Identification of inhibitors of the immunosuppressive enzyme IL4I1

Full text of DOI:10.1016/j.bioorg.2019.103463

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Identification of inhibitors of the immunosuppressive enzyme IL4I1
Marc Presset, Diana Djordjevic, Aurélie Dupont, Erwan Le Gall, Valérie
Molinier-Frenkel, Flavia Castellano
PII:
S0045-2068(19)30917-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103463
YBIOO 103463
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To appear in:
Bioorganic Chemistry
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Revised Date:
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7 June 2019
19 November 2019
20 November 2019
Please cite this article as: M. Presset, D. Djordjevic, A. Dupont, E. Le Gall, V. Molinier-Frenkel, F. Castellano,
Identification of inhibitors of the immunosuppressive enzyme IL4I1, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103463
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Identification of inhibitors of the immunosuppressive enzyme IL4I1
Marc Presset1, Diana Djordjevic2,3, Aurélie Dupont2,3, Erwan Le Gall1, Valérie Molinier-Frenkel2,3,4*,
and Flavia Castellano2,3*
1
Electrochimie et Synthèse Organique, Université Paris Est, ICMPE (UMR 7182), CNRS, UPEC, 2 rue
Henri Dunant, F-94320 Thiais, France ;
2 INSERM, U955, Equipe 09, Créteil, France;
3 Université Paris Est, Faculté de Médecine, Créteil, France;
4 AP-HP, Hôpital H. Mondor - A. Chenevier, Laboratoire d’Immunologie, Créteil, France.
* Equal participation
Corresponding authors: Flavia Castellano and Valérie Molinier-Frenkel, INSERM U955, IMRB eq 09,
Hôpital Henri Mondor, 51 av du Maréchal de Lattre de Tassigny, F-94010 Créteil cedex, France;
flavia.castellano@inserm.fr, valerie.frenkel@inserm.fr, Tel: +33 1 49 813765, Fax: +33 1 49813566.
Running title: identification of IL4I1 inhibitors
Key words: immunosuppressive enzymes, cancer therapy, inhibitors, phenylalanine derivatives
1

1 INTRODUCTION
Recent advances in cancer immunotherapy have shown that limiting local or systemic
cancer-related immunosuppression can result in long-lasting clinical responses. Amino-acid
catabolizing enzymes reduce the availability of essential or semi-essential amino acids, while
producing toxic products. Both effects result in impaired anti-tumor T-cell responses and the
promotion of tumor growth [1]. Interleukin-4 Induced gene 1 (IL4I1) is a secreted L-phenylalanine
oxidase expressed by antigen-presenting cells. In vitro, IL4I1 inhibits T-cell proliferation and cytokine
production and facilitates the differentiation of naïve CD4+ T cells into regulatory T cells, in part via
its capacity to produce H2O2 and deplete phenylalanine from the T-cell microenvironment. IL4I1 is
strongly detected in the tumor bed of most human tumor types and, in some cases, such as certain
B cell lymphomas, it is expressed by the tumor cells themselves [2]. Local production of IL4I1
facilitates tumor growth in mouse models by inhibiting the proliferation and function of anti-tumor
CD8+ T cells and altering the balance of immune cells in the tumor microenvironment towards
immunosuppressive populations [3 , 4]. Moreover, the localization and density of IL4I1-expressing
cells correlates with pejorative clinical parameters and tend to predict shorter survival in patients
suffering from melanoma [5]. A correlation between the presence of the IL4I1 protein or mRNA in
the tumor and prognosis has also been observed in renal carcinoma, glioma (The Human Protein
Atlas) [6], colon cancer [7] and breast cancer [8]. These data all suggest that therapeutic strategies
aimed at inhibiting IL4I1 expression or activity in cancer patients may improve immune control of
the tumor, without harming the host, as the absence of IL4I1 in KO mice does not appear to result in
specific pathologies or premature death [9]. Targeting IL4I1 activity in the context of cancer
treatment could thus become a new immunotherapy approach [10].
Phenylalanine (Phe) is the preferred substrate of IL4I1 catalytic activity. Our group has
developed an approach for straightforward three-component reductive coupling between organic
halides, amines, and aldehyde derivatives to produce -branched amines [11],[12]. Here, we
applied this method to synthesize 21 distinct phenylalanine derivatives and tested them, together
with other 7 commercially available phenylalanine derivatives, as potential inhibitors of IL4I1
activity. Seven molecules inhibited IL4I1 enzymatic activity in the micromolar range and three of
them were not toxic in vitro. One could reverse the negative effect of IL4I1 on T-cell proliferation in
cell cultures. This work paves the way for the future development of clinically effective IL4I1
inhibitors.
2

