The synthesis and characterization of tetramic acid derivatives as Mdm2-p53 inhibitors

Article abstract of DOI:10.1016/j.molstruc.2019.03.089

We present syntheses, prediction of tautomer forms and activities of the second generation of the Mdm2-p53 inhibitors that are based on the tetramic acid scaffold. The inhibitors do not contain 6-chloroindole. Binding of these compounds to Mdm2 was checked by two orthogonal methods: the fluorescence polarization and the ¹H-¹⁵N HSQC NMR titration experiments. We discovered that the 3-phenylthio-substituted tetramic acid derivatives exist in solution solely in their enol forms which is in contrast to the similar 3-aliphatic substituted derivatives. The inhibitory (K_i) and dissociation (K_D) constants are in low micromolar ranges with the best binding compound 9a having K_D = 2.9 μM. Furthermore, our data show that the compounds indeed bind to the p53-binding pocket of Mdm2 and do not cause dimerization of Mdm2. The current work provides solid base for further rational design of the Mdm2/p53 inhibitors.

Full text of DOI:10.1016/j.molstruc.2019.03.089

Accepted Manuscript
The synthesis and characterization of tetramic acid derivatives as Mdm2-p53
inhibitors
Damian Muszak, Beata Łabuzek, Mateusz Z. Brela, Aleksandra Twarda-Clapa,
Miroslawa Czub, Bogdan Musielak, Ewa Surmiak, Tad A. Holak
PII:
S0022-2860(19)30374-6
DOI:
https://doi.org/10.1016/j.molstruc.2019.03.089
MOLSTR 26358
Reference:
To appear in: Journal of Molecular Structure
Received Date: 30 January 2019
Revised Date: 22 March 2019
Accepted Date: 28 March 2019
Please cite this article as: D. Muszak, B. Łabuzek, M.Z. Brela, A. Twarda-Clapa, M. Czub, B. Musielak,
E. Surmiak, T.A. Holak, The synthesis and characterization of tetramic acid derivatives as Mdm2-p53
inhibitors, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.03.089.
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ACCEPTED MANUSCRIPT
The synthesis and characterization of tetramic acid
derivatives as Mdm2-p53 inhibitors
Damian Muszak,† Beata Łabuzek,† Mateusz Z. Brela,‡ * Aleksandra Twarda-Clapa,§
Miroslawa Czub,† Bogdan Musielak,† Ewa Surmiak,†* Tad A. Holak†
† Faculty of Chemistry, Department of Organic Chemistry, Jagiellonian University, Gronostajowa 2, 30-387
Krakow, Poland
‡ Faculty of Chemistry, Department of Physical Chemistry, Jagiellonian University, Gronostajowa 2, 30-387
Krakow, Poland
§ Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387
Krakow, Poland
Corresponding Author
*E-mail: ewa.surmiak@uj.edu.pl, brela@chemia.uj.edu.pl
KEYWORDS
Mdm2-p53 inhibitors, tetramic acid, DFT, tautomerism
HIGHLIGHTS
•
•
Synthesis and activity of the tetramic acid core inhibitors are presented.
1
Binding to Mdm2 was tested by two orthogonal methods: the FP assay and the H-
15N HSQC titration experiments.
•
The tautomeric effect of the 3-phenylthio-substituted tetramic acids were studied by
spectroscopic and DFT methods and showed that they exist in solution in a usually
not-preferred enol form.

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ABSTRACT
We present syntheses, prediction of tautomer forms and activities of the second generation of
the Mdm2-p53 inhibitors that are based on the tetramic acid scafolld. The inhibitors do not
contain 6-chloroindole. Binding of these compounds to Mdm2 was checked by two
1
orthogonal methods: the fluorescence polarization and the H-15N HSQC NMR titration
experiments. We discovered that the 3-phenylthio-substituted tetramic acid derivatives exist
in solution solely in their enol forms which is in contrast to the similar 3-aliphatic substituted
derivatives. The inhibitory (Ki) and dissociation (KD) constants are in low micromolar ranges
with the best binding compound 9a having KD = 2.9 ꢀM. Furthermore, our data show that the
compounds indeed bind to the p53-binding pocket of Mdm2 and do not cause dimerization of
Mdm2. The current work provides solid base for further rational design of the Mdm2/p53
inhibitors
.
1. Introduction
The p53 tumor suppressor protein is inactivated or mutated in nearly all types of human
cancers [1-3]. Restoration of the p53 activity should in principle be a promising strategy
against cancer. The Mdm2 protein is an E3 ubiquitin ligase and a main negative regulator of
the p53 tumor suppressor protein. p53 is inactivated by mutations in over 50% of all cancers.
Most of the remaining malignancies have the wild-type p53 (wt-p54) present but this p53 is
deactivated by overexpression of p53-regulatory proteins, such as Mdm2 and MdmX [2-6]. In
such cases, the restoration of wt-p53 activity is possible by introducing small-molecule
inhibitors, which by binding to Mdm2, can liberate wt-p53 from the Mdm2/p53 complex
[2,4]. Therefore, a low-molecular-weight antagonist could inhibit or reverse tumor formation
and provide a non-genotoxic anticancer therapy [5,6]. The vast majority of the small-molecule
Mdm2 inhibitors are designed using a three-finger-pharmacophore-model, based on the triad
of p53 amino acids: Phe19, Trp23 and Leu26, which inserts deeply into the binding pocket of
Mdm2 [7-9]. Nevertheless, recently several new approaches were proposed which exploit
additional induced pockets-[10-11], dual inhibitors [12,13] or dimerization [14-15].
In dimerization of Mdm2 by 1,5-dihydro-2H-pyrrol-2-ones and 1,5-dihydro-2H-furan-2-
ones described by us, the 6-chloroindole moieties point outside the p53 binding pockets of
Mdm2 [15]. In order to change the physicochemical properties and binding mode of these
compounds, we have decided to obtain a second generation of substituted 1,5-dihydro-2H-

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pyrrol-2-ones (Fig. 1). First and foremost, we decided to replace the 6-chloroindole moiety in
the new scaffold, as there is no inhibitor in the clinical trials with this moiety [8,16]. Even
though the 6-chloroindole usually interacts with the Trp23 pocket of Mdm2, in our 1,5-
dihydro-2H-pyrrol-2-one molecules it caused protein dimerization, which now we wanted to
avoid. Another highly important aspect of our study was shortening and simplifying the
compound preparation. For that reason we decided to proceed with a new scaffold that
comprises the tetramic acid core, which is both capable of providing suitable substituents
arrangement and could be easily synthesized.
Figure 1. Structure of the scaffold based on the 1,5-dihydro-2H-pyrrol-2-one inhibitor (I) and its second
generation (IA). (Numbering of atoms in the central ring of tetramic acid is shown in blue)
The tetramic acid core possessing compounds (2,4-pyrrolidinedione) are of interest of
researchers both due to their biological functions, unusual physical properties and challenging
synthesis. The tetramic acid ring can be found in many naturally occurring products with
biological functions, mostly antibiotic and antiviral [17-18]. Naturally occurring tetramic acid
derivatives are most commonly isolated from marine species and possess a 3-acyl moiety and
5C chirality originating from the amino acid used in their biosynthesis. Examples of such
compounds are: the antibacterial Ravenic acid isolated from Penicillium [19], the antiviral
Trichobotysin A from Trychobotrys effuse [20], the anticancer Cylindramid from the marine
sponge Halichondria cylindrata [21], the cytotoxic Palau’imide from cyanobacteria Lyngbya
[22], an anti-inflammatory lipoxygenase inhibitor Tetrapetalone A from Streptomyces sp.
USF-4727 [23] or the antifungal dihydromaltophilin from Lascobacter enzymogenes [24].

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Tetramic acid can be found in both the ketone and enol states. Traditionally it is often
presented as an enolic derivative, due to its structural similarity to the highly acidic oxygen
analogue - tetronic acid. Although, this is the preferred form only when the compound
structure contains the 3-acyl moiety – in which case four tautomers interconvert slowly (Fig.
S1) [25]. In these compounds, the 4-hydroxyl group is acidic and can be deprotonated even in
physiological conditions, which makes them a perfect chelator for a variety of metals [26].
Moreover, in some natural products the enol form is trapped by the 4-O alkyl ether group, like
for example in the Palau’imide. Even though the enol form of tetramic acid derivatives can be
stable, in most cases the ketone form is predominant which is reflected in their significantly
reduced acidity (pKa≈6.4) [27] when compared to the oxygen-based tetronic acids. The keto-
enol tautomerization equilibrium depends on the various factors, like solvent properties, and
therefore can be shifted toward one of possible forms. Taken together, the tetramic acid
properties and structural identification can be difficult to predict due to the presence of several
tautomeric forms in solution. The knowledge of the compound structure, especially in
aqueous solutions is crucial to their biological activity. Herein we present synthesis, structural
characterization and the Mdm2 binding of a series of 3-phenylthio substituted tetramic acid
derivatives.
2. Experimental and computational details.
2. 1. Synthesis
The compounds were synthesized as depicted in Schemes 1 and 2. Flash chromatography
was performed with a Reveleris® X2 Flash Chromatography, using Grace® Reveleris Silica
flash cartridges. Monitoring of the reactions was carried out using the silica gel TLC plates
silica Merck 60 F254. Spots were visualized by UV light at 254 and 365 nm. All NMR spectra
were recorded on a Bruker Avance 600 MHz. Chemical shifts for 1H NMR were reported as δ
values and coupling constants were in Hertz (Hz). The following abbreviations were used for
spin multiplicity: s = singlet, br = broad, d = doublet, t = triplet, q = quartet, quin = quintet, m
13
= multiplet, sept = septet. Chemical shifts for C NMR were reported in δ calibrated to the
solvent peak. IR absorption spectra were recorded on a Nicolet IR200 spectrometer using
ATR technique. HRMS were carried out by the Laboratory for Forensic Chemistry Faculty of
Chemistry, Jagiellonian University with the microOTOF-QII spectrometer using ESI
ionization technique. The UPLC-MS/MS system consisted of a Waters ACQUITY® UPLC®
(Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer
(electrospray ionization mode ESI-tandem quadrupole). Chromatographic separations were

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carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm,
and 1.7 µm particle size, equipped with Acquity UPLC BEH C18 VanGuard pre-column;
2.1 × 5 mm, and 1.7 µm particle size. The column was maintained at 40°C, and eluted under
gradient conditions using from 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 mL
min-1. Eluent A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v).
The purity of all final compounds, determined using chromatographic LC-MS, was >95%. All
reagents were obtained from commercial sources and used without further purification unless
otherwise stated. Anhydrous solvents were purchased from Sigma-Aldrich or Alfa-Aesar,
anhydrous Et2O and THF were distilled from sodium-benzophenone and stored over
molecular sieves (4Å, 3-5 mm beads).
Scheme 1. Synthesis of compounds 2a-b, 4a-c, and 6a-d. Reagents and conditions: (a) 1a-b (1 eq.), TBAB (0.01
eq), NaOH (2 eq.) bromoacetic acid (1 eq.), THF/H2O (1/2), 0°C to RT, 16 h, yield 90-91%; (b) 3a-c (1 eq.),
SOCl2 (1.5 eq), anh. MeOH, 0°C to RT, 16 h, yield 80-100%; (c) (i) 4a (1 eq.), Et3N (1.1 eq), anh MeOH, RT,
15 min. (ii) 5a-d (1.1 eq), NaBH3CN (1 eq), RT, 16 h, yield 60-79%.
2.2. General synthetic procedure for preparation and analytical data of 2-
(phenylthio)acetic acid derivatives (2a-b)
The corresponding thiophenol (1a-b) (1 eq.), tetrabutylammonium bromide (TBAB) (0.01
eq.) and THF/H2O (1:2 mixture) were placed in a round bottom flask. The reaction mixture
was cooled to 0ºC then NaOH (2 eq.) and bromoacetic acid (1 eq.) were added. The reaction
was stirred at RT overnight (16 h). After this time diethyl ether (20 ml) was added and the
reaction was poured into 1M HCl solution (30 ml) and then extracted with diethyl ether (3 x
20 ml). Organic layers were collected, washed with brine, dried over anhydrous MgSO4 and
evaporated.
2.2.1. 2-(Phenylthio)acetic acid (2a)

