Design, synthesis, and structure-activity relationship studies of tryptanthrins as antitubercular agents

Article abstract of DOI:10.1021/np3007167

The natural product tryptanthrin (1a) represents a potential lead for new tuberculosis (TB) drugs since tryptanthrin and its synthetic analogues possess potent in vitro activity against Mycobacterium tuberculosis (Mtb). However, in spite of their in vitro activity, none of these agents have been shown to be efficacious in vivo against animal models of TB. Described herein are syntheses of new tryptanthrin analogues together with a systematic investigation of their in vitro antitubercular activity and ADME properties followed by pharmacokinetic characterization in rodents for the most promising compounds. Those with the best potency and oral bioavailability were progressed to evaluations of efficacy against acute murine TB. The work aimed to prove the concept that this compound class can limit growth of Mtb during infection as well as to establish the SAR for in vitro activity against Mtb and the range of in vitro ADME parameters for this class of natural products. Novel C-11-deoxy (5b) and A-ring-saturated (6) tryptanthrin analogues were discovered that maintained activity against Mtb and showed improved solubility compared to tryptanthrin as well as evidence of oral bioavailability in rodents. However, neither 5b nor 6 demonstrated efficacy against acute murine TB following administration at doses up to 400 mg/kg daily for 4 weeks. Although 5b and 6 failed to inhibit replication or kill Mtb in vivo, they illuminate a path to new structural variations of the tryptanthrin scaffold that may maximize the potential of this class of compounds against TB.

Full text of DOI:10.1021/np3007167

Article
pubs.acs.org/jnp
Design, Synthesis, and Structure−Activity Relationship Studies of
Tryptanthrins As Antitubercular Agents
Jae-Min Hwang,† Taegwon Oh,‡ Takushi Kaneko,§ Anna M. Upton,§ Scott G. Franzblau,⊥ Zhenkun Ma,§
Sang-Nae Cho,‡ and Pilho Kim*,†,∥
†Cancer and Infectious Diseases Therapeutics Research Group, Korea Research Institute of Chemical Technology, Daejeon 305-600,
Korea
‡Department of Microbiology and Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine,
Seoul 120-752, Korea
§Global Alliance for TB Drug Development, New York, New York 10005, United States
⊥Institute for Tuberculosis Research, University of Illinois, Chicago, Illinois 60612, United States
∥Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology, Daejeon 306-350, Korea
ABSTRACT: The natural product tryptanthrin (1a) represents a
potential lead for new tuberculosis (TB) drugs since tryptanthrin
and its synthetic analogues possess potent in vitro activity against
Mycobacterium tuberculosis (Mtb). However, in spite of their in
vitro activity, none of these agents have been shown to be
efficacious in vivo against animal models of TB. Described herein
are syntheses of new tryptanthrin analogues together with a
systematic investigation of their in vitro antitubercular activity and
ADME properties followed by pharmacokinetic characterization in rodents for the most promising compounds. Those with the
best potency and oral bioavailability were progressed to evaluations of efficacy against acute murine TB. The work aimed to
prove the concept that this compound class can limit growth of Mtb during infection as well as to establish the SAR for in vitro
activity against Mtb and the range of in vitro ADME parameters for this class of natural products. Novel C-11-deoxy (5b) and A-
ring-saturated (6) tryptanthrin analogues were discovered that maintained activity against Mtb and showed improved solubility
compared to tryptanthrin as well as evidence of oral bioavailability in rodents. However, neither 5b nor 6 demonstrated efficacy
against acute murine TB following administration at doses up to 400 mg/kg daily for 4 weeks. Although 5b and 6 failed to inhibit
replication or kill Mtb in vivo, they illuminate a path to new structural variations of the tryptanthrin scaffold that may maximize
the potential of this class of compounds against TB.
out an extensive analogue development study and identified
PA-505 (2) and PA-510 (3) as the most promising compounds.
However, in a subsequent in vivo study little efficacy could be
demonstrated in a mouse acute tuberculosis (TB) infection
model. The reason for the lack of efficacy was not presented or
conjectured on.
TB is a major infectious disease killer, causing an estimated
1.4 million deaths globally in 2010.22 Although the main
burden of TB remains in developing countries, developed
countries are not immune to outbreaks of TB.23 The recent
emergence of multidrug resistant (MDR)- and extensively drug
resistant (XDR)-TB is of particular concern, posing serious
challenges for global TB control and for the treatment of
individual patients. Compounding this issue, no novel TB drugs
have been developed in the past four decades, leaving
healthcare providers with an old and limited TB treatment
armamentarium. Therefore, there is a pressing need to develop
ryptanthrin (1a) is a natural product containing an
Tindolo[2,1-b]quinazoline ring system isolated from the
indigo plant Strobilanthes cusia Kuntze (Acanthaceae) and its
relatives.1 It is also obtained from Candida lipolytica when it is
fermented in a tryptophan-rich medium.2 A number of
biological activities have been reported for tryptanthrin or its
analogues. These compounds exhibit growth inhibition of
Bacillus subtilis,3 permeabilized Escherichia coli,4 methicillin-
resistant Staphylococcus aureus,5 dermatophytic fungal patho-
gens,1 Plasmodium falciparum,6,7 Leishmania donovani,8 Trypa-
nosoma brucei,9 and Toxoplasma gondii.10 Further, cytostatic or
cytotoxic activities have been demonstrated against mammalian
tumor cell lines,11−13 as well as reversal of multidrug resistance
in cancer cell lines.14 Finally, wide-ranging immune-modulatory
effects have been observed, including inhibition of Th2
development, IgE-mediated degranulation, and IL-4 produc-
tion,15 interferon-γ production,16 nitric oxide and prostaglandin
E2 synthesis,17 NF-κB,18 and leukotriene activities,19 and
immune enhancement.20
Special Issue: Special Issue in Honor of Lester A. Mitscher
Received: October 14, 2012
In 1998, Mitscher and Baker reported the antitubercular
activity of tryptanthrin (1a) and its derivatives.21 They carried
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of tryptanthrin (1a) and its analogues.
novel TB drugs to address the substantial burden of both drug-
sensitive and drug-resistant diseases.
RESULTS AND DISCUSSION
Chemistry. At the outset, several previously reported A-
■
In the field of TB drug development, a key goal is the
discovery of compounds that are able to shorten the duration of
combination drug therapy required to achieve relapse-free cure
of both drug-sensitive and drug-resistant diseases. Current TB
treatment comprises 6−9 months of combination therapy for
patients with drug-sensitive disease and 18−24 months for
patients with drug-resistant TB. There are numerous reports
suggesting the opportunity to shorten such regimens through
the inclusion of compounds that are active against both
replicating and nonreplicating Mycobacterium tuberculosis
(Mtb).24 For the past 10 years, the TB drug discovery and
development pipeline has been getting stronger.25 However,
tremendous efforts are needed to further strengthen the
pipeline. Drugs representative of new chemotypes and with
new mechanisms of action are essential to avoid cross-
resistance with existing TB drugs and for use in the multidrug
combinations that are necessary for the successful treatment of
TB.
ring- and D-ring-substituted analogues of tryptanthrin (1a)
were synthesized followed by development of new syntheses of
more extensively modified analogues. Tryptanthrins (1) were
prepared from isatoic anhydride (7) and isatin (8) by a
reference method (Scheme 1A).11 With this chemistry in hand,
Scheme 1. Synthesis of Tryptanthrins
With these circumstances in mind, this work aimed to further
investigate the potential of tryptanthrin and its analogues, a
novel class with respect to currently used TB drugs, for utility
against TB. An exploration of the tryptanthrin scaffold was
conducted by repeating some early, published work, in addition
to synthesizing and testing new analogues. The goal of this
work was demonstration of in vivo efficacy for this class of
compounds against murine acute TB. To achieve this, new
analogues were designed with the aim of maintaining or
improving potency in vitro against Mtb, while also improving
physicochemical properties compared to tryptanthin, in
particular, solubility. It was anticipated that these improved
physicochemical properties would in turn enhance the in vitro
ADME profiles and pharmacokinetic (PK) characteristics of
these compounds compared to tryptanthrin. In addition, to
better understand the bactericidal versus bacteriostatic potential
of these compounds, minimum bactericidal activity (MBC) was
evaluated for a range of the new analogues.