2 METHODS
2.1 Chemical compounds (Cps)
Cp1 (ethyl 2-(4-methylpiperazin-1-yl)-3-phenylpropanoate), Cp2 (ethyl propylphenylalaninate), Cp3
(ethyl 3-(2,6-dichlorophenyl)-2-(piperidin-1-yl)propanoate), Cp4 (ethyl 2-morpholino-3-
phenylpropanoate), Cp5 (ethyl 3-phenyl-2-(piperidin-1-yl)propanoate), Cp6 (ethyl
diethylphenylalaninate), Cp7 (ethyl phenylphenylalaninate), Cp8 (ethyl 2-(allylamino)-3-(3-
(trifluoromethyl)phenyl)propanoate), and Cp9 (ethyl 3-(4-cyanophenyl)-2-
(phenylamino)propanoate) were prepared as described in [C. Haurena, E. Le Gall, S. Sengmany, T.
Martens, M. Troupel, J. Org. Chem. 2010, 75, 2645-2650]. Commercial phenylalanine derivatives : 3-
(4-pyridyl)-L-alanine (Cp10), 1-methyl tryptophane (Cp11), benzoic acid, 3,4-dihydroxy-L-
phenylalanine (Cp12), L-phenylalanine ethyl ester (Cp13), N-acetyl-phenylalanine (Cp14), 2-fluoro-
phenylalanine (Cp15) were purchased from Sigma-Aldrich and used as received.
General procedure: In air, Zn (3.3 equiv) was placed in a round-bottom flask equipped with a stir bar
and the flask closed with a septum and purged with Ar. CH3CN (C = 0.25 M) and TFA (0.1 equiv) were
added and the flask heated to reflux and then cooled to room temperature (RT). Benzyl halide (3.0
equiv) was added and the mixture was stirred at rt (for bromides) or at 60 °C (for chlorides) for 1 h.
In air, a 2-necked round-bottom flask equipped with a stir bar, a condenser and an addition funnel
was charged with ethyl glyoxylate (1.1 equiv) and the amine (1.0 equiv). The solution of organozinc
halide in CH3CN was then added dropwise and the reaction was stirred at rt for 14 h. Then, the
reaction mixture was poured into saturated aqueous NH4Cl and the solution extracted twice with
AcOEt. The combined organic layers were washed with brine, dried (Na2SO4) and evaporated to give
the crude product, which was purified by an acid/base work-up, as described in the above-
mentioned reference.
Cp2-SO4 was prepared as follows. H2SO4 (53 μL, 1.0 mmol, 0.5 equiv) was added to a solution of Cp2
(0.49 g, 2.0 mmol) in anhydrous Et2O (8 mL, C = 0.25 M) and the solution stirred at RT for 1 h. Then,
the supernatant was removed and the paste dried to give the title compound as a sticky paste (333
mg, 50%). 1H NMR (400 MHz, CDCl3): δ 7.21 (s, 5H), 4.12 (dd, J = 10.1, 4.8 Hz, 1H), 4.04 (q, J = 7.1 Hz,
2H), 3.64 (dd, J = 13.4, 6.6 Hz, 1H), 3.26 (dd, J = 13.5, 10.5 Hz, 1H), 3.04-2.93 (m, 2H), 1.87-1.85 (m,
2H), 1.01 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H) [2 NH not detected]. HRMS (ESI/Q-TOF) m/z:
[M+H]+ Calcd for C14H25NO2S 236.1645; Found 236.1645.
3

Cp2-HCl was prepared as follows. A solution of Cp2 (0.5 g, 2.1 mmol) in anhydrous Et2O (20 mL, C =
0.1 M) was saturated with gaseous HCl (prepared by the addition of concentrated H2SO4 to NaCl)
and the solution stirred at RT for 1 h. Then, the suspension was filtered on a fritted funnel and the
1
solid dried to give the title compound as a white solid (0.57 g, 99%). H NMR (400 MHz, MeOD): δ
7.37-7.31 (m, 5H), 4.33 (dd, J = 8.5, 5.4 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.49 (dd, J = 13.6, 5.1 Hz, 1H),
3.18 (dd, J = 13.6, 9.3 Hz, 1H), 3.10-3.00 (m, 2H), 1.84-1.78 (m, 2H), 1.10 (t¸J = 7.1 Hz, 3H), 1.03 (t, J =
7.4 Hz, 3H) [2 NH not detected].
Cp2-TFA was prepared as follows. TFA (0.16 mL, 2.0 mmol, 1.0 equiv) was added to a solution of Cp2
(0.49 g, 2.0 mmol) in anhydrous Et2O (8 mL, C = 0.25 M) and the solution stirred at RT for 1 h. Then,
the suspension was filtered on a fritted funnel and the solid dried to give the title compound as a
1
white solid (154 mg, 20%). H NMR (400 MHz, CDCl3): δ 7.31-7.28 (m, 3H), 7.21-7.19 (m, 2H), 4.13-
4.07 (m, 3H), 3.44 (dd, J = 13.5, 4.8 Hz, 1H), 3.19 (dd, J = 13.4, 10.0 Hz, 1H), 3.08-3.01 (m, 1H), 2.98-
2.91 (m, 1H), 1.83-1.78 (m, 2H), 1.08 (t, J = 7.1 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H) [2 NH not detected].
13C{1H} NMR (100 MHz, CDCl3): δ 168.1 (C), 162.3 (q, J = 35.8 Hz, C), 134.0 (C), 129.1 (2 CH), 128.8 (2
CH), 127.6 (CH), 62.3 (CH2), 61.3 (CH), 48.4 (CH2), 36.0 (CH2), 19.6 (CH2), 13.6 (CH3), 11.0 (CH3) [CF3
not detected]. 19F NMR (376 MHz, CDCl3): δ -75.7 (s, 3F).
Cp7Bis (ethyl (2-(2-ethoxy-1-hydroxy-2-oxoethyl)phenyl)phenylalaninate) was isolated as a side-
1
product of the preparation of Cp7 during flash chromatography on a silica gel. H NMR (400 MHz,
CDCl3): (2 diastereomers) δ 7.29 (s, 8H), 7.18-7.13 (m, 4H), 6,74 (dd, J = 7,3, 7.3 Hz, 2H), 6.60 (dd, J =
8.7, 8.7 Hz, 4H), 5.51 (s, 1H), 5.45 (s, 1H), 4.57 (br s, 2H), 4.42 (br s, 2H), 4.32-4.11 (m, 8H), 3.80 (br s,
1H), 3.73 (br s, 1H), 3.33-3.26 (m, 4H), 1.25-1.15 (m, 12H). 13C{1H} NMR (100 MHz, CDCl3): (2
diastereomers) δ 173.8 (C), 173.8 (C), 173.5 (2 C), 146.6 (C), 146.6 (C), 137.3 (C), 137.2 (C), 135.5 (C),
135.2 (C), 130.8 (CH), 130.6 (CH), 129.2 (2 CH), 129.2 (2 CH), 128.7 (CH), 128.7 (CH), 127.8 (CH),
127.6 (CH), 127.5 (CH), 127.3 (CH), 118.5 (CH), 118.4 (CH), 113.7 (2 CH), 113.6 (2 CH), 70.4 (CH), 70.0
(CH), 62.2 (CH2), 62.2 (CH2), 61.3 (CH2), 61.1 (CH2), 58.4 (CH), 58.3 (CH), 35.7 (CH2), 35.6 (CH2), 14.0
(4 CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C21H26NO5 372.1805; Found 372.1811.
Cp2-pMe (ethyl 2-(propylamino)-3-(p-tolyl)propanoate): The general procedure performed with 4-
methylbenzyl bromide (1.39 g, 7.5 mmol, 3.0 equiv), ethyl glyoxylate (0.64 mL, 50% in toluene,
2.75 mmol, 1.1 equiv) and n-propylamine (0.20 mL, 2.5 mmol, 1.0 equiv) afforded the title
4

