Indoleamine 2,3-dioxygenase inhibitory activity of derivatives of marine alkaloid tsitsikammamine A

Article abstract of DOI:10.1016/j.bmcl.2012.11.036

Tsitsikammamines are marine alkaloids whose structure is based on the pyrroloiminoquinone scaffold. These and related compounds have attracted attention due to various interesting biological properties, including cytotoxicity, topoisomerase inhibition, antimicrobial, antifungal and antimalarial activity. Indoleamine 2,3-dioxygenase (IDO1) is a well-established therapeutic target as an important factor in the tumor immune evasion mechanism. In this preliminary communication, we report the inhibitory activity of tsitsikammamine derivatives against IDO1. Tsitsikammamine A analogue 11b displays submicromolar potency in an enzymatic assay. A number of derivatives are also active in a cellular assay while showing little or no activity towards tryptophan 2,3-dioxygenase (TDO), a functionally related enzyme. This IDO1 inhibitory activity is rationalized by molecular modeling studies. An interest is thus established in this class of compounds as a potential source of lead compounds for the development of new pharmaceutically useful IDO1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.11.036

Bioorganic & Medicinal Chemistry Letters 23 (2013) 47–54
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Indoleamine 2,3-dioxygenase inhibitory activity of derivatives of marine
alkaloid tsitsikammamine A
a,
⇑
Eduard Dolušic , Pierre Larrieu b, Céline Meinguet a, Delphine Colette c, Arnaud Rives d, Sébastien Blanc c,
´
Thierry Ferain c, Luc Pilotte b, Vincent Stroobant b, Johan Wouters a, Benoît Van den Eynde b,
Bernard Masereel a, Evelyne Delfourne d, Raphaël Frédérick a,
⇑
a Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, 61 Rue de Bruxelles, B-5000 Namur, Belgium
b Ludwig Institute for Cancer Research, Brussels Branch, and de Duve Institute, Université Catholique de Louvain, 74 Avenue Hippocrate, UCL 7459, B-1200 Brussels, Belgium
c Euroscreen SA, 47 Rue Adrienne Boland, B-6041 Gosselies, Belgium
d Université Paul Sabatier, UMR CNRS 5068, Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique, 118 Route de Narbonne, F-31062 Toulouse Cédex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Tsitsikammamines are marine alkaloids whose structure is based on the pyrroloiminoquinone scaffold.
These and related compounds have attracted attention due to various interesting biological properties,
including cytotoxicity, topoisomerase inhibition, antimicrobial, antifungal and antimalarial activity.
Indoleamine 2,3-dioxygenase (IDO1) is a well-established therapeutic target as an important factor in
the tumor immune evasion mechanism. In this preliminary communication, we report the inhibitory
activity of tsitsikammamine derivatives against IDO1. Tsitsikammamine A analogue 11b displays submi-
cromolar potency in an enzymatic assay. A number of derivatives are also active in a cellular assay while
showing little or no activity towards tryptophan 2,3-dioxygenase (TDO), a functionally related enzyme.
This IDO1 inhibitory activity is rationalized by molecular modeling studies. An interest is thus established
in this class of compounds as a potential source of lead compounds for the development of new pharma-
ceutically useful IDO1 inhibitors.
Received 28 September 2012
Revised 7 November 2012
Accepted 9 November 2012
Available online 22 November 2012
Keywords:
Pyrroloiminoquinones
Tsitsikammamines
Indoleamine 2,3-dioxygenase
Cancer immunology
Molecular modeling
Ó 2012 Elsevier Ltd. All rights reserved.
Many alkaloids based on the pyrroloiminoquinone scaffold have
been isolated and characterized from marine and other natural
sources in recent years. These natural products of unique struc-
tures have attracted attention due to various interesting biological
properties, such as cytotoxicity, inhibition of topoisomerases, anti-
microbial, antifungal and antimalarial activity.1–8 A subfamily of
this class of compounds, the bispyrroloiminoquinones tsitsikamm-
amine A (1) and B (2, Fig. 1),were first isolated and characterized in
1996 from a sponge of the Latrunculiidae family in the Tsitsikamma
Marine Reserve, South Africa.9 Further search for pyrroloiminoqui-
none metabolites as chemotaxonomic markers yielded two oxime
derivatives 3 and 4 isolated from Triceratium Favus (Fig. 1).10 Prom-
ising anticancer activities of these compounds were reported
HO
R1
N
O
1, R = H, R1 = H
2, R = CH3, R1 = H
3, R = H, R1 = OH
4, R = CH3, R1 = OH
N
N
H
R
Figure 1. Structures of tsitsikammamines 1–4.