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Thiophenol (1a) (2.05 ml, 20.0 mmol), TBAB (0.08 g 0.2 mmol), bromoacetic acid (2.78 g,
20.0 mmol), NaOH (1.60 g, 40.0 mmol) and THF/H2O (10/20 ml) were used. The crude
product was precipitated and washed with petroleum ether giving compound 2a with 91%
yield (3.36 g) as colorless solid. The product was recrystallized from water. The compound is
reported in the literature [28].
IR (ATR): [cm-1]: 3500-2500 (br), 3055, 2903, 2696, 2588, 1704, 1482, 1441, 1427, 1392,
1314, 1198, 900, 736; NMR: 1H (600 MHz, DMSO-d6): δ [ppm] 12.75 (s, 1H), 7.35-7.30 (m,
13
4H), 7.19 (tt, J = 7.1, 1.0 Hz, 1H), 3.79 (s, 2H); C (151 MHz, DMSO-d6): δ [ppm] 170.6,
135.7, 129.0, 127.7, 125.9, 34.9; LC-MS (DAD/ESI): tr = 4.48 min, Calcd for C8H8O2 (m/z):
[M-H]- 167.02, Found: [M-H]- 166.95; HRMS (ESI): Calcd for C8H8O2 (m/z): [M-H]-
167.0167, Found: [M-H]- 167.0161.
2.2.2. 2-((4-Chloro)phenylthio)acetic acid (2b)
4-chlorothiophenol (1b) (2.67 g, 20.0 mmol), TBAB (0.08 g 0.2 mmol), bromoacetic acid
(2.78 g, 20.0 mmol), NaOH (1.60 g, 40.0 mmol) and THF/H2O (10/20 ml) were used. The
crude product was precipitated and washed with petroleum ether giving compound 2b with
90% yield (4.10 g) as colorless solid. The product was recrystallized from water. The
compound is reported in the literature [28].
IR (ATR): [cm-1] 3500-2500 (br), 3001, 2921, 2581, 1694, 1478, 1427, 1390, 1305, 1203,
1097, 1010, 889, 812; NMR: 1H (600 Mhz, DMSO-d6) δ [ppm]:12.80 (s, 1H), 7.39-7.34 (m,
4H), 3.82 (s, 2H); 13C (151 Mhz, DMSO-d6) δ [ppm]: 170.4, 134.9, 130.5, 129.5, 128.9, 34.9;
LC-MS (DAD/ESI): tr = 5.39 min, Calcd for C8H7ClO2S (m/z): [M-H]- 200.98 [M+2-H]-
202.98 , Found: [M-H]- 200.90 [M+2-H]- 202.90; HRMS (ESI): Calcd for C8H7ClO2S (m/z):
[M-H]- 200.9777 [M+2-H]- 202.9748 , Found: [M-H]- 200.9771 [M+2-H]- 202.9742.
2.3. General synthetic procedure for preparation and analytical data of amino acid
methyl ester hydrochloride derivatives (4a-c)
The corresponding amino acid (3a-c) (1 eq.) and anhydrous methanol were placed in round
bottom flask. The reaction mixture was cooled to 0ºC then thionyl chloride (1.5 eq.) was
added dropwise. The reaction was stirred at RT overnight (16 h). After this time solvent was
evaporated. To the residual oil another potion of methanol was added (approx. 30 ml) and
evaporated again. This procedure was repeated twice.
2.3.1 Leucine methyl ester hydrochloride (4a)

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L-leucine (3a) (1.31 g, 10 mmol), SOCl2 (1.09 ml, 15.0 mmol) and methanol (20 ml) were
used. The crude product was precipitated from petroleum ether giving compound 4a with
80% yield (1.46 g). The product was recrystallized from ethyl acetate. The optical purity of
the obtained compound was not established. The compound is reported in the literature [29].
1
IR (ATR): [cm-1] 2958, 1848, 1738, 1507, 1452, 1250, 1227, 1044; NMR: H (600 MHz,
DMSO-d6) δ [ppm] 8.66 (s, 3H), 3.92 (t, J = 7.1 Hz, 1H), 3.73 (s, 3H), 1.79-1.72 (m, 1H),
13
1.70-1.60 (m, 2H), 0.89 (d, J = 6.5 Hz, 6H); C (151 MHz, DMSO-d6) δ [ppm] 170.3, 52.7,
50.5, 23.7, 22.1, 22.0 LC-MS (DAD/ESI): tr = 1.06 min, Calcd for C7H15NO2 (m/z): [M+H]+
146.12, Found [M+H]+ 146.15; HRMS (ESI): Calcd for C7H15NO2 (m/z): [M+H]+ 146.1181,
Found [M+H]+ 146.1176.
2.3.2 Phenylalanine methyl ester hydrochloride (4b)
L-phenylalanine (3b) (6.30 g, 38.0 mmol), SOCl2 (4.15 ml, 57 mmol) and methanol (80 ml)
were used. The crude product was precipitated from petroleum ether giving compound 4b
with quantitative yield (8.20 g). The product was recrystallized from ethyl acetate/methanol
mixture. The optical purity of the obtained compound was not established. The compound is
reported in the literature [30].
1
IR (ATR): [cm-1] 2842, 1744, 1583, 1495, 1448, 1241, 1146, 1084; NMR: H (600 MHz,
DMSO-d6) δ [ppm] 8.75 (s, 3H), 7.33 (t, J = 7.3 Hz, 2H), 7.29-7.26 (m, 1H), 7.24 (d, J = 7.0
Hz, 2H), 4.23 (dd, J = 7.4, 5.8 Hz, 1H), 3.65 (s, 3H), 3.21 (dd, J = 14.0, 5.7 Hz, 1H), 3.10 (dd,
13
J = 14.0, 7.5 Hz, 1H); C (151 MHz, DMSO-d6) δ [ppm] 169.5, 134.7, 129.4, 128.6, 127.3,
53.2, 52.5, 35.8; LC-MS (DAD/ESI): tr = 2.19 min, Calcd for C10H13NO2 (m/z): [M+H]+
180.10, Found: [M+H]+ 180.10; HRMS (ESI): Calcd for C10H13NO2 (m/z): [M+H]+
180.1024, Found: [M+H]+ 180.1018.
2.3.3 (4-Chloro)phenylalanine methyl ester hydrochloride (4c)
4-Chloro-DL-phenylalanine (3c) (2.99 g, 15 mmol), SOCl2 (1.63 ml, 22.5 mmol) and
methanol (25 ml) were used. The crude product was precipitated from petroleum ether giving
compound 4c with quantitative yield (3.73 g). The optical purity of the obtained compound
was not established. The compound is reported in the literature [31].
IR (ATR): [cm-1] 2908 (br), 1741, 1550, 1489, 1239, 1148, 1096, 1018, 948, 858, 805;
NMR: 1H (600 MHz, DMSO-d6) δ [ppm] 8.77 (br, 3H), 7.38 (d, J = 7.4 Hz, 2H), 7.29 (d, J =
7.3 Hz, 2H), 4.28-4.21 (m, 1H), 3.67 (s, 3H), 3.23-3.17 (m, 1H), 3.17-3.10 (m, 1H) 13C (151
MHz, DMSO-d6) δ [ppm] 169.2, 133.8, 132.0, 131.4, 128.5, 53.0, 52.6, 34.9; LC-MS

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(DAD/ESI): tr = 3.01 min, Calcd for C10H12ClNO2 (m/z): [M+H]+ 214.06 [M+2+H]+ 216.06,
Found: [M+H]+ 214.06 [M+2+H]+ 216.06; HRMS (ESI): Calcd for C10H12ClNO2 (m/z):
[M+H]+ 214.0635 [M+2+H]+ 216.0605, Found: [M+H]+ 214.0629 [M+2+H]+ 216.0600.
2.4 General synthetic procedure and analytical data for reductive amination
(compounds 6a-d)
Leucine methyl ester hydrochloride (4a) (1.eq) and anhydrous MeOH (excess) were placed in
a round bottom flask equipped with CaCl2 tube, then triethylamine was added (1.1 eq.) and the
reaction was stirred for 15 minutes. After this time suitable aldehyde (5a-d) (1.1 eq.) and
sodium cyanoborohydride (1 eq.) were added respectively. The reaction was stirred at RT for
3 days. After this time solvent was evaporated. To the residual slurry water was added (50 ml)
and the mixture was extracted with ethyl acetate (3 x 20 ml). Organic layers were collected,
washed with water and brine, dried over anhydrous MgSO4 and evaporated. Crude products
were purified by flash chromatography (SiO2, petroleum ether/ethyl acetate 10:1). After
column chromatography compounds were transformed into their hydrochlorides. The
corresponding amine was put into a round bottom flask with excess of 4M HCl in 1,4-dioxane
and stirred for 5 minutes at RT. After this time solvent was evaporated and final product was
precipitated from petroleum ether.
2.4.1. Methyl N-(4-chlorobenzyl)leucinate hydrochloride (6a)
Leucine methyl ester hydrochloride (4a) (2.50 g, 13.8 mmol), 4-chlorobenzaldehyde (5a)
(2.13 g, 15.1 mmol), triethylamine (2.10 ml, 15.1 mmol), NaBH3CN (0.86 g, 13.8 mmol) and
anhydrous methanol (70 ml) were used. Compound 6a was obtained as a colorless solid, with
a 60% yield (2.55 g). The compound is reported in the literature [32].
IR (ATR): [cm-1] 2957, 2648 (br), 1749, 1542, 1472, 1254, 1204, 1093, 1019, 843, 808;
1
NMR: H (600 MHz, DMSO-d6) δ[ppm]: 10.27 (s, 1H), 9.79 (s, 1H), 7.60 (d, J = 8.4 Hz,
2H), 7.50 (dt, J = 8.5 Hz, 2.0, 2H), 4.22 (d, J = 13.0 Hz, 1H), 4.11 (d, J = 13.0 Hz, 1H), 3.94
(dd, J = 9.2, 4.6 Hz, 1H), 3.73 (s, 3H), 1.88-1.81 (m, 1H), 1.77-1.70 (m, 1H), 1.69, 1.62 (m,
1H), 0.88 (d, J = 6.1 Hz, 6H); 13C NMR (151 MHz, DMSO-d6) δ[ppm]: 160.4, 133.9, 132.5,
130.6, 128.6, 57.4, 53.0, 48.2, 37.9, 24.2, 23.0, 21.2; LC-MS (DAD/ESI): tr = 4.33 min,
Calcd for C14H20NO2 (m/z): [M+H]+ 270.13 [M+2+H]+ 272.12, Found [M+H]+ 270.15
[M+2+H]+272.21; HRMS (ESI): Calcd for C14H20NO2 (m/z): [M+H]+ 270.1261 [M+2+H]+
272.1231, Found [M+H]+ 270.1255 [M+2+H]+272.1222.

