Indoleamine 2,3-dioxygenase is the anticancer target for a novel series of potent naphthoquinone-based inhibitors

Article abstract of DOI:10.1021/jm7014155

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. While small molecule inhibitors of IDO exist, there remains a dearth of high-potency compounds offering in vivo efficacy and clinical translational potential. In this study, we address this gap by defining a new class of naphthoquinone-based IDO inhibitors exemplified by the natural product menadione, which is shown in mouse tumor models to have similar antitumor activity to previously characterized IDO inhibitors. Genetic validation that IDO is the critical in vivo target is demonstrated using IDO-null mice. Elaboration of menadione to a pyranonaphthoquinone has yielded low nanomolar potency inhibitors, including new compounds which are the most potent reported to date (K_i = 61-70 nM). Synthetic accessibility of this class will facilitate preclinical chemical-genetic studies as well as further optimization of pharmacological parameters for clinical translation.

Full text of DOI:10.1021/jm7014155

1
706
J. Med. Chem. 2008, 51, 1706–1718
Indoleamine 2,3-Dioxygenase Is the Anticancer Target for a Novel Series of Potent
Naphthoquinone-Based Inhibitors
†
,†
‡
,†
§
‡
Sanjeev Kumar, William P. Malachowski,* James B. DuHadaway, Judith M. LaLonde,* Patrick J. Carroll, Daniel Jaller,
|
‡,⊥ ,#
,‡,#,∇
Richard Metz, George C. Prendergast,
and Alexander J. Muller*
Department of Chemistry, Bryn Mawr College, Bryn Mawr, PennsylVania 19010, Lankenau Institute for Medical Research,
Wynnewood, PennsylVania 19096, Department of Chemistry, X-ray Crystallography Facility, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104, LIMR DeVelopment, Inc., Wynnewood, PennsylVania 19096, and Departments of Pathology,
Anatomy & Cell Biology, Microbiology and Immunology, Jefferson Medical School, Kimmel Cancer Center, Thomas Jefferson UniVersity,
Philadelphia, PennsylVania 19104
ReceiVed NoVember 9, 2007
Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of
cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. While
small molecule inhibitors of IDO exist, there remains a dearth of high-potency compounds offering in ViVo
efficacy and clinical translational potential. In this study, we address this gap by defining a new class of
naphthoquinone-based IDO inhibitors exemplified by the natural product menadione, which is shown in
mouse tumor models to have similar antitumor activity to previously characterized IDO inhibitors. Genetic
validation that IDO is the critical in ViVo target is demonstrated using IDO-null mice. Elaboration of menadione
to a pyranonaphthoquinone has yielded low nanomolar potency inhibitors, including new compounds which
are the most potent reported to date (K ) 61–70 nM). Synthetic accessibility of this class will facilitate
i
preclinical chemical-genetic studies as well as further optimization of pharmacological parameters for clinical
translation.
Introduction
to drug development is a dearth of potent and bioactive drug-
like molecules that offer translational potential.
Of the major diseases that plague the developed and develop-
ing worlds, many are associated with immunosuppressed states
that can impede effective treatment and recovery. Some
prominent examples are cancer, where, as a consequence of
IDO (EC 1.13.11.42) is a monomeric heme-containing
enzyme that catalyzes tryptophan degradation in the initial step
of the kynurenine pathway, the de noVo biosynthetic route for
6
–8
nicotinamide adenine dinucleotide (NAD) production. IDO
1
2+
immunoediting, tumor cells are under strong selective pressure
is active with the heme iron in the ferrous (Fe ) form and
3
+
to develop the capacity to undermine an effective immune
response, and chronic infections, such as HIV and HCV
infections, where immunosuppression contributes to disease
persistence. A growing body of evidence implicates the involve-
inactive in the ferric (Fe ) form; substrate inhibition of IDO
is believed to result from tryptophan binding to the ferric
9
,10
form.
While the primary catalytic cycle of IDO does not
involve redox changes, IDO is prone to auto-oxidation and so
a reductant is necessary to reactivate the enzyme. In ViVo, IDO
is thought to rely on a flavin or tetrahydrobiopterin cofactor
for which methylene blue and ascorbic acid can be substituted
a
ment of the enzyme indoleamine 2,3-dioxygenase (IDO) in
mediating pathological immunosuppression in such settings,
indicating that IDO may be an attractive therapeutic target for
pharmacological intervention in cancer as well as other diseases
in which effective immunity is impaired. In preclinical models
of cancer, IDO inhibition has been demonstrated to safely and
dramatically improve chemotherapeutic efficacy. However,
while bioactive small molecule inhibitors of IDO exist, they
are low potency and perhaps better suited to proof-of-concept
experiments than clinical translation. Thus, one present barrier
7
in reactions performed with purified IDO enzyme. Using such
an in Vitro assay, the landmark competitive inhibitor 1-meth-
2,3
yltryptophan (1MT, Figure 1) was identified in the early
11,12
1990s.
Widely employed for IDO studies, 1MT is bioactive
4
,5
and selective but is a rather low potency compound (Ki ) 34
µM). Other bioactive but also relatively low potency inhibitors
have been described, all of which retain the indole ring of
tryptophan, including a thiohydantoin derivative of tryptophan
1
3,14
and derivatives of the natural product brassinin.
Metabolic
enzymes often evolve an affinity for their natural substrate that
*
Address correspondence regarding the chemistry to W.P.M. Tel: 610-
15,16
closelymatchesthephysiologicalconcentrationofthesubstrate,
5
26-5016. Fax: 610-526-5086. E-mail: wmalacho@brynmawr.edu. Address
1
7–19
correspondence regarding the biology A.J.M. or G.C.P. Tel: 610-645-8034.
Fax: 610-645-2095. E-mail: mullera@mlhs.org. Address correspondence
regarding the computational chemistry J.M.L. Tel: 610-526-5016. Fax: 610-
which is ∼60 µM for circulating tryptophan.
Thus, IDO
inhibitors with submicromolar potency may be more likely to
arise from structures lacking the indole core of tryptophan.
5
26-5679. E-mail: jlalonde@brynmawr.edu.
†
Department of Chemistry, Bryn Mawr College.
Lankenau Institute for Medical Research.
Department of Chemistry, University of Pennsylvania.
LIMR Development, Inc.
a
‡
Abbreviations: ATP, adenosine 5′-triphosphate; GSH, glutathione; IDO,
§
indoleamine 2,3-dioxygenase; LD50, median lethal dose; MMTV, mouse
mammary tumor virus; 1-MT, 1-methyltryptophan; MTD, maximum
tolerated dose; NAD, nicotinamide adenine dinucleotide; NAD(P)H reduced
nicotinamide adenine dinucleotide phosphate; NOQ1, NAD(P)H:quinone
oxidoreductase 1; NOQ2, NRH:quinone oxidoreductase 2; pDC, plasma-
cytoid dendritic cells; 4-PI, 4-phenyl-imidazole; ROS, reactive oxygen
species.
|
⊥
Departments of Pathology, Anatomy & Cell Biology, Thomas Jefferson
University.
#
Kimmel Cancer Center, Thomas Jefferson University.
Microbiology and Immunology, Thomas Jefferson University.
∇
1
0.1021/jm7014155 CCC: $40.75

2008 American Chemical Society
Published on Web 03/05/2008

Indoleamine 2,3-Dioxygenase as an Anticancer Target
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1707
Table 1. IDO Inhibition from Commercially Available Quinone Structures
compd
IC50 (µM)
E (mV)
compd
IC50 (µM)
E (mV)
23
2
2
1
2
3
4
5
6
2,3-dichloro-1,4-naphthoquinone
2-methoxy-1,4-naphthoquinone
1,4-naphtho-quinone
5-hydroxy-1,4-naphthoquinone
2-methyl-1,4-naphthoquinone
1,2-napthoquinone
0.28
0.72
0.99
1.0
1.1
7.1
-604
7
8
9
10
11
12
2-hydroxy-1,4-naphthoquinone
benzoquinone
2-methyl-1,4-benzoquinone
2-phenyl-1,4-benzoquinone
vitamin K1
∼675
-357
2
4
no activity
no activity
no activity
no activity
no activity
-401
-466
2
2
2
2
5
6
7
5
24
-140
-93
-203
-89
28
-170
chromone
IC50 (inhibitory concentration 50%) is the concentration of compound that inhibits enzyme activity by half. E is the reduction potential for the one
electron reduction of the quinone to the semiquinone.
Table 2. IC50 Values for Glutathione-Conjugated Menadione (Quinone
and Hydroquinone Forms)
Figure 1. Structure of two small molecule inhibitors of IDO: (a)
1
-methyL-tryptophan (1MT), a widely used IDO inhibitor that is
bioactive; (b) annulin B, a potent IDO inhibitor isolated from a marine
invertebrate, which lacks an indole core structure.
Herein, we describe the discovery of highly potent IDO
inhibitors that lack the indole core of IDO’s natural substrate.
These potent inhibitors are inspired by the natural product
2
0,21
annulin B
(Figure 1) and contain naphthoquinone as the
key pharmacophore. In this study, we demonstrate through
mouse tumor models that the naphthoquinone natural product,
menadione, has antitumor activity mediated through IDO
inhibition. Furthermore, we describe the synthesis and charac-
terization of a new structural class of IDO inhibitors based on
the naphthoquinone pharmacophore. The most active compounds
are pyranonaphthoquinones, and they represent the most potent
IDO inhibitors described to date.
This was a specific feature of this vitamin K precursor molecule,
as vitamin K1 (11) lacked activity as an IDO inhibitor. Although
other naphthoquinones in our initial screen were more potent,
menadione is a known anticancer agent and clinical studies
have provided evidence of its activity as a radiosensitizer and
2
9
Results
Naphthoquinone Is the Pharmacophore of Natural
Product Annulin B. Andersen et al. recently described
its ability to cooperate with chemotherapeutic agents, reminis-
2
0,21
4,14
cent of other IDO inhibitors.
Consequently, we chose to
several natural products isolated from a marine hydroid with
potent activity as IDO inhibitors, the most potent of which was
annulin B with a Ki ) 0.12 µM. Most of the marine natural
product inhibitors contained a naphthoquinone core, and by
comparing the structure of 1MT with annulin B (Figure 1), we
hypothesized that the relevant pharmacophore in annulin B was
the naphthoquinone core. In support of the notion that naph-
thoquinone is an indole mimetic, commercially available
compounds containing a quinone structure were screened for
IDO inhibitory activity and several demonstrated micromolar
levels of inhibitory potency (Table 1). The 1,2- or 1,4-
naphthoquinone unit was essential for activity (cf. 1–6 vs 8–10,
explore the in vivo activity of the naphthoquinones with
menadione.
While a variety of hypotheses for the mechanism of action
of menadione have been proposed, a definitive understanding
has yet to emerge. Most studies have focused on its ability to
generate reactive oxygen species (ROS) or to deplete intracel-
lular glutathione through the formation of menadione-glutathione
3
0
and glutathione-glutathione conjugates, which may have
3
1
cytotoxic consequences. Interestingly, we found that the
glutathione conjugate of menadione (both in quinone 13 and
hydroquinone 14 forms) retained IDO inhibitory activity,
exhibiting even greater potencies than the parent compound
(Table 2). In a cell-based assay, the IC50 of menadione for IDO
inhibition was determined to be lower than the LD50 for cellular
cytotoxicity by >4-fold (Figure 2a), indicating that there is a
window between IDO inhibitory activity and general cytotoxicity
for this compound.
1
4
2), and substitution was permitted on either the benzene (e.g.,
) or the quinone ring (e.g., 1, 2, and 5) of the naphthoquinone
core. Exceptions to this principle were found with the phytylated
1
,4-naphthoquinone derivative vitamin K1 and the 2-hydroxy
derivative 7; the latter was particularly noteworthy given the
activity of the structurally analogous 1,2-naphthoquinone 6. All
benzoquinone derivatives were inactive, thereby confirming the
need for a fused benzene-quinone structure for activity as an
IDO inhibitor. Although quinones are well-known oxidants,
there was no apparent correlation between the IDO inhibitory
potency and the reduction potential of these different quinone-
based compounds (Table 1).
IDO Is an Essential Target for the Antitumor Activity
of the Naphthoquinone Menadione. Included among the
naphthoquinone-based compounds evaluated for IDO inhibitory
activity was menadione, also known as vitamin K3 (5), which
exhibited low micromolar potency (IC50 ) 1.0 µM) (Table 1).
Based on available information in the NCI database about
menadione in different mouse models of cancer (http://dtp.n-
ci.nih.gov/dtpstandard/InvivoSummary/index.jsp), we evaluated
whether menadione, administered at levels near the maximum
tolerated dose (MTD), would cooperate with paclitaxel in the
MMTV-Neu transgenic mouse model of breast cancer, an assay
where the antitumor efficacy of various IDO inhibitors has
14,32
previously been demonstrated.
Administration of menadione
alone at 25 mg/kg once a day (qd) resulted in some evidence
of growth inhibition, while the same dose administered twice a
day (bid) was lethal, indicating that no further dose escalation
would be possible. However, like other IDO inhibitors, which