because of their abilities to intercalate into DNA and topoisomer-
ase I inhibition.7,9–13 More recently, the biosynthesis of tsitsi-
kammamines in the latrunculid sponge T. favus has been
suggested to originate from the microbial community associated
with the species.14
These results prompted studies on the synthetic accessibility of
this class of compounds15–17 aiming at an improved therapeutic
value.7,18–20 In this context, our group described a cycloaddition-
based synthesis of aza-analogues of tsitsikammamines (wherein
either of the pyrrole rings was replaced with a pyrazole) and eval-
uated their topoisomerase I and II inhibition as well as their in vitro
Abbreviations: IDO1, indoleamine 2,3-dioxygenase; TDO, tryptophan 2,3-diox-
ygenase; NHDF, normal fibroblast cell lines; MTT, (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; EDTA, ethylenediaminetetraacetic acid; IMAC,
immobilized metal ion affinity chromatography; SDS–PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; HBSS, Hanks balanced salt solution; SAR,
structure–activity relationships.
⇑
Corresponding authors. Tel.: +32 81 72 43 35; fax: +32 81 72 42 99 (E.D.);
tel.:+32 81 72 42 90; fax: +32 81 72 42 99 (R.F.).
E-mail addresses: eduard.dolusic@fundp.ac.be (E. Dolušic´), raphael.frederick@
fundp.ac.be (Raphaël Frédérick).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.036

´
48
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
H
NH2
Ts
N
MeO
O
O
N
NH2
O
N
MeO
N
Ts
O
H
5b
5a
Figure 2. Structures of potential anticancer compounds 5a and 5b; Ts = p-CH3C6H4SO2–.
NHBoc
NHBoc
MeO
NHBoc
MeO
O
O
O
O
O
O
H
N
OH
a
+
+
OH
NH2
N
N
OH
N
N
H
6
Ts
Ts
8a
8b
Ts
MeO
7
b
b
MeO
MeO
NH2
NH2
O
O
O
NH2
NH2
H
H
N
N
O
O
O
N
N
Ts
Ts
O
c
N
H
N
N
H
N
c
Ts
Ts
O
5a
9a
9b
5b
MeO
MeO
d
d
MeO
MeO
N
N
N
N
H
N
H
N
e
e
N
NH
N
NH
Ts
N
H
Ts
N
H
O
O
O
O
11b
10a
10b
11a R = Me
12a R = H
RO
MeO
f
Scheme 1. Synthesis of compounds 8–12. (a) abs EtOH, rt, 2 h; (b) TFA, CH2Cl2, rt, 4 h; (c) MnO2, CH2Cl2, rt, overnight; (d) abs EtOH, 4 Å mol sieves, reflux, 3 h; (e) 1 M NaOH,
dioxane, rt, overnight; (f) BBr3, CH2Cl2, N2, 4h.
antiproliferative activity.21,22 Interestingly, some of these com-
pounds showed an antiproliferative effect while being devoid of
any topoisomerase activity, thus suggesting another mechanism
of cell toxicity for this series. More recently, two regioisomeric ser-
ies of tsitsikammamine analogues along with the corresponding
synthetic intermediates were evaluated in an in vitro antiprolifer-
ative assay against the U373 glioblastoma, A549 non-small-cell-
lung cancer and PC-3 prostate cancer cell lines as well as two
human normal fibroblast cell lines (NHDF and Wi38).23 This work
allowed identifying the cytotoxic synthetic intermediate 5b (Fig. 2)
and its regioisomer 5a. Using the MTT colorimetric assay, com-
pound 5b showed potent cytotoxicity towards all the cell lines in
this study. Isomer 5a displayed somewhat higher IC50 values in this
test but also relative selectivity towards some cell lines. It was sug-
gested that this effect could be associated with a specific antipro-
liferative activity. The conclusion was that compound 5a and its
derivatives represent interesting novel anti-cancer agents with
an unknown mechanism of action.
Cancer immunotherapy, a strategy for cancer treatment consist-
ing of stimulating the immune system to attack and destroy tumor
cells, has so far shown limited efficacy in vivo, the main reason being
the ability of tumors to escape an immune response. Among the var-
ious factors accounting for this phenomenon24–27 are the trypto-
phan-catabolizing enzymes indoleamine 2,3-dioxygenase (IDO1;
EC 1.13.11.52)28–34 and tryptophan 2,3-dioxygenase (TDO; EC
1.13.11.11).35 IDO1 is a monomeric extrahepatic cytosolic enzyme
while TDO is homotetrameric and normally expressed at a high level
in the liver and at low level in the brain.36,37 Both enzymes are heme
dioxygenases catalyzing the oxidation of the indole ring of L-trypto-
phan (Trp) to produce N-formylkynurenine. This reaction is the first
and rate-limiting step of the kynurenine pathway of tryptophan
catabolism. Another indoleamine 2,3-dioxygenase isoform, IDO2,

´
49
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
has been described.38–42 However, since its physiological relevance
remains unclear,43 in this work we focused our efforts on IDO1.