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2.4.2. Methyl N-(4-methoxybenzyl)leucinate hydrochloride (6b)
Leucine methyl ester hydrochloride (4a) (3 g, 16.5 mmol), 4-methoxybenzaldehyde (5c) (2.20
ml, 18.1 mmol), triethylamine (2.53 ml, 18.1 mmol), NaBH3CN (1,04 g, 16,5 mmol) and
anhydrous methanol (70 ml) were used. Compound 6b was obtained as a colorless solid, with
a 79% yield (3.94 g). The compound is reported in the literature [33].
1
IR (ATR): [cm-1] 3456, 2931, 1586, 1418, 1050, 1008, 923; NMR: H (600 MHz, DMSO-
d6) δ[ppm]: 7.17 (dt, J = 8.7, 2.1 Hz, 2H), 6.84 (dt, J = 8.7, 2.1 Hz, 2H), 3.70 (s, 3H), 3.64 (d,
J = 13.2 Hz, 1H), 3.60 (s, 3H), 3.42 (d, J = 13.2 Hz, 1H), 3.12 (t, J = 6.7 Hz, 1H), 2.23 (s,
2H), 1.67 (sept, J= 6.6 Hz, 1H), 1.42-1.37 (m, 1H), 1.35-1.30 (m, 1H), 0.83 (d, J = 6.7 Hz,
3H), 0.76 (d, J = 6.6 Hz, 3H); 13C (151 MHz, DMSO-d6) δ[ppm]: 175.0, 158.5, 132.6, 113.9,
58.6, 55.3, 51.8, 50.8, 42.4, 26.0, 24.8, 23.2, 22.4; HRMS (ESI): Calcd for C15H23NO3 (m/z):
[M+H]+ 266.1756 Found [M+H]+ 266.1750.
2.4.3. Methyl N-(4-fluorobenzyl)leucinate hydrochloride (6c)
Leucine methyl ester hydrochloride (4a) (1.50 g, 8.3 mmol), 4-fluorobenzaldehyde (5b) (0.95
ml, 9.1 mmol), triethylamine (1.27 ml, 9.1 mmol), NaBH3CN (0.52 g, 8.3 mmol) and
anhydrous methanol (40 ml) were used. Compound 6c was obtained as a colorless solid, with
a 68% yield (1.64 g). The compound is reported in the literature [34].
IR (ATR): [cm-1] 2960, 2727 (br), 1750, 1742, 1604, 1513, 1417, 1227, 1056, 1023, 840,
1
822; NMR: H (600 MHz, DMSO-d6) δ[ppm]: 10.42 (s, 1H), 9.83 (s, 1H), 7.66 (dd, J= 8.7,
5.5 Hz, 2H), 7.27 (tt, J= 8.9, 3.0 Hz, 2H), 4.21 (d, J= 13.0 Hz, 1H), 4.11 (d, J= 13.0 Hz, 1H ),
3.92 (dd, J= 9.5, 4.7 Hz, 1H), 3.73 (s, 3H), 1.90-1.86 (m, 1H), 1.76-1.72 (m, 1H), 1.70-1.64
(m, 1H), 0.88 (d, J= 6.5 Hz, 6H); ; 13C (151 MHz, DMSO-d6) δ [ppm]: 169.8, 163.7-162.1 (d,
JFC = 245.6 Hz), 133.4-133.3 (d, JFC = 8.4 Hz), 128.3, 115.9-115.8 (d, JFC = 21.5 Hz), 57.6,
53.3, 48.5, 38.3, 24.7, 23.4, 21.6; HRMS (ESI): Calcd for C14H20FNO2 (m/z): [M+H]+
254.1556 Found [M+H]+ 254.1539.
2.4.4. Methyl N-(isoquinolin-4-ylmethyl)leucinate hydrochloride (6d)
Leucine methyl ester hydrochloride (4c) (1.06 g, 5.9 mmol), 4-isoquinolinecarboxaldehyde
(5d) (1.02 g, 6.5 mmol), triethylamine (0.90 ml, 6.5 mmol), NaBH3CN (0.37 g, 5.9 mmol)
and anhydrous methanol (40 ml) were used. Compound 6d was obtained as a colorless solid,
with a 62% yield (1.18 g).
IR (ATR): [cm-1] 3267, 3027, 2508 (br), 1569, 1394, 1310, 1219, 1150, 1060, 888; NMR: 1H
(600 MHz, DMSO-d6) δ [ppm]: 10.73 (s, 1H), 10.22 (s, 1H), 9.38 (s, 1H), 9.10 (s, 1H), 8.34

10
ACCEPTED MANUSCRIPT
(d, J = 8.3 Hz, 1H) 8.26 (d, J = 7.9 Hz, 1H), 8.08 (t, J = 7.3 Hz, 1H), 7.89 (t, J = 7.2 Hz, 1H),
4.59 (d, J = 13.0 Hz, 1H), 4.48 (d, J = 13 Hz, 1H), 4.17 (dd, J = 8.8, 4.2 Hz, 1H), 3.76 (s, 3H),
1.96-1.90 (m, 1H), 1.84-1.78 (m, 1H), 1.77-1.69 (m, 1H), 0.89 (d, J = 6.4 Hz, 6H); 13C
(NMR, DMSO-d6) δ [ppm]: 169.3, 148.7, 144.7, 140.8, 133.6, 129.1, 129.0, 127.3, 125.7,
123.6, 57.6, 53.1, 46.0, 37.8, 24.3, 23.0, 21.2; LC-MS (DAD/ESI): tr = 3.60 min, Calcd for
C17H22N2O2 (m/z): [M+H]+ 287.18 Found [M+H]+ 287.23; HRMS (ESI): Calcd for
C17H22N2O2 (m/z): [M+H]+ 287.1760 Found [M+H]+ 287.1754;
2.5. General synthetic procedure for coupling reaction and analytical data (compounds
7a-c and 8-d)
The corresponding amino acid methyl ester hydrochloride 4a-c or 6a-d (1 eq.) and anhydrous
dichloromethane (DCM, 40 ml) were placed in round bottom flask equipped with CaCl2 tube.
The reaction mixture was cooled to 0ºC and triethylamine (1 eq.) was added dropwise and
stirred for 15 minutes. Then suitable derivatives of 2-(phenylthio)acetic acid (2a-b) (1 eq.), 4-
(dimethylamino)pyridine (DMAP, 0.2 eq.) and N,N’-diisopropylcarbodiimide (DIC, 1 eq.)
were added respectively with approx. 5 minutes between each reagent. The reaction was
stirred at 0ºC for 1 hour and then at RT overnight (16 h). After this time, water was added (30
ml) and the reaction mixture was extracted with DCM (3 x 15 ml). Organic layers were
collected, washed with citric acid (10%, 2 x 15 ml), saturated NaHCO3 (2 x 15 ml) and water
(1 x 15 ml), then dried over anhydrous MgSO4 and evaporated.
2.5.1. Methyl (2-((4-chlorophenyl)thio)acetyl)leucinate (7a)
Leucine methyl ester hydrochloride (4a) (0.40 g, 2.2 mmol), triethylamine (0.31 ml, 2.2
mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (0.45 g, 2.2 mmol), DIC (0.34 ml, 2.2 mmol),
DMAP (0.05 g, 0.4 mmol) were used. The crude product was purified by flash
chromatography (SiO2, petroleum ether/chloroform 1:1) giving compound 7a as a colorless
oil with a 73% yield (0.53 g).
IR (ATR): [cm-1] 3275, 2963, 1732, 1669, 1653, 1540, 1480, 1397, 1289, 1256, 1097, 1009,
816; NMR: 1H (600 MHz, CDCl3) δ [ppm]: 7.29-7.25 (m, 4H), 6.99 (d, J = 8.2 Hz, 1H), 4.58
(td, J = 9.0; 5.0 Hz, 1H), 3.69 (s, 3H), 3.67 (d, J = 16.9 Hz, 1H), 3.60 (d, J = 16.9 Hz, 1H),
13
1.63-1.57 (m, 1H), 1.50-1.44 (m, 1H), 1.40-1.30 (m, 1H), 0.84 (d, J = 6.6 Hz, 6H); C (151
MHz, CDCl3) δ [ppm]: 173.0, 167.4, 133.1, 133.1, 130.0, 129.5, 52.5, 51.0, 41.5, 37.7, 24.8,
22.9, 21.8; LC-MS (DAD/ESI): tr = 7.06 min, Calcd for C15H20ClNO3S (m/z): [M+H]+
330.09 [M+2+H]+ 332.09, Found [M+H]+ 330.16 [M+2+H]+ 332.16; HRMS (ESI): Calcd for

11
ACCEPTED MANUSCRIPT
C15H20ClNO3SNa (m/z): [M]+ 352.0750 [M+2]+ 354.0721, Found [M]+ 352.0743 [M+2]+
354.0712.
2.5.2. Methyl (2-(phenylthio)acetyl)phenylalaninate (7b)
Phenylalanine methyl ester hydrochloride (4b) (1.10 g, 5.1 mmol), triethylamine (0.71 ml, 5.1
mmol), 2-(phenylthio)acetic acid (2a) (0.86 g, 5.1 mmol), DIC (0.78 ml, 5.1 mmol), DMAP
(0.12 g, 1.0 mmol) were used. The crude product was purified by flash chromatography (SiO2,
petroleum ether/ethyl acetate 3:1) giving compound 7b as colorless solid with a 81% yield
(1.36 g). Product was recrystallized from cyclohexane/ethyl acetate [35].
1
IR (ATR): [cm-1] 3300, 2915, 1745, 1652, 1533, 1436, 1239, 1212, 1175, 981; NMR: H
(600 MHz, CDCl3) δ[ppm] 7.29-7.26 (m, 2H), 7.24-7.18 (m, 6H), 6.98-6.94 (m, 2H), 4.85 (dt,
J = 8.0, 6.0 Hz, 1H), 3.67 (s, 3H), 3.65-3.57 (m, 2H), 3.09-3.02 (m, 2H); 13C (151 MHz,
CDCl3) 171.6, 167.8, 135.6, 134.6, 129.4, 129.2, 128.7 128.5, 127.3, 126.9, 53.5, 52.4, 37.9,
37.6; LC-MS (DAD/ESI): tr = 6.59, Calcd for C18H19NO3S (m/z): [M+H]+ 330.12, Found
[M+H]+ 330.16; HRMS (ESI): Calcd for C18H19NO3SNa (m/z): [M]+ 352.0983, Found [M]+
352.0979.
2.5.3. Methyl (2-((4-chlorophenyl)thio)acetyl)phenylalaninate (7c)
Phenylalanine methyl ester hydrochloride (4b) (1.07 g, 5.0 mmol), triethylamine (0.70 ml, 5.0
mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (1.01 g, 5.0 mmol), DIC (0.77 ml, 5.0 mmol),
DMAP (0.12 g, 1.0 mmol) were used. The crude product was purified by flash
chromatography (SiO2, petroleum ether/chloroform 1:1) giving compound 7c as colorless
solid with a 72% yield (1.32 g). Product was recrystallized from cyclohexane.
IR (ATR): [cm-1] 3297, 2912, 1745, 1653, 1533, 1478, 1435, 1239, 1212, 1174, 891, 821;
NMR: 1H (600 Mhz, CDCl3) δ [ppm]: 7.23-7.18 (m, 5H), 7.13 (tt, J = 8.7, 2.0 Hz, 2H), 7.11
(d, J = 7.7 Hz, 1H), 6.98 (dd, J = 7.4, 2.0 Hz, 2H), 4.8 (ddd, J = 7.8, 6.5, 5.7 Hz, 1H), 3.70 (s,
3H), 3.57 (s, 2H), 3.11 (dd, 14.0, 5.5 Hz, 1H), 3.04 (14.0, 6.6 Hz 1H); 13C (151 MHz, CDCl3)
δ [ppm]: 171.6, 167.5, 135.6, 133.1, 130.0, 129.5, 129.2, 128.8, 127.3, 55.5, 52.5, 37.8, 37.8;
LC-MS (DAD/ESI): tr = 7.17 min, Calcd for C18H18ClNO3S (m/z): [M+H]+ 364.12
[M+2+H]+ 366.12, Found [M+H]+ 364.07 [M+2+H]+ 366.07; HRMS (ESI): Calcd for
C18H18ClNO3SNa (m/z): [M]+ 386.0594 [M+2]+ 388.0564, Found [M]+ 386.0589 [M+2]+
388.0560.
2.5.4. Methyl 3-(4-chlorophenyl)-2-(2-((4-chlorophenyl)thio)acetamido)propanoate (7d)