While the molecular weight (248) and logD (2.29) of
tryptanthrin (1a) appear reasonable as a starting point, the low
solubility of the compound (5.4 μM) and the flatness of the
ring system were considered problematic. To potentially
address these issues, analogues were designed to incorporate
solubilizing groups in ring A or D, A-ring-aza analogues (4), C-
11-deoxy analogues (5), and an A-ring-saturated analogue (6)
to disrupt planarity of the compounds (Figure 1).
relatively simple A-ring- and/or D-ring-substituted tryptanthrin
analogues were prepared from their correspondingly sub-
stituted starting materials. Halogen-, nitro-, amido-, ester-, or
methoxy-substituted analogues were prepared in this fashion
(Table 1). In order to introduce a solubilizing group into
tryptanthrin, analogues containing cyclic amines in the D-ring
(1t−w) were prepared by substituting the chlorine atom of 1m
with appropriate cyclic amines under heating at 70−90 °C in
NMP (Scheme 1B).26 Since Mitscher et al. reported that the A-
ring-aza version of tryptanthrins (4) was effective in increasing
activities as well as demonstrating favorable PK profiles,26 4 was
also prepared. 4- or 2-Aminonicotinic acid or 3-amino-
isonicotinic acid (9) was reacted with isatin (8) in the presence
of HBTU, N-methylmorpholine, and DBU in DMF to give the
2- or 4- or 3-aza version of tryptanthrin (4), respectively
(Scheme 2A).27 Two approaches were taken to improve
solubility compared to tryptanthrin. First, analogues containing
a pyridine moiety in place of the A-ring phenyl group were
synthesized from 4g for 4-aza or 4l for 2-aza analogues and
corresponding cyclic amines by a similar method to that
described above (Scheme 2B). Second, the phenyl A-ring was
replaced with a cyclohexyl ring to alter the flat and aromatic
nature of the molecule. This second approach was considered
advantageous since the flat aromatic ring system of the
B
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Table 1. MIC, LORA, and Cytotoxicity of Tryptanthrins (1)
a
b
c
d
MIC-s: MIC with 40% serum. Cytotoxicity in Vero cells. NA: not available. IS: insoluble.
compound has been postulated to intercalate with DNA, which
could result in undesirable cytotoxicity.27 Thus, C-11-deoxy
tryptanthrins (5)28 were synthesized by tandem reduction of
tryptanthrins 1 or 4 under LiAlH4 in ether at 40 °C to give an
intermediate having both C-6 and C-11 carbonyl groups
reduced and oxidation in the presence of MnO2 in CH2Cl2 to
reoxidize the C-6-carbinol group (Scheme 3). These C-11-
deoxy analogues are of considerable interest because the
stereoelectronic properties of the conjugated system has been
considerably modified compared with that of the parent
compound.8 In order to construct an A-ring-saturated version
of tryptanthrin (6), first, A-ring-saturated isatoic anhydride
(11)29 was prepared from ethyl 2-oxocyclohexanecarboxylate
(10) in the presence of ethyl carbamate and POCl3 (Scheme
4). Subsequently, compound 11 was reacted with isatin (8) to
facilitate 6. The analogues thus prepared were then examined
for in vitro antimycobacterial activity and ADME properties.
In Vitro Activities (MIC, LORA, Cytotoxicity). The
antitubercular activity of each compound against Mtb H37Rv
cultured under aerobic conditions was measured using the
microplate Alamar Blue assay (MABA).30 Aerobically grown
Mtb culture was exposed to serial dilutions of test compounds
for a week in 96-well microplates. The aerobic minimum
inhibitory concentration (MIC) was determined by observing
the color change of the Alamar Blue dye, which indicates the
presence of live bacilli.
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Introduction of a cyclic amine at the C-9-position to
potentially increase solubility (1t, 1u, and 1w) resulted in
MABA MICs far higher than that of tryptanthrin, indicating
that such a group was not tolerated for maintenance of in vitro
antitubercular activity. The MABA MIC of 9-pyrrolidinetryp-
tanthrin (1v) could not be measured due to its low solubility.
Introduction of a carboethoxy group at the C-8-position (1c)
resulted in a compound 2-fold more active than tryptanthrin.
Compound 1c and another carboethoxy-containing analogue
(1n) are of interest as starting points for the preparation of
further derivatives. The potency of 1x, despite its bulky
substituent at the C-8-position, merits special attention. As
mentioned above, 8-nitrotryptanthrin (1b) has a MABA MIC
value of 0.032 μg/mL. This compound also has a LORA MIC
value of 2.4 μg/mL, while the majority of analogues lack LORA
activity. In general, any electron-withdrawing group at the C-8-
position appears to enhance in vitro antitubercular activity.
Following consideration of their MABA MIC, LORA MIC, and
cytotoxicity IC50 values, 1b−d, 1f−h, 1m, 1q−s, and 1x (Table
1) were selected for in vitro ADME studies. Tryptanthrin itself
(1a) was included for the sake of comparison.
Scheme 2. Synthesis of A-Ring-Aza Tryptanthrins
Scheme 3. Synthesis of C-11-Deoxy Tryptanthrins
The MABA MIC, LORA MIC, and cytotoxicity IC50 values
of A-ring-aza tryptanthrins are summarized in Table 2.
Introduction of a nitrogen atom at position 2 or 4 as in 2-aza
(4r) or 4-aza (4c) as compared with 1d indicated that such a
substitution is allowed without affecting the MABA MIC (see
1d). However, their LORA MIC values are far better than that
of 1d. As for the A-ring phenyl analogues, cyclic amines at the
C-9-position (4h−j and 4m−p) produced essentially inactive
analogues. Considering their MABA MIC, LORA MIC, and
cytotoxicity values (Table 2), 4b and 4c were selected for
further in vitro ADME studies.
Scheme 4. Synthesis of A-Ring-Saturated Tryptanthrin (6)
Both C-11-deoxy tryptanthrins (5) and A-ring-saturated
tryptanthrin (6) analogues were designed primarily to reduce
the planar characteristics of tryptanthrins to improve solubility
and address potential genotoxicity issues related to their
planarity. The MABA MIC of C-11-deoxy tryptanthrin (5c) is
as good as that of tryptanthrin (1a), suggesting that the C-11
carbonyl group may not be essential for activity. Moreover, 5c
also has a moderate LORA MIC of 20 μg/mL. However, an A-
ring-pyridine in conjunction with a C-11-deoxy modification as
in 5d and 5g demonstrates reduced potency, with MABA MIC
values of 16 and 32 μg/mL, respectively. Compared with 8-
chlorotryptanthrin (1d), its C-11-deoxy version (5b) exhibits a
MABA MIC value of 0.13 μg/mL as well as a LORA MIC of 24
μg/mL. However, in the case of 2-aza-8-OCF3-tryptanthrin
(4a) and 4-aza-8-chlorotryptanthrin (4c), their C-11-deoxy
versions (5f and 5h, respectively) are more than 10-fold less
active, suggesting that introducing a C-11-deoxy substituent is
not universally well-tolerated in terms of activity. Compared
with tryptanthrin, A-ring-saturated tryptanthrin has a 2-fold
lower MABA MIC value and a moderate LORA MIC value of
10.5 μg/mL. Following consideration of the MABA MIC,
LORA MIC, and cytotoxicity IC50 values for these compounds,
5b and 6 (Table 3) were selected for in vitro ADMETox
profiling.
In addition, the low oxygen recovery assay (LORA)31 was
used to determine the activity of a subset of compounds against
low-oxygen-adapted nonreplicating Mtb. In this case, the MIC
refers to the concentration of compound that inhibits by ≥90%
the ability of low-oxygen-adapted Mtb to recover the ability to
express a reporter gene after 10 days exposure to low-oxygen
conditions. The LORA is designed to detect compounds that
may have the potential for shortening the duration of TB
therapy through more efficient killing of the nonreplicating
population of bacteria believed to persist in the face of clinical
chemotherapy, thus necessitating long durations of treatment.
Finally, the cytotoxicity of each compound was measured
against the Vero cell line.
The in vitro antitubercular activities of A-ring- and/or D-
ring-substituted tryptanthrins are presented along with their
cytotoxicities in Table 1. Tryptanthrin (1a) has a MABA MIC
value of 1 μg/mL. Introduction of a nitro group at the 2-
position of tryptanthrin (1j) resulted in a less potent
compound (MABA MIC: 16 μg/mL), while the corresponding
8-nitro analogue (1b) is highly active (MABA MIC: 0.032 μg/
mL). On the other hand, 2-bromotryptanthrin (1h) exhibits a
4-fold lower MABA MIC than tryptanthrin. Compared with the
A-ring-substituted tryptanthrins, D-ring-substituted tryptan-
thrins are more active. For instance, both 8- and 9-
chlorotryptanthrin (1d and 1m) demonstrated 8-fold lower
MABA MICs than tryptanthrin. Incorporating an additional
substituent on the A-ring while keeping an 8-chloro substituent,
such as 1e−g, did not improve potency. Substitution of the C-8
position with bromo (1q), OCF3 (1r), and fluoro (1s) resulted
in MABA MICs as potent as that of 8-Cl tryptanthrin (1d).
In general, a wide range of in vitro activities against Mtb, as
defined by MABA and LORA MIC values, were observed for
the compounds synthesized, and these values varied with the
structural modifications made to the compounds. As for
cytotoxicity, there seems to be no apparent correlation between
activity against Mtb and cytotoxicity for compounds included in
Tables 1 and 3. However, there may be a closer correlation
D
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Table 2. MIC, LORA, and Cytotoxicity of A-Ring-Aza Tryptanthrins (4)
a
b
c
d
MIC-s: MIC with 40% serum. Cytotoxicity in Vero cells. NA: not available. IS: insoluble.
between those two activities among the aza analogues (see
Table 2). To further explore the in vitro activities of this group
of compounds against Mtb, the ability to kill Mtb, as opposed
to inhibit bacterial growth, was determined for a subset of
analogues selected based on structural diversity and potency.
