = Ac or Et
D
3,7
EtO
Scheme 5. Proposed reaction mechanism.
As seen from the above mechanism, an intramolecular cyclization of starting 4,4-diethoxybutan-1-amine
derivative is a key step of the reaction. A nucleophilicity and a basicity of a nitrogen atom play a crucial role in
this process. Obviously, its nucleophilicity must be high enough for a reaction to proceed. On the other hand,
its basicity should be low to prevent its protonation in the presence of acid at least partially. Thus, the role of
substituent at the nitrogen atom is to keep a required balance between these two characteristics. We
envisioned that electron-withdrawing substituents other than sulfonyl and carbamoyl may also satisfy these
criteria.
Thus, we attempted to further expand N-substituents scope by employing (4,4-diethoxybutyl)phosphoramidate
8 into the reaction with diphenylchlororphosphine. A starting phosphoramidate 8 was obtained by
conventional procedure from 4,4-diethoxybutan-1-amine and appropriate P (V) acid chloride. Pleasingly, the
reaction proceeded smoothly to afford diphosphoryl derivative 9 in 67% yield. The presence of
phosphoramidate and phosphine oxide fragments was straightforwardly confirmed by 31P NMR spectrum, in
3
which two doublets were observed at 32.8 and 0.8 ppm with P-P coupling constant JPP = 21.3 Hz. Notably, no
N-phosphoryl phosphaproline derivatives were reported until now and this reaction may serve as an easy entry
to such compounds.
Scheme 6. Synthesis of diphosphorylpyrrolidine 9
Next, a cytotoxicity of some obtained N-substituted 2-phosphorylpyrrolidines against normal and tumor human
cell lines was tested at concentrations of 1 - 100 µM. Cytotoxic effects of the test compounds on cells were
estimated by means of the multifunctional Cytell Cell Imaging system. Table 2 shows the IC50 data for
compounds 3,7 and 9 and reference drugs Tamoxifen and Doxorubicin. As seen from the table, compounds 3b-
e, 7a-d,f,j and 9 exhibit comparable cytotoxicity against M-Hela tumor cell line (IC50 ≈ 50 µM). A cytotoxicity of
N-sulfonyl-2-phosphorylpyrrolidines 7e,g (Table 2, entries 11,13) is considerably lower, whereas compound 7h
(Table 2, entry 14) was inactive in this concentration range. Thus, a presence of alkyl chain or polyaromatic
7

substituent at a sulfur atom lowers a cytotoxicity of N-sulfonylpyrrolidines 7 significantly. The 2-
phosphorylpyrolidine-1-carboxamide 3i possessing ethylphenylphosphoryl moiety appeared to be the most
active one (Table 2, entry 6). Its cytotoxicity against M-Hela and MCF-7 tumor cell lines is comparable to that of
reference drug Tamoxifen, whereas cytotoxicity against normal cells is twofold lower. Notably, the activity of
the most of tested compounds against tumor cells is approximately twofold lower than the activity of
Tamoxifen. At the same time, they show a complete absence of toxic effects against Chang liver cell line in used
concentration range.
Table 2 The cytotoxicity of 2-phosphorylpyrrolidines 3,7 and 9.a
N
IC50 (µM)
Compound
Cell lines
M-Hela
51.0±4.2
51.5±4.4.
54.9±4.6
51.1±4.1
52.1±4.3
MCF-7 Chang liver
1
2
3a
3b
3c
3d
3e
3i
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3
4
5
6
32.8±2.9 34.7±3.1 86.2±7.8
7
7a
7b
7c
7d
7e
7f
50.1±4.1
56.2±4.7
50.1±4.2
52.1±4.4
63.3±5.5
50.2±4.2
79.4±6.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
91±8.8
>100
>100
>100
>100
>100
>100
>100
8
9
10
11
12
13
14
15
16
7g
7h
7j
56.1±4.8
53.8±4.5
9
Tamoxifen 28.0±2.5 25.0±2.2 46.2±3.5
Doxorubicin 3.0±0.2
3.0±0.1
3.0±0.1
a Experiments were repeated three times
Conclusions
In conclusion, we report a novel method for the synthesis of N-substituted 2-phosphorylpyrrolidines from 4,4-
diethoxybutan-1-amine derivatives and P (III) acid chlorides. The proposed approach benefits from easily
available starting materials, simple reaction and work-up procedures with moderate to high yields and provides
a convenient one-pot entry to target compounds. In vitro cytotoxicity studies indicate that obtained
compounds inhibit M-Hela tumor cells growth at about 50 µM concentration, while Chang liver normal cells are
not affected. The activity of the most potent compound against M-Hela and MCF-7 tumor cells is comparable
to that of drug Tamoxifen, whereas its cytotoxicity towards normal cells is twofold lower. These findings render
N-substituted 2-phosphorylpyrrolidines prominent candidates for further studies. Extension of proposed
strategy to a greater library of 2-phosphorylpyrrolidines and exploration of their anti-cancer activity is
underway.
Acknowledgments
8