12
ACCEPTED MANUSCRIPT
4-Chlorophenylalanine methyl ester hydrochloride (4c) (1.25 g, 5.0 mmol), triethylamine
(0.70 ml, 5.0 mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (1.01 g, 5.0 mmol), DIC (0.77
ml, 5.0 mmol), DMAP (0.12 g, 1.0 mmol) were used. Thecrude product was purified by flash
chromatography (SiO2, petroleum ether/chloroform 1:1) giving compound 7d as colorless
solid with a 69% yield (1.38 g). Product was recrystallized from cyclohexane/acetone.
IR (ATR): [cm-1] 3276, 3074, 1748, 1733, 1667, 1544, 1478, 1377, 1269, 1213, 1178, 1098,
1009, 819, 808; NMR: 1H (600 MHz, CDCl3) δ [ppm]: 7.72-7.23 (m, 2H), 7.18-7.13 (m, 4H),
7.10 (d, J = 7.7 Hz, 1H), 6.90 (d, J = 8.4 Hz, 2H), 4.85-4.79 (m, 1H), 3.71 (s, 3H), 3.63-3.54
(m, 2H), 3.12-3.05 (m, 1H), 3.03-2.97 (m, 1H); 13C (151 MHz, CDCl3) δ [ppm]: 171.3, 167.4,
134.0, 133.2, 133.0, 132.9, 130.4, 129.7, 129.4, 128.8, 53.2, 52.5, 37.5, 37.1; LC-MS
(DAD/ESI): tr = 7.65 min, Calcd for C18H17Cl2NO3S (m/z): [M+H]+ 398.04 [M+2+H]+
400.04, Found [M+H]+ 398.09 [M+2+H]+ 400.08; HRMS (ESI): Calcd for
C18H17Cl2NO3SNa (m/z): [M]+ 420.0204 [M+2]+ 422.0174, Found [M]+ 420.0199 [M+2]+
422.0172.
2.5.5. Methyl N-(4-chlorobenzyl)-N-(2-((4-chlorophenyl)thio)acetyl)leucinate (8a)
Methyl N-(4-chlorobenzyl)leucinate hydrochloride (6a) (1.79 g, 5.8 mmol), triethylamine
(0.82 ml, 5.8 mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (1.18 g, 5.8 mmol), DIC (0.89
ml, 5.8 mmol), DMAP (0.14 g, 1.2 mmol) were used. The crude product was purified by flash
chromatography (SiO2, petroleum ether/chloroform 1:1) giving compound 8a as a colorless
oil with a 61% yield (1.60 g).
IR (ATR): [cm-1] 2955, 2869, 1741, 1652, 1477, 1202, 1095, 1013, 814; NMR: mixture of
1
two rotamers (approx. A/B 2:1) H (600 MHz, DMSO-d6) δ [ppm]: rotamer A 7.44-7.38 (m,
4H), 7.37-7.32 (m, 4H), 4.72 (d, J = 15.8 Hz, 1H), 4.65 (d, J = 17.1 Hz, 1H), 4.14 (s, 1H).
4.01 (d, J = 15.2 Hz, 1H), 3.97 (d, J = 15.2 Hz, 1H), 3.47 (s, 3H), 1.75-1.67 (m, 1H), 1-50-
1.45 (m, 1H), 1.43-1.37 (m, 1H), 0.74 (d, J = 6.5 Hz, 3H), 0.69 (d, J = 6.6 Hz, 3H); rotamer B
7.44-7.48 (m, 4H), 7.28 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 4.82 (dd, J = 9.0, 5.2
Hz, 1H), 4.82 (d, J = 15.8 Hz, 1H), 4.39 (dd, J = 7.9, 6.1 Hz, 2H), 4.17 (d, J = 15.9 Hz, 1H),
3.56 (s, 3H), 1.75-1.67 (m, 1H), 1.61-1.55 (m, 1H), 1.36-1.30 (m, 1H), 0.87 (d, J = 6.5 Hz,
13
3H), 0.60 (d, J = 6.7 Hz, 3H); C (151 MHz, DMSO-d6) δ [ppm]: A and B: 171.3, 171.0,
169.5, 168.4, 137.4, 136.0, 134.6, 134.6, 132.1, 131.2, 130.8, 130.7, 130.2, 129.4, 128.8,
128.8, 128.4, 127.9, 58.7, 56.7, 56.6, 52.3, 50.2, 37.8, 35.9, 24.4, 24.2, 22.5, 22.4, 22.1, 22.0;
LC-MS (DAD/ESI): tr = 9.29 min, Calcd for C22H25Cl2NO3S (m/z): [M+H]+ 454.10
[M+2+H]+ 456.10, Found [M+H]+ 454.12 [M+2+H]+ 456.11; HRMS (ESI): Calcd for

13
ACCEPTED MANUSCRIPT
C22H25Cl2NO3SNa (m/z): [M]+ 476.0830 [M+2]+ 448.0800, Found [M]+ 476.0825 [M+2+H]+
478.0796.
2.5.6. Methyl N-(2-((4-chlorophenyl)thio)acetyl)-N-(4-methoxybenzyl)leucinate (8b)
Methyl N-(4-methoxybenzyl)leucinate hydrochloride (6b) (2.71 g, 9.0 mmol), triethylamine
(1.26 ml, 9.0 mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (1.82 g, 9.0 mmol), DIC (1.38
ml, 9.0 mmol), DMAP (0.22 g, 1.8 mmol) were used. The crude product was purified by flash
chromatography (SiO2, petroleum ether/chloroform 1:1) giving compound 8b as a colorless
oil with a 70% yield (2.83 g).
IR (ATR): [cm-1] 2964, 2934, 1741, 1645, 1513, 1467, 1338, 1250, 1158, 1094, 1023, 815;
NMR: mixture of two rotamers (aprox. A/B 3:1) 1H (600 MHz, DMSO-d6) δ [ppm]: rotamer
A; 7.33 (s, 4H), 7,26 (d, J = 8.7 Hz, 2H), 6.90 (dt, J = 8.7, 3.0 Hz, 2H), 4.62 (d, J = 16.6 Hz,
1H), 4.57 d, J = 16.6 Hz, 1H), 4.34 (dd, J = 7.9, 6.1 Hz, 1H), 4.03 (d, J = 15.2 Hz, 1H), 3.97
(d, J = 15.1 Hz, 1H), 3.72 (s, 3H), 3.46 (s, 3H), 1.73-1.68 (m, 1H), 1.48-1.44 (m, 1H), 1.43-
1.36 (m, 1H), 0.72 (d, J = 6.5 Hz, 3H), 0.67 (d, J = 6.6 Hz, 3H); rotamer B; 7.40 (dt, J = 8.8,
2.4 Hz, 2H), 7.38 (dt, J = 8.8, 2.4 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H),
4.78 (dd, J = 8.9, 5.3 Hz, 1H), 4.60-4.57 (m, 1H), 4.14-4.11 (m, 3H), 3.69 (s, 3H), 3.51 (s,
3H), 1.69-1.65 (m, 1H), 1.63-1.59 (m, 1H), 1.35-1.29 (m, 1H), 0.85 (d, J = 6.6 Hz, 3H), 0.59
(d, J= 6.7 Hz, 3H); 13C (151 MHz, DMSO-d6) δ [ppm]: rotamer A; 171.5, 168.6, 159.1,
135.2, 131.2, 130.6, 129.4, 129.3, 129.2, 114.3, 56.9, 55.6, 52.2, 50.9, 38.3, 36.5, 24.9, 22.9,
22.5; rotamer B; 171.8, 169.7, 158.5, 135.5, 132.3, 130.3, 130.2, 129.3, 129.0, 113.8, 58.9,
55.8, 52.7, 46.4, 40.5, 38.2, 24.6, 22.9, 22.5; LC-MS (DAD/ESI): tr = 8.72 min, Calcd for
C23H28ClNO4SNa (m/z): [M]+ 472.13, Found [M]+ 471.99; HRMS (ESI): Calcd for
C23H28ClNO4SNa (m/z): [M]+ 472.1325, Found [M]+ 472.1318.
2.5.7. Methyl N-(2-((4-chlorophenyl)thio)acetyl)-N-(4-fluorobenzyl)leucinate (8c)
Methyl N-(4-fluorobenzyl)leucinate hydrochloride (6c) (1.30 g, 4.5 mmol), triethylamine
(0.63 ml, 4.5 mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (0,91 g, 4.5 mmol), DIC (0.69
ml, 4.5 mmol), DMAP (0.11 g, 0.9 mmol) were used. The crude product was purified by flash
chromatography (SiO2, petroleum ether/chloroform 1:1) giving compound 8c as an colorless
oil with a 67% yield (1.32 g).
IR (ATR): [cm-1] 2956, 2870, 1741, 1652, 1510, 1223, 1096, 820; NMR: mixture of two
1
rotamers (aprox. A/B 2:1) H (600 MHz, DMSO-d6) δ [ppm]: rotamer A 7.39-7.37 (m, 2H),
7.33 (s, 4H), 7.17 (tt, J = 8.8, 1.9 Hz, 2H), 4.70-4.63 (m, 2H), 4.35 (dd, J= 7.8, 6.1 Hz 1H).

14
ACCEPTED MANUSCRIPT
4.04 (d, J = 15.2 Hz, 1H), 3.98 (d, J = 15.2 Hz, 1H), 3.46 (s, 3H), 1.74-1.69 (m, 1H), 1.48-
1.44 (m, 1H), 1.42-1.35 (m, 1H), 0.72 (d, J = 6.5 Hz, 3H), 0.67 (d, J = 6.6 Hz, 3H); rotamer B
7.41-7.37 (m, 4H), 7.19-7.15 (m, 2H), 7.03 (t, J = 8.8 Hz, 2H), 4.81-4.79 (dd, J = 9.1, 5.2 Hz,
1H), 4.70-4.63 (m, 1H), 4.18 (d, J = 15.7 Hz, 1H), 4.12 (s, 2H), 3.54 (s, 3H), 1.69-1.67 (m,
1H), 1.61-1.57 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (d, J = 6.5 Hz, 3H), 0.58 (d, J = 6.7 Hz, 3H);
13C (151 MHz, DMSO-d6) δ [ppm]: rotamer A; 171.4, 168.73, 162.8-161.2 (d, JFC = 243.5
Hz), 135.15, 133,5 (d, JFC = 2 Hz), 131.2, 130.6, 130.2 (d, JFC = 8.1 Hz), 129.20, 115.7 (d, JFC
= 21.3 Hz), 57.1, 52.2, 50.7, 38.3, 36.5, 24.9, 22.9, 22.4; rotamer B; 171.7, 169.9, 161.5 (d,
JFC = 242.5 Hz), 135.1, 135.9 (d, JFC = 1.6 Hz), 131.2, 130.6, 129.9 (d, JFC = 7.9 Hz), 129.3,
115.1 (d, JFC = 21.1 Hz), 59.1, 52.8, 46.4, 50.5, 24.7, 22.9, 22.5; LC-MS (DAD/ESI): tr =
8.86 min, Calcd for C22H25ClFNO3S (m/z): [M-H]- 436.11 [M+2-Cl]- 438.11, Found [M-H]-
435.97 [M+2-Cl]- 438.03; HRMS (ESI): Calcd for C22H25ClFNO3SNa (m/z): [M]+ 460.1125
[M+2]+ 462.1096, Found [M+H]+ 460.1133 [M+2]+ 462.1115.
2.5.8. Methyl N-(2-((4-chlorophenyl)thio)acetyl)-N-(isoquinolin-4-ylmethyl)leucinate (8d)
Methyl N-(isoquinolin-4-ylmethyl)leucinate hydrochloride (6d) (0.60 g, 1.8 mmol),
triethylamine (0.26 ml, 1.8 mmol), 2-((4-chloro)phenylthio)acetic acid (2b) (0.38 g, 1.8
mmol), DIC (0.28 ml, 1.8 mmol), DMAP (0.05 g, 0.4 mmol) were used. The crude product
was purified by flash chromatography (SiO2, petroleum ether/chloroform 1:1) giving
compound 8d as a colorless oil with a 80% yield (0.70 g).
IR (ATR): [cm-1] 2955, 2915, 1737, 1651, 1497, 1477, 1201, 1095, 1048, 826; NMR:
1
mixture of two rotamers (approx. A/B 3:1) H (600 MHz, DMSO-d6) δ [ppm]: rotamer A;
8.91 (d, J = 2.2 Hz, 1H), 8.29 (d, J = 1.3 Hz, 1H), 8.02-7.94 (m, 2H), 7.55-7.50 (m, 1H), 7.62-
7.57 (m, 1H), 7.37-7.30 (m, 4H), 4.96 (d, J = 17.2 Hz, 1H), 4.89 (d, J = 17.2 Hz, 1H), 4.42
(dd, J = 8.2, 5.9 Hz, 1H), 4.17 (d, J = 15.2 Hz, 1H), 4.11 (d, J = 15.2 Hz, 1H), 3.40 (s, 3H),
1.72-1.70 (m, 1H), 1.56-1.51 (m, 1H), 1.45-1.38 (m, 1H), 0.70 (d, J = 6.5 Hz, 3H), 0.64 (d, J
= 6.6 Hz, 3H); rotamer B; 8.75 (d, J = 2.1 Hz, 1H), 8.21 (s, 1H), 8.02-7.94 (m, 2H), 7.55-7.50
(m, 1H), 7.62-7.57 (m, 1H), 7.41-7.40 (dt, J = 8.7, 2.5 Hz, 2H), 7.33-7.31 (m, 2H), 4.81 (d, J
= 16.0 Hz, 1H), 4.47 (d, J = 16.0 Hz, 1H), 4.18 (m, 1H), 3.61 (s, 2H), 3.50 (s, 3H), 1.79-1.73
(m, 1H), 1.56-1.51 (m, 1H), 1.45-1.36 (m, 1H), 0.99 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.5 Hz,
3H) ; 13C (151 MHz, DMSO-d6) δ [ppm]: rotamer A; 171.5, 168.9, 151.3, 147.4, 135.1,
134.7, 131.2, 130.7, 130.0, 129.2, 129.1, 128.4, 128.1, 127.7, 127.4, 57.1, 52.2, 49.5, 38.2,
36.3, 24.9, 23.0, 22.4; rotamer B; 171.8, 170.2, 152.1, 147.0, 135.2, 134.3, 131.8, 130.5,
130.4, 129.6, 129.3, 128.3, 127.9, 127.7, 127.2, 59.1, 58.9, 52.8, 38.3, 36.2, 23.8, 22.9, 22.5