The ability to kill Mtb is represented by the minimal
bactericidal concentration value, which is defined as the
minimal concentration of an analogue that reduces the number
of colony forming units (CFU) by 90% compared with those
growing on analogue-free medium. These MBC values,
presented in Tables 1 and 2, indicate that the analogues are
in general bactericidal. In addition, to assess the impact of the
addition of physiological levels of protein to the MABA MICs
of this series, MABA MICs were assessed in the presence of
40% serum for a subset of structurally diverse analogues
(Tables 1−3). These data indicate that the addition of
supplemental serum results in a modest increase in MABA
MIC against Mtb for the compounds tested.
tryptanthrin (1a) for comparison (Table 4). In addition, in
vitro permeability, plasma protein binding, metabolic stability,
and lipophilicity were evaluated for a subset of these
compounds (Table 4).
Aqueous solubility was evaluated in three matrixes: buffer at
pH 7.4, and simulated gastric and intestinal fluids (SGF and
SIF) with a view to predicting the potential for solubility and
absorption in the upper and lower gastrointestinal tract. In
buffer at pH 7.4, the solubility of the analogues ranges from <1
μM (below the limit of quantitation) to 64 μM, while solubility
in SIF (pH 7.6) is generally higher than this, ranging from 10.3
to 151.9 μM, most likely due to the presence of protein in SIF.
Solubility in SGF (pH 1.5) is broadly lower than that in SIF
(solubility in SGF: <1 to 79.2 μM). The behavior of individual
compounds differs from each other; for example, 1b is more
soluble in neutral buffer and in SIF than in SGF, while the
opposite is true for the C-11-deoxy compound, 5b. In the case
of 8-bromo derivatives, the 2-aza analogue (4b) is more soluble
than the non-aza analogue (1q), presumably due to increased
hydrogen-bonding feasibility.32 However, in other cases, the
difference between the aza and non-aza analogues was not clear
(e.g., 4c and 1d). Deoxygenation at C-11 appears to increase
solubility under acidic conditions, although solubility under
neutral conditions is minimal (see 5b). An A-ring-saturated
In Vitro ADME. Since a goal of this work was to discover
tryptanthrin analogues with improved PK characteristics
compared to previously described compounds, in particular
by improving their solubility compared to tryptanthrin (1a),
aqueous solubility was assessed for the 15 more potent and
selective analogues selected for progression, along with
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ratios all exceeding 1. However, other compounds displayed
poor permeability in this assay. The favorable permeability of
tryptanthrin (1a) itself suggests that this is not a key
characteristic for improvement in new analogues, but rather it
is important to maintain this behavior when designing new
derivatives.
Table 3. MIC, LORA, and Cytotoxicity of C-11-Deoxy (5)
and A-Ring-Reduced (6) Tryptanthrins
A small subset of compounds exhibited solubility in SIF and
SGF higher than 10 μM accompanied by superior permeability,
suggesting that reasonable absorption may be expected in vivo.
These compounds include tryptanthrin (1a), 5b, 6, and 1x,
with 1s also coming close to meeting these criteria. To predict
in vivo clearance, plasma protein binding and metabolic
stability in human liver microsomes were assessed for several
compounds. The analogues tested ranged broadly in their
metabolic stability, with 4b, 4c, 1g, and 1x providing examples
of highly stable compounds, while tryptanthrin (1a), 1c, and 6
were less stable under the conditions of the assay. Plasma
protein binding was high for all analogues tested, ranging from
94.9% to 99%, and the new analogues did not differ significantly
from tryptanthrin with respect to this property. It is interesting
to note that, based on the assays used in the present study,
tryptanthrin possesses an ADME profile suggestive of
reasonable absorption with rapid clearance.
substituent
a
b
MIC
(μg/
mL)
MIC-s
(μg/
mL)
LORA
(μg/
IC50
(μg/
mL)
compound
X
R
mL)
c
5a
5b
5c
5d
5e
5f
no aza
no aza
no aza
2-N
8-OCF3
8-Cl
H
0.125
0.125
1
0.5
NA
1.3
40
0.125
2
24
20
42
H
16
NA
NA
NA
NA
NA
NA
1
>20
8.3
9.0
NA
NA
NA
10.5
>20
3.7
5.2
4.8
>10
4.4
8.7
2-N
8-Cl
8-OCF3
H
2
2-N
2
5g
5h
5i
4-N
32
4-N
8-Cl
8-OCF3
16
4-N
16
6
0.5
a
b
c
MIC-s: MIC with 40% serum. Cytotoxicity in Vero cells. NA: not
TB is treated with combination therapy, in which multiple
anti-TB drugs are administered daily to limit resistance
development and maximize efficacy. Additionally, TB drugs
are often co-administered with anti-retroviral therapies for
patients co-infected with Mtb and HIV. Therefore, it is
important to understand the potential for drug−drug
interactions for any class of compounds with possible use for
TB treatment. Toward understanding this aspect of the profile
of tryptanthrin and related compounds, a select set of analogues
was screened for their capacity to inhibit the CYP isoenzymes
2C9, 2C19, and 3A4 (Table 5). Moderate inhibition of all three
enzymes is exhibited by 4-aza-8-chlorotryptanthrin (4c), of
CYP2C9 by most analogues tested, and of 2C19 by 8-
chlorotryptanthrin (5b) in addition to 4c, suggesting only
limited potential for this class to cause drug−drug interactions
mediated by inhibition of these enzymes. Tryptanthrin itself
available.
analogue (6) exhibited increased solubility compared to other
compounds prepared under all three conditions, suggesting that
reduced planarity may enhance solubility.33
Lipophilicity was determined for several analogues and
ranged from 1.6 to 3.37; several compounds demonstrated a
lower logD value than tryptanthrin (logD 2.37). The in vitro
permeability of these compounds varied greatly. This property
was assessed across a monolayer of human intestinal epithelial
cells (Caco-2) in both apical to basolateral (A to B) and B to A
directions and revealed good permeability in both directions for
some compounds, notably tryptanthrin (1a), 1g, 1q−s, 1x, 4c,
5b, and 6, which possess A → B permeability up to 85.4 × 10−6
cm/s and B → A rates up to 52.4 × 10−6 cm/s, with A/B:B/A
Table 4. Aqueous Solubility, in Vitro Absorption, and Metabolic Stability of Selected Tryptanthrins
solubility, μM
solubility,
solubility, μM
metabolic stability at 1 h
plasma
protein
Caco-2: A → B
permeability (10 ×
10−6 cm/s)
Caco-2: B → A
permeability (10 ×
10−6 cm/s)
(buffer at pH μM (SIF pH
(SGF pH
in HLM (% parent
a
b
c
compound
7.4)
7.6)
1.5)
logD
2.37
remaining)
binding (%)
1a
1b
1c
1d
1f
5.4
12.9
151.9
11.1
16.7
29.8
10.3
49.4
20.3
92.9
31.3
18.8
27.5
34.4
18.9
19.3
73.4
11.7
5.1
15
95.9
96.9
98.4
ND
NA
NA
ND
99
60.1
22.5
ND
0.8
d
e
64.3
3.8
NA
33
ND
1.5
2.94
2.76
NA
NA
1.69
2.45
NA
NA
NA
NA
1.6
0
4.2
3.3
26.8
BLQ
2.5
ND
63
ND
2.2
ND
2.1
f
BLQ
1.1
1g
1h
1m
1q
1r
1s
1x
4b
4c
5b
6
91
28.5
ND
0.4
12.7
ND
0.1
BLQ
BLQ
2.6
BLQ
71.9
2.3
ND
27
59
NA
NA
NA
98.9
NA
94.9
98.7
95.7
23.8
22
7.6
1
1.5
59
5.8
4.9
8.5
46
66.6
59.9
ND
73.8
60.8
85.4
29.9
26
16.3
35.4
8.0
10.2
45.0
6.0
105
96.0
96
ND
44
1.77
3.37
NA
BLQ
54.9
79.2
53
53
20.8
52.4
18
a
b
c
d
e
SIF: simulated intestinal fluid. SGF: simulated gastric fluid. HLM: human liver microsomes. NA: not available. ND: compound not detectable.
f
BLQ: below limit of quantitation.
F
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overall longer half-life and greater AUC for 6 than for the other
compounds. The apparent lack of correlation between
microsomal stability data and half-life for the tested compounds
suggests that elimination pathways other than or in addition to
hepatic metabolism are significant for this class. Further
investigation of the PK behavior of these compounds may aid
in design of new derivatives with enhanced oral bioavailability.
The superior oral exposure of 6 compared to 5b and
especially to 1x was maintained after oral dosing to mice at 40
mg/kg (Table 6). In an attempt to optimize plasma exposure
following oral dosing to mice of 5b and 6, two formulations of
each compound were used and compared. For 5b, formulation
in 0.1% SLS or in 20% DMSO, 20% PEG400, and 60% (30%
HP-beta-CD, 2-hydroxypropyl-β-cyclodextrin, in water) re-
sulted in similar exposures and PK parameters (Table 6). For
6, formulation in 0.5% methylcellulose resulted in somewhat
superior exposure to formulation in 30% HP-beta-CD.
Due to the encouraging potency, ADME, and PK profiles of
5b and 6, a micro-Ames assay for mutagenic potential was
performed for both of these compounds (Table 7). Owing to
their limited solubility, precipitates were observed at ≥25 μg/
well for 5b and at ≥75 μg/well for 6. In addition, cytotoxicity
was observed for 5b at ≥2.5 μg/well and for 6 at ≥25 μg/well
in TA100, both with and without metabolic activation.
However, neither compound, at any concentration tested,
produced an increase in the number of revertant colonies as
compared with the vehicle control in either strain with or
without metabolic activation. Therefore, both compounds were
classified as nonmutagenic under the conditions of the assay.
