Thiosemicarbazide, a fragment with promising indolamine-2,3-dioxygenase (IDO) inhibition properties

Article abstract of DOI:10.1016/j.ejmech.2014.05.044

With the aim to explore the interest of the thiosemicarbazide scaffold for the inhibition of the indoleamine 2,3-dioxygenase (IDO), a promising therapeutic target for anticancer immunotherapy, a series of 32 phenylthiosemicarbazide derivatives was prepared and their IDO inhibition evaluated. Our study demonstrated that among these derivatives, compound 14 characterized with a 4-cyanophenyl group on the thiosemicarbazide was the more potent IDO inhibitor in this series being endowed with an IC₅₀ of 1.2 μM. The SAR depicted showed that substitution in the 3- and 4-position relative to the phenylthiosemicarbazide are very promising whereas substitution in the 2-position always leads to less potent or inactive derivatives. In fact the study highlighted a novel interesting scaffold for IDO inhibition for further development.

Full text of DOI:10.1016/j.ejmech.2014.05.044

Accepted Manuscript
Thiosemicarbazide, a fragment with promising indolamine-2,3-dioxygenase (IDO)
inhibition properties
Silvia Serra , Laurence Moineaux , Christelle Vancrayenest , Bernard Masereel ,
Johan Wouters , Lionel Pochet , Raphaël Frédérick
PII:
S0223-5234(14)00460-7
10.1016/j.ejmech.2014.05.044
EJMECH 7002
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 April 2014
Revised Date: 30 April 2014
Accepted Date: 14 May 2014
Please cite this article as: S. Serra, L. Moineaux, C. Vancrayenest, B. Masereel, J. Wouters, L. Pochet,
R. Frédérick, Thiosemicarbazide, a fragment with promising indolamine-2,3-dioxygenase (IDO) inhibition
properties, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.044.
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ACCEPTED MANUSCRIPT
Graphical abstract

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Thiosemicarbazide, a fragment with promising indolamine-2,3-
dioxygenase (IDO) inhibition properties
Silvia Serra1, Laurence Moineaux1, Christelle Vancrayenest1, Bernard Masereel1, Johan
Wouters1, Lionel Pochet1*, Raphaël Frédérick2*
1. Namur Medicine & Drug Innovation Center (NAMEDIC-NARILIS), University of Namur
(UNamur), 61 rue de Bruxelles, 5000 Namur Belgium
2. Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute
(LDRI), Université Catholique de Louvain (UCL), 73 avenue Mounier, B1.73.10, 1200
Bruxelles Belgium
* Corresponding author information:
Prof Raphaël Frédérick
Prof Lionel Pochet
Medicinal Chemistry Research Group
(CMFA)
Namur Medicine & Drug Innovation Center
(NAMEDIC-NARILIS)
Louvain Drug Research Institute (LDRI)
Université Catholique de Louvain (UCL)
73 avenue Mounier
University of Namur (UNamur)
61 rue de Bruxelles
5000 Namur Belgium
1200 Bruxelles Belgium
Phone : +32 81 72 42 90
Email: lionel.pochet@unamur.be
Phone : +32 2 764 73 41
Email : raphael.frederick@uclouvain.be
Keywords : indoleamine 2,3-dioxygenase, IDO, thiosemicarbazide, immunotherapy,
molecular modelling

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Abstract
With the aim to explore the interest of the thiosemicarbazide scaffold for the inhibition of the
indoleamine 2,3-dioxygenase (IDO), a promising therapeutic target for anticancer
immunotherapy, a series of 32 phenylthiosemicarbazide derivatives was prepared and their
IDO inhibition evaluated. Our study demonstrated that among these derivatives, compound
14 characterized with a 4-cyanophenyl group on the thiosemicarbazide was the more potent
IDO inhibitor in this series being endowed with an IC50 of 1.2 µM. The SAR depicted showed
that substitution in the 3- and 4-position relative to the phenylthiosemicarbazide are very
promising whereas substitution in the 2-position always leads to less potent or inactive
derivatives. In fact the study highlighted a novel interesting scaffold for IDO inhibition for
further development.
1. Introduction
Immune dysregulation is one of the features of tumor growth and progression, a key event
that allows for tumor evasion of the host immune system. Among the different mechanisms
manipulated by tumors during their growth is the upregulation of the immunoregulatory
enzyme indoleamine 2,3-dioxygenase.1 Indeed, this enzyme plays a dual role in
tumorigenesis by restricting the development of cancers that are driven by chronic
inflammation, such as colon cancer,2 while promoting the development of tumors that are
controlled by inflammation, such as certain types of skin tumors.3
IDO is an extrahepatic cytosolic heme-containing dioxygenase that catalyses the initial and
rate limiting step of -typtophan (L-Trp) along the kynurenine pathway. Through the local Trp
L
depletion and the production of kynurenine and other downstream metabolites, IDO thus
regulates immune response, suppressing effector T-cell functions and favoring the
differentiation of regulatory T cells.4 Moreover, it has been shown that an increased level of
IDO expression in tumor cells is correlated with poor prognosis for survival in cancer. Indeed,
it is now very clear that IDO is a very promising target for anticancer immunotherapy.6-8