15
ACCEPTED MANUSCRIPT
2.6. General synthetic procedure for cyclisation reaction and analytical data
(compounds 9a-d and 10a-d)
The corresponding amide 7a-d or 8a-d (1 eq.) and anhydrous 1,4-dioxane (30 ml) were
placed in a round bottom flask equipped with CaCl2 tube and reflux condenser. Then fresh
potassium tert-butoxide (1.5 eq.) was added in one portion and reaction was heated at 80ºC
for 1.5 hours. After this time reaction was cooled down, poured into 1 M HCl solution (15
ml) and extracted with ethyl acetate (3 x 20 ml). Organic layers were collected, washed with
water and brine, dried over anhydrous MgSO4 and evaporated.
2.6.1. 3-((4-Chlorophenyl)thio)-4-hydroxy-5-isobutyl-1,5-dihydro-2H-pyrrol-2-one (9a)
Compound 7a (0.23 g 0.7 mmol) and tBuOK (0.12 g, 1.1 mmol) were used. The crude product
was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result giving
compound 9d as colorless solid with a 37% yield (0.07 g). The product was recrystallized
from cyclohexane/acetone.
IR (ATR): [cm-1] 3179, 2956, 2932, 2907, 2869, 2617, 1655, 1588, 1476, 1390, 1090, 823;
1
NMR: H (600 MHz, DMSO-d6) δ [ppm]: 12.24 (s, 1H), 7.94 (s, 1H), 7.34-7.30 (m, 2H),
7.12-7.08 (m, 2H), 4.11 (dd, J = 9.8; 2.6 Hz, 1H), 1.86-1.78 (m, 1H), 1.66-1.57 (m, 1H), 1.33-
1.27 (m, 1H), 0.91 (dd, J = 6.5; 4.9 Hz, 6H); 13C (151 MHz, DMSO-d6) δ [ppm]: 181.6,
171.2, 136.6, 129.4, 128.7, 127.2, 92.2, 55.1, 41.7, 24.4, 23.7, 21.5 LC-MS (DAD/ESI): tr =
5.54 min, Calcd for C14H16ClNO2S (m/z): [M-H]- 296.05 [M+2-H]- 298.05, Found [M-H]-
296.00 [M+2-H]- 298.00; HRMS (ESI): Calcd for C14H16ClNO2SNa (m/z): [M]+ 320.0488
[M+2]+ 322.0458, Found [M]+ 320.0483 [M+2]+ 322.0453.
2.6.2. 5-Benzyl-4-hydroxy-3-(phenylthio)-1,5-dihydro-2H-pyrrol-2-one (9b)
t
Compound 7b (0.59 g 1.3 mmol) and BuOK (0.22 g, 2.0 mmol) were used. The crude
product was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result
giving compound 9b as colorless solid with a 83% yield (0.33 g). The product was
recrystallized from cyclohexane/ethyl acetate.
IR (ATR): [cm-1] 3434, 3395, 3216, 3028, 1691, 1626, 1579, 1496, 1241, 1133, 1087, 1024,
904
1
NMR: H (600 MHz, DMSO-d6) δ [ppm]: 12.21 (s, 1H), 7.82 (s, 1H), 7.32-7.26 (m, 3H),
7.25-7.21 (m, 2H), 7.07-7.03 (m, 2H), 7.02-6.96 (m, 1H), 6.49-6.42 (m, 2H), 4.43 (dd, J =
13
4.3, 3.5 Hz, 1H), 3.06 (qd, J = 13.8, 4.3 Hz, 2H); C (151 MHz, DMSO-d6) δ [ppm]: 179.1,
170.9, 137.0, 135.3, 130.0, 128.6, 127.9, 126.6, 124.8, 124.3, 93.6, 56.9, 36.0; LC-MS

16
ACCEPTED MANUSCRIPT
(DAD/ESI): tr = 4.77 min, Calcd for C17H15NO2S (m/z): [M+H]+ 298.09 Found [M+H]+
297.93; HRMS (ESI): Calcd for C17H15NO2SNa (m/z): [M]+ 320.0721 Found [M]+ 320.0719.
2.6.3. 5-Benzyl-3-((4-chlorophenyl)thio)-4-hydroxy-1,5-dihydro-2H-pyrrol-2-one (9c)
Compound 7c (0.45 g 1.2 mmol) and tBuOK (0.21 g, 1.9 mmol) were used. The crude product
was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result giving
compound 9c as colorless solid with a 64% yield (0.26 g). The product was recrystallized
from cyclohexane/acetone [36].
IR (ATR): [cm-1] 3362, 3030, 2577 (br), 1661, 1595, 1472, 1387, 1359, 1224, 1089, 101,
822, 698; NMR: 1H (600 MHz, DMSO-d6) δ [ppm]:12.32 (s, 1H), 7.85 (s, 1H). 7.34-7.27 (m,
3H), 7.24-7.21 (m, 2H), 7.07 (dt, J = 8.6, 2.0, 2H), 4.43 (t, J = 3.9 Hz, 1H), 3.11-2.99 (m,
2H); 13C (151 MHz, DMSO-d6) δ [ppm]: 179.2, 170.6, 136.2, 135.3, 130.0, 128.8, 128.4,
127.9, 126.6, 126.5, 56.9, 35.9; LC-MS (DAD/ESI): tr = 5.42 min, Calcd for C17H14ClNO2S
(m/z): [M-H]- 330.03 [M+2-H]- 332.02, Found [M-H]- 330.04 [M+2-H]- 332.03; HRMS
(ESI): Calcd for C17H14ClNO2SNa (m/z): [M]+ 354.0331 [M+2]+ 356.0302, Found [M]+
354.0327 [M+2]+ 356.0300.
2.6.4. 5-(4-Chlorobenzyl)-3-((4-chlorophenyl)thio)-4-hydroxy-1,5-dihydro-2H-pyrrol-2-
one (9d)
t
Compound 7d (1.12 g 2.8 mmol) and BuOK (0.47 g, 4.2 mmol) were used. The crude
product was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result
giving compound 9d as colorless solid with a 57% yield (0.55 g). The product was
recrystallized from cyclohexane/acetone.
IR (ATR): [cm-1] 3352, 2927, 2584 (br), 1683, 1661, 1595, 1492, 1475, 1387, 1229, 1091,
1
1012, 854, 817, 758; NMR: H (600 MHz, DMSO-d6) δ [ppm]: 12.39 (s, 1H), 7.88 (s, 1H),
7.40-7.33 (m, 2H), 7.25-7.19 (m, 2H), 7.11 (dt, J = 8.7, 2.1 Hz, 2H), 6.43 (dt, J = 8.8, 2.1 Hz,
2H), 4.45 (t, J = 3.9 Hz, 1H), 3.09 (dd, J = 13.8, 4.1 Hz, 1H), 3.01 (dd, J = 13.8, 4.2 Hz, 1H);
13C (151 MHz, DMSO-d6) δ [ppm]: 179.0, 170.5, 136.2, 134.3, 131.9, 131.4, 129.0, 128.3,
128.0, 126.5, 93.5, 56.6, 35.1, 30.7; LC-MS (DAD/ESI): tr = 5.88 min, Calcd for
C17H13Cl2NO2S (m/z): [M-H]- 364.00 [M+2-H]- 366.00, Found [M-H]- 363.99 [M+2-H]-
365.92; HRMS (ESI): Calcd for C17H13Cl2NO2SNa (m/z): [M]+ 387.9942 [M+2]+ 389.9912,
Found [M]+ 387.9935 [M+2]+ 389.9909.

17
ACCEPTED MANUSCRIPT
2.6.5. 1-(4-Chlorobenzyl)-3-((4-chlorophenyl)thio)-4-hydroxy-5-isobutyl-1,5-dihydro-2H-
pyrrol-2-one (10a)
Compound 8a (1.11 g 2.5 mmol) and tBuOK (0.42 g, 3.7 mmol) were used. The crude product
was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result giving
compound 10a as colorless solid with a 57% yield (0.60 g). The product was recrystallized
from cyclohexane/acetone.
1
IR (ATR): [cm-1] 2951, 2925, 2868, 2602, 1640, 1475, 1405, 1382, 1090, 818; NMR: H
(600 MHz, DMSO-d6) δ [ppm]: 12.57 (s, 1H), 7.41 (dt, J = 8.4, 1.9 Hz, 2H), 7.35 (dt, J = 8.7,
2.1 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 7.14 (dt, J = 8.7, 2.0 Hz, 2H), 4.79 (d, J = 15.8 Hz, 1H),
4.24 (d, J = 15.8 Hz, 1H), 3.99 (t, J = 4.8 Hz, 1H), 1.71-1.61 (m, 2H), 1.57 (sept, J = 6.6 Hz,
1H), 0.77 (dd, J = 6.6, 1.5 Hz, 6H); 13C NMR (151, MHz, DMSO-d6) δ [ppm]: 180.1, 169.9,
137.2, 136.3, 131.8, 129.7, 129.5, 128.9, 128.6, 127.5, 92.6, 58.6, 42.9, 23.4, 23.1; LC-MS
(DAD/ESI): tr = 7.98 min, Calcd for C21H21Cl2NO2S (m/z): [M-H]- 419.05 [M+2-H]- 421.05,
Found [M-H]- 419.95 [M+2-H]- 422.01; HRMS (ESI): Calcd for C21H21Cl2NO2SNa (m/z):
[M]+ 444.0568 [M+2]+ 446.0538, Found [M]+ 444.0565 [M+2]+ 446.0542.
2.6.6. 3-((4-Chlorophenyl)thio)-4-hydroxy-5-isobutyl-1-(4-metoxybenzyl)-1,5-dihydro-
2H-pyrrol-2-one (10b)
t
Compound 8b (2.00 g 4.0 mmol) and BuOK (0.67 g, 6.0 mmol) were used. The crude
product was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result
giving compound 10b as colorless solid with a 79% yield (1.32 g). The product was
recrystallized from cyclohexane/acetone.
1
IR (ATR): [cm-1] 2952, 2867, 2837, 2584, 1611, 1513, 1379, 1247, 818; NMR: H (600
MHz, DMSO-d6) δ [ppm]: 12.45 (s, 1H), 7.35 (dt, J = 8.8, 2.8 Hz, 2H), 7.16-7.10 (m, 4H),
6.90 (dt, J = 8.7, 2.9 Hz, 2H), 4.82 (d, J = 15.3 Hz, 1H), 4.08 (d, J = 15.3 Hz, 1H), 3.89 (t, J =
13
4.4 Hz, 1H), 3.73 (s, 3H), 1.68-1.54 (m, 3H), 0.79-0.76 (dd, J = 6.2, 2.7 Hz, 6H); C (151,
MHz, DMSO-d6) δ [ppm]: 181.0, 170.8, 159.5, 137.4, 130.9, 130.8, 130.0, 129.9, 128.5,
115.1, 93.9, 59.1, 56.2, 44.0, 38.3, 24.5, 24.2; LC-MS (DAD/ESI): tr = 7.52 min, Calcd for
C22H24ClNO3S (m/z): [M-H]- 416.11 [M+2-H]- 418.11, Found [M-H]- 416.03 [M+2-H]-
417.89; HRMS (ESI): Calcd for C22H24ClNO3SNa (m/z): [M]+ 440.1063 [M+2]+ 442.1034,
Found [M]+ 440.1058 [M+2]+ 442.1038.
2.6.7. 3-((4-Chlorophenyl)thio)-1-(4-fluorobenzyl)- 4-hydroxy-5-isobutyl-1,5-dihydro-
2H-pyrrol-2-one (10c)