In Vivo Efficacy Studies. The in vivo efficacy of 5b and 6
was evaluated in a murine acute TB infection model. Mice were
infected with Mtb Erdman strain via inhalation and were given
each drug solution daily by gavage for 4 weeks starting on the
10th day postinfection. The numbers of viable bacteria in the
lung and spleen were determined at the end of treatment
(Table 8). Assuming that the exposure achieved following
administration of a single dose at 40 mg/kg (in the PK study
described above) was not the maximum achievable exposure,
much higher dose levels were selected for use in the efficacy
study, up to 400 mg/kg daily. However, even after 4 weeks of
treatment at these high dose levels, both analogues failed to
reduce the viable Mtb counts measurable in either the lungs or
the spleens of the mice. On the other hand, the positive control
compound, rifampicin, dosed at 10 mg/kg daily for 4 weeks was
able to control the growth of Mtb in the lungs of mice, resulting
in lung CFU 1 log lower than in mice receiving vehicle only.
Table 5. Recombinant CYP Inhibition by Selected
Tryptanthrins
CYP2C9
% inhibition
CYP2C19
% inhibition
CYP3A4
% inhibition
compound
1a
1c
1d
1h
1m
4b
4c
5b
25
38
38
20
25
10
54
27
9
10
13
10
4
12
5
10
10
7
16
67
31
14
62
1
demonstrated a low percentage of inhibition of each isoenzyme
(25% or below, at 10 μM),
PK Studies. Due to their potency and selectivity and their
possession of ADME profiles that are consistent with or
improved compared with tryptanthrin (1a), 1x, 5b, and 6 were
selected for progression to in vivo studies of their PK
characteristics. Following iv dosing at 5 mg/kg to rats, 5b
exhibited limited exposure with an AUC0‑inf of 0.93 μg·h/mL
with a high volume of distribution and clearance, resulting in an
elimination half-life of 1.42 h. Compound 1x demonstrates a
similar profile following iv dosing, with an AUC0‑inf of 1.07
μg·h/mL, a moderate volume of distribution, and fast clearance,
with an elimination half-life of 0.88 h. The particularly short
half-life of compound 1x despite its stability in liver microsomes
(Table 4) suggests that this compound is subject to other
elimination pathways besides liver metabolism. Oral dosing of
each compound to rats at 20 mg/kg resulted in low exposures
and oral bioavailabilities of only 8% (5b) and 3.2% (1x) (Table
6). On the other hand, compound 6 exhibited good exposure
following iv dosing at 5 mg/kg to rats with an AUC0‑inf of 16.5
μg·h/mL, a lower volume of distribution than the other
compounds, and low clearance, with an elimination half-life of
3.98 h. This compound also demonstrated reasonable exposure
following oral dosing at 20 mg/kg to rats, with an AUC0‑inf of
19.89 μg·h/mL, resulting in an oral bioavailability of 30.1%. It is
notable that 6 is more soluble in pH 7.4 buffer and in SIF than
the other two compounds and shows a higher rate of
permeability (across a human epithelial cell layer) and slightly
lower human plasma protein binding. Compound 6 is also less
metabolically stable in human liver microsomes than 5b or 1x.
While this may appear contradictory to the longer half-life of 6
in vivo, biphasic elimination, with a fast initial elimination
phase, followed by a slower elimination phase, would be
consistent with the microsomal data and would give rise to an
Table 6. PK Parameters Following Administration of Tryptanthrins to Rats and Mice
oral administration
IV administration
species
rat
compound AUC0‑inf (ug·h/mL) −tmax (h) −Cmax (μg/mL) −t1/2 (h) AUC0‑inf (ug·h/mL) Vd (L/kg) Cl (L/h/kg) t1/2 (h)
%F
1x
5b
6
0.14
0.3
1
0.03
7.34
3.33
6.29
ND
3.08
1.07
0.93
16.50
NA
6.21
11.73
1.62
NA
4.86
5.57
0.34
NA
NA
NA
NA
NA
0.88
1.42
3.98
NA
NA
NA
NA
NA
3.16
8
2
0.05
19.89
1.301
1.4
4.67
2
2.03
30.14
NA
NA
NA
NA
NA
mouse
1x
5b
5b
0.112
0.197
0.371
1.934
2.258
a
b
e
1
NA
NA
f
0.968
5.281
2.571
1.5
1.5
0.5
ND
4.99
ND
NA
NA
NA
NA
c
6
d
NA
6
NA
a
b
c
Formulated in 0.1% SLS. Formulated in 20% DMSO, 20% PEG400, 60% (30% HP-beta-CD in water). Formulated in 0.5% methylcellulose.
d
e
f
Formulated in 30% HP-beta-CD. NA: not available. ND: not determined because insufficient data were available.
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Table 7. Mutagenic Potential of 5b and 6
without S9 activation
with S9 activation
a
a
a
a
concentration (μg/well) TA 98 TA 100
concentration (μg/well) TA 98 TA 100
DMSO
3.5 μL
5.0 × 10−4
5.0 × 10−3
5.0 × 10−2
5.0 × 10−1
1
1
7
8
DMSO
3.5 μL
1
2
7
9
e
f
positive control (−S9) NQNO
positive control (+S9) 2-AA
1.0 × 10−3
g
g
g
g
g
g
g
g
g
4
16
>60
>60
8
1.0 × 10−2
1.0 × 10−1
9
14
>60
>60
8
g
g
42
>60
2
>60
>60
1
1.0
5b (−S9)
6 (−S9)
0.25
5b (+S9)
0.25
2.5
b
b
b
b
b
b
b
b
b
b
2.5
1
2
2
0
12.5
1
1
12.5
1
0
c
c
c
c
25
1
0
25
75
1
0
c
75
1
1
1
1
c
250
1
2
250
1
1
0.25
2
7
6 (+S9)
0.25
2.5
3
6
2.5
12.5
25
2
7
2
7
4
4
12.5
3
7
d
d
d
d
d
d
2
−
−
−
25
75
2
−
−
−
c
c
c
75
1
1
c
250
1
250
2
a
b
Mean revertant colonies from duplicate wells (for DMSO controls, means are from 12 wells). Cytotoxicity (reduction in the mean number of
revertant colonies). Precipitates present. Wells not able to be counted due to a reduced background lawn. NQNO: 4-nitroquinoline-N-oxide. 2-
AA: 2-aminoanthracene. Greater than 6 revertant colonies and an increase over vehicle mean by 2-fold for TA100 and 3-fold for TA98.
c
d
e
f
g
behavior.34 Specifically, after an oral dose of 56 mg/kg, Cmax
Table 8. In Vivo Short-Term Efficacy of Tryptanthrin
a
values of of 2.62 g/mL (in male rats) and 1.89 g/mL (in female
rats) were achieved with t1/2 values of 5.60 h (in male rats) and
4.35 h (female rats). The corresponding values in Table 6
indicate that the Cmax values achieved following oral dosing of
20 mg/kg 5b and 1x to rats were low compared with that of
tryptanthrin (1a), even taking the lower dose administered
during the present studies into account. Although the MABA
MIC values for 5b and 1x are similar and significantly lower
than the MABA MIC of tryptanthrin, respectively (Tables 1
and 3), it appears unlikely, based on the data in hand, that 5b
and 1x possess superior PK properties in conjunction with
superior potency against Mtb and therefore greater potential
for efficacy against murine TB than tryptanthrin itself.
Analogues
group (mg/kg/day)
log10 CFU/lung
log10 CFU/spleen
vehicle
6.49 0.10
5.75 0.16
6.58 0.27
6.64 0.13
6.50 0.27
6.60 0.23
4.79 0.21
4.53 0.07
4.85 0.15
4.89 0.19
4.88 0.29
4.89 0.14
rifampin (10)
5b (200)
5b (400)
6 (200)
6 (400)
a
The resulting values are based on the experimental group of five mice
and are presented as means of log10 CFU standard deviation per
group.
On the other hand, oral dosing of 20 mg/kg of 6 to rats
resulted in a Cmax comparable to that achieved in the recently
published study following oral administration of a tryptanthrin
dose almost 3-fold higher; these limited data are suggestive of
improved bioavailability of 6 compared to tryptanthrin. Given
that the MABA MICs for tryptanthrin and 6 are similar (Tables
1 and 3), 6 appears more likely to be efficacious against murine
TB than tryptanthrin. However, proof of the concept and
understanding of the basal requirements for efficacy against
murine TB remain to be demonstrated for this class of
compounds, and the data in hand are insufficient to explain the
lack of efficacy demonstrated by 6, in particular.
Conclusion. More than 50 analogues of tryptanthrin (1a)
were synthesized and tested for their activity against Mtb.
Electron-withdrawing substituents in ring A or D enhanced
antitubercular activity, while introduction of a cyclic amine
moiety in ring D resulted in generally poor antitubercular
activity. A-Ring-aza-modifications appear to produce slightly
more cytotoxic analogues than their non-aza counterparts. In
handling these analogues, however, it was discovered that many
of them have extremely limited aqueous solubility, with some
possessing lower aqueous solubility than tryptanthrin (Table
4). For selected analogues, in vitro ADME properties were
examined and their PK properties were evaluated in mice and
It is challenging to explain the discrepancy between the in
vitro antitubercular activity of compounds 5b and 6 and their in
vivo efficacy data. The laboratory strain of Mtb used for in vitro
MIC determinations (H37Rv) differed from that used for the in
vivo efficacy study (Erdman), and this may provide a partial
explanation.