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In the last years several small molecular-weight IDO inhibitors have been identified by
natural products isolation,9,11 structure-based modifications,10-12 and in silico drug design.13-16
However, to date, only two compounds have entered clinical trials: the 1-methyl-D-tryptophan
(D-1MT) which was indeed shown not to be an IDO inhibitor in contrast to its L-isomer (L-
1MT), and the hydroxylamidine INCB024360 (structure undisclosed) developed by Incyte
corp.17-18 Therefore, there exists a continuing need for the development of new IDO
inhibitors.
In the present work, we investigated a series of thiosemicarbazide compounds that was
designed by modification of the benzo[d]thiazol-2(3H)thione (BTT), a known IDO inhibitor
(Figure 1).14 Interestingly, the resulting phenylthiosemicarbazide series proved to be potent
IDO inhibitors with inhibitory potency in the low micromolar range.
Figure 1. Structures of benzo[d]thiazole-2(3H)-thione (BTT), a known fragment endowed
with IDO inhibition properties and the N-phenylthiosemicarbazide (1) investigated in this
paper.
2. Results and discussion
As an initial step in this study, the BTT compound that was previously reported as an IDO
inhibitor (IC50 = 50 µM) was docked inside the IDO binding cleft. It should be noted that the
thiazole-thione form was used for docking and not the thiazole-thiol tautomeric form, as in
solution the thiazole-thione should predominate. Interestingly, in our docking experiments
using the automated GOLD docking algorithm, the BTT compound was found deeply
inserted inside the IDO binding cleft, interacting with the heme iron via its thione moiety, in

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contrast to a previously published docking of this compound that predicted a binding
conformation stabilized through interaction of the thiazolic S-atom with the heme iron.14
Moreover our study also showed that in this particular orientation, the substitution of the
benzo[d]thiazole-2(3H)-thione in the 5-position with a chlorine atom is still possible and the
compound very well stabilized inside the IDO binding cleft. This result is in agreement with
previous data showing that the 5-chloro-benzo[d]thiazole-2(3H)-thione was also a good IDO
inhibitor being characterized with an IC50 of 50 µM.14 Indeed, in our modelling study, none of
the conformations generated for the benzo[d]thiazole-2(3H)-thione substituted or not are
found in an orientation stabilized through interaction between the thiazolic S-atom and the
heme iron, as initially suggested. On the contrary these compounds are stabilized through an
interaction between the thione moiety present in the inhibitor and the heme iron that is in
agreement with the higher electron density around the thiole function as compared to the S
atom of the thiazole ring.
Interestingly, during our analysis we observed that in such orientation, the thiazolic S-atom is
just slightly too distant from the Ser167 residue to allow a strong H-bond interaction. We thus
reasoned that extending that part of the molecule with a hydrogen bond donor group such as
an amino could provide a better stabilization inside the IDO active site. This led us to design
the thiosemicarbazide motif which presents some analogy to the thiazole-thione moiety.
Moreover this fragment has known chelating properties and has not yet been investigated on
IDO.
Docking of the phenyl-thiosemicarbazide (1) prototype provided the confirmation that a very
similar binding is obtained when compared to BTT, with an additional stabilization between
the terminal amino group of (1) and the hydroxyl group of Ser167. The overlay also
demonstrates that the thione in BTT and in (1) are very well superimposed and shows that
only small differences appear in the orientation of the aromatic ring.

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(b)
(a)
(c)
(d)
Figure 2. View of (a) benzo[d]thiazole-2(3H)-thione (BTT), (b) phenyl-thiosemicarbazide (1),
(c and d) superimposition of both inside the IDO binding cleft. Pictures were made using
Pymol from Delano Scientific.
Compound 1, which was prepared according to the procedure described hereunder, was
tested for IDO inhibition and displayed a promising IC50 of 50 µM and a very good ligand
efficiency (LE) of 0.55 which prompted us to further investigate this series of compounds.
The synthetic route for the thiosemicarbazide derivatives 1-30 is outlined in Scheme 1. The
thiosemicarbazide moiety was linked with differently substituted aryls, varying the length and
the nature of the substituents (Scheme 1). For the sake of an easily reproducible
methodology, the compounds were prepared in good yields through the nucleophilic addition
reaction of hydrazine hydrate with conveniently substituted corresponding isothiocyanates at

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room temperature under Argon.19 For the synthesis of derivatives 31-32 we used the
commercial 1-methyl-1-phenylhydrazine instead of hydrazine hydrate (Scheme 2). The
structure and purity of all compounds were confirmed by means of 1H and 13C NMR, 19F NMR
(where applicable), LC-MS and elemental analysis (C, H, N, S).
Scheme 1 Synthesis of thiosemicarbazides 1-30
Scheme 2 Synthesis of thiosemicarbazides 31-32
Table 1. IDO inhibition by thiosemicarbazide derivatives (1-32)
Ligand
Efficiencya
(LE) = 1.4 x
pIC50/HAC
IDO IC50
(µM)
Compound
L-1MT
Structure
100
0.35
0.60
BTT
50

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1
2
3
4
~50
0.55
>50
>50
>50
-
-
-
5
6
7
8
2.7
3.7
15
0.65
0.63
0.56
0.58
0.41
3.8
9
41
10
11
>50
1.8
-
0.67

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12
13
14
1.6
3.5
1.2
0.68
0.64
0.64
15
>50
-
16
17
>50
>50
-
-
18
19
>50
34
-
0.48
20
21
18
0.51
>50
-
-
22
>50

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23
24
>50
-
-
>50
13
25
26
27
0.53
0.51
17
>50
-
28
29
34
0.45
>50
-
30
31
>50
>50
-
-