1
708 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Kumar et al.
Figure 2. In ViVo validation of IDO as an essential target of menadione antitumor activity. (a) Cell-based comparison of IDO inhibition and
cytotoxicity of menadione. A clonal T-REx-derived cell line, stably transfected with doxycyclin-inducible IDO, was exposed to a range of menadione
concentrations. The top graph shows the percent inhibition of IDO activity (adjusted for cell viability) based on comparison of kynurenine levels
in the culture supernatant of menadione-exposed cells to that of untreated controls. The bottom graph shows the percent viability of the same cells
used for the IDO inhibition assay based on SRB assay results from menadione-exposed cells compared to untreated controls. IC50 and LD50 values
were determined from the sigmoidal dose–response curves. The assays were performed in triplicate and graphed as means ( SD. (b) Menadione
effectively combines with paclitaxel chemotherapy to regress established breast tumors. Parous MMTV-Neu mice with 0.5–1.0 cm mammary gland
tumors were randomly enrolled for 2-week treatment studies. Tumor volume determinations were made at the beginning and end of the treatment
period. Cohorts receiving menadione (K3) were administered compound i.p. either once a day (qd) or twice a day (bid) as indicated at 25 mg/kg
for 5 consecutive days during the first week of treatment. Paclitaxel (Taxol) was administered to the indicated cohorts i.v. at 13.3 mg/kg qd
3
×/week over the entire course of the 2-week treatment period. Each point represents the fold change in volume for an individual tumor with the
mean ( SEM indicated for each group. The significance of the differences between the paclitaxel alone and the paclitaxel + menadione treatment
groups was assessed using a nonparametric two-tailed Mann–Whitney test to determine the indicated P values. (c) Menadione suppresses outgrowth
of B16-F10 tumors in a T cell and host IDO dependent manner. Menadione treatment, administered i.p. at 25 mg/kg qd 5 days a week until
5
termination of the experiment, was initiated 7 days following s.c. injection of C57BL/6 mice with 1 × 10 B16-F10 melanoma-derived cells.
3
Caliper measurements of tumors were performed biweekly until the control tumors reached a volume of ∼5000 mm . From left to right are the
results obtained from C57BL/6 mice, athymic NCr-nu/nu mice, and C57BL/6-strain, IDO knockout mice as indicated above each graph, plotted as
mean tumor size ( SEM at each time point. At the conclusion of each study, the difference in tumor volumes between the treatment and nontreatment
groups was assessed using a nonparametric two-tailed Mann–Whitney test to determine the P value indicated on each graph.
1
4
also display weak antitumor activity on their own, combining
menadione at the 25 mg/kg qd dose with paclitaxel produced
significant tumor regressions in the model (Figure 2b). Surpris-
ingly, mice receiving the combination of paclitaxel with
menadione at 25 mg/kg bid all survived; however, the antitumor
response was similar irrespective of whether the compound was
administered once or twice daily (Figure 2b).
To validate the requirement of IDO as a target for the
antitumor efficacy of menadione, we compared the activity of
this compound in a mouse model of cancer where we could
genetically assess the consequences of IDO loss. Briefly, tumors
formed by the mouse melanoma cell line B16-F10 do not
33
express IDO in Vitro or in ViVo Nevertheless, growth of tumor
isografts formed by these cells can be suppressed significantly

Indoleamine 2,3-Dioxygenase as an Anticancer Target
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1709
Scheme 1. General Synthetic Path to Pyranonaphthoquinone
Derivatives
Table 3. 6π Electrocyclization Reactions
Figure 3. Docking naphthoquinones at the IDO active site by molecular
modeling. (a) Proposed binding mode of compound 1 in IDO active
site. (b) Proposed binding mode of pyranonaphthoquinone 23 in IDO
active site. Graphics generated with PyMOL 098 (http//www.py-
mol.org), an open-source molecular graphics system developed sup-
ported and maintained by DeLano Scientific LLC (http//www.delano-
scientific.com).
naphthoquinone
X
product
R2
X
yield(%)
7
7
15
16
7
8
19
20
H
H
23
24
25
26
27
28
29
30
31
32
H
H
CH3
77
22
69
76
71
71
75
72
56
52
CO CH
2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3
5-OH
5-OCH3
6-OH
6-OCH3
7-OH
7-OCH3
8-OH
8-OCH3
6-OH
6-OCH3
7-OH
7-OCH3
8-OH
8-OCH3
9-OH
9-OCH3
1
1
3
2
by single agent treatment with an IDO inhibitor, due,
presumably, to inhibition of IDO expressed in tolerogenic
dendritic cells that accumulate in tumor draining lymph nodes
33
2
2
1
2
of the host animal. In this model, we confirmed that the growth
of B16-F10 isograft tumors could be reduced significantly by
single agent menadione treatment (Figure 2c). In contrast, we
detected no growth inhibition of tumor grafts in either athymic
nude mice or syngeneic IDO knockout mice (Figure 2c). These
findings indicate that the antitumor activity of menadione
requires both IDO inhibition and T cell involvement, thereby
validating IDO as a critical therapeutic target for menadionesa
prototypical representative of the naphthoquinone class of IDO
inhibitors.
site with the benzene ring projecting toward the entrance of
the active site (Figure 3a). In contrast, tricyclic pyranonaph-
thoquinones, such as 23, which include the pyran ring of annulin
B, entered with the benzene ring projecting into the posterior
of the active site (Figure 3b). Similarly, docking of annulin B
confirmed that the substituted benzene ring is nestled in the back
of the IDO active site, with the pyran ring located at the opening
of the active site (data not shown). These docking studies
provided an initial working model to direct synthetic modifica-
tions to the naphthoquinone core to improve potency.
Molecular Modeling with Naphthoquinone Leads. We
noted that the commercially available naphthoquinone structures
with activity as IDO inhibitors generally displayed a noncom-
petitive mode of inhibition (Table 1). Noncompetitive or
uncompetitive modes of inhibition usually suggest a basis in
allosteric binding; however, there exists a precedent for IDO
inhibitors to bind at the active site and yet display noncompeti-
tive or uncompetititve kinetics. For example, 4-phenylimidizole
Synthesis of Novel Pyranonaphthoquinone Inhibitors of
IDO. Initial efforts were directed at mimicking the structure of
annulin B, by installing and elaborating the pyran ring through
chemical syntheses. Naphthoquinones 7 and 15–22 were easily
converted to pyranonaphthoquinones 23–32 via a one-pot 6π
electrocyclization reaction in modest to good yield (Scheme 1
(
4-PI) has been reported to bind preferentially to the heme iron
3
4,35
in the inactive ferric form of IDO.
Also, ꢀ-carboline has
40–42
and Table 3).
The naphthoquinones with substituents in the
been reported to compete with oxygen for ferrous heme iron
3
4
benzene ring were synthesized according to literature procedures.
Epoxidation of 23 proceeded with dimethyldioxirane to afford
3, while epoxidation of 25 and 31 was accomplished with
binding. Nonetheless, in a mechanistic study, both 4-PI and
3
4
ꢀ
-carboline demonstrated noncompetitive kinetics, while the
3
original report describing ꢀ-carboline as an IDO inhibitor
36
m-CPBA to provide 34 and 35, respectively. Further derivati-
zation of the pyran ring was accomplished by nucleophilic
substitution of the epoxides (Scheme 1 and Table 4). The cis
and trans diastereomers (36-50) were separable by column
chromatography. Assignment of the cis and trans diastereomers
was based on an X-ray crystal structure of 36 (Supporting
Information) and an analysis of NMR coupling constants.
Hydrogenation, bromohydrin formation, and dihydroxylation
were also employed to selectively modify the pyran alkene
Scheme 2).
reported uncompetitive inhibition. Consequently, noncompeti-
tive inhibition of IDO may not preclude heme iron binding at
the active site by the naphthoquinone derivatives. Monodentate
3
7
heme iron binding by quinones is not common, but several
recent studies of photosynthesis and, particularly one involving
3
8,39
study of cytochrome b6f,
have demonstrated monodentate
iron heme binding by quinones.
Utilizing the recently reported crystal structure of IDO,
4
1
3
5
computational docking studies in the absence of molecular
oxygen placed several naphthoquinones at the active site with
the quinone oxygen coordinated to the heme iron. Furthermore,
the docking studies showed that the orientation of a particular
naphthoquinone will depend on the substituents on the naph-
thoquinone core. With relatively small substituents on the C-2
or C-3 position (e.g., 1), the naphthoquinone entered the active
(
Evaluation of Pyran Ring Derivatives. The docked binding
mode of annulin B, with its pyran ring located at the opening
of the active site, suggested focusing synthesis on the pyran
ring as a means of embellishing the naphthoquinone core and
restoring or enhancing the level of IDO inhibition displayed