18
ACCEPTED MANUSCRIPT
Compound 8c (1.25 g 2.9 mmol) and tBuOK (0.49 g, 4.4 mmol) were used. The crude product
was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result giving
compound 10c as colorless solid with a 81% yield (0.94 g). The product was recrystallized
from cyclohexane/acetone.
IR (ATR): [cm-1]2956, 2868, 2562 (br), 1603, 1508, 1475, 1379, 1225, 1088, 1011, 810;
NMR: 1H (600 MHz, DMSO-d6) δ [ppm]: 12.52 (s, 1H), 7.34 (dt, J = 8.7, 2.8 Hz, 2H), 7.26
(dd, J = 8.6, 5.6 Hz, 2H), 7.17-7.12 (m, 4H), 4.78 (d, J = 15.6 Hz, 1H), 4.21 (d, J = 15.6 Hz,
1H), 3.96 (t, J = 4.7 Hz, 1H), 1.70-1.63 (m, 2H), 1.57 (m, J = 6.6 Hz, 1H), 0.75 (dd, J = 6.5,
13
2.6 Hz, 6H); C (151, MHz, DMSO-d6) δ [ppm]: 180.5, 170.3, 162.6-161.0 (d, JFC = 242.8
Hz), 136.7, 134.7 (d, JFC = 2.1 Hz), 130.2, 130.1, 130.0, 129.4 (d, JFC = 15.2 Hz), 127.9, 115.8
(d, JFC = 21.4 Hz), 93.2, 58.9, 43.3, 24.0, 37.7, 23.5; LC-MS (DAD/ESI): tr = 7.52 min,
Calcd for C21H21ClFNO2S (m/z): [M-H]- 404.09 [M+2-H]- 406.09, Found [M-H]- 404.07
[M+2-H]- 405.93; HRMS (ESI): Calcd for C21H21ClFNO2SNa (m/z): [M]+ 428.0863 [M+2]+
430.0834, Found [M]+ 428.0860 [M+2]+ 430.0836.
2.6.8. 3-((4-Chlorophenyl)thio)-4-hydroxy-5-isobutyl-1-(isoquinolin-4-ylmethyl)-1,5-
dihydro-2H-pyrrol-2-one (10d)
t
Compound 8d (0.18 g 0.4 mmol) and BuOK (0.07 g, 0.6 mmol) were used. The crude
product was purified by flash chromatography (SiO2, ethyl acetate/methanol 1:1) as a result
giving compound 10d as yellowish solid with a 19% yield (0.03 g).
IR (ATR): [cm-1] 2956, 2927, 2616 (br), 1674, 1599, 1474, 1385, 1243, 1089, 1010, 812,
753; NMR: 1H (600 MHz, DMSO-d6) δ [ppm]: 12.59 (s, 1H), 8.83 (d, J = 2.1 Hz, 1H), 8.20
(d, J = 1.4 Hz, 1H), 8.03-7.96 (m, 2H), 7.75 (dt, J = 8.4, 1.4 Hz, 1H), 7.62 (dt, J = 6.9, 1.1 Hz,
1H), 7.33 (dt, J = 8.6, 2.7 Hz, 2H), 7.17 (dt, J = 8.6, 2.6 Hz, 2H), 4.98 (d, J = 15.9 Hz, 1H),
4.50 (d, J = 15.9 Hz, 1H), 4.16 (t, J = 4.4 Hz, 1H), 1.77-1.71 (m, 2H), 1.59 (sept. J= 6.5 Hz,
1H), 0.73 (t, J = 6.1 Hz, 6H); 13C NMR (151, MHz, DMSO-d6) δ [ppm]: 181.3, 171.0, 151.8,
147.7, 137.3, 135.5, 132.4, 130.8, 130.6, 129.9, 129.6, 129.1, 128.6, 128.1, 93.8, 59.8, 42.6,
38.2, 24.5, 24.2; LC-MS (DAD/ESI): tr = 6.05 min, Calcd for C24H23ClN2O2S (m/z): [M-H]-
437.11 [M+2-H]- 439.1061, Found [M-H]- 436.83 [M+2-H]- 438.03; HRMS (ESI): Calcd for
C24H23ClN2O2SNa (m/z): [M]+ 461.1066 [M+2]+ 463.1037, Found [M]+ 461.1041 [M+2]+
463.1017.
2.7. General synthetic procedure for Williamson ether synthesis and analytical data
(compounds 11a-c and 12a-d)

19
ACCEPTED MANUSCRIPT
The corresponding derivatives of tetramic acid 9a or 10a-b (1 eq.), anhydrous potassium
carbonate (2 eq.) and anhydrous acetone (15 ml) were placed in a round bottom flask
equipped with CaCl2 tube and reflux condenser and heated at 60°C for 15 minutes. Then
suitable alkyl bromide (1 eq.) in 5 ml of anhydrous acetone was added. The reaction was
refluxed for 3 hours. After this time the reaction was cooled down, poured into saturated
ammonium chloride solution (20 ml) and extracted with ethyl acetate (3 x 15 ml). Organic
layers were collected, washed with water and brine, dried over anhydrous MgSO4 and
evaporated.
2.7.1. 3-((4-Chlorophenyl)thio)-5-isobutyl-4-(isopentyloxy)-1,5-dihydro-2H-pyrrol-2-one
(11a)
Compound 9a (0.15 g 0.5 mmol), isoamyl bromide (0.06 ml, 0.5 mmol) and potassium
carbonate (0.14 g, 1.0 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 1:1) as a result giving compound 11a as colorless
solid with a 8% yield (0.015 g).
IR (ATR) [cm-1]: 3193, 3077, 2958, 1683, 1596, 1473, 1320, 1089, 1055, 1007, 815; NMR:
1H (600 MHz, DMSO-d6) δ [ppm]: 8.20 (s, 1H), 7.35 (dt, J = 8.7, 2.1 Hz, 2H), 7.14 (dt, J =
8.7, 2.1 Hz, 2H), 4.56 (t, J = 6.7 Hz, 2H), 4.20-4.16 (m, 1H), 1.84-1.76 (m, 1H), 1.67-1.59 (m,
1H), 1.58-1.45 (m, 3H), 1.34 (ddd, J = 13.7, 9.4, 4.5 Hz, 1H), 0.91 (dd, J = 6.6, 2.5 Hz, 6H),
0.81 (t, J = 6.8 Hz, 3H); 13C (151 MHz, DMSO-d6) δ [ppm]: 179.1, 170.5, 136.7, 129.8,
129.0, 127.2, 94.0, 69.9, 55.3, 55.3, 41.9, 37.5, 24.4, 24.3, 23.6, 22.3, 22.1, 21.6; LC-MS
(DAD/ESI): tr = 9.10 min, Calcd for C19H26ClNO2S (m/z): [M+H]+ 368.14 [M+2+H]+ 370.14,
Found [M+H]+ 368.14 [M+2+H]+ 370.14; HRMS (ESI): Calcd for C19H26ClNO2SNa (m/z):
[M]+ 390.1270 [M+2]+ 392.1241, Found [M]+ 390.1265 [M+2]+ 392.1238.
2.7.2.Methyl 2-((4-((4-chlorophenyl)thio)-2-isobutyl-5-oxo-2,5-dihydro-1H-pyrrol-3-
yl)oxy)acetate (11b)
Compound 9a (0.15 g 0.5 mmol), methyl bromoacetate (0.05 ml, 0.5 mmol) and potassium
carbonate (0.14 g, 1.0 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 1:1) as a result giving compound 11b as colorless
solid with a 21% yield (0.04 g).
IR (ATR): [cm-1] 3187, 3080, 2953, 1761, 1682, 1605, 1477, 1338, 1224, 1206, 1090, 1006,
1
820; NMR: H (600 MHz, DMSO-d6) δ [ppm]: 8.32 (s, 1H), 7.35 (dt, J = 8.6, 2.0 Hz, 2H),
7.11 (dt, J = 8.7, 2.0 Hz, 2H), 5.26 (d, J = 16.5 Hz, 1H), 5.18 (d, J = 16.5 Hz, 1H), 4.22 (ddd,

20
ACCEPTED MANUSCRIPT
J = 9.7, 3.4, 1.2 Hz, 1H), 3.49 (s, 3H), 1.89-1.79 (m, 1H), 1.62 (ddd, J = 13.5, 9.9, 3.5 Hz,
13
1H), 1.38 (ddd, J = 13.8, 9.7, 4.3 Hz, 1H), 0.92 (dd, J = 6.6, 2.5 Hz, 3H); C (151 MHz,
DMSO-d6) δ [ppm]: 177.7, 169.9, 167.6, 135.8, 130.0, 128.8, 127.5, 95.2, 66.9, 55.3, 52.0,
42.0, 24.4, 23.6, 21.5; LC-MS (DAD/ESI): tr = 6.70 min, Calcd for C17H20ClNO4S (m/z):
[M+H]+ 370.09 [M+2+H]+ 372.08, Found [M+H]+ 370.11 [M+2+H]+ 372.10; HRMS (ESI):
Calcd for C17H20ClNO4SNa (m/z): [M]+ 392.0699 [M+2]+ 394.0670, Found [M]+ 392.0691
[M+2]+ 394.0664.
2.7.3.4-((4-Chlorobenzyl)oxy)-3-((4-chlorophenyl)thio)-5-isobutyl-1,5-dihydro-2H-
pyrrol-2-one (11c)
Compound 9a (0.15 g 0.5 mmol), 4-chlorobenzyl bromide (0.10 g, 0.5 mmol) and potassium
carbonate (0.14 g, 1.0 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 1:1) as a result giving compound as colorless
solid 16c with a14% yield (0.03 g).
1
IR (ATR): [cm-1] 3203, 3078, 2959, 1683, 1604, 1324, 1089, 807; NMR: H (600 MHz,
DMSO-d6) δ [ppm]: 8.26 (s, 1H), 7.38-7.34 (m, 4H), 7.29 (dt, J = 8.7, 2.8 Hz, 2H), 7.08 (dt, J
= 8.7, 2.8 Hz, 2H), 5.60 (d, J = 12.2 Hz, 1H), 5.57 (d, J = 12.2 Hz, 1H), 4,25 (ddd, J = 9.6,
3.4, 1.2 Hz, 1H), 1.83-1.76 (m, 1H), 1.61-1.57 (m, 1H), 1.38-1.33 (m, 1H), 0.90 (dd, J = 6.7,
1.4 Hz, 6H); 13C NMR (151 MHz, DMSO-d6) δ [ppm]: 178.9, 170.6, 136.6, 135.3, 133.4,
130.3, 129.9, 129.3, 129.0, 128.0, 95.8, 72.1, 55.8, 42.3, 24.9, 24.0, 21.0; LC-MS
(DAD/ESI): tr = 8.79 min, Calcd for C21H21Cl2NO2S (m/z): [M+H]+ 422.07 [M+2+H]+
424.07, Found [M+H]+ 421.88
[M+2+H]+ 423.81; HRMS (ESI): Calcd for
C21H21Cl2NO2SNa (m/z): [M]+ 444.0568 [M+2]+ 446.0538, Found [M]+ 444.0562 [M+2]+
446.0537.
2.7.4. Methyl 2-((1-(4-chlorobenzyl)-4-((4-chlorophenyl)thio)-2-isobutyl-5-oxo-2,5-
dihydro-1H-pyrrol-3-yl)oxy)acetate (12a)
Compound 10a (0.2 g 0.47 mmol), methyl bromoacetate (0.045 ml, 0.47 mmol) and
potassium carbonate (0.13 g, 0.94 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 3:1) as a result giving compound 12a as colorless
solid with a 21% yield (0.05 g).
1
IR (ATR): [cm-1] 2963, 1754, 1682, 1614, 1222, 1090, 813; NMR: H (600 MHz, DMSO-
d6) δ [ppm]: 7.43 (dt, J = 8.5, 1.9 Hz, 2H), 7.38 (dt, J = 8.7, 2.0 Hz, 2H), 7.24 (d, J = 8.5 Hz,
2H), 7.14 (dt, J = 8.8, 2.1 Hz, 2H), 5.27 (d, J = 16.4 Hz, 1H), 5.20 (d, J = 16.4 Hz, 1H), 4.79