The work was supported by a grant from the President of the Russian Federation for support of young Russian
scientists (MD-585.2019.3). The authors are grateful to the Assigned Spectral-Analytical Center of FRC Kazan
Scientific Center of RAS for technical assistance in research. The x-ray measurements were performed using
shared experimental facilities supported by IGIC RAS state assignment.
Supplementary Information
Representative procedures for a synthesis of starting compounds, experimental details and full characterization
data for synthesized 2-phosphorylpyrrolidines, x-ray data and copies of NMR spectra.
Experimental
Chemistry
General. 13C NMR spectra were recorded on Bruker Avance 600 (working frequency 150.9 MHz) spectrometer;
1H NMR spectra were recorded on Bruker Avance 400 (working frequency 400.1 MHz) spectrometer in (CD3)2SO
and CDCl3 relative to the residual solvent protons. MALDI-TOF mass spectra were recorded on a Bruker
ULTRAFLEX III TOF/TOF instrument (with 2,5-dihydroxybenzoic acid matrix) instrument. IR spectra were
obtained with a Bruker Vector 22 spectrometer. Elemental analysis was performed on Carlo Erba EA 1108
instrument. Melting points were determined in glass capillaries with a Stuart SMP 10 apparatus. All solvents
were purified and dried according to standard procedures. N-(4,4-diethoxybutyl)ureas 147 and N-sulfonyl-2-
ethoxpyrrolidines 635 were obtained according to known procedures. The X-Ray diffraction data for crystals of
compounds 3a,b,h, and 7a,d,g were collected at 150K on a Bruker AXS Smart Apex II CCD diffractometer in the
ω and φ-scan modes using graphite monochromated MoKα (λ 0.71073Å) radiation. The structure was solved by
direct method and refined by the full matrix least-squares using SHELXTL48 program. All non-hydrogen atoms
were refined anisotropically. The positions of hydrogen atoms were located from the Fourier electron density
synthesis and were included in the refinement in the isotropic riding model approximation. All figures were
made using OLEX2.49
General method of synthesis of 2-phosphorylpyrrolidines 3,7,9 (A). To a solution of urea 1 (1.5 mmol) or 2-
ethoxypyrrolidine 6 (1.5 mmol) or phosporamidite 8 and chlorophosphine 2 (1.5 mmol) in dry chloroform (10
mL) acetic acid (0.1 mL, 1.75 mmol) was added. A reaction mixture was stirred at room temperature for 24
hours. Then a solvent was removed, residue thoroughly washed with diethyl ether (3x10 mL) and dried in
vacuum to afford target compound as white solid.
General method of synthesis of 2-phosphorylpyrrolidines 3,7 (B). A solution of urea 1 (1.5 mmol) or 2-
ethoxypyrrolidine 6 (1.5 mmol) and chlorophosphine 2 (1.5 mmol) in dry chloroform (10 mL) was stirred at
room temperature for 24 hours. Then a solvent was removed, residue thoroughly washed with diethyl ether
(3x10 mL) and dried in vacuum to afford target compound as white solid.
2-(Diphenylphosphoryl)-
N
-phenylpyrrolidine-1-carboxamide (3а). Yield 69% (0.40 g), method A; m.p. 183оС;
IR (KBr, ν, cm–1): 1349, 1440, 1597, 1655, 2863, 3058; H NMR (400 MHz, DMSO-d6) δ (ppm) 1.72-1.91 (m, 2H,
CH2), 2.00-2.19 (m, 2H, CH2), 3.22-3.32 (m, 1H, CH2), 3.50-3.59 (m, 1H, CH2), 5.20-5.28 (m, 1H, CH), 6.91 (t, 1H, J
= 7.3 Hz, Ar-H), 7.19 (t, 2H, J = 7.9 Hz, Ar-H), 7.30 (d, 2H, J = 7.6 Hz, Ar-H), 7.44-7.55 (m, 3H, Ar-H), 7.56-7.66 (m,
3H, Ar-H), 7.78-7.86 (m, 2H, Ar-H), 7.88-7.96 (m, 2H, Ar-H), 8.84 (s, 1H, NH); 13C NMR (151 MHz, DMSO-d6) δ
(ppm) 24.74, 26.96, 47.93, 57.08 (d, J = 81.9 Hz), 119.82, 122.24, 128.68, 128.76, 129.21 (d, J = 10.6 Hz), 131.82
(d, J = 18.1 Hz), 131.83, 132.40 (d, J = 49.9 Hz), 140.62, 155.39. 31P NMR (161.9 MHz, DMSO-d6) δ (ppm) 32.46.
Found (%): C, 71.01; H, 6.14; N, 7.46; P, 8.23. Calc. for C23H23N2O2P (%): C, 70.76; H, 5.94; N, 7.18; P, 7.93.
MALDI-TOF (m/z): 391 [M + H]+, 413 [M + Na]+.
1
9