1
710 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Kumar et al.
Table 4. Epoxide Opening Reactions
Table 6. IC50 Values of Benzene Ring Derivatives of Naphthoquinone
IDO Inhibitors
compd
IC50 (µM)
compd
IC50 (µM)
25
26
27
28
0.190
2.13
5.52
3.02
29
30
31
2.05
0.933
0.121
2.92
a
32
a
Natural product commonly referred to as R-caryopterone.
supports the notion that the region of the IDO enzyme occupied
by these groups is not constrained, such as found at the opening
of the active site.
Evaluation of Benzene Ring Derivatives. The study of the
benzene ring of annulin B focused on the position and the nature
of the oxygen substituent which was viewed as the most
important functionality for intermolecular interactions in the
active site. A distinct preference was observed with the C-6-
and C-9 hydroxy-substituted pyranonaphthoquinones 25 and 31,
demonstrating between 5- and 45-fold greater potency than the
other oxygen-substituted annulin B derivatives (Table 6).
Nevertheless, the activity of 25 and 31 is roughly equal to that
of the unsubstituted parent compound 23. Consequently, the C-6
or C-9 hydroxyl substitution is permissible but does not appear
to lead to any favorable interaction with IDO. Conversely, larger
substitution in C-6 or C-9 or substituents in C-7 or C-8 clearly
have a detrimental effect, probably due to steric interactions.
After an exploration of the optimal elements in the pyran
ring (Table 5) and the benzene ring (Table 6), we synthesized
two inhibitors (49 and 50) that combined the best elements of
both ring substitutions. These compounds were also highly
potent with IC50’s of 0.058 and 0.059, respectively, thereby
demonstrating that substitution is permitted in both rings.
Although the substitution pattern of annulin B suggested this
was possible, it was not to the extent demonstrated with the
benzyl amine in 49 and 50.
a
Reaction in 2-propanol without indium(III) chloride, InCl3.
Table 5. IC50 Values of Pyran Ring Derivatives of Naphthoquinone
IDO Inhibitors
compd
IC50 (µM)
compd
IC50 (µM)
compd
IC50 (µM)
a
2
2
3
3
3
3
3
a
3
4
3
6
7
8
9
0.214
0.247
4.95
0.055
0.252
0.186
0.183
40
41
42
43
44
45
0.130
0.082
1.10
0.361
0.976
3.96
46
47
48
51
52
1.09
3.45
2.12
4.34
0.512
1.50
b
53
b
Natural product commonly referred to as dehydro-R-lapachone. Natural
product commonly referred to as R-lapachone.
Scheme 2
Further Evaluation of Most Active Inhibitors. Three of
the most potent pyranonaphthoquinone derivatives, based on
IC50 values, were further analyzed to determine their inhibition
constants and mode of inhibition. The inhibition constants for
3
6, 41 ,and 50 were determined to be 70, 61, and 66 nM,
respectively. All three are more potent than annulin B (Ki )
2
0,21
1
20 nM
) and thus represent the highest potency IDO
inhibitors reported to date. Each of these compounds is also
roughly 500-fold more potent than the most commonly em-
ployed IDO inhibitor 1MT. Interestingly, all three compounds
exhibited reversible uncompetitive kinetics of inhibition (Sup-
porting Information). Preincubation of three pyranonaphtho-
quinones (31, 36, and 41) with IDO failed to demonstrate any
irreversible inhibition.
with the more complex marine natural product. To begin, we
synthesized and tested the simplified pyranonaphthoquinone 23
(
Table 5), where the tricylic structure was found to restore
Due to the surprising activity of the hydroquinone derivative
14, we attempted to generate and evaluate a hydroquinone
derivative of one of the potent pyranonaphthoquinone deriva-
tives. However, the rapid aerobic oxidation of each compound
tested precluded analysis of the inhibitory activity of the
hydroquinone form. Evaluation of the effect of the isolated
enzyme assay reduction system (ascorbic acid/methylene blue)
on these pyranonaphthoquinones did reveal evidence of hyd-
roquinone formation (data not shown). Consequently, it is likely
that both forms are present under the normal assay conditions,
and it is possible that both are relevant to IDO inhibition as
was witnessed with 13 and 14.
essentially all the activity of annulin B. Notably, antitumor
activity has been associated with compound 23, which is also
known as dehydro-R-lapachone.
4
3,44
Incorporation of the ester
from annulin B onto the pyran ring (24) had little effect on the
potency of the pyranonaphthoquinone nucleus. However, reduc-
tion of the pyran ring (51) resulted in a dramatic loss in activity.
Similarly, oxidation of the alkene to an epoxide (33) also
dramatically reduced the activity of this tricyclic inhibitor. Based
on the docking model of 23 (Figure 3b) and the hypothesized
exposure of the pyran ring to solvent, we expected elaboration
of the pyran ring to be permissible and therefore analyzed a
selection of compounds with functionalized pyran rings. We
found the most potent of these pyran ring derivatives to be 1,2-
amino-alcohol derivatives (i.e., 36 and 41). The absence of
consistent differences between the cis and trans diastereomers
Analysis of compounds 31, 36, and 41 in the same cell-based
assay used to analyze menadione showed an attenuation of their
activity versus the isolated enzyme assay (Table 7). However,
unlike menadione which demonstrated clear cellular cytotoxicity

Indoleamine 2,3-Dioxygenase as an Anticancer Target
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1711
Table 7. IC50 Values of Pyranonaphthoquinones Tested in Cell-Based
Assay
inhibit IDO in order to manifest antitumor activity, providing
genetic validation of the concept that IDO is an essential target
of menadione.
compd
IC50 (µM)
3
3
4
1
6
1
69
6.8
87
Ingested phylloquinone (vitamin K1 produced by plants) is
substantially converted to circulating menadione in humans.
4
9
As a vitamin K precursor, circulating menadione may be a
significant source of menaquinone biosynthesis (vitamin K2
produced by bacteria and animals) in extra-hepatic tissues
through uptake and prenylation. It remains to be determined
whether levels of menadione achieved through dietary intake
or supplementation of vitamin K are sufficient to have a
meaningful effect on IDO activity. In a mouse lung tumor
isograft model in which menaquinone supports metastasis
through its role in the coagulation system (e.g., by impacting
(
Figure 2a), compounds 31, 36, and 41 demonstrated minimal
impact on cell viability at 100 µM after 24 h. Future studies
will endeavor to improve the cell-based activity of the pyra-
nonaphthoquinones.
Discussion
Focusing on the naphthoquinone core of the complex natural
product annulin B, we have identified commercially available
compounds with naphthoquinone core structures that display
potent IDO inhibitory activity. Notably, some of these com-
pounds were up to ∼100-fold more potent than the commonly
used IDO inhibitor 1MT. Reinforcing the definition of this series
as a potentially important class of IDO inhibitors, the majority
of high potency hits identified in a recently conducted screen
of the NCI compound collection included either a naphtho-
quinone core or mimetic (unpublished results). We have
established the applicability of IDO inhibition by compounds
in this structural class to cancer treatment through in ViVo
evaluation of the representative bioactive compound menadione
and followed with the development of novel pyranonaptho-
quinone-based IDO inhibitors exhibiting submicromolar poten-
cies produced from commercially available materials in a short
number of synthetic steps.
Although the antitumor properties of menadione have long
been recognized, this is the first report to demonstrate that IDO
inhibition is an important mechanism of action. Previous studies
of menadione antitumor activity have focused on oxidative stress
as the primary mechanism of action. Intracellular redox cycling
of menadione is catalyzed by bioreductive enzymes such as
NAD(P)H:quinone oxidoreductase 1 (NOQ1), NRH:quinone
oxidoreductase 2 (NOQ2), and cytochrome P450 reductase.
50
Factor X activation ), the pro-metastatic effect of menaquinone
can be combated with compounds that target the regenerative
vitamin K cycle such as warfarin, which inhibits the enzyme
vitamin K epoxide reductase. Interestingly, warfarin has also
been shown to block both the in Vitro and in ViVo conversion
51,52
of menadione to menaquinone.
Taken together, this suggests
that by inhibiting the conversion of endogenous menadione to
menaquinone, anticoagulants could potentially leverage IDO
inhibition by menadione while concomitantly interfering with
the ability of menaquinone to support metastasis. Given the
importance of both immune escape and metastasis in the
pathophysiology of advanced cancers, further study in this area
seems warranted.
One concern regarding the proposed mechanism of action of
menadione was how the metabolism of this compound might
affect its ability to inhibit IDO in cells. Menadione is sufficiently
hydrophilic to be soluble in aqueous solution, but it also is
sufficiently hydrophobic to diffuse across the plasma mem-
5
3
brane. Once menadione has entered a cell, it is rapidly
conjugated to glutathione through nucleophilic addition to form
1
3 (quinone form) and14 (hydroquinone form), which are no
longer cell permeable and in fact are actively transported out
30,54
ofthecell.
Nevertheless,wefoundthatthemenadione-glutathione
conjugated compounds 13 and 14 were no less potent inhibitors
of IDO than menadione itself despite the large size of the
conjugated glutathione moeity. Since 13/14 are actively trans-
ported out of the cell, counteracting this (e.g., by inhibiting the
ATP-dependent pump responsible for removing glutathione-
conjugated menadione) might increase intracellular retention,
thereby lowering the effective antitumor dose and perhaps also
mitigating glutathione depletion (a side effect implicated in
4
5
Additional studies have also implicated nitric oxide synthases,
which are potentially interesting insofar as NO is known to
4
6
directly antagonize IDO activity. Depletion of glutathione
GSH) through direct conjugate formation and active export has
also been proposed as a mechanism for menadione-mediated
(
4
7
cytotoxicity. More recently, menadione has been suggested
to act by disrupting signaling pathways as an alternative to
31
2
9
endothelial barrier damage ). Although the synthetic inhibitors
reported in this study can also undergo redox cycling similar
to menadione, they are chemically incapable of conjugation with
biochemical cytotoxic mechanisms. In particular, treatment
with menadione has been correlated with changes in the
expression of molecules involved in controlling cell cycle
5
5
4
8
glutathione since they are tetra-substituted quinones. Conse-
quently, glutathione processing is irrelevant to the cellular
chemistry of the pyranonaphthoquinone-based IDO inhibitors.
progression. All of these proposed mechanisms of action are
based on the assumption that the antitumor activity of menadione
is mediated through direct cytotoxicity to the tumor target.
However, we have demonstrated here that the dramatic sup-
pression of B16-F10 tumor growth that was elicited by
menadione treatment in wild type mice was completely abol-
ished in T cell-deficient nude mice. These data argue against
direct cytoxicity as the operative mechanism of action, instead
implying that a T cell dependent, immune-mediated mechanism
is crucial to the antitumor activity of menadione. In the B16-
F10 tumor model, IDO is expressed not in the melanoma-derived
tumor cells but rather in highly toleragenic, plasmacytoid
In this study, we have identified the pyranonaphthoquinone
moiety as the IDO inhibitory pharmacophore in the complex
natural product annulin B, but the mechanism by which this
structure achieves inhibition remains somewhat unclear. The
quinone core is clearly important for IDO inhibition and the
quinone oxygen may be the iron-binding group seen in previous
inhibitor designs, most notably ꢀ-carboline, 4-phenylimidazole,
and dithiocarbamates. Quinones are one of nature’s privileged
structures, performing essential roles as biological oxidants, e.g.
vitamin K, vitamin E, ubiquinone, and plastoquinone. The
unique nature of IDO as an oxidoreductase that is inactive in
33
dendritic cells (pDCs) within the tumor-draining lymph nodes.
Since the antitumor activity of menadione was also abolished
in tumor-bearing, IDO nullizygous mice, where no IDO was
present in the system, it is also evident that menadione must
5
6,57
the ferric state and its sensitivity to inhibition by H2O2,
combined with the oxidation potential of the quinone structure,