Our group showed that many human tumors express IDO1 in a
constitutive manner.44 It has been suggested that IDO1 exerts its
immunosuppressive effects on T-lymphocytes in a twofold man-
ner: by depleting Trp locally and by action of toxic Trp catabo-
lites.24,27,31,43,45,46 TDO has also recently been shown to be
expressed constitutively in human glioblastomas where it pro-
motes tumor progression by action of a downstream tryptophan
catabolite kynurenine which acts as endogenous ligand of the aryl
hydrocarbon receptor.47 We showed that TDO inhibition reverses
tumoral immune resistance in vivo.48 Tryptophan catabolites have
also been connected to other pathological conditions, particularly
in the central nervous system (CNS).49–52
Not surprisingly then, the recent years witnessed many at-
tempts to discover and develop IDO153–57 and TDO58,59 inhibitors.
However, with only two IDO1 inhibitors to have entered clinical
trials so far, D-1-methyltryptophan32 and INCB024360,60 there is
still an urgent need for the discovery and development of new anti-
cancer compounds with this mode of action.
hydroid Garveia annulata are submicromolar IDO1 inhibitors in vi-
tro.64 Their skeleton was pharmacomodulated to afford novel pyr-
anonaphthoquinones possessing nanomolar potencies in vitro.65
Mitomycin C, an indoloquinone from Streptomyces, is an uncom-
petitive IDO1 inhibitor with a Ki of approx. 25 lM. The mechanism
by which such compounds inhibit the enzyme is debated. For in-
stance, binding into an inhibitory substrate binding site (Si site)
has been suggested, although mitomycin C was the only qui-
none-type compound for which such type of binding has been pro-
posed.66 Also, a recent study by Pearson and colleagues on the
IDO1 inhibitory potency of menadione, another quinone-type com-
pound, suggested the possibility that its redox potential could
interfere with the reductant cofactor in vitro (methylene blue)
and thus lead to irrelevant IDO1 inhibition data, although other
mechanisms were also suggested in the original study to account
for this phenomenon.67 In any case, some of these naphthoqui-
nones have also been shown to possess some IDO1 inhibitory
potency in a cellular assay that does not involve methylene blue,
thus adding confidence to the potential of this type of
compounds.65
Some of the best IDO1 inhibitors are marine and other natural
products that contain an oligocyclic quinone scaffold.61 Indoloqui-
none exiguamine A, isolated from the marine sponge Neopetrosia
exigua, was shown to possess a Ki of 210 nM for IDO1 inhibition
in vitro62 (Ki = 41 nM in Ref. 63), establishing exiguamine A as
one of the most potent IDO1 inhibitors known. Synthetic analogues
of simplified structures retaining nanomolar potency were de-
scribed in the following study.63 The structures of these com-
Prompted by the tsitsikammamine derivatives’ anticancer
activity and their relative structural likeness to quinone-containing
IDO1 inhibitors, we decided to undertake an inhibitory study of
tsitsikammamine derivatives and synthetic intermediates on
IDO1 while also performing a counter screen on TDO.
The compounds were synthesized as described23 in Schemes 1
and 2. A Michael addition of amino alcohol 6 to the indolequinone
7 gave two isomeric products, which were further elaborated to
the final tsitsikammamine derivatives by acid-mediated cycliza-
tion/deprotection, oxidative aromatization, a second cyclization
followed by the removal of the protecting tosyl group and the final
aromatic methyl ether demethylation effected by BBr3 (Scheme 1).
pounds can be regarded as being partially derived from
a
tryptamine and partially from a quinone moiety; this also applies
to the tsitsikammamine derivatives described herein. A naphtho-
quinone pharmacophore-containing annulins from the marine
MeO
MeO
O
O
O
O
O
H
N
OH
a
+
+
OH
NH2
N
N
OH
N
N
H
6
Ts
Ts
14a
14b
O
Ts
MeO
13
b
b
MeO
MeO
O
O
O
H
H
N
N
O
O
O
N
N
Ts
Ts
O
c
N
H
N
N
H
N
c
Ts
Ts
O
16a
15a
15b
16b
MeO
MeO
d
d
O
H
RO
N
NH
O
17b R = Me
18b R = H
O
e
NH
17a
N
H
O
MeO
Scheme 2. Synthesis of compounds 14–18. (a) abs EtOH, rt, 2 h; (b) TFA, CH2Cl2, rt, 4 h; (c) MnO2, CH2Cl2, rt, overnight; (d) 1 M NaOH, dioxane, rt, overnight; (e) BBr3, CH2Cl2,
N2, 4h.