More probably, the limit of absorption of these analogues
may be much lower than was anticipated; it is possible that
most of the high dose administered during the efficacy study
was not in fact absorbed, resulting in subefficacious exposure.
Since the pharmacodynamics driver of efficacy for this class is
unknown, a critical length of time or magnitude of AUC or Cmax
above MIC may not have been achieved. It is also possible that
the PK of these compounds is affected by TB infection (in the
mice used in the efficacy study) or that the compounds
distributed poorly to the lungs and spleens of the infected mice;
further efficacy studies that include evaluation of plasma and
tissue concentrations of each compound over the course of
treatment may allow identification of one or more of these
limitations that later analogue development work may seek to
improve upon.
The PK properties of this class are not yet well understood.
Rat PK data for tryptanthrin itself (1a), following oral dosing,
were published very recently and indicated not unexpected PK
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growth. Mtb viability was then evaluated via determination of the
ability to produce a luminescent signal after a 28 h “recovery” in
ambient oxygen and exposure to the n-decanal (luciferase) substrate.
The LORA MIC is defined as the lowest concentration of compound
that reduces luminescence by 90% compared to untreated controls.
In Vitro ADMETox Assays. a. Aqueous Solubility. Aqueous
solubility was measured for each compound,36 in phosphate buffered
saline (PBS, pH 7.4), simulated intestinal fluid (35 mM NaCl, 85 mM
HCl, 2000 units/mL pepsin), and simulated gastric fluid (50 mM
KH2PO4, 38 mM NaOH, 10 mg/mL pancreatin, pH 7.5). Briefly, each
analogue was incubated at a final concentration of 200 μM in 2%
DMSO plus the appropriate aqueous medium, with shaking, for 24 h
at room temperature. Compound detection was performed via HPLC
with photodiode array detection where the detection wavelength was
230 nm. All tests were performed in duplicate, and mean values are
presented.
b. In Vitro Absorption. In vitro absorption or transport across a
human intestinal epithelial cell line (TC7, a subclone of Caco-2,
CEREP) monolayer was determined as previously described.37,38 In
brief, to determine permeability in the apical (A) to basolateral (B)
direction, each analogue was provided to the donor (A) side of the
monolayer at 10 μM in 1% DMSO in Hank’s balanced salt solution
(Invitrogen) plus 5 mM 2-(N-morpholino)ethanesulfonic acid
(Sigma) (HBSS-MES) at pH 6.5; the receiver (B) compartment was
maintained at pH 7.4. Samples were taken from the A and B
compartments at the start of the assay and from the B compartment
following incubation with shaking at 37 °C for 1 h. Sample analysis
was via HPLC-MS/MS. For measurements of permeability in the B to
A direction each analogue was provided to the B compartment at 10
μM in 1% DMSO in HBSS plus 2-(2-hydroxyethyl)piperazine-N′-(2-
ethanesulfonic acid) (HEPES, Sigma) pH 7.4; the A compartment was
maintained at pH 6.5. Samples were taken from the A and B
compartments at the start of the assay and from the A compartment
following incubation with shaking at 37 °C for 40 min. Sample analysis
was via HPLC-MS/MS. Measurements were made in duplicate, and
mean values are presented.
rats. Compound 6 exhibited MICs of 0.5 μg/mL (MABA) and
11.5 μg/mL (LORA) and an oral bioavailability of 30% in rats,
the highest among the analogues evaluated. Compound 5b
demonstrated MICs of 0.14 μg/mL (MABA) and 24.0 μg/mL
(LORA) and an oral bioavailability of 8% in the rat. Both
compounds failed to demonstrate efficacy against acute murine
TB at doses up to 400 mg/kg daily, given orally for 4 weeks.
The exact cause of this gap between the in vitro and in vivo
activities is not yet known but may be due to insufficient plasma
and/or target tissue concentrations. Despite the lack of efficacy
of the compounds tested, we have shown that it is possible to
synthesize compounds in this series with superior potency
against Mtb than tryptanthrin (1a) and with a range of ADME
and PK profiles compared with tryptanthrin. Further, it has
been shown for the first time that ring A can be saturated or the
C-11 carbonyl can be converted to a methylene group while
retaining antitubercular activity. This may shed light on the
possible mode of action of this class of compounds against Mtb
since the stereoelectronic properties of the molecules were
altered considerably. Against permeabilized E. coli, the
tryptanthrin analogues were mainly bacteriostatic at 6−40
μM, although cell viability decreased with prolonged exposure.4
In spite of the numerous biological activities reported as
mentioned earlier, a careful review of the published data
indicates there are only a handful of cases where in vivo efficacy
was observed when a tryptanthrin analogue was given
orally.11,15
EXPERIMENTAL SECTION
■
MIC Determinations. The minimal inhibitory concentration of
each compound against Mtb H37Rv was determined by the microplate
Alamar Blue assay.35 Mtb was grown in 10 mL of Middlebrook 7H9
broth (Difco) supplemented with 0.2% (v/v) glycerol (Sigma), 1.0 g
of Casitone (Difco) per liter, 10% (v/v) OADC (oleic acid, albumin,
dextrose, catalase; Difco), and 0.05% (v/v) Tween 80 (Sigma), until
its optical density at 600 nm reached 0.4. Initial compound solutions
were prepared in DMSO (Sigma), and 2-fold serial dilutions were
made in 7H9 broth in microplates. The culture was diluted 1:50 in
7H9 and was inoculated to yield 2 × 105 CFU/mL in plate wells. The
plates containing diluted compounds and Mtb inoculum were
incubated at 37 °C for 7 days, and then 20 μL of Alamar Blue
solution (Serotec) was added to each well. A color change from blue
to pink was observed after 24 h incubation, and the MIC was defined
as the lowest concentration of compounds that inhibited a color
change.
c. LogD. The partition coefficient between n-octanol and
phosphate-buffered saline pH 7.4 was measured for each analogue39
following 1 h incubation with shaking at room temperature. Analysis
was by HPLC with photodiode assay detection. LogD was calculated
as the log10 of the amount of compound in the organic phase divided
by the amount of compound in the aqueous phase.
d. Metabolic Stability in Human Liver Microsomes. Metabolic
stability in human liver microsomes was determined as described
previously.40 Each analogue was incubated at 1 μM with pooled, mixed
gender human liver microsomes that contained 0.3 mg/mL protein
(Xenotech), 1 mM NADP (Sigma), 5 mM D-glucose-6-phosphate
(Sigma), 1 U/mL glucose-6-phosphate dehydrogenase (Sigma), 0.6%
methanol, and 0.6% acetonitrile in phosphate buffer pH 7.4 for 1 h at
37 °C. The parent compound concentration was determined at the
start and end of the incubation period by HPLC-MS/MS. Measure-
ments were performed in duplicate, and mean values are presented.
e. Inhibition of CYP Isoenzymes 2C9, 2C19, and 3A4. Inhibition of
human recombinant cytochrome P450 (CYP) isoenzymes 2C19,
2C19, and 3A4 by each analogue was determined.41−43 Specifically,
each analogue, at 10 μM, was incubated at 37 °C with either CYP2C9
for 80 min (15 pmol/mL, BD Biosciences), CYP 2C19 for 60 min (10
pmol/mL, BD Biosciences), or CYP3A4 for 30 min (2.5 pmol/mL,
Invitrogen) in 1.3 mM NADP (Sigma), 3.3 mM D-glucose-6-
phosphate (Sigma), 0.4 U/mL glucose-6-phosphate dehydrogenase
(Sigma), and 0.4 mg/mL bovine serum albumin. Probe substrates
included in the assays were 50 μM 7-methoxy-4-trifluoromethylcou-
marin (Sigma) for CYP2C9; 25 μM 3-cyano-7-ethoxycoumarin
(Molecular Probes) for CYP2C19, and 50 μM 7-benzyloxy-4-
(trifluoromethyl)coumarin (Discovery Labware) for CYP3A4. Follow-
ing the incubation period, fluorimetric quantitation of the following
metabolites was performed and % inhibition of each CYP by each test
analogue calculated: 7-hydroxy-4-trifluoromethylcoumarin (Sigma) for
CYP2C9 and CYP3A4; 3-cyano-7-hydroxycoumarin (Molecular
Probes) for CYP2C19.
Serum Shift MIC. The human serum (PAA Laboratories) was
added to Middlebrook 7H9 medium to make 40% serum 7H9 broth.
The MIC at 40% serum was determined by the same method as above
except that the incubation period was 14 days. The minimal
bactericidal concentration was determined by the agar dilution
susceptibility test. Twofold serial dilutions of test compounds were
made in Middlebrook 7H10 agar medium (Difco) in plastic quadrant
Petri dishes, and separate 10−2 and 10−4 dilutions of the frozen Mtb
H37Rv stock of known CFU were prepared. The diluted stocks were
inoculated onto the control quadrants and onto each of the
compound-containing quadrants. The plates were incubated at 37
°C for 3 weeks; then the colonies were counted and the MBCs were
determined.