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32
>50
-
(a) HAC = Heavy Atom Count (number of non-hydrogen atoms)
All the synthesized thiosemicarbazide compounds 1-32 were evaluated for their capacity to
inhibit IDO. To this end, a colorimetric in vitro IDO inhibition assay was used as previously
described.20-21 Briefly, the inhibitors were incubated 5 min at 37 °C with IDO and -Trp, the
L
natural substrate. Trichloroacetic acid (TCA) was then added to quench the reaction and the
N-formylkynurenine generated was hydrolysed to kynurenine during the next 30 min. After
that time, p-dimethylaminobenzaldehyde (pDMAB) was added to the reaction mixture to form
a Schiff base with kynurenine. The IDO residual activity was measured at 490 nM which
corresponds to absorption of the Schiff base formed. The IDO inhibitory potency of the
compounds is indicated by IC50 values that were calculated by nonlinear regression analysis
of the concentration-response curves obtained for each compound, in triplicate. The results
are reported in Table 1. The compounds 1-methyl-tryptophan (1-MT) and BTT were used as
reference IDO inhibitors.
A number of thiosemicarbazide compounds exhibit good inhibition of IDO activity. Among
them, compound 14 displayed the most potent IDO inhibitory activity (IC50 = 1.2 µM) that is a
substantial improvement compared to the positive control 1MT (IC50 = 100 µM) and a 41-fold
improvement compared to BTT and compound 1, our starting point in this study.
Structure-activity relationships in these thiosemicarbazide derivatives (1-30) demonstrated
that the substitution of the aromatic ring in the 2-position (compounds 2, 3, 4) always leads to
non-potent IDO inhibitors (IC50 > 50 µM) whereas the substitution of the aromatic ring in the
3- (compounds 5, 6, 7, 8 and 9) or 4-positions (compounds 11, 12, 13, 14 and 19) with
relatively small substituents are well tolerated and potent IDO inhibitors are obtained.
Particularly, introducing a bromine, a fluorine or a cyano group in the 4-position of the aryl

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ring (compounds 11, 12 and 14) leads to potent IDO inhibitors with IC50 of 1.8, 1.6 and 1.2
µM, respectively. On the contrary, introduction of larger groups is detrimental for IDO
inhibition (15, 16, 17, 18, 19). Regarding the di-substitution, apart from compound 20
characterized by a 2,5-dichloro substitution which retains some IDO inhibition (IC50 = 18 µM),
the presence of a substituent in the 2-position of the aromatic ring again leads to inactive
compounds (21, 22, 23, 24, 29, 30). Indeed only compounds possessing substituents in the
3,4- or 3,5-positions retain some IDO inhibition (25, 26, 28), although there are less potent
than the monosubstituted derivatives.
The influence of the substitution of the terminal amino group of thiosemicarbazide was also
investigated and showed that this substitution leads to inactive compounds (compare 31 and
25, and 32 and 28, respectively). This result is in agreement with the proposed binding
orientation which involves the interaction of the terminal amino group of the
thiosemicarbazide motif with the Ser167 residue thus preventing any substitution at this
position.
3. Conclusions
In the present work, we aimed at exploring the interest of the thiosemicarbazide scaffold as
an isostere of the thiazole-2(3H)-thione motif present in BTT, a known IDO inhibitor.
Interestingly, our study confirms that potent IDO inhibitors can be obtained with appropriate
substitution of the aromatic ring bearing the thiosemicarbazide motif. Substitution in the 4-
position relative to the thiosemicarbazide, notably with groups such as the nitrile in
compound 14, the more potent compound in the present study, or halogens (11, 12, 13)
seems very promising whereas substitution in the 2-position always leads to less potent
derivatives. Interestingly, owing to their small size, these compounds are characterized with
excellent ligand efficiencies (LE around 0.6 for the best derivatives) thus suggesting that
these derivatives are very interesting starting points for further optimizations.
4. Experimental protocols

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4.1 Materials and measurements
All chemical reagents and solvents were used as obtained from commercial sources (Sigma
Aldrich, Acros, Maybridge, Apollo, Fisher Scientific). All reactions were performed under an
inert argon (Alphagaz 2) atmosphere, unless stated otherwise. Melting points were
determined with a Buchi B-540 capillary melting point apparatus in open capillaries and are
uncorrected. Thin layer chromatography (TLC) was performed on silica gel plates (60F254,
1
0.2 mm thick, Merck) with visualization under ultraviolet light (254 and 365 nm). H NMR
spectra were recorded in DMSO-d6 solution on a Jeol JNM EX 400 spectrometer at 400 MHz
with tetramethylsilane (TMS) as internal standard. 13C spectra were recorded on the same
spectrometer in DMSO-d6 solution at 100 MHz. Chemical shifts (δ) are expressed in ppm
downfield from tetramethylsilane. Elemental analyses (C, H, N, S) were performed on a
Thermo Finnigan- FlashEA 1112 apparatus. Analytical LC/MS analyses were performed on
an Agilent 1100 series HPLC coupled with an MSD Trap SL system using UV detection at
254 and 361 nm. Mass spectra were recorded using electron spray ionization (ESI) operating
in positive mode, unless stated otherwise. The following method was applied: injection of 10
µL of a 20 µg.mL-1 acetonitrile solution onto a C18 3.5 µm Zorbax SB column (100 mm x 3
mm); separation using a gradient (flow rate of 0.5 mL.min-1) of acetonitrile in acetic acid
(0.1% v/v in water) from 5% to 95% acetonitrile over 5 min, holding for 3 min, then reversing
to 5% acetonitrile within 0.1 min and holding for an additional 5.4 min. Automated flash
chromatography was performed on a Biotage AB SP1 system equipped with prepacked flash
KP-Sil silica cartridges. The following gradient of ethyl acetate in cyclohexane (unless stated
otherwise) was used for elution: the elution started with an ethyl acetate/cyclohexane ratio of
12/88 for one column volume (CV); the ratio increased to 62/38 over 5 CV, kept for 1 CV,
then increased to 100/0 over 7 CV, and finally kept at this value over 5 CV. The product
detection was by UV absorption at 254 and 320 nm. The products were precipitated by
concentration of the pooled column fractions combined with the addition of cyclohexane. The
precipitate formed was filtered off, washed twice with cyclohexane, and dried at 40 °C in

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vacuum to yield an analytically pure sample. All new compounds were determined to be
>95% pure by LC/MS.
4.2. General procedure for the synthesis of thiosemicarbazide derivatives (1-30).
To a stirred solution of hydrazine hydrate (85%, 2.4 mmol) in 20 mL of isopropanol,
appropriately substituted isothiocyanate (2.0 mmol) was added at room temperature.
Precipitate was formed immediately. Stirring was continued for 3 hours. Then the mixture
was filtered and the precipitate was washed with isopropanol three times to give the desired
product.
4.2.1 4-Phenylthiosemicarbazide (1)
1
Yield 91%. Mp. 138-141°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.80 (s, 1H, NH), 9.24
(s, 1H, NH), 7.57-7.54 (m, 2H, H3’, H5’), 7.26 (t, J = 7.7 Hz, 2H, H2’, H6’), 7.05 (t, J = 7.2 Hz,
1H, H4’), 5.10 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 181.7, 139.2, 128.9,
128.8, 124.5, 121.6, 121.3. LC-MS Rt 4.7 min; m/z [MH+] 168. Anal. Calcd. for C7H9N3S: C,
50.28; H, 5.42; N, 25.13; S, 19.17. Found: C, 49.83; H, 5.31; N, 25.34; S, 19.19.4.2.
4.2.2 4-(2-Fluorophenyl)-3-thiosemicarbazide (2)
1
Yield 77%. Mp. 164-166 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.33 (s, 1H, NH), 8.02
(s, 1H, NH), 7.43-7.34 (m, 1H, H6’), 7.23-7.10 (m, 3H, H3’, H4’, H5’), 4.89 (s, 2H, NH2). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) = 182.7, 155.8, 127.8, 127.3, 126.5, 124.2, 115.7. LC-
MS Rt 4.7 min; m/z [MH+] 186. Anal. Calcd. for C7H8FN3S: C, 45.39; H, 4.35; N, 22.69; S,
17.31. Found: C, 45.45; H, 4.52; N, 22.57; S, 17.88.
4.2.3 4-(2-Chlorophenyl)-3-thiosemicarbazide (3)
1
Yield 89%. Mp. 131-133°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.43 (s, 1H, NH), 9.15
(s, 1H, NH), 8.36 (d, J = 8.2 Hz, 1H, H3’), 7.45 (d, J = 8.0 Hz, 1H, H6’), 7.28 (t, J = 7.8 Hz,
1H, H5’), 7.14 (t, J = 7.7 Hz, 1H, H4’), 4.98 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6):

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δ (ppm) = 179.6, 136.5, 129.8, 129.6, 127.3, 126.6, 126.1. LC-MS Rt 5.2 min; m/z [MH+] 202.
Anal. Calcd. for C7H8BrN3S: 41.69; H, 4.00; N, 20.84; S, 15.90. Found: C, 41.53; H, 3.38; N,
20.65; S, 16.08.
4.2.4 4-(2-Bromophenyl)-3-thiosemicarbazide (4)
1
Yield 87%. Mp. 103-105°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 8.82 (s, 1H, NH), 7.51
(s, 1H, NH), 7.35-7.31 (m, 2H, H3’, H5’), 7.20-7.17 (m, 2H, H2’, H6’), 5.00 (s, 2H, NH2). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) = 181.8, 139.3, 129.6, 125.7, 122.8, 121.6, 121.2. LC-
MS Rt 5.4 min; m/z [MH+] 246; 248. Anal. Calcd. for C7H8BrN3S: 34.16; H, 3.28; N, 17.07; S,
13.03. Found: C, 35.03; H, 3.38; N, 17.34; S, 13.08.
4.2.5 4-(3-Bromophenyl)-3-thiosemicarbazide (5)
1
Yield 87%. Mp. 103-105°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 8.82 (s, 1H, NH), 7.51
(s, 1H, NH), 7.35-7.31 (m, 2H, H3’, H5’), 7.20-7.17 (m, 2H, H2’, H6’), 5.00 (s, 2H, NH2). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) = 181.8, 139.3, 129.6, 125.7, 122.8, 121.6, 121.2. LC-
MS Rt 5.5 min; m/z [MH+] 246; 248. Anal. Calcd. for C7H8BrN3S: 34.16; H, 3.28; N, 17.07; S,
13.03. Found: C, 35.03; H, 3.38; N, 17.34; S, 13.08.
4.2.6 4-(3-Fluorophenyl)-3-thiosemicarbazide (6)
1
Yield 86%. Mp. 135-138 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.27 (s, 1H, NH), 7.82
(s, 1H, NH), 7.51-7.40 (m, 1H, H5’), 7.24-7.30 (m, 1H, H2’), 7.20-7.10 (m, 1H, H6’), 6.98-6.87
(m, 1H, H4’), 4.87 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 179.5, 161.3,
140.2, 130.1, 119.4, 110.7, 110.1. LC-MS Rt 5.0 min; m/z [MH+] 186. Anal. Calcd. for
C7H8FN3S: C, 45.39; H, 4.35; N, 22.69; S, 17.31. Found: C, 45.45; H, 4.54; N, 22.97; S,
17.41.
4.2.7 4-(3-Chlorophenyl)-3-thiosemicarbazide (7)

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1
Yield 66%. Mp. 170-173 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.27 (s, 1H, NH), 7.96
(s, 1H, NH), 7.53 (s, 1H, H6’), 7.29 (t, J = 15.3 Hz, 1H, H3’), 7.24-7.20 (m, 1H, H2’), 7.11-
7.09 (d, J = 6.4 Hz, 1H, H4’), 4.99 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) =
179.7, 141.1, 132.6, 130.1, 124.2, 123.3, 122.4. LC-MS Rt 6.5 min; m/z [MH+] 202. Anal.
Calcd. for C7H8ClN3S: C, 41.69; H, 4.00; N, 20.84; S, 15.90. Found: C, 41.45; H, 3.63; N,
20.57; S, 15.28.
4.2.8 4-(3-Cyanophenyl)-3-thiosemicarbazide (8)
1
Yield 76%. Mp. 152-154°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.38 (s, 1H, NH), 8.21
(s, 1H, NH), 7.97 (s, 1H, H2’), 7.91-7.88 (m, 1H, H4’), 7.60-7.64 (m, 2H, H5’, H6’), 4.98 (s,
2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 179.8, 140.8, 130.0, 129.0, 128.0,
127.1, 119.3, 111.1. LC-MS Rt 4.7 min; m/z [MH+] 193. Anal. Calcd. for C8H8N4S: C, 49.98;
H, 4.19; N, 29.14; S, 16.68. Found: C, 49.61; H, 4.14; N, 28.89; S, 16.69.
4.2.9 4-[3-(Trifluoromethyl)phenyl]-3-thiosemicarbazide (9)
1
Yield 65%. Mp. 117-120°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.33 (s, 1H, NH), 8.22
(s, 1H, NH), 7.97 (s, 1H, H2’), 7.98-7.86 (m, 1H, H4’), 7.50 (t, J = 15.57 Hz, 1H, H5’), 7.38 (d,
J = 6.64 Hz, 1H, H6’), 4.98 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.8,
140.8, 129.8, 129.0, 128.0, 127.1, 123.3, 121.1. LC-MS Rt 5.7 min; m/z [MH+] 236. Anal.
Calcd. for C8H8F3N3S: C, 40.85; H, 3.43; N, 17.86; S, 13.63. Found: C, 40.43; H, 3.36; N,
17.84; S, 13.16.
4.2.10 4-(4-Chlorophenyl)-3-thiosemicarbazide (10)
1
Yield 81%. Mp. 178-181°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 8.77 (s, 1H, NH), 7.56
(s, 1H, NH), 7.51 (s, 1H, H2’), 7.30-7.27 (m, 1H, H4’), 7.23-7.21 (m, 1H, H6’), 7.12-7.09 (m,
1H, H5’), 4.79 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 181.7, 146.9, 133.5,
133.2, 130.5, 126.6, 126.2. LC-MS Rt 5.4 min; m/z [MH+] 202. Anal. Calcd. for C7H8ClN3S:
41.69; H, 4.00; N, 20.84; S, 15.90. Found: C, 41.83; H, 4.38; N, 20.91; S, 15.78.

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4.2.11 4-(4-Fluorophenyl)-3-thiosemicarbazide (11)
1
Yield 96%. Mp. 175-178 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.67 (s, 1H, NH), 9.11
(s, 1H, NH), 7.78-7.38 (m, 2H, H2’, H6’), 7.09 (t, J = 8.8 Hz, 2H, H3’, H5’), 4.73 (s, 2H, NH2).
13C NMR (100 MHz, DMSO-d6): δ (ppm) = 180.2, 159.5, 135.7, 126.5, 126.3, 115.4, 114.9.
LC-MS Rt 4.8 min; m/z [MH+] 186. Anal. Calcd. for C7H8FN3S: C, 45.39; H, 4.35; N, 22.69; S,
17.31. Found: C, 45.45; H, 4.47; N, 22.70; S, 17.86.
4.2.12 4-(4-Bromophenyl)-3-thiosemicarbazide (12)
1
Yield 90%. Mp. 166-169 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 10.27-9.37 (m, 1H,
NH), 9.21 (s, 1H, NH), 7.62 (d, J = 7.2 Hz, 2H, H2’, H6’), 7.42 (d, J = 8.7 Hz, 2H, H3’, H5’),
4.80 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 176.3, 138.0, 131.3, 131.2,
125.3, 125.2, 115.0. LC-MS Rt 5.5 min; m/z [MH+] 246; 248. Anal. Calcd. for C7H8BrN3S: C,
34.16; H, 3.28; N, 17.07; S, 13.03. Found: C, 34.46; H, 3.38; N, 17.17; S, 13.33.
4.2.13 4-(4-Iodophenyl)-3-thiosemicarbazide (13)
1
Yield 89%. Mp. 182-184 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.11 (s, 1H, NH), 7.98
(s, 1H, NH), 7.34-7.29 (m, 2H, H3’, H5’), 7.24-7.20 (m, 2H, H2’, H6’), 4.92 (s, 2H, NH2). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) = 176.7, 139.7, 137.2, 137.1, 122.5, 121.3, 96.4. LC-MS
Rt 5.7; m/z [MH+] 295. Anal. Calcd. for C7H8IN3S: C, 28.68; H, 2.75; N, 14.34; S, 10.94.
Found: C, 28.82; H, 2.86; N, 14.11; S, 10.73.
4.2.14 4-(4-Cyanophenyl)-3-thiosemicarbazide (14)
1
Yield 80%. Mp. 175-177°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.47 (s, 1H, NH), 9.23
(s, 1H, NH), 8.02 (d, J = 8.1 Hz, 2H , H3’, H5’), 7.70 (d, J = 8.4 Hz, 2H, H2’, H6’), 5.00 (s, 2H,
NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.3, 142.4, 131.5, 131.3, 120.8, 120.4,
119.3, 102.2. LC-MS Rt 6.0 min; m/z [MH+] 193. Anal. Calcd. for C8H8N4S: C, 49.98; H, 4.19;
N, 29.14; S, 16.68. Found: C, 49.53; H, 4.36; N, 28.84; S, 16.19.

ACCEPTED MANUSCRIPT
4.2.15 4-[(4-Dimethylamino)phenyl]-3-thiosemicarbazide (15)
1
Yield 71%. Mp. 190-192°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.33 (s, 1H, NH), 8.83
(s, 1H, NH), 7.27 (d, J = 8.5, 2H, H2’, H6’), 6.69-6.55 (m, 2H, H3’, H5’), 4.56 (s, 2H, NH2),
2.83 (s, 6H, 2 X CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.3, 148.1, 132.2, 127.2,
126.9, 113.2, 113.2, 41.9, 41.8. LC-MS Rt 2.7 min; m/z [MH+] 211. Anal. Calcd. for C9H14N4S:
51.40; H, 6.71; N, 26.64; S, 15.24. Found: C, 51.83; H, 6.58; N, 26.54; S, 15.58.
4.2.16 4-(4-Nitrophenyl)-3-thiosemicarbazide (16)
1
Yield 76%. Mp. 188-190 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.52 (s, 1H, NH), 8.95
(s, 1H, NH), 7.50-7.44 (m, 2H, H2’, H6’), 4.75 (s, 2H, NH2), 3.70 (s, 3H, OCH3). 13C NMR
(100 MHz, DMSO-d6): δ (ppm) = 183.0, 143.7, 143.2, 125.1, 125.0, 121.0, 120.9. LC-MS Rt
4.3 min; m/z [MH+] 198. Anal. Calcd. for C8H11N3OS: C, 48.71; H, 5.62; N, 21.30; S, 16.25.
Found: C, 48.76; H, 5.57; N, 21.50; S, 16.26.
4.2.17 4-(4-Methoxyphenyl)-3-thiosemicarbazide (17)
1
Yield 86%. Mp. 154-156 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.61 (s, 1H, NH), 9.33
(s, 1H, NH), 8.20-8.09 (m, 4H, H2’, H3’, H5’, H6’), 4.73 (s, 2H, NH2). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 182.7, 154.8, 133.2, 122.4, 122.3, 114.2, 114.1, 56.2. LC-MS Rt 4.3
min; m/z [MH+] 198. Anal. Calcd. for C7H8N4O2S: C, 39.62; H, 3.80; N, 26.40; S, 15.11.
Found: C, 39.45; H, 3.57; N, 26.50; S, 15.26.
4.2.18 4-(4-Methylphenyl)-3-thiosemicarbazide (18)
1
Yield 90%. Mp. 142-144 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.70 (s, 1H, NH), 9.02
(s, 1H, NH), 7.45 (d, J = 7.1, 2H, H2’, H6’), 7.06 (d, J = 8.1 Hz, 2H, H3’, H5’), 4.67 (s, 2H,
NH2), 2.23 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.8, 137.7, 132.7,
130.6, 130.3, 121.0, 120.9, 21.1. LC-MS Rt 4.5 min; m/z [MH+] 182. Anal. Calcd. for

ACCEPTED MANUSCRIPT
C8H11N3S: C, 53.01; H, 6.12; N, 23.18; S, 17.69. Found: C, 53.45; H, 6.27; N, 23.30; S,
17.86.
4.2.19 [4-(4-Methylthio)phenyl]-3-thiosemicarbazide (19)
1
Yield 96%. Mp. 166-167°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.42 (s, 1H, NH), 9.10
(s, 1H, NH), 7.55 (d, J = 7.5 Hz, 2H, H3’, H5’), 7.25-7.10 (m, 2H, H2’, H6’), 4.91 (s, 2H, NH2),
2.43 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.7, 136.9, 133.4, 124.3,
124.2, 123.8, 123.7, 16.5. LC-MS Rt 4.7 min; m/z [MH+] 214. Anal. Calcd. for C8H11N3S2: C,
45.04; H, 5.20; N, 19.70; S, 30.06. Found: C, 45.45; H, 5.47; N, 19.70; S, 30.36.
4.2.20 4-(2,5-Dichlorophenyl)-3-thiosemicarbazide (20)
1
Yield 79%. Mp. 156-158°C. H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.64 (s, 1H, NH), 8.70
(s, 1H, NH), 7.50 (d, J = 8.6 Hz, 1H, H4’), 7.25-7.11 (m, 2H, H3’, H6’), 4.97 (s, 2H, NH2). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) = 184.6, 138.2, 132.8, 130.2, 128.9, 124.8. 123.5. LC-
MS Rt 5.7 min; m/z [MH+] 236. Anal. Calcd. for C7H7Cl2N3S: C, 35.61; H, 2.99; N, 17.80; S,
13.58. Found: C, 35.94; H, 3.00; N, 17.96; S, 12.98.
4.2.21 4-(2,3-Dichlorophenyl)-3-thiosemicarbazide (21)
1
Yield 96%. Mp. 123-126°C. H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.60-9.47 (m, 1H, NH),
8.22 (s, 1H, NH), 7.48-7.20 (m, 3H, H4’, H5’, H6’), 4.42 (s, 2H, NH2). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 184.1, 136.4, 132.9, 127.8, 127.4, 126.5. 122.4. LC-MS Rt 5.8 min; m/z
[MH+] 236. Anal. Calcd. for C7H7Cl2N3S: C, 35.61; H, 2.99; N, 17.80; S, 13.58. Found: C,
35.89; H, 3.07; N, 17.61; S, 13.04.
4.2.22 4-(5-Chloro-2-methoxyphenyl)-3-thiosemicarbazide (22)
1
Yield 97%. Mp. 167-170°C. H NMR (400 MHz, DMSO-d6) δ (ppm) = 10.05 (s, 1H, NH), 9.41
(s, 1H, NH), 9.02 (s, 1H, H4’), 7.12-6.98 (m, 2H, H3’, H6’), 4.88 (s, 2H, NH2), 3.82 (s, 3H,
OCH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 178.4, 148.3, 129.8, 123.8, 123.2, 119.8.

ACCEPTED MANUSCRIPT
118.4. 112.8. LC-MS Rt 5.9 min; m/z [MH+] 232. Anal. Calcd. for C8H10ClN3OS: C, 41.97; H,
4.35; N, 18.14; S, 13.84. Found: C, 40.92; H, 4.28; N, 17.84; S, 13.76.
4.2.23 4-(2-Chloro-4-nitrophenyl)-3-thiosemicarbazide (23)
1
Yield 84%. Mp. 191-194°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.95 (s, 1H, NH), 9.16
(d, J = 9.2 Hz, 1H, H3’), 8.36 (d, J = 2.6 Hz, 1H, H5’), 8.19 (dd, J = 9.2, 2.7 Hz, 2H, NH, H6’ ),
5.00 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 184.8, 142.5, 138.8, 126.0,
124.4, 124.3, 119.7. LC-MS Rt 5.8 min; m/z [MH+] 247. Anal. Calcd. for C7H7ClN4O2S: C,
34.08; H, 2.86; N, 22.71; S, 13.00. Found: C, 34.98; H, 2.99; N, 22.91; S, 12.78.
4.2.24 4-(2,4-Dichlorophenyl)-3-thiosemicarbazide (24)
1
Yield 64%. Mp. 169-171 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.50 (s, 1H, NH), 8.37
(s, 1H, NH), 7.63 (s, 1H, H3’), 7.38-7.36 (m, 2H, H5’, H6’), 4.99 (s, 2H, NH2). 13C NMR (100
MHz, DMSO-d6): δ (ppm) = 179.7, 135.9, 134.1, 129.9, 127.9, 127.5, 126.3. LC-MS Rt 5.9
min; m/z [MH+] 236. Anal. Calcd. for C7H7Cl2N3S: C, 35.61; H, 2.99; N, 17.80; S, 13.58.
Found: C, 35.49; H, 2.99; N, 17.84; S, 13.33.
4.2.25 4-(3,5-Dichlorophenyl)-3-thiosemicarbazide (25)
1
Yield 77%. Mp. 157-159°C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.43 (s, 1H, NH), 7.93
(s, 1H, NH), 7.24 (s, 1H, H4’), 7.00-6.97 (m, 2H, H2’, H6’), 4.88 (s, 2H, NH2). 13C NMR (100
MHz, DMSO-d6): δ (ppm) = 179.5, 142.4, 133.5, 133.3, 123.5, 121.8, 119.5. LC-MS Rt 6.1
min; m/z [MH+] 236. Anal. Calcd. for C7H7Cl2N3S: C, 35.61; H, 2.99; N, 17.80; S, 13.58.
Found: C, 35.69; H, 3.03; N, 17.85; S, 13.96.
4.2.26 4-(3,4-Dichlorophenyl)-3-thiosemicarbazide (26)
1
Yield 79%. Mp. 169-172 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 8.82 (s, 1H, NH), 7.52
(s, 1H, NH), 7.36 (s, 1H, H2’), 7.30-7.27 (m, 1H, H5’), 7.16-7.12 (m, 1H, H6’) 4.79 (s, 2H,
NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.3, 141.6, 132.7, 131.9, 126.2, 123.6,

ACCEPTED MANUSCRIPT
120.1. LC-MS Rt 5.9 min; m/z [MH+] 236. Anal. Calcd. for C7H7Cl2N3S: C, 35.61; H, 2.99; N,
17.80; S, 13.58. Found: C, 35.63; H, 3.01; N, 17.75; S, 13.66.
4.2.27 4-[4-Chloro-3-(trifluoromethyl)phenyl]-3-thiosemicarbazide (27)
1
Yield 89%. Mp. 151-154°C. H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.42 (s, 1H, NH), 8.36
(s, 1H, NH), 7.98 (d, J = 7.5 Hz, 1H, H5’), 7.61 (s, 1H, H2’), 7.27-7.25 (m, 1H, H6’), 4.89 (s,
2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 179.6, 137.7, 130.7, 127.5, 125.1,
123.8. 120.4. LC-MS Rt 6.1 min; m/z [MH+] 272. Anal. Calcd. for C8H7ClF3N3S: C, 35.63; H,
2.62; N, 15.58; S, 11.89. Found: C, 35.91; H, 3.00; N, 15.77; S, 11.76.
4.2.28 4-[(3-Chloro-4-cyano)phenyl]-3-thiosemicarbazide (28)
1
Yield 88%. Mp. 194-197°C. H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.66 (s, 1H, NH), 8.46
(s, 1H, NH), 8.15 (s, 1H, H2’), 7.99 (d, J = 8.9 Hz, 1H, H5’), 7.82 (d, J = 8.7 Hz, 1H, H6’),
6.46 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.3, 142.7, 139.3, 132.6,
121.4, 118.5, 114.7, 99.9, 13.6. LC-MS Rt 5.6 min; m/z [MH+] 227. Anal. Calcd. for
C8H7ClN4S: C, 42.39; H, 3.11; N, 24.72; S, 14.14. Found: C, 42.66; H, 3.38; N, 24.54; S,
14.51.
4.2.29 4-(2,3,4-Trichlorophenyl)-3-thiosemicarbazide (29)
1
Yield 74%. Mp. 170-172°C. H NMR (400 MHz, DMSO-d6) δ (ppm) = 9.69 (s, 1H, NH), 9.12
(s, 1H, NH), 8.84 (s, 1H, H3’), 7.88 (s, 1H, H6’), 4.80 (s, 2H, NH2). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 176.9, 137.1, 130.1, 129.7, 129.1, 128.8. LC-MS Rt 6.5 min; m/z [MH+]
272. Anal. Calcd. for C7H6Cl3N3S: C, 31.07; H, 2.24; N, 15.53; S, 11.85. Found: C, 31.74; H,
2.21; N, 15.51; S, 12.07.
4.2.30 4-(2,3,4,5,6-Pentafluorophenyl)-3-thiosemicarbazide (30)
1
Yield 63%. Mp. 184-186 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.62 (s, 1H, NH), 8.98
(s, 1H, NH), 6.40 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 182.1, 150.0,

ACCEPTED MANUSCRIPT
149.3, 139.2, 138.5, 138.3, 119.3. 19F NMR (376 MHz, DMSO-d6): δ (ppm) = 164.7 (t, J =
22.0, 2F), 157.2 (t, J = 22.0, 1F), 144.5 (d, J = 22.0, 2F). LC-MS Rt 5.5 min; m/z [MH+] 258.
Anal. Calcd. for C7H4F5N3S: C, 32.69; H, 1.57; N, 16.34; S, 12.47. Found: C, 33.05; H, 1.46;
N, 16.40; S, 12.67.
4.3. General procedure for the synthesis of thiosemicarbazide derivatives (31-32).
To a stirred solution of 1-methyl-1-phenylhydrazine (2.4 mmol) in 20 mL of isopropanol,
appropriately substituted isothiocyanate (2.0 mmol) was added at room temperature.
Precipitate was formed immediately. Stirring was continued for 3 hours. Then the mixture
was filtered and the precipitate was washed with isopropanol three times to give the desired
product.
4.3.1 4-(3,5-Dichlorophenyl)-1-(methylphenyl)-3-thiosemicarbazide (31)
1
Yield 89%. Mp. 177-179 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 10.08 (s, 2H, 2NH),
7.80 (s, 2H, H2’, H6’), 7.32 ( s, 1H, H4’), 7.27 (t, J = 7.7 Hz, 2H, H3’’, H5’’), 6.87 (dd, J = 15.7
Hz, 8.0 Hz, 3H, H2’’, H4’’, H6’’), 3.03 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) =
181.7, 149.5, 140.4, 133.3, 133.2, 129.6, 129.5, 123.5, 121.1, 119.2, 119.1, 114.3, 114.1,
44.8. LC-MS Rt 7.4 min; m/z [MH+] 326. Anal. Calcd. for C14H13Cl2N3S: C, 51.54; H, 4.02; N,
12.88; S, 9.83. Found: C, 52.05; H, 4.05; N, 12.70; S, 9.87.
4.3.2 4-(3,5-Dichlorophenyl)-1-(methylphenyl)-3-thiosemicarbazide (32)
1
Yield 89%. Mp. 177-179 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) = 10.08 (s, 2H, 2NH),
7.80 (s, 2H, H2’, H6’), 7.32 ( s, 1H, H4’), 7.27 (t, J = 7.7 Hz, 2H, H3’’, H5’’), 6.87 (dd, J = 15.7
Hz, 8.0 Hz, 3H, H2’’, H4’’, H6’’), 3.03 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) =
181.7, 149.5, 140.4, 133.3, 133.2, 129.6, 129.5, 123.5, 121.1, 119.2, 119.1, 114.3, 114.1,
44.8. LC-MS Rt 7.4 min; m/z [MH+] 326. Anal. Calcd. for C14H13Cl2N3S: C, 51.54; H, 4.02; N,
12.88; S, 9.83. Found: C, 52.05; H, 4.05; N, 12.70; S, 9.87.
4.4 Enzymatic assay

ACCEPTED MANUSCRIPT
The IDO inhibition assay was performed using a reported procedure.12,15
4.5 Molecular modelling
Molecular modelling experiments were performed on a windows workstation.22 The
compounds were built using the Chembiodraw 3D module and minimized using the MMFF94
forcefield. The GOLD docking software was used using the IDO X-ray structure (pdb code
2D0T) with an active site definition of 10Å around PIM chosen as center of the active site.
For each compounds 30 conformations were generated and ranked according to the
GOLDSCORE.
Ackowledgments
We gratefully acknowledge the FUNDP-CERUNA fund (UNamur), the FRS-FNRS as well as
the Télévie (7.4.543.07F) for funding this project. The author’s thank Mr Arnaud Lamourette
for technical assistance.
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ACCEPTED MANUSCRIPT
Ligand
Efficiencya
(LE) = 1.4 x
pIC50/HAC
IDO IC50
(µM)
Compound
L-1MT
Structure
100
0.35
BTT
1
50
0.60
~50
>50
>50
>50
0.55
2
-
-
-
3
4
5
6
7
8
2.7
3.7
15
0.65
0.63
0.56
0.58
3.8

ACCEPTED MANUSCRIPT
9
41
0.41
10
11
12
13
14
>50
1.8
1.6
3.5
1.2
-
0.67
0.68
0.64
0.64
15
>50
-
16
17
>50
>50
-
-
18
19
>50
34
-
0.48

ACCEPTED MANUSCRIPT
20
21
18
0.51
>50
>50
-
-
22
23
24
>50
>50
-
-
25
26
27
13
17
0.53
0.51
>50
-
28
29
34
0.45
>50
-

ACCEPTED MANUSCRIPT
30
31
32
>50
-
-
-
>50
>50

ACCEPTED MANUSCRIPT