´
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
50
Table 1
Biological activity and cell viability data for the tsitsikammamine derivatives 5a–18b
Compound Structure
Mean IC50
(l
M) in the enzymatic mIDO1 cell. Assay inhib.
@ 10 M (%)
mTDO cell. assayinhib.
@ 10 M (%)
Cell viability @
10 M (%)
test on hIDO1a
l
l
l
OH
L-1-MT
138.5
14.5 @ 22
lM
NIb @ 22
lM
>90
>90
N
O
H2N
NH
O
5a
11.9
26.0
NI
MeO
O
O
NH2
N
Ts
NH2
Ts
MeO
5b
5.4
15.6
NI
80
N
N
O
H
O
HO
NHBoc
NHBoc
H
N
8a
10.1
26.9
17.4
31.3
NI
NI
>90
>90
N
Ts
MeO
O
O
MeO
8b
N
Ts
N
H
HO
O
H
N
O
9a
5.6
2.8
4.2
40.2
26.1
NDc
4.7
>90
NH2
O
N
MeO
MeO
Ts
NH2
Ts
O
9b
NI
83
N
N
O
H
H
N
N
10a
ND
<50
O
N
MeO
MeO
Ts
N
N
10b
2.2
7.1
32.0
ND
3.1
ND
>90
<50
Ts
N
O
H
H
N
N
11a
O
MeO
N
H

´
51
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
MeO
N
NH
11b
12a
0.9
7.0
19.4
NI
NI
>90
>90
N
O
N
H
H
N
5.3
O
N
HO
H
O
HO
H
N
14a
14b
8.8
2.5
37.3
24.3
1.1
NI
>90
>90
N
Ts
MeO
MeO
O
O
O
N
N
H
Ts
HO
H
O
N
15a
15b
16b
5.6
54.2
40.2
19.6
3.2
2.5
NI
>90
>90
>90
N
O
O
MeO
MeO
Ts
N
Ts
1.8
O
N
H
MeO
O
d
N
Ts
N
O
H
H
N
O
17a
17b
18b
22.4
ND
ND
NI
ND
ND
NI
<50
<50
>90
O
N
H
MeO
MeO
O
NH
12.6
O
N
H
HO
O
NH
d
N
H
O
a
b
c
Result = mean of two independent determinations. Error between experiments is no more than 10%.
NI = no inhibition.
ND = not determined because of cellular toxicity of the compound.
<50% Inhibition at maximal concentration.
d
Analogous chemistry was applied for the synthesis of the deriv-
atives 14–18 lacking the iminoquinone motif (Scheme 2).
murine cancer cell lines overexpressing mIDO1 and mTDO and cell
viabilities are presented in Table 1.
Protein expression and purification, cell line constructions and
enzymatic and cellular assays were carried out as described previ-
ously.54,56,58 Inhibitory activities of synthetic derivatives of tsitsi-
kammamines on purified recombinant hIDO1 as well as on
In all cases except for the intermediates 8, the b regioisomer
(leading to the ‘natural geometry’ of the final tetracyclic product)
is at least twice as active on IDO1 in the enzymatic assay as the
corresponding a regioisomer. Similar tendencies were reported

´
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
52
Figure 3. View of (a) 11b and (c) 11a docked within the IDO1 active site (PDB code 2D0T) showing Pocket A, and (b) 11b and (d) 11a showing Pocket B. Pictures made with
Pymol (Delano Scientific).
earlier23 regarding their in vitro activity in an MTT colorimetric as-
say. On the contrary, a reverse pattern is seen in the cellular test.
Deletion of the conjugated double bond bearing the anisyl moi-
ety in the tricyclic series improves the activity in the enzymatic as-
say (compd 5–9 and 15b–16b). This effect persists in the cellular
assay, where three out of the four compounds possessing this
structural feature (9a, 15a and 15b) are the most potent com-
N-tosylated analogue 10b (which is somewhat less potent in the
enzymatic assay). Again, compound 11b might be too polar to dis-
play good membrane permeability. This could also be the reason
for the even poorer cellular activity of the yet more polar tsitsi-
kammamine unnatural regioisomer 12a.
It should be noted that, in contrast to the enzymatic test, the
cell-based assay does not involve the reductant cofactor methylene
blue to activate IDO1, thus strengthening the hypothesis of IDO1
inhibition not based on a redox mechanism.
pounds with 40% or more IDO1 inhibition at 10 lM.
Interestingly, deleting the aminoethyl side chain, N-protected
or not, does not seem to have a significant effect on the potency
in the enzymatic assay (compd 8a–14a, 9a–15a, 9b–15b and 5b–
16b), except in one case (8b and 14b). This is in good agreement
with the results for exiguamine A derivatives published in 2008
by Carr et al.63 The compounds described in Carr’s work share
some structural features with the tsitsikammamine-related com-
pounds studied here; both classes can be seen as being partially
derived from a tryptamine and from a quinone moiety. The pres-
ence of a side chain in the compounds described in this work does,
however, in most cases (and certainly for the free primary amines)
cause a drop in potency in the cellular assay. This could be a con-
sequence of lower cell membrane permeability due to the presence
of a protonated amino group.