1
compound (426 mg, 68%) as a yellow oil. H NMR (400 MHz, CDCl3): δ 7.05 (s, 4H), 4.07 (q, J = 7.1
Hz, 2H), 3.45 (t, J = 7.0 Hz, 1H), 2.89 (dd, J = 8.8, 7.1 Hz, 2H), 2.53 (ddd, J = 11.1, 8.1, 6.7 Hz, 1H), 2.42
(ddd, J = 11.1, 8.1, 6.2 Hz, 1H), 2.28 (s, 3H), 1.62 (br s, 1H), 1.47-1.40 (m, 2H), 1.13 (t, J = 7.0 Hz, 3H),
0.85 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (100 MHz, CDCl3): δ 174.5 (C), 135.8 (C), 134.0 (C), 128.8 (2 CH),
128.8 (2 CH), 62.9 (CH), 60.2 (CH2), 49.8 (CH2), 39.1 (CH2), 23.0 (CH2), 20.8 (CH3), 13.9 (CH3), 11.4
(CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C15H24NO2 250.1801; Found 250.1801.
Cp2-mMe (ethyl 2-(propylamino)-3-(m-tolyl)propanoate): The general procedure performed with 3-
methylbenzyl bromide (1.0 mL, 7.5 mmol, 3.0 equiv), ethyl glyoxylate (0.64 mL, 50% in toluene,
2.75 mmol, 1.1 equiv) and n-propylamine (0.20 mL, 2.5 mmol, 1.0 equiv) afforded the title
compound (432 mg, 69%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.15 (dd, J = 7.4, 7.4 Hz, 1H),
7.02-6.97 (m, 3H), 4.09 (q, J = 7.0 Hz, 2H), 3.48 (t, J = 7.0 Hz, 1H), 2.96-2.84 (m, 2H), 2.57-2.51 (m,
1H), 2.43 (ddd, J = 11.1, 8.1, 6.3 Hz, 1H), 2.31 (s, 3H), 1.64 (br s, 1H), 1.49-1.42 (m, 2H), 1.14 (t, J =
7.0 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (100 MHz, CDCl3): δ 174.7 (C), 137.7 (C), 137.2 (C),
129.9 (CH), 128.1 (CH), 127.2 (CH), 126.1 (CH), 63.0 (CH), 60.3 (CH2), 49.9 (CH2), 39.6 (CH2), 23.1
(CH2), 21.2 (CH3), 14.1 (CH3), 11.5 (CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C15H24NO2
250.1801; Found 250.1802.
Cp2-sulfamide (ethyl N-(methylsulfonyl)-N-propylphenylalaninate) was prepared as follows.
Triethylamine (0.3 mL, 2.0 mmol, 2.0 equiv) and mesyl chloride (0.1 mL, 1.1 mmol, 1.1 equiv) was
added to a solution of Cp2 (235 mg, 1.0 mmol) in CH2Cl2 (2 mL) at 0 °C under Ar and the reaction
stirred at 0°C for 2 h. Then, the reaction mixture was poured into saturated aqueous NH4Cl (10 mL)
and extracted twice with CH2Cl2 (2x10 mL). The combined organic layers were dried (Na2SO4) and
evaporated. Purification of the residue by flash chromatography gave the title compound as a
1
colorless oil (176 mg, 56%). H NMR (400 MHz, CDCl3): δ 7.32-7.25 (m, 5H), 4.74 (t, J = 7.4 Hz, 1H),
4.19 (q, J = 7.0 Hz, 2H), 3.38 (dd, J = 14.1, 7.4 Hz, 1H), 3.28-3.20 (m, 1H), 3.13 – 3.03 (m, 2H), 2.73 (s,
3H), 1.691.58 (m, 2H), 1.26 (t, J = 7.0 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (100 MHz, CDCl3):
δ 170.8 (C), 137.1 (C), 129.3 (2 CH), 128.6 (2 CH), 127.0 (CH), 61.8 (CH), 61.7 (CH2), 48.0 (CH2), 39.9
(CH3), 37.0 (CH2), 23.4 (CH2), 14.1 (CH3), 11.4 (CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for
C15H24NO4S 314.1420; Found 314.1417.
Cp2-sulfamic acid (1-ethoxy-1-oxo-3-phenylpropan-2-yl)(propyl)sulfamic acid) was prepared as
follows. Chlorosulfonic acid (25 μL, 0.38 mmol, 1.1 equiv) was added to a solution of Cp2 (82 mg,
5