21
ACCEPTED MANUSCRIPT
(d, J = 15.8 Hz, 1H), 4.26 (d, J = 15.8 Hz, 1H), 4.10 (t, J = 4.8 Hz, 1H), 3.49 (s, 3H), 1.78-
1.71 (m, 1H), 1.68-1.61 (m, 1H), 0.81 (dd, J = 6.3, 3.0 Hz, 6H); 13C NMR (151 MHz,
DMSO-d6) δ [ppm]: 176.9, 169.0, 168.1, 137.1, 135.9, 132.4, 130.8, 129.9, 129.4, 129.1,
128.2, 95.8, 67.8, 58.9, 52.4, 43.4, 38.1, 24.2, 23.4; LC-MS (DAD/ESI): tr = 8.91 min, Calcd
for C24H25Cl2NO4S (m/z): [M+H]+ 494.09 [M+2+H]+ 496.09, Found [M+H]+ 494.13
[M+2+H]+ 496.12; HRMS (ESI)(: Calcd for C24H25Cl2NO4SNa (m/z): [M]+ 516.0779
[M+2]+ 518.0750, Found [M]+ 516.0775 [M+2]+ 518.0760.
2.7.5. 1-(4-Chlorobenzyl)-4-((4-chlorobenzyl)oxy)-3-((4-chlorophenyl)thio)-5-isobutyl-
1,5-dihydro-2H-pyrrol-2-one (12b)
Compound 10a (0.18 g 0.43 mmol), 4-chlorobenzyl bromide (0.09 g, 0.43 mmol) and
potassium carbonate (0.12 g, 0.86 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 3:1) as a result giving compound 12b as colorless
solid with a 35% yield (0.034 g).
1
IR (ATR): [cm-1] 2960, 2932, 1695, 1608, 1319, 1087, 806; NMR: H (600 MHz, DMSO-
d6) δ [ppm]: 7.42-7.38 (m, 4H), 7.37-7.34 (m, 4H), 7.24 (d, J = 8.4 Hz, 2H), 7.16 (dt, J = 8.6,
1.9 Hz, 2H), 5.61 (q, J = 12.1 Hz, 2H), 4.78 (d, J = 15.8 Hz, 1H), 4.25 (d, J = 15.8 Hz, 1H),
4.16 (dd, J = 5.4, 4.0 Hz, 1H), 1.75-1.69 (m, 1H), 1.65-1.60 (m, 1H), 1.60-1.53 (m, 1H), 0.76
(dd, J = 6.5, 2.9 Hz, 6H); 13C (151 MHz, DMSO-d6) δ [ppm]: 177.6, 169.4, 137.2, 136.3,
135.0, 133.5, 132.3, 130.7, 130.1, 130.0, 129.5, 129.1, 129.0, 128.3, 96.0, 72.6, 59.1, 43.4,
37.8, 24.0, 23.4; LC-MS (DAD/ESI): tr = 10.40 min, Calcd for C28H26Cl3NO2S (m/z):
[M+H]+ 546.08 [M+2+H]+ 548.08, Found [M+H]+ 546.11[M+2+H]+ 548.10; HRMS (ESI):
Calcd for C28H26Cl3NO2SNa (m/z): [M]+ 568.0648 [M+2]+ 570.0618, Found [M]+
568.0643[M+2]+ 570.0619.
2.7.6.4-((4-Chlorobenzyl)oxy)-3-((4-chlorophenyl)thio)-5-isobutyl-1-(4-methoxybenzyl)-
1,5-dihydro-2H-pyrrol-2-one (12c)
Compound 10b (0.2 g 0.48 mmol), 4-chlorobenzyl bromide (0.1 g, 0.48 mmol) and potassium
carbonate (0.13 g, 0.96 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 3:1) as a result giving compound 12c as colorless
solid with a 58% yield (0.15 g).
IR (ATR): [cm-1] 3000, 2964, 2921, 1691, 1615, 1510, 1318, 1247, 818, 805; NMR: 1H (600
MHz, DMSO-d6) δ [ppm]: 7.38-7.33 (m, 6H), 7.16-7.11 (m, 4H), 6.90 (dt, J= 8.7, 2.9 Hz,
2H), 5.61 (d, J= 12.0 Hz, 1H), 5.57 (d, J= 12.0 Hz, 1H), 4.79 (d, J= 15.3 Hz, 1H), 4.11 (d, J=

22
ACCEPTED MANUSCRIPT
15.3 Hz, 1H), 4.06-4.04 (m, 1H), 3.72 (s, 3H), 1.73-1.69 (m, 1H), 1.64-1.55 (m, 2H), 0.76
(dd, J= 8.9, 6.3 Hz, 6H); 13C (151 MHz, DMSO-d6) δ [ppm]: 177.4, 169.3, 159.0, 136.4,
135.1, 133.5, 130.7, 130.1, 129.9, 129.5, 129.4, 128.9, 128.4, 114.5, 96.0, 72.6, 58.8, 55.6,
43.4, 37.8, 24.1, 23.6, 23.4; LC-MS (DAD/ESI): tr = 10.40 min, Calcd for C29H29Cl2NO3S
(m/z): [M+H]+ 542.13[M+2+H]+ 544.13, Found [M+H]+ 542.05[M+2+H]+ 543.91; HRMS
(ESI): Calcd for C29H29Cl2NO3SNa (m/z): [M]+ 564.1143 [M+2]+ 566.1113, Found [M]+
564.1137[M+2]+ 566.1118.
2.7.7. 3-((4-Chlorophenyl)thio)-5-isobutyl-1-(4-methoxybenzyl)-4-phenethoxy-1,5-
dihydro-2H-pyrrol-2-one (12d)
Compound 10b (0.2 g 0.48 mmol), 2-phenylethyl bromide (0.07 ml, 0.48 mmol) and
potassium carbonate (0.13 g, 0.96 mmol) were used. The crude product was purified by flash
chromatography (SiO2, hexane/ethyl acetate 3:1) as a result giving compound 12d as colorless
solid with a 63% yield (0.158 g).
IR (ATR): [cm-1] 2956, 1684, 1596, 1512, 1473, 1406, 1316, 1247, 1173, 1087, 1039, 1007,
1
812; NMR: H (600 MHz, DMSO-d6) δ [ppm]: 7.36 (dt, J = 5.7, 2.8 Hz, 2H), 7.23 (m, 2H),
7.18-7.14 (m, 5H), 7.10 (dt, J = 8.6, 2.8 Hz, 2H), 6.88 (dt, J = 8.7, 2.9 Hz, 2H), 4.80-4.78 (m,
1H), 4.76 (d, J = 15.3 Hz, 1H), 4.73-4.69 (m, 1H), 4.06 (d, J = 15.3 Hz, 1H), 3.87 (m, 1H),
3.71 (s, 3H), 2.95-2.89 (m, 2H), 1.58-1.53 (m, 1H), 1.51-1.43 (m, 2H), 0.69 (d, J = 6.4 Hz,
3H), 0.59 (d, J = 6.3 Hz, 3H); 13C (151 MHz, DMSO-d6) δ [ppm]: 177.9, 169.4, 159.0, 137.9,
136.8, 130.6, 129.9, 129.5, 129.4, 129.4, 128.8, 128.0, 126.9, 114.5, 94.6, 72.8, 58.7, 55.6,
43.4, 40.5, 37.8, 35.6, 24.0, 23.5, 23.0; LC-MS (DAD/ESI): tr = 9.87 min, Calcd for
C30H32ClNO3S (m/z): [M+H]+ 522.19[M+2+H]+ 524.18, Found [M+H]+ 522.11b [M+2+H]+
524.04; HRMS (ESI): Calcd for C30H32ClNO3SNa (m/z): [M]+ 544.1689 [M+2]+ 546.1660,
Found [M]+ 544.1681[M+2]+ 546.1659.
2.8. DFT calculations
DFT calculations based on the Becke88 exchange [37] and Perdew86 correlation [38,39]
functional (BP86) were performed using the Amsterdam Density Functional (ADF) program,
versions 2014 [40-42]. A standard triple-ζ STO basis containing one set of polarization
functions (TZP) as well as standard triple-ζ STO basis containing a double set of polarization
functions (TZP), was adopted for all elements. The scalar relativistic effects by employing
the Zero Order Regular Approximation (ZORA) were considered [43]. In the geometry
optimization, the following convergence criteria were applied: -0.001 au and 0.0003 au/A° for

23
ACCEPTED MANUSCRIPT
changes in the energy gradients. The integration accuracy was set to 5. Geometry optimization
was performed for the three depicted in Fig. 2 tautomers of tested compound 9a.
Vibrational frequencies were obtained through numerical differentiation of the analytical
gradients [40-42]. Free energy (ꢁG) at 298.15K were calculated from electronic bond
energies (ꢁE) and vibrational frequencies using standard thermochemistry relations for ideal
gas [44]. Table 3 shows the relative energy and free energy of each tautomers as well as the
relative population pi.
2.9. Protein Expression and Purification.
Two variants of the N-terminal domain of human Mdm2 (1-118 and 18-125) were cloned
into pET-20 vector (Novagen) and expressed in the Escherichia coli BL21(DE3) as described
previously [44]. In brief, cells were grown at 37°C and induced with 1 mM isopropyl β-D-1-
thiogalactopyranoside (IPTG) at OD600 of 0.6-0.8 and grown for an additional 5 h at 37°C.
Cells were harvested by centrifugation (4000g for 15 min). Then cells were resuspended in
phosphate-buffered saline (PBS) supplemented with protease inhibitor cocktail and lysed by
sonication until the lysate was homogeneous. Centrifugation of the lysate at 20000g for 15
min yielded the inclusion body pellet. The pellet was washed in ice-cold PBS containing
0.05% Triton-X100 for three times and then in PBS for the next three times. Each wash was
followed by centrifugation (20 000g, 15 min). After the final centrifugation the inclusion body
pellet was solubilized in 10-20 ml of a solution comprising 6 M guanidine hydrochloride, 100
mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol and slowly rotated at 4oC
for at least 2h. The protein was centrifuged at 20 000g for 20 min to remove insoluble
material and then dialyzed against 4 M guanidine hydrochloride (pH 3.5), 10 mM 2-
mercaptoethanol overnight. The protein was refolded by dropwise addition into 10 mM Tris-
HCl (pH 7.0), 1 mM EDTA, 10 mM 2-mercaptoethanol and slowly stirred at 4°C for 16 h.
Then ammonium sulphate was added to the final concentration of 1.5 M and the solution was
stirred for additional 2h at 4oC and centrifuged at 20 000g for 10 min. The refolded protein
was recovered on Butyl Sepharose 4 Fast Flow (GE Healthcare). The resign was washed with
10 mM Tris-HCl (pH 7.0), 1.5M ammonium sulphate, 1 mM EDTA, 10 mM 2-
mercaptoethanol, and the protein was eluted using 100 mM Tris-HCl (pH 7.2), 5 mM 2-
mercaptoethanol and further purified by gel filtration using HiLoad 16/600 Superdex75 (GE
Healthcare).
2.10. FP measurements

24
ACCEPTED MANUSCRIPT
Fluorescence Polarization (FP) assay was used to monitor interactions between Mdm2 and
its antagonists. For each assay, fresh protein stocks of Mdm2 (1-118) were thawed and the
protein concentration was determined using the Bradford method. Assay buffer contained 50
mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA and 5 % DMSO.
The binding affinity of P2 peptide (sequence: LTFEHYWAQLTS labeled with
carboxyfluorescein) towards Mdm2 was first determined. For this purpose, 10 nM of the
fluorescent P2 peptide was contacted with serial dilutions of tested protein (range from 750
to 0.012 nM) in a final volume of 100 ꢀl and fluorescence polarization was determined. Kd
was established by fitting the curve described by the below equation to experimental data:
ꢀ
ꢁ
FPmax − FPmin ∙ c
FP = FPmin +
Kꢂ + c
where FP is the determined value of fluorescence polarization, FPmin- fluorescence
polarization for ligand only, FPmax- fluorescence polarization at protein concentration
saturating the ligand, and c – protein concentration. Competition binding assay was performed
using 10 nM fluorescent P2 peptide and optimal protein concentration for the measurement
calculated based on determined Kd according to Huang, 2003 (f0 = 0.8) [45]. Tested
compounds were dissolved in DMSO at 50 µM. Serial dilutions (50 ꢀM to 0.05 ꢀM) were
prepared in DMSO. Nutlin-3a was used as positive controls for Mdm2 (Table S2). All the
experiments were prepared in duplicates.
Fluorescence polarization was determined using Tecan InfinitePro F200 plate reader with
the 485 nm excitation and 535 nm emission filters. The fluorescence intensities, parallel and
perpendicular to the plane of excitation, were determined in Corning black 96-well NBS assay
plates at room temperature. Fluorescence polarization values were expressed in
millipolarization units (mP). Inhibition curves were fitted to obtain Ki values.
2.11. 1H-15N HSQC Measurements for KD determination
.
Uniform 15N isotope labeling was achieved by expression of the protein in the M9 minimal
media containing 15NH4Cl as the sole nitrogen source. The final step of purification of Mdm2
(residues 1-118) [46] consisted of gel filtration into the NMR buffer (50 mM phosphate buffer
pH 7.4 containing 150 mM NaCl, 5 mM DTT). 10% (v/v) of D2O was added to the samples to
provide lock signal. Water suppression was carried out using the WATERGATE sequence
[47]. All the spectra were recorded at 300 K using a Bruker Avance 600 MHz spectrometer.
1H-15N heteronuclear correlations were obtained using the fast HSQC pulse sequence [48].
Assignment of the amide groups of Mdm2 was obtained after Stoll et al. [49].