In Vitro Activity against Nonreplicating Mtb. MIC values
against nonreplicating Mtb, under low oxygen conditions, were
measured using the low oxygen recovery assay.31 Briefly, Mtb H37Rv
transfected with the plasmid pFCA-luxAB was adapted to low oxygen
through gradual, monitored, self-depletion of oxygen during culture in
a sealed flask with slow stirring, then exposed for 10 days to test
compounds in 96-well microplates that were maintained under an
anaerobic environment using an Anoxomat system, thus precluding
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f. MicroAmes Reverse Mutation Assay. A MicroAmes assay was
performed for each compound to determine their potential to induce a
positive response in a full Ames test. Two Ames Salmonella tester
strains were used: TA98, which detects frameshift mutations, and
TA100, which detects base pair substitutions. Briefly, each tester strain
was preincubated both with and without an Aroclor-induced rat liver
S9 fraction in duplicate in multiwell plates with six concentrations
(0.25−250 μg equivalents/well) of each compound. Four concen-
trations of each positive control mutagen (in duplicate) and six
replicates of vehicle controls (DMSO) were also used. The positive
control without S9 was 4-nitroquinoline-N-oxide. The positive control
with S9 was 2-aminoanthracene. Following preincubation, treated
bacteria were added to the agar wells and incubated. Cultures were
examined for signs of cytotoxicity (decreased spontaneous revertant
colonies and/or background lawn), compound precipitation, and
number of mutant colonies. A positive response was defined as the
appearance of at least 2-fold revertant colonies for TA100 and at least
3-fold for TA98 over the control revertant colonies, where there were
at least six colonies per well. Concentration−response relationships
were also taken into account.
g. Plasma Protein Binding. The proportion of each analogue
bound to human plasma proteins was determined via equilibrium
dialysis as described previously.44 In brief, each analogue was
incubated at 10 μM in 1% DMSO for at least 8 h at 37 °C in
human plasma (Bioreclamation) on the sample side of a 12−14 K Mw
cutoff dialysis membrane, with 0.05 M phosphate buffer pH 7.5 at the
dialysate side. Following incubation, sample analysis was via HPLC
with photodiode assay detection at 230 nm. Measurements were made
in duplicate, and means are presented.
serial dilutions on the Middlebrook 7H11 agar (Difco). For
inoculation of Mtb into mice, bacterial suspensions were sonicated
briefly in a sonic bath and diluted with PBS to reach the desired
numbers.
c. Mtb Infection and Bacterial Counts. Mice were challenged by
aerosol exposure with Mtb H37Rv using an inhalation device (Glas-
Col, Terre Haute, IN, USA) calibrated to deliver about 300 bacteria
into the lungs. Five mice were sacrificed for bacterial count the next
day to count initial infection dose per organ. Drug treatment started
on the 10th day postinfection. Compound 6 was suspended in 30%
HP-beta-CD in sterile water, and 5b was suspended in DMSO
(Sigma)/PEG400 (polyethylene glycol 400, Sigma)/HP-beta-CD
(30% in sterile water) (2:2:6) solution. Rifampin (Sigma) was
dissolved in sterile water. The drug was given to mice daily by gavage 5
days a week for 4 weeks. The mice of 400 mg/kg/day dose groups
were given a 200 mg/kg dose twice daily. Mice were sacrificed for
bacterial count at the end of a 4-week drug treatment. The numbers of
viable bacteria in the lung and spleen were determined by plating serial
dilution of whole organ homogenates on Middlebrook 7H11 agar.
Colonies were counted after 3−4 weeks of incubation at 37 °C.
Chemistry. General Procedure for the Synthesis of
Tryptanthrins (1). A solution of isatoic anhydride (1 mmol), isatin
(1 mmol), and Et3N (5 mmol) in toluene (5 mL) was refluxed for 5−6
h. The reaction mixture was quenched with H2O and then extracted
with EtOAc (2×). The organic layers were dried with MgSO4, filtered,
and concentrated. The reaction mixture was purified via column
chromatography (eluted with EtOAc/hexanes) to give tryptanthrins.
Indolo[2,1-b]quinazoline-6,12-dione (1a): yellow solid (83%);
1H NMR (300 MHz, DMSO-d6) δ 7.47 (1H, t, J = 7.4 Hz), 7.70−7.76
PK Studies in Rats. Plasma exposure to each analogue was
evaluated following both intravenous (iv) and oral (po) administration
of single doses of each formulated compound to male Sprague−
Dawley rats (n = 3). For iv administration, compounds were prepared
as solutions in polyethyleneglycol 300/DMSO (50:50, v/v) and
administered to achieve a single 5 mg/kg body weight dose. For po
administration, compounds were prepared as suspensions or solutions
in 0.5% carboxymethylcellulose/Tween 80, and a single 20 mg/kg
body weight dose was delivered via oral gavage. Blood was collected at
0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h (iv) and at 0.25, 0.5, 1, 1.5, 2, 4, 8,
and 24 h (po) and processed to plasma. Bioanalysis was performed via
LC-MS, and PK analyses of the plasma concentration−time
relationships for the compounds were performed using PK Solutions
(Summit PK). A noncompartmental model was used to calculate PK
parameters, which are reported in Table 5 as means.
PK Studies in Mice. Plasma levels of each analogue were measured
following po administration of single doses of each formulated
compound to female C57BL/6 mice, with three mice used per
compound per time point. Compounds were prepared in 0.1% sodium
lauryl sulfate and in 20% DMSO, 20% polyethylene glycol 400
(PEG400), and 60% (30% HP-beta-CD) (for 5b); in 0.5% methyl
cellulose and in 30% HP-beta-CD (for 6); or in 0.5% methyl cellulose
only (for 1x) and administered via oral gavage to achieve a single dose
of 40 mg/kg body weight. Blood was collected at 30 min and 1, 1.5, 2,
4, 8, and 24 h and processed to plasma. Bioanalysis was performed via
LC-MS, and PK analyses of the plasma concentration−time
relationships for the compounds were performed using PK Solutions
(Summit PK). A noncompartmental model was used to calculate PK
parameters, which are reported in Table 5 as means.
In Vivo Efficacy Determination. a. Mice. Specific pathogen-free
female C57BL/6 mice at 5−6 weeks of age were purchased from Japan
SLC, Inc. (Shijuoka, Japan), maintained under barrier conditions in a
BL-3 biohazard animal room at Yonsei University Medical Research
Center, and fed a sterile commercial mouse diet and water ad libitum.
b. Bacteria. Mtb Erdman (ATCC 35801) was used and grown for
about 10 days at 37 °C as a surface pellicle on Sauton medium
enriched with 0.4% sodium glutamate (Sigma) and 3.0% glycerin
(Sigma). The surface pellicles were collected and disrupted with 6 mm
glass beads by gentle vortexing. After the clumps settled, the upper
suspension was collected and aliquots were stored at −70 °C until
used. After thawing, viable organisms were then counted by plating
(1H, m), 7.83−7.89 (2H, m), 7.94 (2H, d, J = 3.6 Hz), 8.31 (1H, d, J =
7.8 Hz), 8.47 (1H, d, J = 7.8 Hz); 13C NMR (75 MHz, CDCl3) δ
117.9, 121.9, 123.7, 125.3, 127.1, 127.5, 130.2, 130.7, 135.1, 138.2,
144.3, 146.3, 146.6, 158.0, 182.5; HRESIMS m/z 248.0583 [M+]
(calcd for C15H8N2O2, 248.0586).
8-Nitroindolo[2,1-b]quinazoline-6,12-dione (1b): yellow solid
1
(79%); H NMR (300 MHz, DMSO-d6) δ 7.76−7.81 (1H, m), 8.01
(2H, d, J = 3.7 Hz), 8.36 (1H, d, J = 7.7 Hz), 8.56 (1H, s), 8.66−8.75
(2H, m); HRESIMS m/z 293.0419 [M+] (calcd for C15H7N3O4,
293.0437).
Ethyl 6,12-dioxo-6,12-dihydroindolo[2,1-b]quinazoline-8-
carboxylate (1c): yellow solid (34%); 1H NMR (300 MHz,
CDCl3) δ 1.43 (3H, t, J = 7.1 Hz), 4.43 (2H, q, J = 7.1 Hz), 7.67−
7.73 (1H, m), 7.85−7.90 (1H, m), 8.04 (1H, dd, J = 8.0, 0.6 Hz),
8.44−8.50 (2H, m), 8.58−8.58 (1H, m), 8.71 (1H, dd, J = 8.4, 0.5
Hz); HRESIMS m/z 320.0794 [M+] (calcd for C18H12N2O4,
320.0797).
8-Chloroindolo[2,1-b]quinazoline-6,12-dione (1d): yellow
solid (87%); 1H NMR (300 MHz, DMSO-d6) δ 7.72−7.78 (1H,
m), 7.92 (1H, dd, J = 8.5, 2.2 Hz), 7.95−7.97 (3H, m), 8.33 (1H, dt, J
= 7.8, 0.9 Hz), 8.47 (1H, d, J = 8.4 Hz); HRESIMS m/z 282.0208
[M+] (calcd for C15H7ClN2O2, 282.0196).
8-Chloro-2-((2-(dimethylamino)ethyl)(ethyl)amino)indolo-
[2,1-b]quinazoline-6,12-dione (1e). To a solution of 1f (200 mg,
0.670 mmol) and K2CO3 (180 mg, 1.33 mmol) in DMF (10 mL) was
added N,N-dimethyl-N′-ethylethylenediamine (0.13 mL, 0.80 mmol),
and the reaction mixture was refluxed for 14 h. The reaction mixture
was quenched with H2O and then extracted with EtOAc (3×). The
organic layers were dried with MgSO4, filtered, and concentrated. The
reaction mixture was purified via column chromatography (eluted with
5% MeOH in CH2Cl2) to give the title compound as a yellow solid
(140 mg, 53%). 1H NMR (300 MHz, MeOH-d4) δ 1.17 (3H, t, J = 6.9
Hz), 2.23 (6H, s), 2.2−2.47 (2H, m), 3.54−3.59 (4H, m), 7.29 (1H,
dd, J = 9.5, 3.9 Hz), 7.38 (1H, d, J = 3.0 Hz), 7.74 (1H, d, J = 9.0 Hz),
7.86−7.90 (2H, m), 8.47 (1H, d, J = 9.2 Hz); LC-MS m/z 397.11 [M
+ H+].
8-Chloro-2-fluoroindolo[2,1-b]quinazoline-6,12-dione (1f):
1
yellow solid (76%); H NMR (300 MHz, DMSO-d6) δ 7.85 (1H,
td, J = 8.6, 2.8 Hz), 7.91−7.97 (2H, m), 8.02−8.08 (2H, m), 8.46 (1H,
d, J = 8.6 Hz); HRESIMS m/z 300.0075 [M+] (calcd for
C15H6ClFN2O2, 300.0102).
J
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8-Chloro-3-fluoroindolo[2,1-b]quinazoline-6,12-dione (1g):
yellow solid (77%); H NMR (300 MHz, DMSO-d6) δ 7.59−7.65
(1H, m), 7.83 (1H, dd, J = 7.7, 2.2 Hz), 7.90−8.01 (2H, m), 8.38 (1H,
dd, J = 8.9, 6.1 Hz), 8.45 (1H, d, J = 8.6 Hz); 13C NMR (75 MHz,
CDCl3) δ 116.52 (d, JC−F = 22.5 Hz), 118.86 (d, JC−F = 23.0 Hz),
119.18, 120.25, 122.87, 130.12 (d, JC−F = 10.25 Hz), 133.49, 137.87,
added ammonium formate (430 mg, 6.82 mmol), and the reaction
mixture was degassed under an argon atmosphere. To the reaction
mixture was added Pd(OH)2 on carbon (200 mg, 1.70 mmol), and the
reaction mixture was stirred at rt for 2 h. The reaction mixture was
filtered and then extracted with EtOAc (3×). The organic layers were
dried with MgSO4, filtered, and concentrated. The reaction mixture
was purified via column chromatography (eluted with 5% MeOH in
CH2Cl2) to give the title compound as a black solid (540 mg, 60%):
1H NMR (300 MHz, DMSO-d6) δ 5.66 (2H, s), 6.96−7.00 (2H, m),
1
144.40, 144.90, 148.78 (d, JC−F = 12.62 Hz), 157.15, 166.71 (d, JC−F
=
255.8 Hz), 181.17; HRESIMS m/z 300.0092 [M+] (calcd for
C15H6ClFN2O2, 300.0102).
2-Bromoindolo[2,1-b]quinazoline-6,12-dione (1h): yellow
solid (90%); 1H NMR (300 MHz, DMSO-d6) δ 7.47−7.52 (1H,
m), 7.88 (1H, s), 7.91 (2H, d, J = 2.1 Hz), 8.12 (1H, dd, J = 8.5, 2.3
Hz), 8.40 (1H, d, J = 2.2 Hz), 8.46 (1H, d, J = 7.8 Hz); HRESIMS m/
z 325.9694 [M+] (calcd for C15H7BrN2O2, 325.9691).
7.67−7.72 (1H, m), 7.88−7.90 (1H, m), 7.90 (1H, d, J = 1.0 Hz), 8.13
(1H, d, J = 9.1 Hz), 8.25−8.28 (1H, m); HRESIMS m/z 263.0693
[M+] (calcd for C15H9N3O2, 263.0695).
N-(6,12-Dioxo-6,12-dihydroindolo[2,1-b]quinazolin-8-yl)-
methanesulfonamide (1p). To a solution of 1o (540 mg, 2.05
mmol) in MeOH (20 mL) was added Et3N (430 mg, 6.82 mmol) and
MsCl (0.20 mL, 2.5 mmol) at 0 °C. The reaction mixture was stirred
at rt for 2 h, quenched with H2O, and then extracted with EtOAc
(3×). The organic layers were dried with MgSO4, filtered, and
concentrated. The reaction mixture was purified via column
chromatography (eluted with 3% MeOH in CH2Cl2) to give the
8-Methoxyindolo[2,1-b]quinazoline-6,12-dione (1i): red solid
1
(73%); H NMR (300 MHz, DMSO-d6) δ 3.89 (3H, s), 7.28−7.33
(1H, m), 7.38 (1H, d, J = 2.5 Hz), 7.64−7.69 (1H, m), 7.81−7.86
(1H, m), 8.02 (1H, d, J = 8.2 Hz), 8.41−8.44 (1H, m), 8.52 (1H, d, J =
8.8 Hz); HRESIMS m/z 278.0694 [M+] (calcd for C16H10N2O3,
278.0691).
1
2-Nitroindolo[2,1-b]quinazoline-6,12-dione (1j): yellow solid
(82%); 1H NMR (300 MHz, CDCl3) δ 7.53 (1H, t, J = 7.5 Hz), 7.88−
7.96 (2H, m), 8.18 (1H, d, J = 8.8 Hz), 8.49 (1H, d, J = 7.7 Hz), 8.67
(1H, dd, J = 8.8, 2.7 Hz), 8.96 (1H, d, J = 2.7 Hz); HRESIMS m/z
293.0431 [M+] (calcd for C15H7N3O4, 293.0437).
title compound as a gray solid (850 mg, 72%): H NMR (300 MHz,
DMSO-d6) δ 3.58 (3H, s), 7.73−7.78 (1H, m), 7.95−7.97 (2H, m),
8.02 (1H, dd, J = 8.5, 2.3 Hz), 8.13 (1H, d, J = 2.1 Hz), 8.34 (1H, d, J
= 7.8 Hz), 8.55 (1H, d, J = 8.6 Hz); HRESIMS m/z 341.0457 [M+]
(calcd for C16H11N3O4S, 341.0470).
9-(Piperazin-1-yl)indolo[2,1-b]quinazoline-6,12-dione (1k).
Step 1: To a solution of 1m (260 mg, 0.920 mmol) in 1-methyl-2-
pyrrolidinone (NMP; 5 mL) was added tert-butyl-1-piperazine
carboxylate (210 mg, 1.11 mmol), and the reaction mixture was
heated at 70 °C for 2 h. The reaction mixture was quenched with H2O
and then extracted with EtOAc (3×). The organic layers were dried
with MgSO4, filtered, and concentrated. The reaction mixture was
purified via column chromatography (eluted with 5% MeOH in
CH2Cl2) to give a Boc-protected version of the title compound as a
yellow solid (320 mg, 80%). Step 2: To a solution of the Boc-
protected title compound (300 mg, 0.790 mmol) in CH2Cl2 (10 mL)
was added TFA (5 mL), and the reaction mixture was stirred at rt for
30 min. The reaction mixture was quenched with NaHCO3 and then
extracted with CHCl3 (3×). The organic layers were dried with
MgSO4, filtered, and concentrated. The reaction mixture was purified
via column chromatography (eluted with 5% MeOH in CH2Cl2) to
give the title compound as a red solid (160 mg, 69%): 1H NMR (300
MHz, DMSO-d6) δ 3.35−3.63 (8H, m), 6.88 (1H, dd, J = 8.9, 2.1 Hz),
7.67 (1H, d, J = 8.8 Hz), 7.91−7.97 (4H, m), 8.28 (1H, d, J = 7.5 Hz);
HRESIMS m/z 332.1250 [M+] (calcd for C19H16N4O2, 332.1273).
9-(Piperazin-1-yl)indolo[2,1-b]quinazoline-6,12-dione hy-
drochloride (1l). To a solution of 1k (100 mg, 0.300 mmol) in
MeOH/CH2Cl2 (1:1; 40 mL) was added a 4 M solution of HCl in
dioxane (0.11 mL, 0.44 mmol), the reaction mixture was stirred at rt
for 3 days, and a precipitate was formed. The solid precipitate was
filtered and washed with ether. The title compound was obtained as a
red solid (93 mg, 84%): 1H NMR (300 MHz, D2O) δ 3.20−3.34 (8H,
m), 5.99−6.03 (1H, m), 6.71 (1H, s), 6.83 (1H, d, J = 8.9 Hz), 7.37
(3H, m), 7.69 (1H, t, J = 6.9 Hz); LC-MS m/z 333.0 [M + H+].
9-Chloroindolo[2,1-b]quinazoline-6,12-dione (1m): brown
8-Bromoindolo[2,1-b]quinazoline-6,12-dione (1q): yellow
solid (67%); H NMR (300 MHz, CDCl3) δ 7.66−7.71 (1H, m),
7.83−7.90 (2H, m), 8.01−8.04 (2H, m), 8.42 (1H, dd, J = 7.8, 1.0
Hz), 8.52 (1H, d, J = 8.5 Hz); 13C NMR (75 MHz, CDCl3) δ 119.5,
120.7, 123.3, 123.6, 127.6, 128.2, 130.5, 130.9, 135.3, 140.6, 143.7,
144.9, 146.5, 157.9, 181.2; HRESIMS m/z 325.9689 [M+] (calcd for
C15H7BrN2O2, 325.9691).