0.35 mmol) in CH2Cl2 (2 mL) at 0°C under Ar and the reaction stirred at 0°C for 2 h. Then, the
reaction mixture was evaporated and Et2O (10 mL) added. The resulting mixture was stirred
overnight at RT and the suspension filtered on a fritted funnel and the solid dried to give the title
1
compound (89 mg, 80%) as a white solid. H NMR (400 MHz, CDCl3): δ 8.96 (s, 1H), 7.29-7.24 (m,
5H), 4.19 (br s, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.63 (dd, J = 13.1, 4.6 Hz, 1H), 3.29 – 3.23 (m, 1H), 3.11
(br s, 1H), 2.98 (br s, 1H), 1.93-1.86 (m, 2H), 1.03 (t, J = 7.1 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H). 13C{1H}
NMR (100 MHz, CDCl3): δ 168.2 (C), 134.2 (C), 129.5 (2 CH), 128.8 (2 CH), 127.6 (CH), 62.6 (CH), 61.9
(CH2), 49.4 (CH2), 36.4 (CH2), 19.6 (CH2), 13.8 (CH3), 11.0 (CH3). HRMS (ESI/Q-TOF) m/z: [M–H]– Calcd
for C14H20NO5S 314.1067; Found 314.1066.
Cp16 (ethyl 3-(2,6-dichlorophenyl)-2-(diethylamino)propanoate): The general procedure performed
with 2,6-dichlorobenzyl bromide (3.6 g, 15 mmol, 3.0 equiv), ethyl glyoxylate (1.3 mL, 50% in
toluene, 5.5 mmol, 1.1 equiv) and diethylamine (0.52 mL, 5 mmol, 1.0 equiv) afforded the title
compound (1.064 g, 67%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.25 d, J = 7.9 Hz, 2H), 7.06 (t,
J = 7.9 Hz, 1H), 4.11 (q, J = 7.0, 2H), 3.92 (t, J = 7.6 Hz, 1H), 3.39–3.23 (m, 2H), 2.79–2.74 (m, 2H),
2.58–2.53 (m, 2H), 1.18 (t, J = 7.0 Hz, 3H), 0.93 (t, J = 6.9 Hz, 6H). 13C{1H} NMR (100 MHz, CDCl3): δ
172.6 (C), 136.0 (2 C), 135.2 (C), 128.0 (2 CH), 127.8 (CH), 60.7 (CH2), 60.2 (CH), 44.3 (2 CH2), 31.5
(CH2) 14.3 (CH3), 13.8 (2 CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C15H22Cl2NO2 318.1022;
Found 318.1020.
Cp17 (ethyl diethylphenylalaninate): The general procedure performed with benzyl chloride (1.9 mL,
15 mmol, 3.0 equiv), ethyl glyoxylate (1.3 mL, 50% in toluene, 5.5 mmol, 1.1 equiv) and
diethylamine (0.52 mL, 5 mmol, 1.0 equiv) afforded the title compound (0.82 g, 65%) as a yellow oil.
1H NMR (400 MHz, CDCl3): δ 7.36–7.11 (m, 5H), 4.10 (ddd, J = 6.8, 4.5, 2.1 Hz, 2H), 3.68–3.59 (m,
1H), 3.15–3.04 (m, 1H), 2.91 (dd, J = 13.4, 6.2 Hz, 1H), 2.81 (ddd, J = 14.5, 8.2, 6.4 Hz, 2H), 2.57 (ddd,
J = 13.3, 6.9, 1.9 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H), 1.05 (t, J = 7.1 Hz, 5H). 13C{1H} NMR (100 MHz,
CDCl3): δ 172.6 (C), 138.8 (C), 129.3 (2 CH), 128.2 (2 CH), 126.2 (CH), 65.0(CH), 60.0 (CH2), 44.5 (2
CH2), 36.4 (CH2), 14.3 (CH3), 13.8(2 CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C15H24NO2
250.1801; Found 250.1800.
Cp18 (ethyl 3-(2,6-dichlorophenyl)-2-(propylamino)propanoate): The general procedure performed
with 2,6-dichlorobenzyl bromide (3.6 g, 15 mmol, 3.0 equiv), ethyl glyoxylate (1.3 mL, 50% in
toluene, 5.5 mmol, 1.1 equiv) and n-propylamine (0.4 mL, 5 mmol, 1.0 equiv) afforded the title
compound (0.58 g, 38%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.25 (d, J = 7.8 Hz, 2H), 7.07 (t, J
6

= 7.8 Hz, 1H), 4.08 (q, J = 7.4 Hz, 2H), 3.64–3.61 (m, 1H), 3.26–3.22 (m, 2H), 2.60–2.35 (m, 2H), 1.71
(br s, 1H), 1.50–1.35 (m, 2H), 1.11–1.08 (m, 3H), 0.84 (t, J = 9.4 Hz, 3H). 13C{1H} NMR (100 MHz,
CDCl3): δ 174.5 (C), 136.0 (2 C), 134.3 (C), 128.1 (CH), 128.1 (2 CH), 60.8 (CH2), 60.0 (CH), 49.9 (CH2),
35.2 (CH2), 23.2 (CH2), 14.0 (CH3), 11.6 (CH3). HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C14H20Cl2NO2
304.0865; Found 304.0865.
Cp19 (ethyl 3-phenyl-2-(piperidin-1-yl)propanoate): The general procedure performed with benzyl
chloride (1.9 mL, 15 mmol, 3.0 equiv), ethyl glyoxylate (1.3 mL, 50% in toluene, 5.5 mmol, 1.1 equiv)
and piperidine (0.5 mL, 5 mmol, 1.0 equiv) afforded the title compound (0.78 g, 59%) as a yellow oil.
1H NMR (400 MHz, CDCl3): δ 7.27–7.21 (m, 5H), 4.08–4.06 (m, 2H), 3.42 (dd, J = 9.9, 5.4 Hz, 1H),
3.12–3.06 (m, 1H), 2.97 (dd, J = 13.1, 4.9 Hz, 1H), 2.75–2.55 (m, 4H), 1.70–1.35 (m, 6H), 1.13 (t, J =
7.1 Hz, 3H). 13C{1H} NMR (100 MHz, CDCl3): δ 171.4 (C), 138.4 (C), 129.3 (2 CH), 128.2 (2 CH), 126.3
(CH), 70.4 (CH), 59.9 (CH2), 51.1 (CH2), 35.9 (CH2), 26.4 (3 CH2), 24.6 (CH2), 14.4 (CH3). HRMS (ESI/Q-
TOF) m/z: [M+H]+ Calcd for C16H24NO2 262.1801; Found 262.1800.
Cp20 (methyl propylphenylalaninate) was prepared as follows. A solution of Cp2 (235 mg, 1.0 mmol)
and Sc(OTf)3 (98 mg, 0.2 mmol, 0.2 equiv) in CH3OH (10 mL) was heated under reflux for 18 h. Then,
the reaction mixture was evaporated. Purification of the residue by flash chromatography gave the
1
title compound as a yellow oil (176 mg, 79%). H NMR (400 MHz, CDCl3): δ 7.30–7.18 (m, 5H), 3.64
(s, 3H), 3.54 (t, J = 6.9 Hz, 1H), 2.97 (t, J = 7.0 Hz, 2H), 2.59–2.42 (m, 2H), 1.49–1.46 (m, 3H), 0.88 (t, J
= 7.3 Hz, 3H). 13C{1H} NMR (100 MHz, CDCl3): δ 175.1 (C), 137.2 (C), 129.0 (2 CH), 128.3 (2 CH), 126.6
(CH), 63.0 (CH), 51.5 (CH3), 50.0 (CH2), 39.6 (CH2), 23.1 (CH2), 11.5 (CH3). HRMS (ESI/Q-TOF) m/z:
[M+H]+ Calcd for C13H20NO2 222.1488; Found 222.1487.
2.2 Sample preparation for in vitro assays
Stock solutions to be used for the enzymatic and cellular tests were prepared by suspending the
compounds in DMSO at a concentration of 100 mM. Working solutions of the compounds were
diluted in DMSO and stored at -20°C. Potential inhibitors were added at a 1:1000 dilution to the
enzyme solution or culture medium.
2.3 Cells
Peripheral blood mononuclear cells (PBMCs), obtained from healthy donors by cytapheresis, were
provided by the Etablissement Français du Sang (EFS) and separated using Unisep tubes filled with
lymphocyte-separation medium (EuroBio, France). The Jurkat T cell line and human PBMCs were
7

cultivated in RPMI 1640 medium containing 10% fetal calf serum, 50 U/mL penicillin, and 50 g/mL
streptomycin. For measures of cell growth, Jurkat cells were seeded at 0.1 x 106/mL in a 24-well
plate and counted on days 1, 2, 3, and 6 in disposable Fast-read 10 chambers after dilution in Trypan
blue. THP1 and THP1 cells stably expressing IL4I1 (THP1-IL4I1) have been described previously [13]
and were used in proliferation experiments after irradiation at 200 Gy.
2.4 Determination of Ki.
Recombinant human IL4I1 (from R&D Systems, Inc) was diluted in PBS at 100 ng/mL and pre-
incubated for 30 min with various amounts of each inhibitor dissolved in DMSO (50, 25, and 2.5
μM) or DMSO as a control. The activity was tested in the presence of various amounts of substrate
(0.25, 0.5, 1, 2, or 10 mM phenylalanine) using the fluorometric IL4I1 assay described by
Carbonnelle-Puscian et al. [2]. The Ki for competitive inhibition was calculated using Graph Pad
Prism software.
2.5 Metabolism-based cell proliferation assay.
Jurkat cells were seeded at 0.1 x 106/mL in a 96-well flat-bottom plate and incubated at 37°C in 5%
CO2. At successive time points, cells were centrifuged, the medium removed, and CellTiter 96® Non-
Radioactive Cell Proliferation Assay reagent (Promega) added. The cells were then incubated an
additional 4 h before measuring the optical density at 570 nm in a plate reader (BMG Labtech
Fluostar Optima).
2.6 Apoptosis assay.
Cells were seeded at 5 x 105/mL in 24-well plates, with or without the compounds at 100 M or 10
M etoposide (VP16) as a positive control, and incubated at 37°C for 24 h. Cells were then fixed and
permeabilized using cytox/cytoperm solution (BD Bioscience), followed by the addition of anti-
activated caspase 3-AlexaFluor 647 (Rabbit anti-active Caspase 3 clone C92-605, BD Pharmingen).
Data acquisition was performed on a Cyan ADP LX7 (Beckman Coulter) or Fortessa X20 (BD
Bioscience), followed by analysis using Flowjo 10 software.
2.7 Flow cytometry analysis of cell proliferation.
Jurkat cells and PBMCs were labeled with 2 M Cell Proliferation Dye (CPD) eFluor670 (Thermo
Fisher) for 10 min at 37°C. Each compound or control DMSO was added to the cells before seeding
them at 2 x 105/well in 96-well U-shaped plates. PBMCs were stimulated in plates pre-coated
overnight with an anti-CD3 antibody (clone OKT3, eBioscience) at 0.5 g/mL in PBS. Cells were
collected on day 0, 3, and 6 for fluorescence acquisition using a Cyan ADP LX7 (Beckman Coulter).
8

The reversion of the effect of IL4I1 on T cell proliferation was tested by incubating PBMCs labeled
with CPD (2 x 105/well) with irradiated THP1 or THP1-IL4I1 cells (2.5 x 105/well) in the presence of
various concentrations of Cp2-SO4 dissolved in DMSO (100, 200, or 400 M) or the equivalent
volume of DMSO. Proliferation was induced by the addition of soluble OKT3 (20 ng/mL). Cells were
collected on day 5 for flow cytometry acquisition on a Fortessa X20 (BD Bioscience). FlowJo 10
software was used for all data analyses and the calculation of proliferation and expansion indices.
Statistical analysis (paired Student t-test) was performed using Prism 6.0.
3 RESULTS
3.1 Synthesis of the phenylalanine derivatives
IL4I1 is an immunosuppressive enzyme and the ability to control its enzymatic activity could
be beneficial in situations in which its ectopic or deregulated activity limits the immune response, as
in cancer. We generated potential inhibitors of IL4I1 by modifying the backbone of its preferential
substrate, phenylalanine, using an expedient three-component Mannich-type reaction using
aldehyde derivatives of organic halides to produce α-branched amines. We also tested 7
commercially available phenylalanine derivatives. The various compounds tested and their
structures are shown in Table I.
Compound structure
Compound number
Inhibitory activity and KI (M)
N
1
N.I.
N
CO2Et
H
N
2-SO4
2-HCl
21.2  5.9
N.I.
. H2SO4
CO2Et
H
N
N
. HCl
CO2Et
H
2-TFA
2-pMe
N.I.
. CF3CO2H
CO2Et
H
N
N.I.
CO2Et
9

H
S
S
N
N
2-mMe
N.I.
N.I.
N.I.
CO2Et
CO2Et
CO2Et
O
2-sulfamide
2-sulfamic acid
O
O
OH
N
O
Cl
N
3
4
9.8  4.6
CO2Et
Cl
O
N
N.I.
CO2Et
5
6
7
N.I.
N
N
CO2Et
CO2Et
102.0  29.7
26.8  6.9
H
N
CO2Et
OH
EtO2C
H
N
7bis
8
24.8  12.2
CO2Et
CF3
H
N
N.I.
CO2Et
NC
H
9
N.I.
N
CO2Et
N
NH2
CO2H
10
2800  310
10

N
NH2
CO2H
11
N.I.
N.I.
HO
HO
NH2
CO2H
12
13
NH2
3100 560
2100  696
CO2Et
O
HN
14
15
CO2H
NH2
N.I.
N.I.
CO2H
F
Cl
N
16
CO2Et
Cl
N
17
18
N.I.
CO2Et
CO2Et
Cl
HN
49.9  20.9
Cl
19
20
N
333.9  198.0
CO2Et
HN
1320
CO2Me
Table 1 – Structure of the phenylalanine derivatives tested as inhibitors of IL4I1. Compounds are
listed with numbers. Ki of inhibition were obtained using Graph Pad Prism software and using 3
different concentrations of each compound (50, 25 and 2.5 μM) in the modified enzymatic test
described by Carbonelle-Puscian [14] Tests were performed 3 times for Cp1 to Cp17, two times for
Cp18 and Cp19 and once for Cp20. N. I. means no inhibition. Values are reported in μM ±S.D.
3.2 Characterization of the IL4I1 inhibitory capacity of the phenylalanine derivatives
We first determined the inhibitory properties of the 21 phenylalanine derivatives synthetized
using our method and 7 commercially available compounds on the enzymatic activity of IL4I1.
11

Purified recombinant human IL4I1 enzyme was pre-incubated for 30 min with decreasing amounts
of each inhibitor and the IL4I1 enzymatic activity tested in the presence of variable amounts of
substrate to perform a Michaelis-Menten reaction, using a variation of the fluorometric method
described by Carbonnelle-Puscian [2]. Most compounds, such as Cp4, Cp5, Cp8, Cp9, Cp11, Cp15,
Cp16, Cp17 and all Cp2 derivatives except Cp2-SO4, did not interfere with IL4I1 activity. Cp1, which
is modified on the amino group by an aromatic CH2CH2N(CH3)CH2CH2 group, and the di-phenyl-
hydroxy-phenylalanine did not block the activity and behaved as a slightly more efficient substrate
of IL4I1 than phenylalanine (data not shown). Benzoic acid has been previously suggested as an
inhibitor of IL4I1 [15]. We indeed measured diminished IL4I1 activities in the presence of benzoic
acid but this was due to its effect on the peroxidase used in the indirect enzymatic test to measure
H2O2 production. Seven compounds (Cp2-SO4, Cp3, Cp6, Cp7, Cp7Bis, Cp18 and Cp19) showed
inhibitory activity in the micromolar range, while Cp10, Cp13, Cp14 and Cp20 inhibited IL4I1 activity
in the millimolar range, although Cp14 later became a substrate for IL4I1. The competitive inhibition
of human IL4I1 activity and their Kis are reported in Figure 1 for Cp2-SO4, Cp3, Cp6, Cp7 and Cp7Bis
and in Table 1 for all compound tested, respectively. Amongst the most effective compounds, Cp6,
Cp18 and Cp19 displayed the weakest inhibitory activity. Cp2-SO4, Cp7, and Cp7Bis showed a similar
inhibitory capacity, with Ki of 21.26 ± 5.9, 26.89 ± 6.91, and 24.82 ± 12.1 μM, respectively. Cp3,
characterized by two Cl groups on the aromatic ring of phenylalanine and a CH2CH2CH2CH2CH2 group
on the lateral chain, showed the strongest inhibitory capacity with a Ki of 9.8 ± 4.6 μM. Some of the
Cps, although active against the human enzyme, did not inhibit mouse IL4I1 (data not shown).
3.3 Cp2-SO4 and Cp6 are not toxic
We next sought to verify whether the active inhibitors interfere with cell viability and
proliferation (Figures 2, 3, & 4). Since IL4I1 acts on T lymphocytes, we first chose the human T-cell
line Jurkat for our tests. Cells were cultivated in the presence of 100 M of each inhibitor and
compared to cells to which an equivalent volume of DMSO, the vehicle of the inhibitors (1:1000
maximum), had been added. We initially counted the viable cells at various time points to generate
growth curves (Figure 2A). Compounds such as Cp10 were extremely toxic even at doses largely
inferior to the Ki values (data not shown) excluding them from any possible consideration and
further work in cellular systems. Cp3 and Cp7 significantly slowed the growth of Jurkat cells, as early
as one day after the beginning of the culture, with no more viable cells detectable by day 6. On the
contrary, Cp2-SO4, Cp6, and Cp7Bis did not substantially decrease the growth of Jurkat cells, even
after six days. We confirmed these results using a metabolism-based cell proliferation assay: while
12

Cp3 and Cp7 strongly limited Jurkat cell proliferation, Cp2-SO4 and Cp6 had no effect (Figure 2B).
Finally, we analyzed the effect of Cp2-SO4 and Cp6 on the proliferation of both Jurkat cells (Figure
3A) and primary human T cells stimulated with an anti-CD3 antibody (Figure 3B). The cells were
labeled with a fluorescent cell-proliferation dye (CPD) on day 0 and the dilution of the dye induced
by cell division was measured by flow cytometry during the next several days. As expected, the two
compounds had no effect on the proliferation of Jurkat cells relative to cells treated with DMSO.
Interestingly, the proliferation of primary T-cells was also not affected, as established by calculation
of the expansion and proliferation indices (Figure 3C).
Thus, the candidate inhibitors Cp2-SO4 and Cp6 did not inhibit cell proliferation. However,
they may still induce apoptotic cell death. We evaluated this possibility by first incubating Jurkat
cells overnight with various compounds, including Cp2-SO4, Cp6, Cp7Bis or etoposide (VP16, a
clinical chemotherapy drug known for its proapoptotic properties [16]), as a positive control. We
then measured the appearance of the cleaved form of caspase 3, which results from its activation
during the early stages of apoptosis (Figure 4). Cp3 and Cp7 both induced the activation of caspase 3
in Jurkat cells, with Cp3 displaying a proapoptotic effect equivalent to that of VP16. In contrast,
neither Cp2-SO4 nor Cp6 and Cp7Bis induced significant caspase 3 activation, i.e. above the levels
detected in DMSO-treated control Jurkat cells. Cp2-SO4 and Cp6 also did not induce the apoptosis of
primary lymphocytes. Thus, our data suggest that the phenylalanine derivatives Cp2-SO4 and Cp6
are non-toxic in vitro for human primary cells.
3.4 Reversion of the inhibitory effect of IL4I1 by Cp2-SO4.
IL4I1 is an immunosuppressive enzyme, which mainly functions as an inhibitor of T-cell proliferation.
We thus decided to test whether Cp2-SO4 can reverse the antiproliferative effect of IL4I1 on T cells.
We stimulated PBMCs with an anti-CD3 antibody, with or without IL4I1, using the THP1 and THP1-
IL4I1 cell lines as described by Marquet et al. [13]. As expected, polyclonal T-cell stimulation was
inhibited in the presence of IL4I1 (Figure 5A). Both the mean division number, evaluated by the
proliferation index (Figure 5B), and the total population amplification, evaluated by the expansion
index (Figure 5C), were affected. This inhibition was reversed in a dose dependent manner by the
addition of Cp2-SO4. A proliferation level similar to that of PBMCs stimulated without IL4I1 was
obtained with doses of the inhibitor of 200 and 400 M. Thus, our data identify the first non-toxic
IL4I1 inhibitor that is effective in the micromolar range to reverse IL4I1 inhibitory effect and that
could be used to block IL4I1-induced immunosuppression.
13

4 DISCUSSION
IL4I1 has been implicated in tumor escape from the immune response and represents a
promising candidate for cancer immunotherapy. No specific inhibitors have been described for this
enzyme to date. Here, we characterize in vitro, for the first time, small phenylalanine derivatives
with IL4I1 inhibitory activity without an apparent toxic effect on human cells. One of the compounds
can reverse the blocking effect of IL4I1 on T cell proliferation, thus displaying the expected biological
effect.
Among the 28 molecules tested, only 7 showed inhibitory activity in the micromolar range.
Their comparison allowed us to identify several chemical features that are important for the
inhibitory effect and those to avoid reducing cell toxicity. Indeed, all compounds, except Cp3 and
Cp18, which are modified on the aromatic group of phenylalanine (i.e. Cp8, Cp9, Cp2-pMe, and Cp2-
mMe) did not show inhibitory activity, suggesting that the aromatic group may be important to
enter the catalytic groove of IL4I1. IL4I1 is an L-amino acid oxidase similar to those found in snake
venom [17]. Many of these enzymes have a strong preference for hydrophobic amino acids which
perfectly suit the hydrophobic binding pocket in their catalytic cavity, as shown by crystallization
studies. Substitutions at the phenylalanine ring may create steric hindrance at the active site [18]
and thus interfere with the capacity of the inhibitor to compete with the natural substrate. An
exception appears to be the presence of Cl groups on the aromatic ring. Indeed, these groups seem
to confer strong inhibitory activity to Cp3, which otherwise shares the same structure with the non-
active Cp5.
Surprisingly, Cp2-SO4 showed an inhibitory activity while Cp2 associated with different
counter ions was not effective. One hypothesis is that the counter ion can change the solubility of
the compound or its stability, thus facilitating or prolonging its inhibitory activity. This would be
consistent with recently reported data on two different salts of an inhibitor of the human  tryptase
[19]. Alternatively, the counter ion may directly participate to the inhibitory effect. This second
hypothesis is supported by the structural studies of Pawelek of the L-Amino acid oxidase from
Calloselasma rhodostoma which is highly homologous to IL4I1 [20]. Indeed, the α carbon atom of
the phenylalanine substrate forms a strong salt bridge interaction with the guanidinium group of
Arg90 (equivalent to Arg97 in human IL4I1) of the catalytic site. Thus, the H2SO4 counter ion may be
more efficient in competing for this interaction than the others we tested.
We tested the toxicity of the active molecules in vitro on a human T-cell line and primary
human PBMCs. The most active compound, Cp3, showed strong toxicity. Cp7 was also toxic, as seen
14

in both cell proliferation and the induction of apoptosis. Modification of the R3 position by a C6H5
group in the Cp7Bis molecule reduced this toxicity to values comparable to the control, while almost
keeping the same Ki. These observations will be important for future improvements of the active
compounds.
We tested one of the nontoxic active compounds, Cp2-SO4, for its ability to reverse the
immunosuppressive action of IL4I1 in vitro. Indeed, the compound was able to restore T-cell
proliferation, even with a Ki that is only in the micromolar range. This suggests that improving the
backbone of any of our nontoxic compounds to more strongly inhibit the enzymatic activity of IL4I1
could be a valid approach to develop clinically amenable anticancer drugs.
5 CONCLUSION
In this work we have identified easily synthesizable phenylalanine derivatives endowed with IL4I1
inhibitory activity. Even if the non-toxic Cp2-SO4 had a Ki in the micromolar range, it was able to
reverse IL4I1 inhibitory effect in vitro on human T cells. Since IL4I1 plays a role in tumor escape from
the immune system, such inhibitors may be envisioned as a basis for future development of anti-
cancer drugs.
6 AKNOWLEDGEMENTS
This work was conducted with the support of Bristol-Myers Squibb Foundation for Research in
Immuno-Oncology, with the support of the Institut National du Cancer grant PLBIO18-255, and a
pre-maturation grant from SATT-idf-Innov/UPEC/IMRB/IPCME. We thank Allan Thiolat for help in
cell irradiation. This work has been revised by a professional English editing company (Alex Edelman
& associates).
7 CONFLICT OF INTEREST
The authors declare no commercial or financial conflict of interest.
8 AUTHORS’ CONTRIBUTIONS
Chemical synthesis: Marc Presset, Erwan Le Gall
Conception, design and supervision: Flavia Castellano and Valérie Molinier-Frenkel
Development of methodology: Flavia Castellano
15

Acquisition of the data: Aurelié Dupont, Diana Djordjevic, Flavia Castellano
Analysis and interpretation of data: Flavia Castellano and Valérie Molinier-Frenkel
Writing, review, and/or revision of the manuscript: Valérie Molinier-Frenkel, Erwan Le Gall, Marc
Presset and Flavia Castellano
9 FIGURE LEGENDS
Figure 1. Michaelis-Menten plots of the molecules tested. Serial dilutions of the compounds (2.5,
25, and 50 M) dissolved in DMSO were tested on the activity of a 100 ng/mL solution of
recombinant human IL4I1 using various concentrations of the enzyme substrate. Controls (0 M)
contained an equal volume (1:1000) of DMSO. The data shown are mean  SEM of two to four
independent experiments.
Figure 2. Cps 2-SO4, 6, and 7Bis do not decrease Jurkat cell growth. A) 105 Jurkat cells per mL were
cultured in the presence of 100 M of each compound or an equivalent volume of DMSO (control).
An aliquot was taken at various times (1,2,3, and 6 days) and the Trypan blue-negative cell number
evaluated and reported as the percentage of control cell value. The results shown are the average of
three independent experiments. B). 105 Jurkat cells per mL were seeded in a 96 well plate for 24 h
before the addition of the Vibrant MTT reagent and measurement of metabolic proliferation by
spectrometry. Results are reported as the percentage of the control value (cells grown in the
presence of DMSO) and are the mean of three independent experiments. *p < 0.01, **p < 0.001,
*** p < 0.0001, paired t-test.
Figure 3. Cp2-SO4 and Cp6 do not alter cell proliferation. A) 105 Jurkat cells and B) PBMC were
labeled with Cell Proliferation Dye 670 (CPD) and grown for up to several days in complete medium
containing DMSO or 100 M Cp2-SO4 or Cp6. Cells were analyzed each day by flow cytometry.
Representative histograms are shown. C) The proliferation index (left) and expansion index (right)
were evaluated on day 3 for stimulated PBMC using the proliferation module of FlowJo.
Figure 4. Cp2-SO4, Cp6, and Cp7Bis do not induce apoptosis. Activated caspase 3 was measured by
flow cytometry after exposition of cells to the compounds at 100 M for 24h. A & B) Jurkat cells. A)
Representative histograms. B) Mean of three independent experiments  SEM. C) PBMC. Mean of
three independent experiments  SEM. *p < 0.01, **p < 0.001, *** p < 0.0001, paired t-test.
Figure 5. Cp2-SO4 reverses IL4I1-dependet inhibition of T-cell proliferation. 2 x 105 CPD Alexa
Flour670-labeled PBMCs were incubated with 0.25 x 106 irradiated THP1 or THP1-IL4I1 cells in the
presence of various concentrations of Cp2-S04 dissolved in DMSO (100, 200, or 400 M). Control
THP1 or THP1-IL4I1 were treated with equivalent volumes of DMSO. T cell proliferation was induced
by the addition of anti-CD3 antibody and evaluated on day 5 by flow cytometry. A) Representative
histogram with Cp2-SO4 at 400 M. B) Proliferation index, and C) expansion index. B & C,
cumulative results from four independent experiments using cells of three different donors are
shown as mean  SEM. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001, paired t-test.
16

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11 GLOSSARY
IL4I1 Interleukin-induced gene 1
PBMC Periferal Blood Mononuclear Cells
18

GA
19

20

21

22

23

24

Highlights
 Identification of easily synthesizable phenylalanine derivatives inhibitors of the
immunosuppressive enzyme Interleukin 4-induced gene 1 (IL4I1).
 reversion of IL4I1 inhibitory effect on primary T lymphocytes proliferation by the compound
2-SO4
25

Conflict of interest
All the authors declare no conflict of interest
26