1
712 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Kumar et al.
suggests that redox chemistry might be involved in the mech-
anism of inhibition. However, the absence of any correlation
between inhibitor potency and the oxidation potential of the
quinones (Table 1) tends to argue against such a mechanism as
the primary basis for inhibition.
test (APT) sequence have multiplicities reported as δ
u
(up) for
methyl and methine and δ (down) for methylene and quaternary
d
carbons. Normal-phase HPLC (NP-HPLC) analysis was performed
with UV detection at 254 nm and a 5 µm silica gel column (250–4.6
mm) eluted with 90:10 or 85:15 n-hexane/IPA at 0.5 or 1 mL/min.
Reversed-phase HPLC (RP-HPLC) analysis was performed with
Indeed, the structure–activity relationships that we discovered
in the preliminary screen (Table 1) and subsequent structural
modifications (Tables 2-6) support a more complex interaction
between IDO and the quinone-based inhibitors. Particularly
intriguing in this regard is the potent IDO inhibitory activity
exhibited by the menadione-glutathione conjugates 13 and 14
despite the fact that the hydroquinone could in theory replace
ascorbic acid as a reductant, thereby activating IDO. There is
strong evidence for phenols, such as in hydroquinone, to be
UV detection at 254 nm and a 5 µm Eclipse XDB-C
8
column
(
250–4.6 mm) eluted with 50:50 solvent A/solvent B; solvent A,
4
0% acetonitrile in water; solvent B, 0.1 M ammonium acetate
adjusted to pH 5.3 with glacial acetic acid. IR data were obtained
with an FT-IR spectrometer. MS data were recorded with atmo-
spheric pressure chemical ionization (APCI) or atmospheric pressure
electrospray ionization (APESI) mode.
(S)-2-Amino-5-((R)-1-(carboxymethylamino)-3-(3-methyl-1,
-dioxo-1,4-dihydronaphthalen-2-ylthio)-1-oxopropan-2-ylamino)-
4
5
8–64
5-oxopentanoic Acid (13). Prepared according to the literature
monodentate ligands for iron,
in line with speculation that
6
5
procedure with a minor modification. To a solution of 2-methyl-
,4-naphthoquinone (200 mg, 1.16 mmol) in dimethyl sulfoxide
iron binding by this moiety is important for IDO inhibition.
One possible interpretation of this model is that the quinone 13
may be acting as a prodrug for the hydroquinone 14 since,
presumably, under the assay conditions some of the quinone is
reduced by ascorbic acid/methylene blue to the hydroquinone.
This hypothesis is consistent with the observation that 14 is a
more potent IDO inhibitor than 13.
1
(
6 mL) and 95% ethanol (6 mL) at 0 °C was added L-glutathione
(
178 mg, 0.581 mmol) as a solution in water (2 mL). After the
reaction mixture was stirred for 1 h, the reaction was diluted with
ethyl acetate (50 mL) and filtered. The precipitate (220 mg) was
boiled with water (30 mL) and filtered; the filtrate was diluted with
ethanol (15 mL) and left undisturbed overnight. The precipitated
product was isolated as a yellow solid (83 mg) in 30% yield, mp
Future experiments will endeavor to understand the role of
the quinone structure as well as redox chemistry in the inhibition
of IDO. Based on the structure–activity relationships in the
pyranonaphthoquinones, structural complementarity between the
inhibitor and IDO clearly has an important role in inhibition as
well. Moreover, computational docking predicted binding at the
active site and rationalized many of the successful structural
modifications. The uncompetitive mode of inhibition displayed
by the most potent inhibitors (34, 39, and 50) would normally
point to allosteric binding and regulation; however, other IDO
inhibitors with a similar mode of inhibition have been shown
to actually bind at the active site. Detailed kinetic analysis may
shed further light on the precise molecular mechanism of IDO
inhibition by this class of compounds. In addition, studies will
also focus on enhancing the cell-based potency of this intriguing
and highly potent class of IDO inhibitors.
)
195–197 °C dec. The product has poor solubility in many
solvents, which made it difficult to obtain NMR information: TLC
1
R
(
(
f
) 0.40 (30% H
CDCl + TFA) δ 10.99 (s, 1H), 8.49–8.07 (m, 2H), 7.86–7.78
m, 4H), 4.84 (dd, 1H, J ) 5.55, 2.75 Hz), 4.35 (m, 1H), 4.19 (d,
H, J ) 1.02 Hz), 3.46 (dd, 1H, J ) 8.55, 5.58 Hz), 3.34 (dd, 1H,
J ) 7.68, 6.3 Hz), 2.83–2.87 (m, 2H), 2.45–2.36 (m, 2H), 2.40 (s,
2 3 2
O/MeOH with 0.1% CF CO H); H NMR
3
2
1
3
3
6 u
H); C NMR (DMSO-d ) δ 134.4, 134.2, 126.9, 126.5, 53.6,
15.6 (2C); δ 182.3, 180.8, 172.5, 171.4, 171.0, 170.6, 148.3, 145.3,
d
132.8, 131.8, 41.5, 35.6, 31.8, 26.9; IR (KBr) 3353, 1682, 1642,
1511 cm- ; APESI-MS m/z 500 (M+ + Na, 55), 478 (M + 1,
1
+
1
00); RP-HPLC t
mL/min).
S)-2-Amino-5-((R)-1-(carboxymethylamino)-3-(1,4-dihydroxy-
-methylnaphthalen-2-ylthio)-1-oxopropan-2-ylamino)-5-oxopen-
R
) 6.14 min (50:50; solvent A/solvent B, 0.5
(
3
6
5
tanoic Acid (14). Prepared according to the literature procedure
with a minor modification. To a solution of 2-methyl-1,4-naphtho-
quinone (100 mg, 0.581 mmol) under nitrogen in 95% ethanol (10
mL) at 0 °C was added L-glutathione (178 mg, 0.581mmol) as a
solution in water (2 mL). After being stirred overnight at rt, the
precipitated product was filtered and washed with water. The crude
product (210 mg) was boiled with water (2 – 20 mL) and filtered
while hot to afford the product as a violet solid in 57% yield: mp
Experimental Section
General Procedures. All reactants and reagents were com-
mercially available and were used without further purification unless
otherwise indicated. Anhydrous CH Cl , benzene, and 2-propanol
2 2
were obtained by distillation from calcium hydride under nitrogen.
Anhydrous MeOH was obtained by distillation from Mg metal
under nitrogen. All reactions were carried out under an inert
atmosphere of argon or nitrogen unless otherwise indicated.
Concentrated refers to the removal of solvent with a rotary
evaporator at normal water aspirator pressure followed by further
evacuation with a two-stage mechanical pump. Thin-layer chro-
matography was performed using silica gel 60 Å precoated glass
or aluminum-backed plates (0.25 mm thickness) with fluorescent
indicator, which were cut. Developed TLC plates were visualized
) 216–217 °C dec; TLC R
f
) 0.40 (30% H
2
O/MeOH with 0.1%
1
CF
3
CO
2
H); H NMR (DMSO-d ) δ 8.51 (d, 1H, J ) 7.35 Hz),
6
8.38 (s, 1H), 7.98–7.94 (m, 2H), 7.35–7.24 (m, 2H), 4.12 (m, 1H),
3.51 (d, 2H, J ) 4.5 Hz), 3.32–3.25 (m, 1H), 2.90–2.74 (m, 2H),
2.33 (s, 3H), 2.22–2.07 (m, 2H), 1.82 (m, 2H); C NMR (DMSO-
1
3
d
6
u d
) δ 126.3, 124.7, 122.7, 121.9, 53.2, 52.8, 14.9; δ 172.2, 171.0,
170.8, 170.7, 148.8, 142.5, 127.1, 123.0, 122.1, 113.1, 41.2, 37.5,
-
1
31.5, 26.7; IR (KBr) 3410, 3350, 1687, 1629, 1512 cm ; APESI-
MS m/z 502 (M+ + Na, 15), 480 (M + 1, 100); RP-HPLC t
+
)
R
with UV light (254 nm), iodine, or KMnO
4
. Flash column
6.06 min (50:50; solvent A/solvent B, 0.5 mL/min).
chromatography was conducted with the indicated solvent system
using normal-phase silica gel 60 Å, 230–400 mesh. Yields refer to
chromatographically and spectroscopically pure (>95%) com-
pounds, except as otherwise indicated. All new compounds were
determined to be >95% pure by NMR, HPLC, and/or GC as
indicted. Melting points were determined using an open capillary
8-Methoxy-2-(phenylamino)-1,4-naphthaquinone. Prepared from
5-methoxy-1,4-naphthoquinone according to the literature pro-
66
6
7
cedure in 77% yield: mp ) 150–151 °C (lit mp 152 °C); TLC R
f
1
) 0.45 (10% MeOH/CHCl
) 6.58, 1.11 Hz), 7.71–7.66 (m, 2H), 7.43–7.38 (m, 2H), 7.27–7.17
(m, 3H), 6.35 (s, 1H), 4.03 (s, 3H); C NMR (CDCl
129.8, 125.7, 122.9, 119.2, 116.4, 102.0, 56.6; δ 183.7, 180.4,
160.4, 145.9, 137.9, 135.7, 118.3; IR (KBr) 3302, 3275, 1670, 1616
3 3
); H NMR (CDCl ) δ 7.78 (dd, 1H, J
1
3
3 u
) δ 136.3,
1
13
and are uncorrected. H and C NMR spectra were recorded at
00 and 75 MHz, respectively. Chemical shifts are reported in δ
d
3
-
1
values (ppm) relative to an internal reference (0.05% v/v) of
tetramethylsilane (TMS) for H NMR and the solvent peak in
NMR, except where noted. Peak splitting patterns in the H NMR
are reported as follows: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; br, broad. C experiments with the attached proton
cm .
1
13
68
C
2-Hydroxy-8-methoxy-1,4-naphthoquinone (22 ). A mixture
of 8-methoxy-2-(phenylamino)-1,4-naphthaquinone (0.500 g, 1.79
mmol) was heated to reflux for 5 h in concd HCl (15 mL). The
reaction mixture was allowed to cool to rt, diluted with water (20
1
1
3