The tetracyclic iminoquinone motif, as present in the natural
scaffold, enhances the potencies in both tests (compd 5–10 and
11–17) but also provokes enhanced cellular toxicity, especially in
the a series (Table 1).
Removing the tosyl group from the ‘eastern’ pyrrole ring
(compd 10–11 and 16b–17b) or the methyl group from the anisole
moiety (compd 11a–12a or 17b–18b) does not seem to exert a sig-
nificant effect on the potency in the enzymatic assay. Nevertheless,
in contrast to the phenolic compounds, their O-methylated coun-
terparts are cytotoxic. Despite being the most potent compound
Finally, hardly any TDO inhibitory activity is detected in the cel-
lular assay on this series, showing its selectivity for IDO1.
In order to understand the affinity observed, we studied the
binding mode of compounds 11a and 11b within IDO1 by means
of docking using the automated GOLD program.68 For each com-
pound, 20 docking solutions were generated and ranked according
to the the GoldScore scoring function. The key interactions stabiliz-
ing the compounds within the IDO binding cleft are depicted in
Figure 3. First, regarding the more potent compound 11b, the 20
solutions obtained are found in the same orientation and a very
high score was obtained (GoldScore of the best pose = 55.80). In
this orientation, the pyrroloiminoquinone moiety is deeply in-
serted inside pocket A of the IDO1 active site (Fig. 3a), interacting
via an H-bond to the Ser167 residue and complexing the heme iron
by its carbonyl function (distance C@OÁ Á ÁFe = 2.89 Å, angle ꢀ120°).
The compound is also well stabilized by hydrophobic interactions
with the Tyr126, Phe164 and Phe163 (omitted for clarity) residues.
Interestingly, a nice shape complementarity occurs between the
pyrroloiminoquinone scaffold and pocket A of IDO1. On the con-
trary pocket B (Fig. 3b) is only partially filled by the methoxy-
phenyl side chain. This suggests that the introduction of larger
side chains in this position could possibly enhance IDO1 inhibition.
Then, regarding the less potent compound 11a, one preferential
orientation was obtained (Fig. 3c,d; 10 solutions out of the 20 gen-
erated) with a GoldScore for the best pose of 47.27. Two other
in the enzymatic assay (IC50 = 0.9
lM), pyrroloiminoquinone
11b’s cellular activity is almost halved compared to that of its

´
53
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
14. Walmsley, T. A.; Matcher, G. F.; Zhang, F.; Hill, R. T.; Davies-Coleman, M. T.;
Dorrington, R. A. Mar. Biotechnol. 2012.
orientations with an even lower stabilization were also suggested.
In the preferred orientation (Fig. 3c,d) the compound seems to
adopt a reverse binding compared to the compounds of the ‘b-ser-
ies’ (Fig. 3c), probably to allow a better accommodation of the
methoxyphenyl sidechain. In this orientation, the pyrroloimino-
quinone interacts with the heme via its nitrogen (distance
N|Á Á ÁFe = 4.0 Å) rather than the carbonyl function and the hydrogen
bonding to the Ser167 residue is lost. Indeed the nice fit with high
stabilization (high GoldScore) of the pyrroloiminoquinone of the
‘b-series’ and the IDO1 active site can certainly account for their
usually higher IDO1 inhibition compared to compounds of the
‘a-series’ (for instance compare compounds 10a vs 10b, 11a vs
11b, 14a vs 14b, 15a vs 15b and 17a vs 17b).
15. Murugesan, S.; Nadkarni, D. H.; Velu, S. E. Tetrahedron Lett. 2009, 50, 3074.
16. Harayama, Y.; Kita, Y. Curr. Org. Chem. 2005, 9, 1567.
17. Rives, A.; Delaine, T.; Legentil, L.; Delfourne, E. Tetrahedron Lett. 2009, 50, 1128.
18. Shinkre, B. A.; Raisch, K. P.; Fan, L.; Velu, S. E. Bioorg. Med. Chem. Lett. 2007, 17,
2890.
19. Shinkre, B. A.; Raisch, K. P.; Fan, L.; Velu, S. E. Bioorg. Med. Chem. 2008, 16, 2541.
20. Passarella, D.; Belinghieri, F.; Scarpellini, M.; Pratesi, G.; Zunino, F.; Gia, O. M.;
Via, L. D.; Santoro, G.; Danieli, B. Bioorg. Med. Chem. 2008, 16, 2431.
21. Legentil, L.; Benel, L.; Bertrand, V.; Lesur, B.; Delfourne, E. J. Med. Chem. 2006,
49, 2979.
22. Legentil, L.; Lesur, B.; Delfourne, E. Bioorg. Med. Chem. Lett. 2006, 16, 427.
23. Rives, A.; Le Calvé, B.; Delaine, T.; Legentil, L.; Kiss, R.; Delfourne, E. Eur. J. Med.
Chem. 2010, 45, 343.
24. Munn, D. H.; Shafizadeh, E.; Attwood, J. T.; Bondarev, I.; Pashine, A.; Mellor, A.
L. J. Exp. Med. 1999, 189, 1363.
25. Tiwari, M. J. Cancer Res. Ther. 2010, 6, 427.
In conclusion, derivatives of natural products tsitsikammam-
ines have previously been shown to be potent anticancer agents.
Here we show for the first time that these compounds exert inhib-
itory activity on indoleamine 2,3-dioxygenase, an enzyme involved
in tumoral immune resistance. Although the previously demon-
strated anticancer activity cannot probably be attributed to their
IDO1 inhibitory potency, the present work sheds light on one pos-
sible mechanism of anticancer activity in this series. Some of these
compounds were also shown to possess reasonable activities on
IDO1 in a cell-based assay, thus strengthening their potential for
future development in the field of anticancer immunotherapy.
They also display a selectivity for IDO1 versus tryptophan 2,3-diox-
ygenase. A molecular modeling study of compounds 11a–b in the
IDO1 active site suggested that the pyrroloiminoquinone was dee-
ply inserted in the cavity A, leaving pocket B only partially filled. In
the more potent ‘b-series’, introduction of larger side chains in or-
der to fill this other pocket can thus be proposed as a potential
means of enhancing the IDO1 inhibitory potency.
26. Whiteside, T. L.; Mandapathil, M.; Szczepanski, M.; Szajnik, M. Bull. Cancer
2011, 98, E25.
27. Munn, D. H.; Zhou, M.; Attwood, J. T.; Bondarev, I.; Conway, S. J.; Marshall, B.;
Brown, C.; Mellor, A. L. Science 1998, 281, 1191.
28. Muller, A. J.; DuHadaway, J. B.; Donover, P. S.; Sutanto-Ward, E.; Prendergast, G.
C. Nat. Med. 2005, 11, 312.
29. Löb, S.; Königsrainer, A.; Rammensee, H. G.; Opelz, G.; Terness, P. Nat. Rev.
Cancer 2009, 9, 445.
30. Prendergast, G. C. Oncogene 2008, 27, 3889.
31. Munn, D. H.; Mellor, A. L. J. Clin. Invest. 2007, 117, 1147.
32. Katz, J. B.; Muller, A. J.; Prendergast, G. C. Immunol. Rev. 2008, 222, 206.
33. Muller, A. J.; Prendergast, G. C. Curr. Cancer Drug Targets 2007, 7, 31.
34. Liu, X.; Newton, R. C.; Friedman, S. M.; Scherle, P. A. Curr. Cancer Drug Targets
2009, 9, 938.
35. Van den Eynde, B.; Pilotte, L.; De Plaen, E. WO2010008427 A1 2010.
36. Rafice, S. A.; Chauhan, N.; Efimov, I.; Basran, J.; Raven, E. L. Biochem. Soc. Trans.
2009, 37, 408.
37. Miller, C. L.; Llenos, I. C.; Dulay, J. R.; Barillo, M. M.; Yolken, R. H.; Weis, S.
Neurobiol. Dis. 2004, 15, 618.
38. Metz, R.; Duhadaway, J. B.; Kamasani, U.; Laury-Kleintop, L.; Muller, A. J.;
Prendergast, G. C. Cancer Res. 2007, 67, 7082.
39. Lob, S.; Konigsrainer, A.; Schafer, R.; Rammensee, H. G.; Opelz, G.; Terness, P.
Blood 2008, 111, 2152.
40. Lob, S.; Konigsrainer, A.; Zieker, D.; Brucher, B. L.; Rammensee, H. G.; Opelz, G.;
Terness, P. Cancer Immunol. Immunother. 2009, 58, 153.
41. Meininger, D.; Zalameda, L.; Liu, Y.; Stepan, L. P.; Borges, L.; McCarter, J. D.;
Sutherland, C. L. Biochim. Biophys. Acta 2011, 1814, 1947.
42. Yuasa, H. J.; Ball, H. J.; Austin, C. J.; Hunt, N. H. Comp. Biochem. Physiol. B.
Biochem. Mol. Biol. 2010, 157, 10.
43. Platten, M.; Wick, W.; Van den Eynde, B. J. Cancer Res. 2012, 72, 5435.
44. Uyttenhove, C.; Pilotte, L.; Theate, I.; Stroobant, V.; Colau, D.; Parmentier, N.;
Boon, T.; Van den Eynde, B. J. Nat. Med. 2003, 9, 1269.
Acknowledgments
This work is supported by the FNRS (Belgium) and the Walloon
region (CANTOL grant no 5678). R.F. is Associate Researcher from
the FRS-FNRS (Belgium).
45. Hwu, P.; Du, M. X.; Lapointe, R.; Do, M.; Taylor, M. W.; Young, H. A. J. Immunol.
2000, 164, 3596.
46. Terness, P.; Bauer, T. M.; Rose, L.; Dufter, C.; Watzlik, A.; Simon, H.; Opelz, G. J.
Exp. Med. 2002, 196, 447.
Supplementary data
47. Opitz, C. A.; Litzenburger, U. M.; Sahm, F.; Ott, M.; Tritschler, I.; Trump, S.;
Schumacher, T.; Jestaedt, L.; Schrenk, D.; Weller, M.; Jugold, M.; Guillemin, G. J.;
Miller, C. L.; Lutz, C.; Radlwimmer, B.; Lehmann, I.; von Deimling, A.; Wick, W.;
Platten, M. Nature 2011, 478, 197.
48. Pilotte, L.; Larrieu, P.; Stroobant, V.; Colau, D.; Dolušic´, E.; Frédérick, R.; De
Plaen, E.; Uyttenhove, C.; Wouters, J.; Masereel, B.; Van den Eynde, B. J. Proc.
Natl. Acad. Sci. U.S.A. 2012, 109, 2497.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.
036.
References and notes
49. Schwarcz, R. Curr. Opin. Pharmacol. 2004, 4, 12.
50. Dantzer, R.; O’Connor, J. C.; Freund, G. G.; Johnson, R. W.; Kelley, K. W. Nat. Rev.
Neurosci. 2008, 9, 46.
1. Hu, J. F.; Fan, H.; Xiong, J.; Wu, S. B. Chem. Rev. 2011, 111, 5465.
2. Wada, Y.; Harayama, Y.; Kamimura, D.; Yoshida, M.; Shibata, T.; Fujiwara, K.;
Morimoto, K.; Fujioka, H.; Kita, Y. Org. Biomol. Chem. 2011, 9, 4959.
3. Ding, Q.; Chichak, K.; Lown, J. W. Curr. Med. Chem. 1999, 6, 1.
4. Urban, S.; Hickford, S. J. H.; Blunt, J. W.; Munro, M. H. G. Curr. Org. Chem. 2000,
4, 765.
51. Stone, T. W.; Darlington, L. G. Nat. Rev. Drug Disc. 2002, 1, 609.
52. Schwarcz, R.; Bruno, J. P.; Muchowski, P. J.; Wu, H. Q. Nat. Rev. Neurosci. 2012.
53. Röhrig, U. F.; Awad, L.; Grosdidier, A.; Larrieu, P.; Stroobant, V.; Colau, D.;
Cerundolo, V.; Simpson, A. J.; Vogel, P.; Van den Eynde, B. J.; Zoete, V.;
Michielin, O. J. Med. Chem. 2010, 53, 1172.
54. Dolušic´, E.; Larrieu, P.; Blanc, S.; Sapunaric, F.; Pouyez, J.; Moineaux, L.; Colette,
D.; Stroobant, V.; Pilotte, L.; Colau, D.; Ferain, T.; Fraser, G.; Galleni, M.; Frere, J.
M.; Masereel, B.; Van den Eynde, B.; Wouters, J.; Frédérick, R. Eur. J. Med. Chem.
2011, 46, 3058.
55. Macchiarulo, A.; Camaioni, E.; Nuti, R.; Pellicciari, R. Amino Acids 2009, 37, 219.
56. Dolušic´, E.; Larrieu, P.; Blanc, S.; Sapunaric, F.; Norberg, B.; Moineaux, L.;
Colette, D.; Stroobant, V.; Pilotte, L.; Colau, D.; Ferain, T.; Fraser, G.; Galleni, M.;
Frere, J. M.; Masereel, B.; Van den Eynde, B.; Wouters, J.; Frédérick, R. Bioorg.
Med. Chem. 2011, 19, 1550.
5. Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1.
6. Antunes, E. M.; Copp, B. R.; Davies-Coleman, M. T.; Samaai, T. Nat. Prod. Rep.
2005, 22, 62.
7. Delfourne, E. Anticancer Agents Med. Chem. 2008, 8, 910.
8. Davis, R. A.; Buchanan, M. S.; Duffy, S.; Avery, V. M.; Charman, S. A.; Charman,
W. N.; White, K. L.; Shackleford, D. M.; Edstein, M. D.; Andrews, K. T.; Camp, D.;
Quinn, R. J. J. Med. Chem. 2012, 55, 5851.
9. Hooper, G. J.; Davies-Coleman, M. T.; Kelly-Borges, M.; Coetzee, P. S.
Tetrahedron Lett. 1996, 37, 7135.
57. Röhrig, U. F.; Majjigapu, S. R.; Grosdidier, A.; Bron, S.; Stroobant, V.; Pilotte, L.;
Colau, D.; Vogel, P.; Van den Eynde, B. J.; Zoete, V.; Michielin, O. J. Med. Chem.
2012, 55, 5270.
10. Antunes, E. M.; Beukes, D. R.; Kelly, M.; Samaai, T.; Barrows, L. R.; Marshall, K.
M.; Sincich, C.; Davies-Coleman, M. T. J. Nat. Prod. 2004, 67, 1268.
11. Bénéteau, V.; Besson, T. Tetrahedron Lett. 2001, 42, 2673.
12. Bénéteau, V.; Pierre, A.; Pfeiffer, B.; Renard, P.; Besson, T. Bioorg. Med. Chem.
Lett. 2000, 10, 2231.
´
58. Dolušic, E.; Larrieu, P.; Moineaux, L.; Stroobant, V.; Pilotte, L.; Colau, D.; Pochet,
L.; Van den Eynde, B.; Masereel, B.; Wouters, J.; Frédérick, R. J. Med. Chem. 2011,
54, 5320.
13. Kokoshka, J. M.; Capson, T. L.; Holden, J. A.; Ireland, C. M.; Barrows, L. R.
Anticancer Drugs 1996, 7, 758.
59. Moineaux, L.; Laurent, S.; Reniers, J.; Dolušic´, E.; Galleni, M.; Frère, J. M.;
Masereel, B.; Frédérick, R.; Wouters, J. Eur. J. Med. Chem. 2012, 54, 95.

´
E. Dolušic et al. / Bioorg. Med. Chem. Lett. 23 (2013) 47–54
54
60. Koblish, H. K.; Hansbury, M. J.; Bowman, K. J.; Yang, G.; Neilan, C. L.; Haley, P. J.;
Burn, T. C.; Waeltz, P.; Sparks, R. B.; Yue, E. W.; Combs, A. P.; Scherle, P. A.;
Vaddi, K.; Fridman, J. S. Mol. Cancer Ther. 2010, 9, 489.
61. Delfourne, E. Mini. Rev. Med. Chem. 2012.
62. Brastianos, H. C.; Vottero, E.; Patrick, B. O.; Van Soest, R.; Matainaho, T.; Mauk,
A. G.; Andersen, R. J. J. Am. Chem. Soc. 2006, 128, 16046.
63. Carr, G.; Chung, M. K.; Mauk, A. G.; Andersen, R. J. J. Med. Chem. 2008, 51, 2634.
64. Pereira, A.; Vottero, E.; Roberge, M.; Mauk, A. G.; Andersen, R. J. J. Nat. Prod.
2006, 69, 1496.
65. Kumar, S.; Malachowski, W. P.; DuHadaway, J. B.; LaLonde, J. M.; Carroll, P. J.;
Jaller, D.; Metz, R.; Prendergast, G. C.; Muller, A. J. J. Med. Chem. 2008, 51, 1706.
66. Lu, C.; Lin, Y.; Yeh, S. R. J. Am. Chem. Soc. 2009, 131, 12866.
67. Pearson, J. T.; Siu, S.; Meininger, D. P.; Wienkers, L. C.; Rock, D. A. Biochemistry
2010, 49, 2647.
1699 South Hanley Rd., St. Louis, Missouri, 63144, USA). Docking was
performed using the 3D coordinates of human IDO1 (PDB ID: 2D0T) with the
help of the automated GOLD program (Jones, G.; Willett, P.; Glen, R. C.; Leach,
A. R.; Taylor, R. J. Mol. Biol. 1997, 267, 727). For the docking, the default
parameters were used, without constraints and using an active site definition
of 7 Å around phenyl imidazole taken as references. For each compound, 20
solutions were gernerated and ranked according to the GoldScore scoring
function. In order to take protein flexibility into account, the enzyme-inhibitor
complexes were optimized in SYBYL using the MINIMIZE module. The
minimization process uses the Powell method (Powell, M. J. D. Math.
Program 1977, 12, 241) with the Tripos force field (Clark, M.; Cramer, R. D.
III; Van Opdenbosch, N. J. Comp. Chem. 1989, 10, 982) (dielectric constant = 1Ãr)
to reach a final convergence of 0.01 kcal/mol. Pictures were generated with
Pymol from Delano Scientific.
68. The study was carried out on a Linux workstation. Compound 11a and b were
built using the SKETCH module implemented in SYBYL (Sybyl 8.1, Tripos Inc.,