Synthesis of indoleamine 2,3-dioxygenase inhibitory analogues of the sponge alkaloid exiguamine A

Article abstract of DOI:10.1021/jm800143h

Synthetic analogues of the sponge natural product exiguamine A (3) have been prepared and evaluated for their ability to inhibit indoleamine 2,3-dioxygenase in vitro.

Full text of DOI:10.1021/jm800143h

2634
J. Med. Chem. 2008, 51, 2634–2637
Letters
Table 1. In Vitro Inhibition of IDO
Synthesis of Indoleamine 2,3-Dioxygenase
Inhibitory Analogues of the Sponge Alkaloid
Exiguamine A
Gavin Carr,† Marco K. W. Chung,‡ A. Grant Mauk,*,‡ and
Raymond J. Andersen*,†
Departments of Chemistry and Earth & Ocean Sciences,
UniVersity of British Columbia, VancouVer,
British Columbia V6T 1Z1, Canada, and Department of
Biochemistry and Molecular Biology, UniVersity of British
Columbia, VancouVer, British Columbia V6T 1Z3, Canada
ReceiVed February 10, 2008
Abstract: Synthetic analogues of the sponge natural product exigua-
mine A (3) have been prepared and evaluated for their ability to inhibit
indoleamine 2,3-dioxygenase in vitro.
Indoleamine 2,3-dioxygenase (IDOa) catalyzes the conversion
of tryptophan into N-formylkynurenine in the first and rate
limiting step in the catabolism of this essential amino acid.1 A
link between IDO and immune tolerance was first established
by Munn et al. when they showed that treatment of pregnant
mice with the IDO inhibitor 1-methyltryptophan (1) removed
the toleragenic state protecting fetal tissue from the maternal
immune system.2 In environments where tryptophan concentra-
tion has been depleted by IDO, killer T cells cannot be activated
by antigens, and they undergo G1 cell cycle arrest leading to
apoptosis and immunosuppression.3 One of the hallmarks of
solid tumor cancers is immune escape, and it has been proposed
that IDO contributes to this process by depleting local concen-
trations of tryptophan, much as it does in protecting fetal tissue.4
Consistent with this hypothesis is the observation that IDO is
overexpressed in many tumor cell types4c and that increased
expression of IDO in tumor cells is correlated with poor
prognosis for survival in patients with serious ovarian and
colorectal cancers.5,6
Recently, there has been considerable interest in evaluating
the potential of IDO inhibitors to mobilize the body’s immune
system against solid tumors. Muller et al. found that the IDO
inhibitor 1 used in combination with a cytotoxic agent led to
regression of tumors in mouse models that showed no response
to the cytotoxin administered alone.7 A similarly positive in
vivo response has been obtained by using siRNA to silence the
IDO gene in B16F10 tumor-bearing mice.8
Most of the experimental studies looking at small molecule
IDO inhibitors have focused on the properties of 1, which is
not very water soluble or potent (Ki ≈ 62 µM, Table 1). Despite
its limitations, 1 is in preclinical development at NCI as part of
their RAID program.9 In an attempt to find better IDO inhibitors
that could be used as experimental tools and drug leads, we
have screened a library of marine natural product extracts against
recombinant human IDO in vitro. One of the first compounds
discovered using the in vitro screen was the hydroid metabolite
annulin C (2), which has a Ki of ≈ 140 nM, making it much
more potent than 1.10 Exiguamine A (3), an even more potent
IDO inhibitor having a Ki of 41 ( 3 nM, was subsequently
obtained from the marine sponge Neopetrosia exigua.11
* To whom correspondence should be addressed. Phone: 604 822 4511.
Fax: 604 822 6091. E-mail: randersn@interchange.ubc.ca.
†
Chemistry and EOS.
Biochemistry and Molecular Biology.
‡
a Abbreviations: IDO, indoleamine 2,3-dioxygenase; Cbz, carbobenzy-
loxy; DDQ, dichlorodicyanoquinone; siRNA, small interfering ribonucleic
acid.