Indoleamine 2,3-Dioxygenase as an Anticancer Target
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1713
mL), and extracted with CHCl
3
. The organic extract was dried
(d, 1H, J ) 1.83 Hz), 7.05 (dd, 1H, J ) 6.27, 2.19 Hz), 6.59 (dd,
(
8
Na SO ) and concentrated to afford a brownish solid: 325 mg in
9% yield; mp ) 202–207 °C dec (lit. mp ) 211–214 °C dec;
2
4
1H, J ) 7.89, 2.1 Hz), 5.67 (d, 1H, J ) 9.99 Hz), 1.54 (s, 3H);
6
8
13
C NMR (CDCl
3
3 u
+ CD OD) δ 130.3, 129.5, 120.2, 115.4, 112.7,
1
TLC R
7
(
1
f
) 0.18 (10% MeOH/CHCl
3
); H NMR (CDCl
3
) δ
28.4; δ 182.6, 179.1, 163.2, 153.3, 134.0, 124.0, 117.5, 80.8. IR
d
-1
+
.80–7.71 (m, 3H), 7.28 (d, 1H, J ) 1.23 Hz), 6.29 (s, 1H), 4.04
(KBr) 3343, 3246, 1738, 1660, 1630 cm ; APCI-MS m/z 256 (M ,
1
3
15), 255 (M+ - 1, 100); NP-HPLC t
) 8.4 min (85:15; n-hexane/
s, 3H); C NMR (CDCl
84.9, 180.3, 160.6, 157.0, 135.5; IR (KBr) 3206, 1662 cm
,8-Dihydroxy-1,4-naphthoquinone (21). Prepared from 22
3
) δ
u
137.0, 119.7, 117.1, 108.7, 56.7; δ
d
R
-1
.
IPA, 0.5 mL/min).
2
7-Methoxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (28).
67
according to the literature procedure in 73% yield: mp ) 214–216
Pyranonaphthoquinone 28 was synthesized from 2-hydroxy-6-
6
7
°
C dec (lit. mp ) 210–215 °C dec; TLC R
f
) 0.33(20% MeOH/
73
methoxy-1,4-naphthoquinone 18 and 3-methylcrotonaldehyde
1
CHCl
dd, 1H, J ) 6.33, 1.62 Hz), 6.23 (s, 1H); C NMR (CDCl
CD OD) δ 137.2, 123.3, 118.7, 111.7; δ 185.9, 185.1, 161.3,
59.2, 132.4, 113.8.
General Procedure for the Synthesis of Pyranonaphtho-
3
); H NMR (CDCl
3
+ CD
3
OD) δ 7.67–7.57 (m, 2H), 7.21
according to the general procedure to afford 28 as a yellow solid
1
3
(
3
+
1
in 71% yield: TLC R
CDCl
J ) 6.06, 2.25 Hz), 6.63 (d, 1H, J ) 9.96 Hz), 5.67 (d, 1H, J )
f
) 0.43 (20% EtOAc/hexanes); H NMR
3
u
d
(
) δ 8.02 (d, 1H, J ) 8.61 Hz), 7.53 (s, 1H), 7.12 (dd, 1H,
3
1
1
3
9
1
1
.99 Hz), 3.93 (s, 3H), 1.54 (s, 6H); C NMR (CDCl
29.0, 119.4, 115.7, 110.4, 56.1, 28.6; δ 181.9, 179.1, 164.6, 153.1,
34.1, 125.2, 117.7, 80.7; IR (KBr) 1663, 1646 cm ; APCI-MS
3 u
) δ 130.4,
quinones by the 6π Electrocyclization Reaction. A solution of
the appropriate 1,4-naphthoquinone (1.00 mmol) and R,ꢀ-unsatur-
ated aldehyde (1.25 mmol), ꢀ-alanine (0.15 mmol), and acetic acid
d
-1
+
+
m/z 272 (M + 2, 20), 271 (M + 1, 100); NP-HPLC t
min (90:10; n-hexane/IPA, 0.5 mL/min).
R
) 8.4
(
6.0 mmol) in benzene (15 mL) was heated to reflux for 18 h. The
reaction mixture was then concentrated in Vacuo. Flash chroma-
tography afforded the desired products.
8-Hydroxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (29).
Pyranonaphthoquinone 29 was synthesized from 2,7-dihydroxy-
2
,2-Dimethyl-2H-benzo[g]chromene-5,10-dione (23). Pyra-
73
1
,4-naphthoquinone 19 and 3-methylcrotonaldehyde according to
the general procedure to afford a 75% yield of 29, an orange-red
solid: mp ) 206–210 °C dec; TLC R ) 0.50 (5% MeOH/CHCl );
) δ 7.94 (d, 1H, J ) 8.46 Hz), 7.42 (d, 1H, J )
.4 Hz), 7.10 (dd, 1H, J ) 6.03, 2.43 Hz), 6.62 (d, 1H, J ) 9.96
nonaphthoquinone 23 was synthesized from 2-hydroxy-1,4-naph-
thoquinone 7 and 3-methylcrotonaldehyde according to the general
procedure to yield 68%: mp ) 142–143 °C (lit. mp ) 145–146
f
3
1
H NMR (CDCl
3
°
C). The product matched previously reported analytical data in
2
41
the literature.
13
Hz), 5.74 (d, 1H, J ) 9.99 Hz), 1.54 (s, 6H); C NMR (CDCl
CD OD) δ 131.2, 129.1, 120.9, 115.7, 112.6, 28.3; δ 181.7, 180.7,
62.3, 152.2, 133.5, 123.9, 118.0, 80.3; IR (KBr) 3354, 1673, 1638,
3
+
Methyl 2-Methyl-5,10-dioxo-5,10-dihydro-2H-benzo[g]chromene-
-carboxylate (24). Pyranonaphthoquinone 24 was synthesized from
-hydroxy-1,4-naphthoquinone 7 and fumaraldehydic acid methyl
3
u
d
2
1
1
2
ester
-1
+
+
570 cm ; APCI-MS m/z 256 (M , 20), 255 (M - 1, 100); NP-
69,70
according to the general procedure to afford 24 as a yellow
HPLC t
R
) 8.6 min (85:15; n-hexane/IPA, 0.5 mL/min).
solid in 22% yield: mp ) 131–132 °C; TLC R
f
) 0.43 (20% EtOAc/
1
8-Methoxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (30).
Pyranonaphthoquinone 30 was synthesized from 2-hydroxy-7-
hexanes); H NMR (CDCl
3
) δ 8.14–8.08 (m, 2H), 7.76–7.70 (m,
2
3
1
1
H), 6.79 (d, 1H, J ) 9.84 Hz), 5.88 (d, 1H, J ) 9.84 Hz), 3.77 (s,
7
3
13
methoxy-1,4-naphthoquinone 20 and 3-methylcrotonaldehyde
according to the general procedure to afford 30 as a yellow solid
H), 1.84 (s, 3H); C NMR (CDCl
26.2, 118.0, 53.3, 25.5; δ 181.8, 178.9, 170.8, 152.6, 131.6, 131.5,
17.9, 81.1; IR (KBr) 1749, 1671, 1651 cm ; APCI-MS m/z 284
3 u
) δ 134.3, 133.7, 126.6, 126.5,
d
-
1
in 72% yield. mp)130 °C. TLC R
f
) 0.43 (20% EtOAc/Hexanes);
1
+
H NMR (CDCl ) δ 8.02 (d, 1H, J ) 8.58 Hz), 7.53 (d, 1H, J )
3
(
R
M , 100); NP-HPLC t ) 7.1 min (85:15; n-hexane/IPA, 0.5 mL/
2
.31 Hz), 7.15 (dd, 1H, J ) 6.09, 2.49 Hz), 6.64 (d, 1H, J ) 9.96
13
min).
Hz), 5.70 (d, 1H, J ) 9.96 Hz), 3.93 (s, 3H), 1.54 (s, 6H);
C
6-Hydroxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (25).
3 u
NMR (CDCl ) δ 131.1, 128.8, 120.2, 115.9, 110.2, 56.1, 28.5;
Pyranonaphthoquinone 25 was synthesized from 2,5-dihydroxy-
71
δ 181.5, 180.1, 163.9, 152.4, 133.7, 125.1, 118.0, 80.4. IR (KBr)
d
1
,4-naphthoquinone 15 and 3-methylcrotonaldehyde according to
1
673, 1641, 1595, 1578 cm ; APCI-MS m/z 271 (M+ + 1, 20),
-1
the general procedure to afford 25 as an orange-red solid in 69%
+
7
2
270 (M , 100); NP-HPLC t
R
) 8.0 min (85:15; n-hexane/IPA, 0.5
yield: mp ) 159–160 °C (lit. mp ) 156–158 °C); TLC R )
f
1
mL/min).
0
(
1
.65 (20% EtOAc/hexanes); H NMR (CDCl
dd, 1H, J ) 6.18, 1.26 Hz), 7.53 (t, 1H, J ) 8.22 Hz), 7.22 (dd,
H, J ) 7.08, 1.26 Hz), 6.60 (d, 1H, J ) 10.02 Hz), 5.73 (d, 1H,
3
) δ 12.2 (s, 1H), 7.61
9
-Hydroxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (31).
Pyranonaphthoquinone 31 was synthesized from 2,8-dihydroxy-
,4-naphthoquinone 21 and 3-methylcrotonaldehyde according to
the general procedure to afford 31 as an orange solid in 56% yield:
1
3
1
J ) 10.05 Hz), 1.56 (s, 6H); C NMR (CDCl
3
u
)δ 135.5, 130.9,
1
1
25.1, 119.3, 114.7, 28.6; δ
d
187.6, 179.3, 161.3, 153.2, 131.6,
7
2
74
-
1
mp ) 155–157 °C (lit. mp ) 160–165 °C; lit. mp )
17.7, 113.8, 81.1; IR (KBr) 3452, 1671, 1620 cm ; APCI-MS
1
+
+
1
43.5–145.5 °C dec); TLC R
NMR (CDCl ) δ 11.86 (s, 1H), 7.62–7.54 (m, 2H), 7.19 (dd, 1H,
J ) 5.70, 1.95 Hz), 6.61 (d, 1H, J ) 10.02 Hz), 5.72 (d, 1H, J )
f
) 0.57 (25% EtOAc/hexanes); H
m/z 258 (M + 2, 15), 257 (M + 1, 100); NP-HPLC t
min (85:15; n-hexane/IPA, 0.5 mL/min).
R
) 7.4
3
6-Methoxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (26).
1
3
1
1
1
0.02 Hz), 1.55 (s, 6H); C NMR (CDCl
19.2, 115.6, 28.6; δ 184.9, 181.2, 161.7, 152.3, 131.7, 118.7,
14.7, 80.9; IR (KBr) 3418, 1641, 1624, 1577 cm ; APCI-MS
3 u
)δ 136.9, 131.5, 124.1,
Pyranonaphthoquinone 26 was synthesized from 2-hydroxy-5-
methoxy-1,4-naphthoquinone 16 and 3-methylcrotonaldehyde
according to the general procedure to afford 26 as a yellow solid
7
1
d
-1
+
+
m/z 257 (M + 1, 15), 226 (M , 100); NP-HPLC t
85:15; n-hexane/IPA, 0.5 mL/min).
9-Methoxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (32).
R
) 7.5 min
in 76% yield: mp ) 126–127 °C; TLC R
f
) 0.45 (40% EtOAc/
)δ 7.78 (dd, 1H, J ) 6.57, 1.08 Hz),
.61 (t, 1H, J ) 8.40 Hz), 7.30 (d, 1H, J ) 0.90 Hz), 6.67 (d, 1H,
J ) 9.96 Hz), 5.72 (d, 1H, J ) 9.99 Hz), 4.0 (s, 3H), 1.53 (s, 6H);
1
(
hexanes); H NMR (CDCl
3
7
Pyranonaphthoquinone 32 was synthesized from 2-hydroxy-8-
methoxy-1,4-naphthoquinone 22 and 3-methylcrotonaldehyde ac-
cording to the general procedure to afford 32 as a yellow solid in
1
3
C NMR (CDCl
3 u
) δ 134.3, 131.4, 119.2, 118.6, 116.0, 56.7, 28.4;
δ
d
181.6, 180.2, 159.7, 150.8, 133.9, 119.5, 119.4, 79.9; IR (KBr)
-
1
+
75
76
1
716, 1670, 1644 cm ; APCI-MS m/z 271 (M + 1, 25), 270
52% yield: mp 135–136 °C (lit. mp ) 139.5–141.5 °C; lit. mp
+
1
(
M , 100); NP-HPLC t
R
) 11.8 min (85:15; n-hexane/IPA, 0.5
) 132–134 °C); TLC R ) 0.40 (40% EtOAc/hexanes); H NMR
f
mL/min).
(CDCl ) δ 7.75 (dd, 1H, J ) 6.81, 0.78 Hz), 7.63 (t, 1H, J ) 8.19
3
7-Hydroxy-2,2-dimethyl-2H-benzo[g]chromene-5,10-dione (27).
Hz), 7.23 (d, 1H, J ) 8.43 Hz), 6.60 (d, 1H, J ) 9.93 Hz), 5.65 (d,
1
3
Pyranonaphthoquinone 27 was synthesized from 2,6-dihydroxy-
1H, J ) 9.93), 3.98 (s, 3H), 1.52 (s, 6H); C NMR (CDCl
3
) δ
181.8, 178.8, 160.0,
153.5, 134.2, 119.6, 116.2, 80.6. IR (KBr): 1734, 1671, 1644, 1583
u
73
1
,4-naphthoquinone 17 and 3-methylcrotonaldehyde according to
135.2, 130.2, 119.2, 117.7, 115.5, 56.7, 28.5; δ
d
the general procedure to afford 27 as an orange-red solid in 71%
1
cm ; APCI-MS m/z 272 (M+ + 2, 15), 271 (M + 1, 100); NP-
-1
+
yield: mp ) 195 °C dec; TLC R
f
3
) 0.50 (5% MeOH/CHCl ); H
NMR (CDCl + CD OD) δ 7.96 (dd, 1H, J ) 6.09, 2.40 Hz), 7.38
3
3
HPLC t ) 12.3 min (85:15; n-hexane/IPA, 0.5 mL/min).
R

1
714 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
,2-Dimethyl-3,4-epoxy-2H-naphtho[2,3-b]pyran-5,10-dione
Kumar et al.
2
(3R,4S and 3S,4R)-4-(Allylamino)-3-hydroxy-2,2-dimethyl-
3,4-dihydro-2H-benzo[g]chromene-5,10-dione (39). Compound
39 was synthesized using the general procedure with allylamine.
Chromatographic separation afforded pure trans diastereomer 39
f
4
1
(
33). The compound was synthesized via the reported procedure
41
to afford a 62% yield: mp ) 138–139 °C (lit. mp 139–140 °C).
The spectroscopic data matched the reported information in the
literature.
as a yellow solid in 29% yield: mp ) 131–132 °C; TLC R ) 0.60
1
5
-Hydroxy-2,2-dimethyl-1aH-benzo[g]oxireno[2,3-c]chromene-
,9(2H,9bH)-dione (35). Alkene 31 (150 mg, 0.585 mmol) was
dissolved in CH Cl , cooled to 0 °C, and treated with mCPBA (152
(10% MeOH/CHCl ); H NMR (CDCl ) δ 8.12–8.06 (m, 2H),
3
3
4
7.77–7.67 (m, 2H), 5.94–5.81 (m, 1H), 5.18 (dd, 1H J ) 15.6,
1.53 Hz), 5.13 (dd, 1H, J ) 8.91, 1.32 Hz), 3.88 (d, 1H, J ) 8.64
Hz), 3.76 (d, 1H, J ) 8.64 Hz), 3.20 (dd, 1H, J ) 7.98, 5.70 Hz),
2
2
7
7
mg, 0.878 mmol). The reaction was stirred overnight at 0 °C.
The solvent was removed in Vacuo, and the crude product was
chromatographed on silica gel to afford 84 mg of the epoxide 35
1
3
3.01 (dd, 1H, J ) 7.59, 6.03 Hz), 1.65 (s, 3H), 1.32 (s, 3H);
C
NMR (CDCl ) δ 136.3, 134.5, 133.6, 126.7, 126.3, 70.1, 54.9,
3
u
(
53% yield), a yellow solid. Unreacted 31 was also recovered (48
d
26.2, 19.2; δ 184.9, 179.5, 155.5, 132.3, 131.2, 119.2, 116.7, 82.3,
-1
mg). Characterization data for 35: mp ) 145–150 °C; yellow solid;
TLC R
46.6; IR (KBr) 3319, 3149, 1678, 1634, 1621 cm ; APCI-MS
1
m/z 315 (M+ + 2, 20), 314 (M + 1, 100); NP-HPLC t ) 10.5
+
f
) 0.33 (20% EtOAc/hexanes); H NMR (CDCl
3
) δ 11.74
R
(
s, 1H), 7.70-7.61 (m, 2H), 7.27–7.22 (m, 1H), 4.33 (d, 1H, J )
min (85:15; n-hexane/IPA, 0.5 mL/min).
4
.41 Hz), 3.55 (d, 1H, J ) 4.44 Hz), 1.71 (s, 3Η), 1.46 (s, 3Η);
(
3S,4S)-4-(Butylamino)-3-hydroxy-2,2-dimethyl-3,4-dihydro-
H-benzo[g]chromene-5,10-dione (40). Compound 40 was syn-
thesized using the general procedure with allylamine. Chromato-
graphic separation afforded pure cis diastereomer 40 as a yellow
) 0.60 (5% MeOH/
) δ 8.11–8.05 (m, 2H), 7.76–7.66 (m,
H), 3.85 (d, 1H, J ) 4.50 Hz), 3.70 (d, 1H, J ) 4.50 Hz),
.86–2.68 (m, 2H), 1.68 (s, 3H), 1.62–1.38 (m, 4H), 1.29 (s, 3H),
1
3
C NMR (CDCl
3 u
) δ 137.3, 124.3, 119.3, 61.6, 43.8, 25.3, 23.5;
2
δ
3
2
d
184.1, 182.4, 162.1, 153.7, 131.9, 118.1, 114.5, 78.5; IR (KBr)
-
1
+
421, 1644, 1612 cm ; APCI-MS m/z 305 (M + MeOH, 100),
73 (M+ + 1, 18).
solid in 58% yield: mp ) 120–121 °C; TLC R
CHCl ); H NMR (CDCl
3 3
f
General Procedure for the Epoxide-Opening Reaction. To a
1
solution of epoxide 33 (256 mg, 1.0 mmol) in CH
0
appropriate nucleophile (4 equiv), and the reaction mixture was
allowed to warm to rt and stirred for 1–3 h. The solvent was
evaporated, and the crude product was chromatographed on silica
to give the desired products. The relative stereochemical conforma-
tion was assigned based on the coupling constant of the methine
2 2
Cl (10 mL) at
2
2
0
1
1
1
°C was added InCl (0.05 mmol) followed by the addition of the
3
1
3
.96 (t, 3H, J ) 7.11 Hz); C NMR (CDCl
3 u
) δ 134.4, 133.5,
26.6, 126.3, 67.0, 52.5, 24.9, 22.4, 14.2; δ 185.8, 179.5, 155.0,
d
32.5, 131.2, 117.3, 80.8, 47.6, 32.4, 20.6; IR (KBr) 3335, 3281,
-1
680, 1629, 1602, 1575 cm ; APCI-MS m/z 331 (M+ + 2, 25),
+
1
330 (M + 1, 100); NP-HPLC t ) 10.07 min (85:15; n-hexane/
R
protons in H NMR and confirmed in the case of 34 by an X-ray
crystal structure.
IPA, 0.5 mL/min).
(
3S,4S and 3R,4R)-4-(Benzylamino)-3-hydroxy-2,2-dimethyl-
(3R,4S and 3S,4R)-4-(Butylamino)-3-hydroxy-2,2-dimethyl-
3,4-dihydro-2H-benzo[g]chromene-5,10-dione (41). Compound
41 was synthesized using the general procedure with allylamine.
Chromatographic separation afforded pure trans diastereomer 41
3
3
,4-dihydro-2H-benzo[g]chromene-5,10-dione (36). Compound
6 was synthesized using the general procedure with benzylamine.
Chromatographic separation afforded pure cis diastereomer 36 as
a yellow solid in 53% yield: mp ) 155 °C; TLC R ) 0.30 (25%
) δ 8.07–8.01 (m, 2H), 7.72–7.63
m, 2H), 7.45 -7.25 (m, 5H), 4.66 (br s, 1H), 3.99–3.88 (m, 3H),
f
as a yellow solid in 16% yield: mp ) 103–104 °C; TLC R
f
) 0.60
1
1
EtOAc/hexanes); H NMR (CDCl
3
(10% MeOH/CHCl ); H NMR (CDCl ) δ 8.11–8.05 (m, 2H),
3
3
(
7.75–7.66 (m, 2H), 3.81 (d, 1H, J ) 9.00 Hz), 3.72 (d, 1H, J )
8.97 Hz), 2.52–2.44 (m, 1H), 2.31–2.23 (m, 1H), 1.65 (s, 3H),
3
3
1
1
.72 (d, 1H, J ) 4.47 Hz), 3.32 (br s, 1H), 1.67 (s, 3H), 1.25 (s,
13
13
H); C NMR (CDCl
26.3, 66.9, 51.6, 24.8, 22.5; δ
31.0, 117.3, 80.7, 51.9; IR (KBr) 3342, 1681, 1643, 1612, 1578
3
) δ
u
134.4, 133.5, 128.8, 128.5, 127.7, 126.6,
1.43–1.30 (m, 4H), 1.30 (s, 3H), 0.86 (t, 3H, J ) 7.20 Hz);
C
d
185.7, 179.4, 155.0, 139.1, 132.4,
3 u
NMR (CDCl ) δ 134.4, 133.5, 126.7, 126.3, 69.9, 55.1, 26.3, 18.9,
14.1; δ 184.9, 179.6, 155.5, 132.4, 131.4, 119.6, 82.2, 43.2, 32.9,
d
-
1
+
+
-1
cm ; APCI-MS m/z 365 (M + 2, 25), 364 (M + 1, 100); NP-
HPLC t ) 7.6 min (85:15; n-hexane/IPA, 0.5 mL/min).
3R,4S and 3S,4R)-4-(Benzylamino)-3-hydroxy-2,2-dimethyl-
20.5; IR (KBr) 3210, 1681, 1637, 1612 cm ; APCI-MS m/z 331
(M + 2, 20), 330 (M + 1, 100); NP-HPLC t ) 9.8 min (85:
15; n-hexane/IPA, 0.5 mL/min).
+
+
R
R
(
3
3
,4-dihydro-2H-benzo[g]chromene-5,10-dione (37). Compound
7 was synthesized using the general procedure with benzylamine.
(
3S,4S and 3R,4R)-3-Hydroxy-2,2-dimethyl-4-morpholino-
3
4
,4-dihydro-2H-benzo[g]chromene-5,10-dione (42). Compound
2 was synthesized using the general procedure with morpholine.
Chromatographic separation afforded pure trans diastereomer 37
as a yellow solid in 37% yield: mp ) 88–89 °C; TLC R ) 0.50
) δ 8.12–8.08 (m, 2H),
.77–7.67 (m, 2H), 7.33–7.19 (m, 5H), 3.90 (d, 1H, J ) 8.58 Hz),
.79 (d, 1H, J ) 8.55 Hz), 3.68 (d, 1H, J ) 12.39 Hz), 3.53 (d,
f
Chromatographic separation afforded pure cis diastereomer 42 as
a yellow solid in 57% yield: mp ) 103–104 °C; TLC R ) 0.44
) δ 8.09 (d, 2H, J ) 7.59
Hz), 7.77–7.69 (m, 2H), 3.67 (t, 4H, J ) 4.47 Hz), 3.57 (s, 2H),
.06 (m, 2H), 2.94 (s, 1H), 2.65–2.58 (m, 2H), 1.64 (s, 3H), 1.34
1
(
3 3
5% MeOH/CHCl ); H NMR (CDCl
f
7
3
1
1
(
3 3
5% MeOH/CHCl ); H NMR (CDCl
1
3
H, J ) 12.36 Hz), 2.97 (br s, 1H), 1.65 (s, 3H), 1.31 (s, 3H);
) δ 134.4, 133.5, 128.7, 128.4, 127.4, 126.7, 126.3,
0.0, 55.3, 26.1, 19.3; δ 184.9, 179.6, 155.5, 140.0, 132.4, 131.3,
19.5, 82.2, 48.3; IR (KBr) 3343, 1723, 1683, 1640, 1607, 1577
C
3
NMR (CDCl
3
u
13
(s, 3H); C NMR (CDCl
3
) δ
u
134.5, 133.4, 126.6, 71.7, 62.1, 26.4,
7
1
d
1
9.6; δ 184.9, 179.6, 155.9, 132.5, 131.1, 119.8, 81.9, 68.3, 50.7;
d
-
1
-
1
+
+
IR (KBr) 3500, 2938, 2854, 2819, 1666, 1645, 1611, 1581 cm ;
cm ; APCI-MS m/z 365 (M + 2, 25), 364 (M + 1, 100); NP-
HPLC t ) 8.0 min (85:15; n-hexane/IPA, 0.5 mL/min).
3S,4S and 3R,4R)-4-(Allylamino)-3-hydroxy-2,2-dimethyl-
APCI-MS m/z 345 (M+ + 2, 20), 344 (M + 1, 100); NP-HPLC
) 13.3 min (85:15; n-hexane/IPA, 0.5 mL/min).
3R,4S and 3S,4R)-3-Hydroxy-2,2-dimethyl-4-morpholino-
,4-dihydro-2H-benzo[g]chromene-5,10-dione (43). Compound
+
R
t
R
(
(
3
3
,4-dihydro-2H-benzo[g]chromene-5,10-dione (38). Compound
8 was synthesized using the general procedure with allylamine.
3
Chromatographic separation afforded pure cis diastereomer 38 as
a yellow solid in 58% yield: mp ) 127–128 °;. TLC R ) 0.60
) δ 8.10–8.04 (m, 2H),
.76–7.66 (m, 2H), 6.06–5.93 (m, 1H), 5.31 (dd, 1H J ) 15.66,
.51 Hz), 5.20 (dd, 1H, J ) 8.97, 1.26 Hz), 3.94 (d, 1H, J ) 4.53
43 was synthesized using the general procedure with morpholine.
Chromatographic separation afforded pure trans diastereomer 43
f
1
(
7
1
5% MeOH/CHCl
3
); H NMR (CDCl
3
as a yellow solid in 14% yield: mp ) 157–158 °C; TLC R
f
) 0.70
1
(5% MeOH/CHCl ); H NMR (CDCl ) δ 8.13–8.09 (m, 2H),
3
3
7.79–7.68 (m, 2H), 4.17 (s, 1H), 3.95 (d, 1H, J ) 6.15 Hz),
Hz), 3.67 (d, 1H, J ) 4.53 Hz), 3.43–3.40 (m, 2H), 1.67 (s, 3H),
3.73–3.64 (m, 5H), 2.99 (m, 2H), 2.73–2.66 (m, 2H), 1.51 (s, 3H),
13
13
1
6
1
.28 (s, 3H); C NMR (CDCl
6.8, 51.3, 24.8, 22.5; δ 185.6, 179.3, 155.1, 132.5, 131.0, 117.4,
17.2, 80.7, 50.1; IR (KBr) 3355, 1681, 1641, 1609 cm ; APCI-
)δ
3 u
135.9, 134.3, 133.4, 126.6, 126.3,
1.44 (s, 3H); C NMR (CDCl
3
) δ
u
134.5, 133.6, 126.8, 126.6, 70.6,
56.6, 26.4, 22.2; δ
d
185.1, 179.3, 155.9, 132.1, 131.2, 118.3, 81.5,
d
-1
-1
67.9, 52.8; IR (KBr) 3487, 2990, 2852, 1679, 1638, 1578 cm
;
+
+
APCI-MS m/z 345 (M+ + 2, 25), 344 (M + 1, 100); NP-HPLC
+
MS m/z 315 (M + 2, 20), 314 (M + 1, 100); NP-HPLC t
0.0 min (85:15; n-hexane/IPA, 0.5 mL/min).
R
)
1
t ) 13.1 min (85:15; n-hexane/IPA, 0.5 mL/min).
R

Indoleamine 2,3-Dioxygenase as an Anticancer Target
3S,4S and 3R,4R)-3-Hydroxy-4-methoxy-2,2-dimethyl-3,4-
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1715
1
3
(
J ) 4.21 Hz), 1.67 (s, 3H), 1.26 (s, 3H); C NMR (CDCl
3
) δ
u
dihydro-2H-benzo[g]chromene-5,10-dione (44). Compound 44
135.6, 128.9, 128.5, 127.8, 125.5, 119.6, 66.9, 51.4, 24.8, 22.6; δ
d
was synthesized using the general procedure with methanol as
191.5, 161.4, 155.9, 139.0, 131.1, 116.9, 81.2, 52.0; APCI-MS m/z
41
381(M+ + 2, 20), 380 (M + 1, 100); NP-HPLC t
+
previously described in the literature. Chromatographic separation
R
) 4.0 min
1
3
afforded pure cis diastereomer 44 in 54% yield: H NMR (CDCl )
(85:15; n-hexane/IPA, 1 mL/min).
δ 8.10–8.07 (m, 2H), 7.73–7.67 (m, 2H), 4.35 (d, 1H, J ) 3.0 Hz),
(
3S,4S)-4-(Benzylamino)-3,9-dihydroxy-2,2-dimethyl-3,4-di-
3
3
.91 (br s, 1H), 3.64 (s, 3H), 1.88 (br s, 1H), 1.55 (s, 3H), 1.50 (s,
hydro-2H-benzo[g]chromene-5,10-dione (50). To a solution of
epoxide 35 (50 mg, 0.184 mmol) in 2-propanol (5 mL) at rt was
added benzylamine (0.734 mmol), and the reaction mixture was
stirred for 30 min. The solvent was removed in Vacuo, and the
crude was chromatographed on silica gel to afford the desired cis
isomer 50 as a yellow solid (40 mg, 58% yield): mp ) 140–141
C; TLC R
41
H). The analytical data matched the literature report.
3R,4S and 3S,4R)-3-Hydroxy-4-methoxy-2,2-dimethyl-3,4-
(
dihydro-2H-benzo[g]chromene-5,10-dione (45). Compound 45
was synthesized using the general procedure with methanol as
41
previously described in the literature. Chromatographic separation
1
afforded pure trans diastereomer 45 in 28% yield: H NMR (CDCl
3
)
1
°
1
f
) 0.30 (20% EtOAc/hexanes); H NMR (CDCl
3
) δ
δ 8.21–8.05 (m,2H), 7.79–7.73 (m, 2H), 7.01 (d, 1H, J ) 2.49
Hz), 4.29 (d, 1H, J ) 4.89 Hz), 3.17 (s, 3H), 2.02 (d, 1H, J ) 5.61
Hz), 1.49 (s, 3H), 1.44 (s, 3H). The analytical data matched the
1.70 (s, 1H), 7.59–7.18 (m, 8H), 3.99–3.88 (m, 3H), 3.71 (d, 1H,
13
J ) 4.26 Hz), 1.67 (s, 3H), 1.26 (s, 3H); C NMR (CDCl
3
) δ
u
1
1
37.1, 128.9, 128.6, 127.8, 124.0, 119.2, 66.9, 51.7, 24.9, 22.6; δ
d
4
1
literature report.
84.8, 184.2, 161.9, 154.8, 139.0, 132.5, 118.1, 114.2, 81.0, 51.9;
-1
+
(
3S,4S and 3R,4R)-4-(Benzyloxy)-3-hydroxy-2,2-dimethyl-
IR (KBr) 3340, 1636, 1603 cm ; APCI-MS m/z 381(M + 2,
5), 380 (M+ + 1, 100); NP-HPLC t
) 7.8 min (85:15; n-hexane/
IPA, 0.5 mL/min).
,2-Dimethyl-3,4-dihydro-2H-benzo[g]chromene-5,10-dione
3
4
,4-dihydro-2H-benzo[g]chromene-5,10-dione (46). Compound
6 was synthesized using the general procedure with benzyl alcohol.
2
R
Chromatographic separation afforded pure trans diastereomer 46
as a yellow solid in 48% yield: mp ) 149–150 °C; TLC R ) 0.30
) δ 8.05 (t, 2H,J ) 7.50
2
f
41
1
(51). Prepared according to the literature procedure to afford 92%
yield of 51: mp ) 114–115 °C (lit. mp ) 113–114 °C); H NMR
CDCl
) 6.66 Hz), 1.83 (t, 2H, J ) 6.60 Hz), 1.44 (s, 6H). The product
matched previously reported analytical data in the literature.
(
20% EtOAc/hexanes); H NMR (CDCl
3
41 1
Hz), 7.72–7.62 (m, 2H), 7.38–7.26 (m, 5H), 4.97 (d, 1H, J ) 11.28
(
3
) δ 8.10-8.05 (m, 2Η), 7.73-7.63 (m, 2Η), 2.62 (t, 2H, J
Hz), 4.84 (d, 1H, J ) 11.30 Hz), 4.60 (d, 1H, J ) 2.88 Hz), 3.85
1
3
(
d, 1H, J ) 2.94 Hz), 2.18 (s, 1H), 1.55 (s, 3H), 1.52 (s, 3H);
C
41
NMR (CDCl
7
1
3
) δ
2.4, 71.5, 24.0, 23.7; δ
18.4, 81.4, 74.0; IR (KBr) 3481, 1635, 1591 cm ; APCI-MS
u
134.5, 133.3, 128.6, 128.1, 128.0, 126.6, 126.4,
(
3R,4S and 3S,4R)-3-Bromo-4-hydroxy-2,2-dimethyl-3,4-di-
d
184.4, 180.1, 154.0, 138.6, 132.5, 131.2,
-
1
hydro-2H-benzo[g]chromene-5,10-dione (52). Prepared according
4
1
+
+
to the literature procedure to afford 26% yield of 52: mp )
m/z 366 (M + 2, 10), 365 (M + 1, 35), 257 (100); NP-HPLC
) 8.3 min (85:15; n-hexane/IPA, 0.5 mL/min).
3R,4S and 3S,4R)-4-(Benzylthio)-3-hydroxy-2,2-dimethyl-
4
1
1
1
(
4
1
3
74–175 °C (lit. mp ) 176 °C); H NMR (CDCl ) δ 8.14–8.09
t
R
m, 2H), 7.79–7.72 (m, 2H), 5.09 (dd, 1H, J ) 5.31, 1.62 Hz),
(
.16 (d, 1H, J ) 6.93 Hz), 4.04 (d, 1H, J ) 1.5 Hz), 1.73 (s, 3H),
3
4
,4-dihydro-2H-benzo[g]chromene-5,10-dione (47). Compound
7 was synthesized using the general procedure with benzyl
.58 (s, 3H). The product matched previously reported analytical
4
1
data in the literature.
mercaptan. Chromatographic separation afforded pure cis diaste-
reomer 47 as a yellow solid in 45% yield: mp ) 151 °C; TLC R
f
(3S,4S and 3R,4R)-3,4-Dihydroxy-2,2-dimethyl-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione (53). Prepared according to the
1
)
0.30 (25% EtOAc/hexanes); H NMR (CDCl
H), 7.72–7.61 (m, 2H), 7.41–7.15 (m, 5H), 4.30 (d, 1H, J ) 13.08
Hz), 4.05 (d, 1H, J ) 13.08 Hz), 3.65–3.58 (m, 2H), 2.32 (d, 1H,
3
) δ 8.06–8.01 (m,
4
1
2
literature procedure to afford 53% yield of 53: mp ) 168–169
1
°C; TLC R ) 0.20 (25% EtOAc/hexanes); H NMR (CDCl ) δ
f
3
1
3
J ) 3.96 Hz), 1.49 (s, 3H), 1.29 (s, 3H); C NMR (CDCl
3
) δ
34.3, 133.3, 129.4, 128.9, 127.6, 126.5, 126.4, 74.5, 42.4, 25.5,
0.8; δ 183.9, 179.5, 153.2, 138.6, 132.6, 131.1, 121.4, 81.1, 38.8;
u
8.12–8.06 (m, 2H), 7.77–7.68 (m, 2H), 5.01 (d, 1H, J ) 4.47 Hz),
4.80 (s, 1H), 3.84 (d, 1H, J ) 4.44 Hz), 3.12 (s, 1H), 1.63 (s, 3H),
1
2
13
d
3 u
1.37 (s, 3H); C NMR (CDCl ) δ 134.5, 133.9, 126.9, 126.2, 70.1,
-
1
IR (KBr) 3453, 1673, 1645, 1603, 1574 cm ; APCI-MS m/z 382
63.0, 23.9, 23.6; δ 187.2, 179.4, 154.1, 132.2, 131.3, 117.2, 82.0;
d
+
+
(
M
+ 2, 25), 381 (M + 1, 100); NP-HPLC t
5; n-hexane/IPA, 0.5 mL/min).
3S,4S and 3R,4R)-4-(Benzylthio)-3-hydroxy-2,2-dimethyl-
R
) 7.5 min (85:
NP-HPLC t ) 9.9 min (85:15; n-hexane/IPA, 1 mL/min).
R
1
Biochemical Assays. Recombinant human IDO was expressed
7
8
(
and purified as described. The IC50 inhibition assays were
performed in a 96-well microtiter plate as described by Littlejohn
3
4
,4-dihydro-2H-benzo[g]chromene-5,10-dione (48). Compound
8 was synthesized using the general procedure with benzyl
7
8
et al. with some modification. Briefly, the reaction mixture
contained 50 mM potassium phosphate buffer (pH 6.5), 40 mM
ascorbic acid, 400 µg/mL catalase, 20 µM methylene blue, and ∼27
nM purified recombinant IDO per reaction. The reaction mixture
was added to the substrate, L-tryptophan (L-Trp), and the inhibitor.
The inhibitors were serially diluted in 3-fold increments ranging
from 100 µM to 1.69 nM, and the L-Trp was tested at 100 µM (Km
) 80 µM). The reaction was carried out at 37 °C for 60 min and
stopped by the addition of 30% (w/v) trichloroacetic acid. The plate
was incubated at 65 °C for 15 min to convert N-formylkynurenine
to kynurenine and was then centrifuged at 1250g for 10 min. Lastly,
100 µL of supernatant from each well was transferred to a new
96-well plate and mixed at equal volume with 2% (w/v) p-
dimethylaminobenzaldehyde in acetic acid. The yellow color
generated from the reaction with kynurenine was measured at 490
nm using a Synergy HT microtiter plate reader (Bio-Tek, Winooski,
VT). The data were analyzed using Graph Pad Prism 4 software
(Graph Pad Software Inc., San Diego, CA). For the K
tions of 36, 41, and 50, tryptophan concentrations were varied from
25 to 200 µM (K ) 42 µM), and inhibitor concentrations were
varied between 3-fold above and below the calculated IC50
Otherwise, reaction conditions were exactly as described above.
Data were analyzed with the Enzyme Kinetics module in SigmaPlot
version 10.
mercaptan. Chromatographic separation afforded pure trans dias-
tereomer 48 as a yellow solid in 27% yield: mp ) 127–128 °C;
1
TLC R
8
(
)
f
) 0.46 (20% EtOAc/hexanes); H NMR (CDCl
3
) δ
.15–8.07 (m, 2H), 7.77–7.66 (m, 2H), 7.45–7.26 (m, 5H), 4.26
d, 1H, J ) 12.69 Hz), 4.14 (d, 1H, J ) 12.69 Hz), 4.09 (d, 1H, J
6.15 Hz), 3.74 (dd, 1H, J ) 6.15, 3.39 Hz), 2.88 (d, 1H, J )
1
3
9
1
1
3
2
.57 Hz), 1.50 (s, 3H), 1.24 (s, 3H); C NMR (CDCl
33.5, 129.5, 129.1, 127.9, 126.7, 126.5, 69.6, 42.6, 26.5, 20.3; δ
83.8, 179.6, 152.9, 138.1, 132.5, 131.2, 121.8, 81.7, 40.2; IR (KBr)
3 u
) δ 134.4,
d
421, 1681, 1645, 1609, 1574 cm ; APCI-MS m/z 382 (M+ + 2,
-1
5), 381 (M+ + 1, 100); NP-HPLC t
) 8.0 min (85:15; n-hexane/
R
IPA, 0.5 mL/min).
3S,4S)-4-(Benzylamino)-3,6-dihydroxy-2,2-dimethyl-3,4-di-
hydro-2H-benzo[g]chromene-5,10-dione (49). Alkene 25 (50 mg,
.195 mmol) was dissolved in CH Cl (3 mL), cooled to 0 °C, and
(
0
2
2
7
7
treated with mCPBA (50.0 mg, 0.290 mmol). The reaction was
stirred overnight at 0 °C. The solvent was removed in Vacuo, and
the crude solid epoxide product 34 was treated with benzylamine
i
determina-
(
0.975 mmol) in 2-propanol (3 mL). After the reaction was stirred
m
for 30 min, the solvent was removed in Vacuo, and the crude was
purified by preparative TLC to afford 25 mg of product 49 in 34%
yield: TLC R
1
.
1
f
) 0.30 (25% EtOAc/hexanes); H NMR (CDCl
3
) δ
2.27 (s, 1H), 7.64–7.22 (m, 8H), 4.01–3.90 (m, 3H), 3.72 (d, 1H,

1
716 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Kumar et al.
Cell-Based IDO Inhibition and Cytotoxicity Assays. T-REx
using a quadratic force constant (100) to tether the atoms to their
starting geometries; for each subsequent minimization, the force
constant was reduced by a half-until zero.
7
9
cells containing an inducible human INDO cDNA were seeded
in a 96-well plate at a density of 20000 cells per well in 100 µL of
DMEM + 10% FBS. IDO expression was induced for 72 h by the
addition of 100 µL of media containing 20 ng/mL doxycycline.
The media was then discarded, the wells rinsed once, and serial
dilutions of menadione in 200 µL of phenol red-free DMEM +
Docking Calculations. The 2-[N-cyclohexylamino]ethanesulfo-
nic acid and 4-phenyl-1-imidazole ligands were removed from the
active site prior to docking. Preliminary docking calculations
87
performed with annulin B were carried out using MolDock. Gold
8
8,89
90
1
0% FBS was added in triplicate and incubated for 18 h. The
(version 3.1)
and AutoDock (version 3.05) were used with
reaction was stopped by the addition of 40 µL of 50% (w/v) TCA
default parameters and reproduced the crystallographic position of
4-phenyl-1-imidazole binding to the heme. Docking of the
napthquinone series of compounds using AutoDock and Gold
produced a top scoring binding pose with a ketone oxygen within
coordination distance to the heme iron.
to each well, and the cells were fixed by incubating for 1 h at
4
°C.
To Assess IDO Activity. Following the TCA fixation step, the
supernatants were transferred to a round-bottomed 96-well plate
and incubated at 65 °C for 15 min. The plates were then centrifuged
at 1250g for 10 min, and 100 µL of clarified supernatant was
transferred to a new flat-bottomed 96-well plate and mixed at equal
volume with 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid.
The yellow reaction was measured at 490 nm using a Synergy HT
microtiter plate reader (Bio-Tek, Winooski,VT).
Acknowledgment. We thank Ms. Erika Sutanto-Ward for
excellent technical assistance. Dr. Mike McLeish of the
University of Michigan is gratefully acknowledged for guidance
with the enzyme inhibition kinetics analysis. We are also grateful
to Dr. Andrew Mellor for generously providing IDO knockout
mice used in this study. Financial support for this work was
provided by the National Institutes of Health (NCI R01
CA109542). A.J.M. is also the recipient of grants from the DoD
Breast Cancer Research Program (BC044350), the State of
Pennsylvania Department of Health (CURE/Tobacco Settlement
Award), the Lance Armstrong Foundation, and the Concern
Foundation. G.C.P. is the recipient of NIH Grant Nos. CA82222
and CA100123. Additional support for this project was provided
by grants to G.C.P. from the Charlotte Geyer Foundation and
the Lankenau Hospital Foundation.
To Assess Cell Viability. The TCA-fixed cells remaining in the
9
6-well plate following transfer of the media were processed
80
essentially as described. Fixed cells were washed four times in
tap water, blotted, air-dried, and treated for 15 min at room
temperature with 100 µL of 0.4% (w/v) sulfarhodamine B (SRB)
(
Sigma-Aldrich, St. Louis, MO) prepared in 1% acetic acid. Wells
were then rinsed four times in 1% acetic acid, air-dried, and
developed by adding 200 µL of 10 mM unbuffered Tris-HCl and
incubating for 15 min at room temperature with gentle shaking.
Staining intensity, proportional to cell number, was determined by
reading the absorbance at 570 nm on a plate reader. Data were
collected and analyzed using Excel software (Microsoft).
1
13
Tumor Formation and Drug Response. FVB-strain MMTV-
Neu transgenic mice were obtained from the Jackson Laboratory.
C57BL/6 and athymic NCr-nu/nu (nude mice) were obtained from
NCI-Frederick. IDO knockout mice have previously been de-
Supporting Information Available: Copies of H and C NMR
spectra and liquid chromatograms for compounds 13, 14, 24-32,
1
13
36-43, 46-50, and 53. Copies of H and C NMR spectra for
1
35. Copies of H NMR spectra for previously synthesized com-
81
scribed. Studies involving mice were approved by the institutional
animal use committee of the Lankenau Institute for Medical
Research. For autochthonous mammary gland tumor treatment
studies, parous, FVB-strain MMTV-Neu mice expressing the wild
type form of the rat HER2/Neu proto-oncogene were used as
pounds 45, 51, and 52. X-ray crystal structure of 36 and
Hanes-Woolf plots for 36, 41, and 50. This material is available
free of charge via the Internet at http://pubs.acs.org.
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