25
ACCEPTED MANUSCRIPT
1
For each compound, the two-dimensional H-15N correlated NMR spectrum was recorded
at 7-9 different ligand/protein ratios, ranging from 0:1 to 5:1, respectively. The samples were
prepared by adding small amounts of a 50 mM ligand stock solution in DMSO to the protein
solution (0.12 ml) containing the 15N labeled Mdm2 fragment at a concentration of 0.16 –
0.30 mM. The acquisition parameters for each HSQC spectrum was as follows: the size of the
FID F2: 2048, F1: 100, and a number of scans: 10. Spectra were visualized using TopSpin
4.0.2 (Fig. 3, Fig. S7, Fig. S8). For the determination of dissociation constants (KD), nonlinear
fits of chemical shifts of single residue versus ligand ratio were performed using the program
OriginPro (version 9.1) according to the following equation [50,51]:
ꢐ
ꢀꢈ ꢊ
ꢈ ꢊ
ꢁ
ꢈ ꢊ
ꢈ ꢊ
ꢋ
ꢉ + ꢌ + ꢍꢎ − ꢏꢀ ꢉ + ꢌ + ꢍꢎꢁ − 4ꢈꢉꢊꢋꢈꢌꢊꢋ
ꢋ
ꢋ
ꢋ
ꢃꢄꢅꢆ = ∆ꢇ
2ꢈꢉꢊꢋ
Where
[L]0 – ligand concentration
[P]0 – protein concentration
∆
obs – observed chemical shift perturbation normalized according to the Pythagoras
formula with 15N weighting factor of 0.2.
∆M –chemical shift for single residue obtained with maximum ligand concentration
KD – dissociation constants
The final value of KD was calculated as a weighted mean value of KD’s obtained for at
least four most perturbed residues which undergo fast chemical exchange: Gly58, Tyr60,
Lys94, Leu57, His73, Tyr93, Thr63, Val108, Thr23.
Reference 1H-15N HSQC spectra of MDM2 titrated with Nutlin-3a can be found in
Krajewski et al. [52]
3. Results and discussion
3.1. Synthesis of tetramic acid derivatives
We first focused on the synthesis of the derivatives of tetramic acid (IA). There are
numerous methods of the syntheses of the tetramic acid derivatives, out of which, the ones
based on the Dieckmann condensation, the Lacey-Dieckmann condensation or the Meldrum’s
acid ring opening are among the most often used [18,53].
For the synthesis of the compounds, we chose to use the Dieckmann condensation of the
N-alkoxycarbonylacety derivatives [54]. We obtained the desired compounds starting from
the amino acids methyl esters (4a-c, 6a-d) and substituted 2-(phenylthio)acetic acids (2a-b)

26
ACCEPTED MANUSCRIPT
(Scheme 1, Scheme 2). Further, these reactants were condensed with the appropriate amides
(7a-d, 8a-d), using the carbodiimide coupling agent: diisopropylcarbodiimide (DIC),
supported by a catalytic amount of DMAP (4-dimethylaminopyridine). Although the N-
substituted tetramic acid is usually obtained using the Meldrum’s acid, we decided to proceed
with the modification made in reactants that were used in the Dieckmann condensation. We
introduced a suitable substituent before the coupling step, which was done by reductive
amination of appropriate amino acid esters.
O
S
OH
R1
2a-b
R3
NH2Cl
NH3Cl
a
a
R2 CO2Me
R2 CO2Me
6a-d
4a-c
O
CO2Me
R2
O
CO2Me
R2
S
S
N
N
H
R1
R3
R1
8a-d
7a-d
b
b
R3
H
N
R2
O
R2
O
N
R1
R1
HO
S
HO
S
9a-d
10a-d
R4Br
R4Br
R3
c
c
H
N
R2
N
R2
O
O
R1
R1
O
R4
S
O
R4
S
11a-c
12a-d
Scheme 2. Synthesis of tetramic acid derivatives. Reagents and conditions: (a) (i) 4a-c or 6a-d (1 eq.), Et3N (1
eq.), anh. DCM, 0°C, 15 min (ii) 2a-b (1 eq.), DMAP (0.2 eq.), DIC (1 eq), 0°C to RT, 16 h; (b) 7a-d or 8a-d (1
eq.), tBuOK (1.5 eq.), anh. 1,4-dioxane, 80°C, 1.5 h; (c) (i) 9a or 10a-b (1 eq.), anh. K2CO3 (2 eq.), anh. acetone,
60°C, 15 min (ii) alkyl bromide (1 eq.), reflux, 3h.
Amides 7a-d, 8a-d were subjected to the Dieckmann condensation carried out with
strong bases, leading to the tetramic acid derivatives 9a-d and 10a-d. The best yields were
obtained using the freshly distilled anhydrous 1,4-dioxane as a solvent and the potassium tert-
butoxide as a base. This is consistent with the literature findings, which reported that addition
of water promotes dimerization of tetramic acid [55]. The yields vary from moderate to high
(Table 1). Significantly lower yields were observed for cyclisation with the leucine amino
acid derivative leading to compound 9a - as compared to the aromatic amino acid derivatives

27
ACCEPTED MANUSCRIPT
(leading to 9b-d). With the exception of the isoquinoline derivative 10d, the obtained yields
were better when the N-alkylated compounds were used (10a-c).
Table 1. Structures, Dieckmann condensation yields and activities measured by fluorescence polarization
(FP) assay (Ki) of compounds 9a-d and 10a-d.
Yield Ki FP
No
R1
R2
-
[%]
[µM]
Cl
H
37
2.9
9a
9b
83
55
57
>100
9.0
Cl
Cl
9c
5.8
9d
Yield Ki FP
No
R1
R2
R3
[%]
[µM]
Cl
57
35.7
10a
Cl
Cl
79
81
26.3
10b
10c
>100
Cl
19
>100
10d
To introduce another substituent to our scaffold, we decided to proceed with the
Williamson ether synthesis [56] which gave the O-substituted products (11a-c,12a-d) with a
rather low yield, probably due to the modest acidity of the 4-hydroxyl group (Table 2). The
formation of the O-alkylated product rather than C-alkylated is confirmed by analysis of the
chemical shift of the methylene protons of R4 substituent. For example in the compound 11c
and 12b, these proton signals appear at 5.60 ppm and 5.61 ppm respectively. In this case such
kind of downfield presence of aliphatic signals can be caused only by the deshielding effect of
the neighboring oxygen. Surprisingly the N-alkylated compounds (10a and 10c), seem to be
more prone to alkylation than those without substituents on the nitrogen atom (9a).
Table 2. Structures, alkylation yield, and activity of compounds 11a-c and 12a-d.a
Yield
[%]
No
R1
Cl
Cl
Cl
R2
R4
-
-
-
-
8
11a
11b
11c
21
14
Cl
Yield
[%]
No
R1
R2
R3
R4
Cl
21
12a

28
ACCEPTED MANUSCRIPT
Cl
Cl
Cl
Cl
35
58
63
12b
12c
12d
Cl
a Ki (FP) obtained for these compounds were above 100 ꢀM
3.2. Tautomerization of 3-phenylthio- tetramic acid derivative 9a
Having in mind a possible tautomerization of the tetramic acid derivatives and as a
consequence its chemical and biochemical properties, we decided to determine whether the
ketone or enol forms are predominant in our compounds (Fig. 2). To achieve this goal, we
both analyzed spectral data and carried out theoretical calculations for compound 9a.
Figure 2. Possible tautomeric forms of 9a. (Numbering of atoms in the central ring of tetramic acid is shown in
blue)
1H NMR spectra for 9a show a proton signal at 12.24 ppm which is a region
characteristic for enol forms of tetronic and tetramic acids. This suggested that the main form
present in solution (DMSO) is the enolic one. To further verify this assumption, we decided to
measure the DEPT-135 13C NMR (Fig. S2). The signals originating from carbons C(2) –
171.2 ppm, C(3) – 92.2 ppm and C(4) – 181.6 ppm (taken from 13C spectrum) are not present
at DEPT-135 spectrum. The absence of signal originating from C(3) is due to lack of
hydrogen attached to C(3) – situation present only in ketone form (the signal assignment was
done using 2D NMR: COSY, HSQC and HMBC experiments; Table S1, Fig. S3-5). Further,
the chemical shift value of C(4) more likely originates from an enolic carbon atom (enol A
form) than the ketone one (enol B form). Moreover, an infrared spectrum of 9a shows the
C=O stretching vibration at 1655 cm-1 which is characteristic for an amide carbonyl group, in
contrast to ketone forms for which the values 1750-1700 cm-1 are expected (Fig. S6). A
similar situation is present in all compounds, which suggest the main tautomer in all cases is
the enol A form.
Furthermore, to verify this hypothesis we performed DFT calculations of energy for all
possible tautomeric forms of 9a. The energy, as well as free energy, are collected in Table 3.
The COSMO model has been applied for the consideration of the solvent effects. The various

29
ACCEPTED MANUSCRIPT
solvent with the different character has been used. These calculations clearly show that the
enol A form is preferred over both the ketone and enol B form. The energy of the ketone is
around 2 kcal higher and the energy in the gas phase of enol B is around 1 kcal higher than
the one for the enol A. The calculation with taking into account the solvation effect shows
even bigger preference for the enol form. The observed probability, based on the
thermodynamics, for the enol A form is bigger than for the ketone and for the enol B forms. It
should be pointed out that in cyclohexane the ketone is preferred over enol B form. However
the enol A is still the most probable form.
Table 3. The energy difference, the free energy difference as well as population (pi) of the alternative
tautomers of the compound 9a.
Gas phase
Chloroform
cycohexane
DMSO
Ethanol
forms:
ꢃ E
ꢃ G
ꢃ G
ꢃ G
ꢃ G
ꢃ G
pi
pi
pi
pi
pi
pi
[kcal/mol]
[kcal/mol]
[kcal/mol]
[kcal/mol]
[kcal/mol]
0.00
[kcal/mol]
0.00
0.00
2.18
0.950
0.024
0.00
0.811
0.032
0.00
2.31
0.944
0.019
0.00
3.18
0.994
0.005
0.944
0.009
0.940
0.020
enol A
ketone
1.92
0.97
2.74
2.07
2.24
1.90
2.13
0.026
0.157
1.93
0.038
3.82
0.001
0.029
0.040
enol B
3.3. Biological evaluation
To evaluate the potency of obtained inhibitors, we checked their activity in the
fluorescence polarization (FP) and 1H-15N HSQC NMR titration assays (Table 1).
The FP method show moderate activities of our compounds. The series of the di-
substituted tetramic acid (9a-d) shows the best binding to Mdm2. These results show that it is
beneficial to introduce a chlorine atom to the molecule structure of designed inhibitor. This
trend is illustrated by increasing binding affinity: from the not binding 9b (without the
chlorine atoms) to the good affinity of 9d that has two chlorine atoms. From the other series,
only the N-p-chlorobenzylated tetramic acid 10a and 10b show binding towards Mdm2. None
of the O-alkylated compounds (11a-c, 12a-d) was found active. Such situation can be caused
by either the importance of the 4-OH substituent for the binding, relative positions of
substituents in the central core or the decreasing compound solubility.
To confirm the activities of compounds found in the FP assay, we proceeded with the
1H-15N HSQC NMR titration experiments for selected compounds: 9d and 10a. The method
used is based on monitoring of chemical shift changes in protein amide backbone resonances
upon titration with an increasing amount of the tested compound [50,51,57]. Moreover, since
all cross peaks in the Mdm2 spectrum were assigned to particular amino acid residues [49], it