10.1021/jm800143h CCC: $40.75
2008 American Chemical Society
Published on Web 04/08/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2635
Scheme 1
Exiguamine A (3), which is the most potent IDO inhibitor
reported to date and combines structural features found in 1
(tryptamine) and annulin C (2) (quinone), is a particularly
appealing lead structure for the development of synthetic IDO
inhibitors. We envisioned that the tryptamine quinone substruc-
ture comprised the core IDO inhibitory pharmacophore in
exiguamine A (3). However, it seemed unlikely that an
unsubstituted tryptamine quinone would be stable. In exiguamine
A (3), the hydantoin substituent at C-6 and the phenyl substituent
at C-7 presumably stabilize the core pharmacophore by acting
as steric impediments to the intermolecular Michael addition
and intramolecular iminoquinone-forming reactions expected to
take place with an unsubstituted tryptamine quinone.12 At the
outset, we also assumed that the positively charged quaternary
ammonium ion in exiguamine A might prevent the molecule
from crossing cell membranes. On the basis of the above
analysis, we set out to prepare uncharged derivatives of
tryptamine quinone that were substituted with hydantoin and
various other moieties in an attempt to find easily accessible
stable analogues of 3 having potent IDO inhibitory properties
against the pure enzyme.
Scheme 2
Scheme 3
Our synthesis of the tryptamine quinone fragment followed
an elegant sequential oxidative strategy developed by Tatsuta
et al. for the preparation of indole quinone intermediates used
in the synthesis of lymphostin.13 Tryptamine 4 was first
protected as its Cbz derivative 5 and then oxidized to the ketone
6 in good yield with DDQ (Scheme 1). Regioselective thallation
of 6 at C-4 by reaction with Tl(OCOCF3)3 followed by treatment
with CuSO4 ·5H2O gave phenol 7. Ketophenol 7 was deoxy-
genated with NaBH4 in the presence of BF3 etherate to give 8,
which was oxidized with Fremy’s salt [NO(SO3K)2] to give Cbz-
protected tryptamine quinone 9 in good yield.
Reaction of quinone 9 with dodecylamine (10) at rt gave the
Michael addition product 11. This was consistent with our
observation that deprotection of 9 by hydrogenolysis failed to
give any isolatable products. The deprotected tryptamine
quinone presumably polymerizes by intermolecular nucleophilic
attack of the primary C-2′ amine at C-5.
A literature report that the readily deprotonated 2-phenyl-
1,3-indanedione 12 adds to electrochemically generated 2,3-
dimethylparaquinone in the presence of sodium acetate served
as a model for our approach to adding a hydantoin substituent
to the quinone moiety in 9.14 We found that 12 reacted smoothly
with the protected tryptamine quinone 9 to give the C-5 Michael
adduct 13 in excellent yield (Scheme 2). The ability of 12 to
act as an effective carbanion nucleophile in a Michael addition
to 9 suggested that a readily enolizabile hydantoin of the general
structure 14 might serve as a synthon for adding the desired
hydantoin substituent to C-5 of indole quinones.
Hydantoin 14a was routinely prepared in good yield from
2-(N-methylamino)dimethylmalonate (17) and n-propyl isocy-
anate using a modification of a literature procedure as shown
in Scheme 3.15 n-Propyl isocyanate was used instead of methyl
isocyanate for ease of handling. Reaction of hydantoin 14a and
indolequinone 19, prepared by Fremy’s salt oxidation of the
commercially available 4-hydroxyindole (18), gave the Michael
adduct 20 in good yield (Scheme 4). Similarly, reaction of 14a
with the Cbz-protected tryptamine quinone 9 gave the Michael
adduct 21. Deprotection of 21 with BBr3 removed the Cbz group
and the methyl ester resulting in spontaneous decarboxylation
to give the desired C-5-substituted tryptamine quinone 22.
Compound 22 was stable to normal organic chemistry manipu-
lations, indicating that the hydantoin substituent at C-5 was

2636 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Letters
Scheme 4
Figure 1. Steady-state kinetic analysis of recombinant human IDO
by (A) 20 (inhibitor concentrations: open circle, no inhibitor; open
square, 0.16 µM; closed square, 0.65 µM) and (B) 25 (inhibitor
concentrations: open circle, no inhibitor; open square, 0.2 µM; closed
square, 0.4 µM; triangle, 0.6 µM.) Error bars represent the standard
deviation of each measurement for those cases where they exceed the
dimensions of the symbols.
Scheme 5
Ki determined for inhibition by 1, the natural product exiguamine
A (3), and the synthetic analogues of the putative pharmacophore
of exiguamine A. It should be noted that the Ki of 41 nM
reported here for exiguamine A (3) is lower than the value of
≈200 nM reported when the natural product structure was first
described.11 The difference in Kis for exiguamine A (3) results
from the use of slight modifications to the data acquisition and
analysis protocol in the two studies. In the current work, the Ki
for 1 was redetermined with the revised protocol as a form of
validation, and the result is comparable to the values reported
previously (34 µM7 and 62 µM18). Representative double-
reciprocal kinetics plots (for 20 and 25, Figure 1) are consistent
with uncompetitive inhibition of IDO. Similar, uncompetitive
inhibitory kinetics were observed for the other compounds
included in Table 1.
The data in Table 1 shows that unsubstituted indole quinone
(19), with a Ki of 190 nM, is a very good inhibitor of IDO in
vitro and is roughly 300-fold more potent than 1. In contrast,
4-hydroxyindole (18) is completely inactive, demonstrating that
the quinone functionality in 19 is critical for activity. The
reactivity of indole quinones (i.e., 9, Scheme 1) toward Michael
addition of amines at C-5 means that, even though indolequinone
(19) is a potent inhibitor of IDO in vitro, it would be expected
to have significant off target toxicity in vivo and it is not a
good candidate for use in animal models. Simply blocking C-5
with a hydantoin substituent to give 20 should prevent Michael
additions, but it results in a 2-fold reduction in potency. Adding
a Cbz-protected ethylamine side chain at C-3 of indolequinone
to give 9 reduced the potency 8-fold, and further substitution
at C-5 with the 2-phenyl-1,3-indanedione moiety to give 13 led
sufficient to block further Michael additions at C-6 and cyclic
iminoquinone formation.
As an alternate approach to blocking the potential Michael
addition and intramolecular iminoquinone-forming reactions of
tryptamine quinone, compound 24 was prepared (Scheme 5).
Reaction of quinone 9 with 2,5-diphenylisobenzofuran gave the
Diels-Alder adduct 23,16 which was not isolated. Treatment
of 23 with BBr3 converted it to the desired product 24, which
was also stable to routine chemical manipulations.
The ability of the compounds prepared in this study to inhibit
purified recombinant human IDO was evaluated with a steady-
state spectrophotometric assay17 using microtiter plates and a
plate reader (Tecan Infinite M200). All assays were performed
in triplicate, and the resulting double-reciprocal plots were
analyzed by weighted linear-least-squares fits to the data. The
results of these assays are provided in Table 1 and include the

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2637
Colau, D.; Parmentier, N.; Boon, T.; van den Eynde, B. Evidence for
a tumoral immune resistance mechanism based on tryptophan degrada-
tion by indoleamine 2,3-dioxygenase. Nat. Med. 2003, 9, 1269–1274.
(5) Okamoto, A.; Nikaido, T.; Ochiai, K.; Takakura, S.; Saito, M.; Aoki,
Y.; Ishii, N.; Yanaihara, N.; Yamada, K.; Takikawa, O.; Kawaguchi,
R.; Isonishi, S.; Tanaka, T.; Urashima, M. Indoleamine 2,3-dioxyge-
nase serves as a marker of poor prognosis in gene expression profiles
of serous ovarian cancer cells. Clin. Cancer Res. 2005, 11, 6030–
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(6) Brandacher, G.; Perathoner, A.; Ladurner, R.; Schneeberger, S.; Orbist,
P.; Winkler, C.; Werner, E. R.; Werner-Felmayer, G.; Weiss, H. G.;
Gobel, G.; Margreiter, R.; Konigsrainer, A.; Fuchs, D.; Amberger, A.
Prognostic value of indoleamine 2,3-dioxygenase expression in
colorectal cancer: effect on tumor-infiltrating T cells. Clin. Cancer
Res. 2006, 12, 1144–1151.
(7) Muller, A. J.; Duhadaway, J. B.; Donover, P. S.; Sutanto-Ward, E.;
Prendergast, G. C. Inhibition of indoleamine 2,3-dioxygenase, an
immunoregulatory target of the cancer suppression gene Bin1,
potentiates cancer chemotherapy. Nat. Med. 2005, 11, 312–319.
(8) Zheng, X.; Koropatnick, J.; Li, M.; Zhang, X.; Ling, F.; Ren, X.; Hao,
X.; Sun, H.; Vladau, C.; Franek, J. A.; Febg, B.; Urquhart, B. L.;
Zhong, R.; Freeman, D. J.; Garcia, B.; Min, W.-P. Reinstalling
antitumor immunity by inhibiting tumor-derived immunosuppressive
molecule IDO through RNA interference. J. Immunol. 2006, 177,
5639–5646.
(9) Muller, A. J. and Scherle, P. A. Targeting the mechanisms of tumoral
immune tolerance with small-molecule inhibitors. Nat. ReV. Cancer
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(10) Pereira, A.; Vottero, E.; Roberge, M.; Mauk, A. G.; Andersen, R. J.
Indoleamine 2,3-dioxygenase inhibitors from the northeastern Pacific
marine hydroid GarVeia annulata. J. Nat. Prod. 2006, 69, 1496–1499.
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T.; Mauk, A. G.; Andersen, R. J. Exiguamine A, an indoleamine 2,3-
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to another 7-fold reduction in potency compared with 9.
Nevertheless, compound 13 is still more potent than 1.
Combining the C-5 hydantoin substituent in 20 with a Cbz-
protected ethylamine side chain at C-3 to give compound 21
produced an analogue that was only slightly less active than
indole quinone 19. Replacing the Cbz group in 21 with a
phenylpropionamide residue to give 25 produced no change in
potency, as exected. Removing the Cbz-protecting group in 21
to liberate a C-3 ethylamine side chain and at the same time
removing the methyl ester fragment from the hydantoin moiety
by decarboxylation to give 22, gave an analogue that recaptured
the potency of indolequinone 19. The Diels-Alder adduct 24,
which has the C-5 and C-6 positions blocked with a fused
aromatic ring, was completely inactive. It is noteworthy that a
combination of the hydantoin and tryptaminequinone substruc-
tures present in the natural product exiguamine A (3) was
required to give the potent synthetic analogue 22.
In summary, starting from the lead structure of the sponge
natural product exiguamine A (3), we have prepared the simpler
synthetic analogue 22 that is a potent uncompetitive in vitro
inhibitor of IDO. Although 22 is somewhat less active than the
natural product 3, it is readily accessible via synthesis and is
roughly 300-fold more potent than 1, which is currently
undergoing development for clinical evaluation.9 Compound 22
should be a useful new tool to study IDO in vitro and also for
whole cell and animal model evaluations of the potential of IDO
as a drug target in cancer and other human diseases.
(12) Kita, Y. Y.; Tohma, H.; Inagaki, M.; Hatanaka, K.; Yakura, T. Total
synthesis of discorhabdin-C: a general aza spiro dienone formation
from O-silylated derivatives using a hypervalent iodine reagent. J. Am.
Chem. Soc. 1992, 114, 2175–2180.
Acknowledgment. Financial support was provided by
NSERC (R.J.A.), NCIC (R.J.A.), CIHR (A.G.M.), and a CRC
Chair (A.G.M.).
(13) Tatsuta, K.; Imamura, K.; Itoh, S.; Kasai, S. The first total synthesis
Supporting Information Available: Synthetic procedures and
1H NMR spectra for compounds 9, 13, 19, 20, 21, 22, 24, and 25.
This material is available free of charge via the Internet at http://
pubs.acs.org.
of lymphostin. Tehrahedron Lett. 2004, 45, 2847–2850.
(14) Davarani, S. S. H.; Nematollahi, D.; Shamsipur, M.; Najafi, N. M.;
Masoumi, L.; Ramyar, S. Electrochemical oxidation of 2,3-dimeth-
ylhydroquinone in the presence of 1,3-dicarbonyl compounds. J. Org.
Chem. 2006, 71, 2139–2142.
(15) Li, J. P. Synthesis of 1-substituted and 1,3-disubstituted 5-hydanto-
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