Biological studies
Cytotoxicity assay. Cytotoxic effects of the test compounds on human cancer and normal cells were estimated
by means of the multifunctional Cytell Cell Imaging system (GE Health Care Life Science, Sweden) using the Cell
Viability Bio App which precisely counts the number of cells and evaluates their viability from fluorescence
intensity data. Two fluorescent dyes that selectively penetrate the cell membranes and fluoresce at different
wavelengths were used in the experiments. A low-molecular-weight 4′,6-diamidin-2-phenylindol dye (DAPI) is
able to penetrate intact membranes of living cells and color nuclei in blue. High-molecular propidium iodide
dye penetrates only dead cells with damaged membranes, staining them in yellow. As a result, living cells are
painted in blue and dead cells are painted in yellow. DAPI and propidium iodide were purchased from Sigma.
The M-Hela clone 11 human, epithelioid cervical carcinoma, strain of Hela, clone of M-Hela from the Type
Culture Collection of the Institute of Cytology (Russian Academy of Sciences) and Chang liver cell line (Human
liver cells) from N. F. Gamaleya Research Center of Epidemiology and Microbiology were used in the
experiments. The cells were cultured in a standard Eagle’s nutrient medium manufactured at the Chumakov
Institute of Poliomyelitis and Virus Encephalitis (PanEco company) and supplemented with 10% fetal calf serum
and 1% nonessential amino acids. The cells were plated into a 96-well plate (Eppendorf) at a concentration of
100000 cells/mL, 150 μL of medium per well, and cultured in a CO2 incubator at 37°C. Twenty-four hours after
seeding the cells into wells, the compound under study was added at a preset dilution, 150 μL to each well. The
dilutions of the compounds at concentrations of 1 - 100 µM were prepared immediately in nutrient media; 5%
DMSO that does not induce the inhibition of cells at this concentration was added for better solubility. The
experiments were repeated three times. Intact cells cultured in parallel with experimental cells were used as a
control.
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12

A novel method of synthesis of N-substituted 2-phopshorylpyrrolidines is developed
Some of obtained 2-phopshorylpyrrolidines inhibit M-Hela and MCF-7 tumor cells growth
In contrast to Tamoxifen and Doxorubicin tested compounds do not affect normal cells

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: