N 1-fluoroalkyltryptophan analogues: Synthesis and in vitro study as potential substrates for indoleamine 2,3-dioxygenase

Article abstract of DOI:10.1021/ml500385d

Indoleamine 2,3-dioxygenase (hIDO) is an enzyme that catalyzes the oxidative cleavage of the indole ring of l-tryptophan through the kynurenine pathway, thereby exerting immunosuppressive properties in inflammatory and tumoral tissues. The syntheses of 1-(2-fluoroethyl)-tryptophan (1-FETrp) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-tryptophan, two N¹-fluoroalkylated tryptophan derivatives, are described here. In vitro enzymatic assays with these two new potential substrates of hIDO show that 1-FETrp is a good and specific substrate of hIDO. Therefore, its radioactive isotopomer, 1-[¹⁸F]FETrp, should be a molecule of choice to visualize tumoral and inflammatory tissues and/or to validate new potential inhibitors.

Full text of DOI:10.1021/ml500385d

Letter
pubs.acs.org/acsmedchemlett
N1‑Fluoroalkyltryptophan Analogues: Synthesis and in vitro Study
as Potential Substrates for Indoleamine 2,3-Dioxygenase
Jean Henrottin,*,†,‡ Astrid Zervosen,† Christian Lemaire,† Frederic Sapunaric,§ Sophie Laurent,§
́
́
Benoit Van den Eynde,∥ Serge Goldman,⊥,# Alain Plenevaux,† and Andre Luxen†
́
‡
§
†Cyclotron Research Center, Department of Chemistry, and Macromolec
́
ules Biologiques, Center for Protein Engineering,
Universite de Liege, Sart-Tilman, B-4000 Liege, Belgium
́
̀
̀
∥Ludwig Institute for Cancer Research, Brussels Branch and de Duve Institute, Universite
Belgium
́
Catholique de Louvain, B-1200 Brussels,
⊥PET/Biomedical Cyclotron Unit and Department of Nuclear Medicine, Erasme Hospital, Universite
B-1070 Brussels, Belgium
́
Libre de Bruxelles,
#Center for Microscopy and Molecular Imaging, Rue Adrienne Bolland 8, B-6041 Gosselies, Belgium
S
* Supporting Information
ABSTRACT: Indoleamine 2,3-dioxygenase (hIDO) is an
enzyme that catalyzes the oxidative cleavage of the indole
ring of L-tryptophan through the kynurenine pathway, thereby
exerting immunosuppressive properties in inflammatory and
tumoral tissues. The syntheses of 1-(2-fluoroethyl)-tryptophan
(1-FETrp) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)-
methyl)-tryptophan, two N1-fluoroalkylated tryptophan de-
rivatives, are described here. In vitro enzymatic assays with
these two new potential substrates of hIDO show that 1-FETrp is a good and specific substrate of hIDO. Therefore, its
radioactive isotopomer, 1-[18F]FETrp, should be a molecule of choice to visualize tumoral and inflammatory tissues and/or to
validate new potential inhibitors.
KEYWORDS: Tryptophan, indoleamine 2,3-dioxygenase, kynurenine, 1-(2-fluoroethyl)-tryptophan, tryptophan 2,3-dioxygenase,
positron emission tomography
a
Scheme 1. The Two First Steps of the Kynurenine Pathway
L-Tryptophan (Trp), the least abundant of the essential amino
acids, is used in three biological processes: (i) the serotonin
pathway, which converts only tiny amounts (about 1%) of
dietary Trp into serotonin, (ii) protein synthesis, and (iii) the
kynurenine pathway, metabolizing more than 95% of Trp and
leading to the biosynthesis of kynurenine (KYN), nicotinamide
adenine dinucleotide (NAD+), kynurenic acid (KYNA),
3-hydroxyanthranilic acid (3-HAA), etc.1−4 The initial and
rate-limiting step of the kynurenine pathway requires the
involvement of indoleamine 2,3-dioxygenase (hIDO), or
tryptophan 2,3-dioxygenase (hTDO; mainly expressed in
liver). These structurally different heme-containing enzymes
catalyze the C2−C3 oxidative cleavage of the pyrrole ring of the
indole nucleus of Trp, yielding to the N-formyl-L-kynurenine
(Scheme 1).5−8 hIDO expression is strongly stimulated by
interferon-γ (IFN-γ) and also, to some extent, by cytokines
such as interleukins (IL-1β, IL-2) and tumor necrosis factor
(TNF-α).6,9−15
aL-Kynurenine, the key intermediate of the kynurenine pathway, is
formed from L-tryptophan in two steps catalyzed by indoleamine
2,3-dioxygenase (or tryptophan 2,3-dioxygenase) and formamidase,
respectively.
accumulation of immunosuppressive tryptophan catabolites,
both of which result in a local suppression of the immune
response by blocking T-lymphocyte proliferation in the G1
phase of the cell cycle.1,5,7,16,17
Recently, in order to improve cancer immunotherapy and to
counteract the local immunosuppression that protects malig-
nancies from endogenous and drug-induced destruction, a
number of hIDO inhibitors have been developed.18 To facilitate
hIDO is mainly and highly expressed (i) in placenta, prevent-
ing the rejection of allogeneic fetuses,5 (ii) in inflammatory
tissues,11,12 and (iii) in human tumors such as mammary,
prostatic, colorectal, pancreatic, cervical, and endometrial
carcinomas.4,5,11−15 In these various tissues, this high expres-
sion of hIDO creates a depletion of tryptophan and an
Received: September 22, 2014
Accepted: January 25, 2015
© XXXX American Chemical Society
A
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Scheme 2. General Scheme for the Syntheses of 1-(2-Fluoroethyl)-tryptophan (5) and 1-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-
yl)methyl)-tryptophan (10)
(i) the preclinical and clinical development and validation
of such compounds and (ii) the clinical detection of hIDO
expressing cells in cancer imaging, the synthesis (and the
radiosynthesis) of a PET imaging tracer could prove to be a
highly desirable and valuable tool.
for 5 h in DMF (93%). This result was in good accordance with
that obtained by Schultz et al.25 The 1,2,3-triazole ring was
formed by a Huisgen 1,3-dipolar cycloaddition (also known as a
Click reaction), usually catalyzed by copper(I) salt (directly
introduced or generated in situ) in conjunction with an added
organic or inorganic base.26−28 This synthesis allowed an easy
introduction of the fluorine, leading to a longer and more rigid
monofluorinated chain, due to a triazole ring formation between
7 and the fluorinated prosthetic group 8 (synthesized
beforehand in one step from 3 and sodium azide), at room
temperature for 7 h. The reusable polymer-supported Amberlyst
A-21·CuI, which associates an organic base and Cu(I) salt,
should facilitate Cu(I) elimination.26 However, even though the
yields obtained were good (73%), after an extended stirring,
copper was partially released into the solution. Copper(I) iodide
offered the desired product but with a small amount of 5-iodo-
1,2,3-triazole derivative as an unwanted side-product. The
[α/β-11C]-L-Tryptophan,19,20 5-hydroxy-[α/β-11C]-L-trypto-
phan,20,21 and α-[11C]methyl-L-tryptophan ([11C]AMT)15,21−23
are radiotracers used essentially in positron emission tomography
(PET) studies of the in vivo transport and brain serotonin
metabolism of L-tryptophan in health and disease.
Inasmuch as following the metabolization of a radioactive
substrate of hIDO through the kynurenine pathway could
highlight the presence of this enzyme, the introduction into
tryptophan of a radionucleide with a longer half-life time, such
as [18F]fluorine (t1/2 = 109.7 min), seems to be better adapted
than [11C]carbon (t1/2 = 20.3 min) (in terms of [18F]fluorine
production, radiotracer synthesis, biological studies over longer
periods, etc.).
Hunig’s base and tetrakis(acetonitrile)copper(I) hexafluoro-
̈
In order to achieve these objectives, two molecules alkylated
on the nitrogen atom of the indole ring, 1-(2-fluoroethyl)-
tryptophan (5) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-
yl)methyl)-tryptophan (10) (Scheme 2), were synthesized
using two different approaches (including a “Click” reaction),
both easily transposable for the radiosynthesis.
phosphate (Cu(CH3CN)4PF6), which is much more soluble
than CuI,28 gave higher yields (81%) while limiting the number
of side-products. Nevertheless, an inert atmosphere is essential
to limit the formation of the 5-hydroxy-1,2,3-triazole analogue.
Finally, hydrolysis of 9 in hydrochloric acid (6 N) and
1,4-dioxane (1/1 (v/v)) offered 1-((1-(2-fluoroethyl)-1H-
1,2,3-triazol-4-yl)methyl)-tryptophan as its hydrochloride salt
(10·HCl) (83%). A complete racemization of the alpha-amino
acid product was also observed.
In vitro enzymatic assays with 5 and 10 were performed in
order to characterize the affinity and the specificity of these
new potential substrates for recombinant human indoleamine
2,3-dioxygenase (rhIDO) (Scheme 1; see Supporting Information
for experimental details). This reaction has been widely studied,
and substrates have been characterized by kinetic constants
determined through the use of a range of analytical methods,
observing either the formation of N-formyl-L-kynurenine
derivatives (direct method)29,30 or an increase in the concen-
tration of L-kynurenine analogues after incubation with perchloric
acid (indirect method).31−33
The synthesis of the key intermediate 2, used for those of 5
and 10 (Scheme 2), involved the protection of the carboxylic
acid function of the commercially available Boc-L-tryptophan 1
with tert-butyl ester (27%). The synthesis of 5, allowing the
introduction in two steps of a fluoroethyl group, one of the
smallest monofluoroalkyl chains, required the N1-alkylation
of 2 with 1-fluoro-2-tosyloxyethane (3) (1.1 equiv; synthesized
beforehand from 2-fluoroethanol 6 (95%)) with sodium
hydride (2.5 equiv) at 0 °C in DMF. These conditions were
crucial to minimize side reactions while maximizing the yield
of the N1-alkylation (52%). Hydrolysis of the intermediate 4 in
hydrochloric acid (6 N) and 1,4-dioxane (1/1 (v/v)) then gave
1-(2-fluoroethyl)-tryptophan as its hydrochloride salt (5·HCl;
93%). The enantiomeric excess was determined for 5 by a chiral
HPLC analysis. A racemization of the alpha-amino acid product
was observed. However, our method using sodium hydride
offered a better yield (52%) compared with that recently
obtained by Sun et al. (NaOH, 30%). Moreover, epimerization
was not evaluated in this paper.24
The formation of the product, N-formyl-L-kynurenine, was
followed by measuring its absorbance at 321 nm.30−32
Nevertheless, as mentioned briefly by Chauhan et al.,31 the
UV-absorbance of the N-formyl-L-kynurenine derivative
decreased significantly when the nitrogen in the substrate was
alkylated, as for 1-Me-L-Trp (12). This decrease was also
observed here for 1-FETrp (5), where the low UV-absorption
The synthesis of 10 began with the N1-alkylation of 2 with pro-
pargyl bromide (5 equiv) and sodium hydride (5 equiv) at 0 °C
B
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Table 1. Enzymatic Assays Carried out under Different Conditions with Some Substrates and NON Substrates of rhIDO
a
b
c
d
e
f
From Aldrich. Racemate. Acros Organics. Iris Biotech Gmbh. Percentage determined by UV detection method. Percentage obtained by
fluorescence detection method. All assays were repeated two to three times. nd: not determined.
of the product did not allow the study of the reaction by this
method. An indirect method was therefore investigated. Moreover,
it was observed that N-alkylated-N-formyl-kynurenine derivatives
were hydrolyzed into N-alkylated-kynurenine analogues much
more slowly (for at least 30 min in HClO4). The substrates
and their products were separated by HPLC or UPLC and
detected by UV (method A, less sensitive; [S] ≥ 200 μM) or
by fluorescence detection (method B; used to detect only the
substrates; [S] ≥ 2 μM).34
The oxidative conditions of the enzymatic reaction were first
improved to allow a complete oxidation of the natural substrate:
L-tryptophan (11) (Table 1). Under these optimal conditions,
C
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Letter
compounds were therefore found not to be substrates of this
enzyme. In our experimental conditions, the opening of the
indole ring was also not observed for α-Me-DL-Trp (AMT, 14).
Thus, our results showed that AMT was not metabolized by
rhIDO through the kynurenine pathway, contrary to the findings
reported in the literature.35
The incubation conditions (substrate concentration, enzyme
concentration, and time) were then changed in order to compare
the substrates. Compound 5 was identified as a good substrate
of this enzyme, but it was found to be oxidized more slowly than
5-HO-L-Trp (around 2 times), 1-Me-L-Trp (around 4 times),
and L-Trp, which is known as the best substrate for rhIDO
(Table 1).
The fluorescence method was used to determine the initial
rates, allowing the determination of the Km and kcat values. An
inhibition by the substrate was observed for L-Trp and 1-Me-L-
Trp, as described in the literature,30 but not for 5-HO-L-Trp and
1-FETrp under the conditions studied. However, even though
the concentration of rhIDO was reduced as much as possible,
the reaction was still too fast to allow an accurate determination
Table 2. In vitro Enzymatic Assays Performed with rhIDO
(37 °C, pH 6.5)
a
substrate
Km
kcat/Km
b
b
b
L-Trp (11)
15 2 μM
62 9 μM
2100.102 M−1 s−1
1-Me-L-Trp (12)
5-HO-L-Trp (13)
(38 17).102 M−1 s−1
(21 4).102 M−1 s−1
(5.8 0.7).102 M−1 s−1
c
17 1.1 μM
d
e
1-FETrp (5)
70 30 μM
a
Ratios determined by measurement of the substrate concentration
b
in relation to the incubation time (method B). Values determined by
Lu et al.30 Values determined by Basran et al.29 Racemate. Constant
c
d
e
obtained by initial-rate determination (method B).
12, 13, and 5 were also completely oxidized. These results were
in contrast to those found with the following six compounds:
10 (which probably had a too long and rigid substituent to fit
adequately into the catalytic cavity of the enzyme); α-Me-DL-
Trp (14); tryptamine (15); α-Me-DL-tryptamine (16); Boc-L-
Trp (17); and Ac-L-Trp (18) (whose NH3+ group is protected
and no longer available for H-bonding interaction with the
catalytic center facilitating the ring-opening reaction7). These six
Scheme 3. General Enzymatic Oxidative Indole Ring Opening of rhIDO Substrates and Hydrolysis in Perchloric Acid
Table 3. HPLC Analyses of the Enzymatic Assays of L-Tryptophan and Derivatives with rhIDO (Retention Times and MS)
a
Retention times determined by HPLC analyses (or UPLC analyses; see Supporting Information). Elution conditions: CH3CN/ammonium acetate
b
c
d
(10 mM), 10/90 (v/v), flow 0.7 mL/min. UV detection (method A) at 221 and 284 nm. UV detection (method A) at 321 or 360 nm. Mass
spectra (see Supporting Information). Retention times determined by HPLC analyses (or UPLC analyses; see Supporting Information). Elution
conditions: CH3CN/ammonium acetate (10 mM), 5/95 (v/v), flow 0.7 mL/min. Racemate. n.o.: not observed because hydrolysis of the formyl
e
f
substituent was too fast in these conditions.
D
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of the initial rates. Nonetheless, these results still allowed an
estimation of the Km value (70 30 μM for 1-FETrp).
Because of the difficulties involved in determining accurately
these kinetic constants, the kcat/Km ratio, a second order constant,
characterizing the reaction rate for substrate concentrations
smaller than Km (values summarized in Table 2), was determined
via method B.29,30 The oxidation rate of 5 was smaller than for 13
(around 3 times), 12 (6 times), and 11 (360 times) (Table 2;
Supporting Information), suggesting that, having a more sterically
hindered structure than L-Trp, 5 has lesser conformational
freedom in the catalytic cavity, reducing the flexibility of its
complex with rhIDO and undoubtedly conducing to a slower
enzymatic reaction.
isotopomer, currently being developed in our laboratory, could
be a valuable tool for the preclinical and the clinical validation of
new potential inhibitors. Furthermore, 1-[18F]FETrp could also
facilitate a more specific detection of hIDO expressing cells in
cancer imaging.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental sections, enzymatic equations, enzymatic curves
obtained with 5, NMR analyses of some compounds (4, 5, 9,
and 10), UPLC chromatograms, and mass spectra of the rhIDO
substrates. This material is available free of charge via the
Internet at http://pubs.acs.org.
HPLC and UPLC analyses were performed, and the two
product peaks appearing on the chromatograms for 11, 12, 13
(only one peak), and 5 (see Supporting Information)
were collected and analyzed in mass spectrometry (Table 3).
These analyses confirmed that the products formed were
kynurenine and N-formyl-kynurenine derivatives (Scheme 3).
Accurate mass determination was performed on N-fluoroethyl-
N-formyl-kynurenine (19) (FT-MS (positive): m/z =
283.108862 (calculated), 283.108707 (measured) [M + H])
and N-fluoroethyl-kynurenine (20) (FT-MS (positive): m/z =
255.113947 (calculated), 255.113825 (measured) [M + H])
confirming the nature of these compounds.
Similar in vitro enzymatic assays with recombinant human
tryptophan 2,3-dioxygenase (rhTDO; Scheme 1) were per-
formed to determine the specificity of the substrates (11, 12,
13, and 5) for rhIDO.36,37 The solutions obtained after
incubation and the addition of perchloric acid were analyzed by
HPLC with fluorescence detection. As expected, L-tryptophan
was found to be an excellent substrate of rhTDO, while 5-HO-
L-Trp was a poor substrate. 1-FETrp and 1-Me-L-Trp, however,
were found not to be substrates (Table 4). For the highest
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jean.henrottin@ulg.ac.be. Phone: (+32)43663383.
Fax: (+32)43662946.
Funding
The authors gratefully acknowledge the Region Wallonne
(Keymarker Project and Cantol Project (BioWin)) for their
financial support. Alain Plenevaux is a senior research associate
from FRS-FNRS Belgium.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
AMT, α-methyl-L-tryptophan; 1-FETrp, 1-(2-fluoroethyl)-
tryptophan; 3-HAA, 3-hydroxyanthranilic acid; hIDO, human
indoleamine 2,3-dioxygenase; IFN-γ, interferon-γ; KYN,
kynurenine; KYNA, kynurenic acid; NAD+, nicotinamide
adenine dinucleotide; hTDO, human tryptophan 2,3-dioxygenase;
Trp, L-tryptophan
Table 4. In vitro Enzymatic Assays Performed with rhTDO
(37 °C, pH 7.5)
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DOI: 10.1021/ml500385d
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