1
8-(Trifluoromethoxy)indolo[2,1-b]quinazoline-6,12-dione
1
(1r): yellow solid (67%); H NMR (300 MHz, CDCl3) δ 7.60−7.63
(1H, m), 7.66−7.71 (1H, m), 7.75 (1H, s), 7.83−7.89 (1H, m), 8.02
(1H, d, J = 8.0 Hz), 8.42 (1H, dd, J = 7.9, 0.9 Hz), 8.68 (1H, d, J = 8.7
Hz); 13C NMR (75 MHz, CDCl3) δ 117.6, 119.4, 123.0, 123.5, 127.6,
130.5, 130.6, 130.9, 135.3, 144.0, 144.3, 146.4, 147.7, 157.8, 181.4;
HRESIMS m/z 332.0408 [M+] (calcd for C16H7F3N2O3, 332.0409).
8-Fluoroindolo[2,1-b]quinazoline-6,12-dione (1s): yellow
1
solid (74%); H NMR (300 MHz, CDCl3) δ 7.48 (1H, dd, J = 8.6,
2.7 Hz), 7.55−7.58 (1H, m), 7.65−7.71 (1H, m), 7.85 (1H, dt, J = 7.6,
1.4 Hz), 8.02 (1H, dd, J = 8.0, 0.5 Hz), 8.42 (1H, dd, J = 7.9, 0.9 Hz),
8.63 (1H, dd, J = 8.8, 4.0 Hz); 13C NMR (75 MHz, CDCl3) δ 112.0
(d, JC−F = 24.3 Hz), 119.0 (d, JC−F = 7.5 Hz), 123.4 (d, JC−F = 7.6 Hz),
124.8 (d, JC−F = 23.8 Hz), 127.5, 130.5, 130.8, 135.2, 142.4, 144.2,
146.4, 157.8, 161.1 (d, J
= 248.8 Hz), 181.7; HRESIMS m/z
C−F
266.0493 [M+] (calcd for C15H7FN2O2, 266.0492).
9-(4-Hydroxypiperidin-1-yl)indolo[2,1-b]quinazoline-6,12-
dione (1t). To a solution of 1m (340 mg, 1.21 mmol) in NMP (5
mL) was added 4-hydroxypiperidine (150 mg, 1.45 mmol) at rt. The
reaction mixture was stirred at 90 °C for 2 h. The reaction mixture was
concentrated and dissolved in MeOH. To the reaction mixture was
added ether to precipitate solids, which were filtered and dried to give
the title compound as a red solid (320 mg, 76%): 1H NMR (300 MHz,
CDCl3) δ 1.99−2.02 (2H, m), 2.00−2.08 (2H, m), 3.41−3.50 (2H,
m), 3.90−3.98 (2H, m), 4.05−4.10 (1H, m), 6.72 (1H, dd, J = 8.8, 2.4
Hz), 7.63 (1H, t, J = 7.1 Hz), 7.75 (1H, d, J = 8.9 Hz), 7.80−7.85 (1H,
m), 8.02 (1H, d, J = 9.4 Hz), 8.12 (1H, d, J = 2.0 Hz), 8.38−8.40 (1H,
m); HRESIMS m/z 347.1269 [M+] (calcd for C20H17N3O3,
347.1270).
9-(3-Hydroxypyrrolidin-1-yl)indolo[2,1-b]quinazoline-6,12-
dione (1u). Using 3-pyrrolidinol instead of 4-hydroxypiperidine, the
procedure for 1t was adopted to give the title compound as a red solid
(72%): 1H NMR (300 MHz, CDCl3) δ 1.99−2.04 (1H, m), 2.19−2.24
(1H, m), 3.54−3.75 (4H, m), 4.71 (1H, s), 6.38−6.42 (1H, m), 7.58−
7.64 (1H, m), 7.72 (2H, d, J = 8.8 Hz), 7.79−7.84 (1H, m), 8.01 (1H,
d, J = 7.6 Hz), 8.36 (1H, d, J = 8.5 Hz); HRESIMS m/z 333.1110
[M+] (calcd for C19H15N3O3, 333.1113).
1
solid (77%); H NMR (300 MHz, CDCl3) δ 7.41 (1H, dd, J = 8.1,
1.6 Hz), 7.67−7.72 (1H, m), 7.83−7.90 (2H, m), 8.03 (1H, d, J = 8.1
Hz), 8.44 (1H, dd, J = 7.8, 0.9 Hz), 8.69 (1H, d, J = 1.2 Hz);
HRESIMS m/z 282.0179 [M+] (calcd for C15H7ClN2O2, 282.0196);
anal. C 63.73, H 2.50, N 9.91%, calcd for C15H7ClN2O2, C 63.67, H
2.48, N 9.77%.
Ethyl 4-methoxy-6,12-dioxo-6,12-dihydroindolo[2,1-b]-
quinazoline-8-carboxylate (1n): yellow solid (33%); 1H NMR
(300 MHz, CDCl3) δ 1.42 (3H, t, J = 7.1 Hz), 4.07 (3H, s), 4.42 (2H,
q, J = 7.1 Hz), 7.32 (1H, d, J = 9.2 Hz), 7.63 (1H, t, J = 8.0 Hz), 8.01
(1H, dd, J = 7.9, 1.1 Hz), 8.47 (1H, dd, J = 8.4, 1.7 Hz), 8.57 (1H, d, J
= 1.5 Hz), 8.70 (1H, d, J = 8.4 Hz); HRESIMS m/z 350.0903 [M+]
(calcd for C19H14N2O5, 350.0903).
8-Aminoindolo[2,1-b]quinazoline-6,12-dione (1o). To a sol-
ution of 1b (1.00 g, 3.41 mmol) in MeOH/THF (4:1; 25 mL) was
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9-(Pyrrolidin-1-yl)indolo[2,1-b]quinazoline-6,12-dione (1v).
8-Fluoroindolo[2,1-b]-4-azaquinazoline-6,12-dione (4f): yel-
low solid (33%); H NMR (300 MHz, DMSO-d6) δ 7.69−7.76 (2H,
m), 7.82 (1H, dd, J = 7.1, 2.7 Hz), 8.30 (1H, dd, J = 8.8, 4.1 Hz), 8.69
(1H, dd, J = 7.9, 1.9 Hz), 9.06 (1H, dd, J = 4.5, 1.9 Hz); HRESIMS m/
z 267.0442 [M+] (calcd for C14H6FN3O2, 267.0444).
9-Chloroindolo[2,1-b]-4-azaquinazoline-6,12-dione (4g): yel-
low solid (54%); 1H NMR (300 MHz, CDCl3) δ 7.42−7.46 (1H, m),
7.60−7.65 (1H, m), 7.86−7.89 (1H, m), 8.65 (1H, s), 8.73−8.78 (1H,
m), 9.09−9.12 (1H, m); HRESIMS m/z 283.0145 [M+] (calcd for
C14H6ClN3O2, 283.0149).
1
Using pyrrolidine instead of 4-hydroxypiperidine, the procedure for
1
1t was adopted to give the title compound as a red solid (73%): H
NMR (300 MHz, CDCl3) δ 2.10 (4H, s), 3.53 (4H, s), 6.39 (1H, d, J
= 8.8 Hz), 7.60 (1H, t, J = 7.1 Hz), 7.70 (2H, d, J = 8.7 Hz), 7.80 (1H,
t, J = 7.0 Hz), 7.99 (1H, d, J = 7.7 Hz), 8.36 (1H, d, J = 7.3 Hz);
HRESIMS m/z 317.1162 [M+] (calcd for C19H15N3O2, 317.1164).
9-(Piperidin-1-yl)indolo[2,1-b]quinazoline-6,12-dione (1w).
Using piperidine instead of 4-hydroxypiperidine, the procedure for
1
1t was adopted to give the title compound as a red solid (71%): H
NMR (300 MHz, CDCl3) δ 1.71 (6H, s), 3.57 (4H, s), 6.62 (1H, d, J
= 8.6 Hz), 7.59 (1H, t, J = 7.4 Hz), 7.67 (1H, d, J = 8.7 Hz), 7.79 (1H,
d, J = 7.4 Hz), 7.99 (2H, d, J = 7.8 Hz), 8.34 (1H, d, J = 7.8 Hz); 13C
NMR (75 MHz, CDCl3) δ 24.2, 25.5, 48.6, 100.5, 110.2, 110.8, 123.3,
127.1, 127.7, 129.2, 130.1, 134.8, 146.9, 149.1, 156.8, 158.6, 177.7;
HRESIMS m/z 331.1304 [M+] (calcd for C20H17N3O2, 331.1321).
8-(Piperidine-1-carbonyl)indolo[2,1-b]quinazoline-6,12-
dione (1x) (ref 45): yellow solid (50%); 1H NMR (300 MHz,
CDCl3) δ 1.46−1.70 (6H, m), 3.32−3.75 (4H, m), 7.65−7.71 (1H,
m), 7.84−7.71 (3H, m), 8.04 (1H, d, J = 8.0 Hz), 8.44 (1H, d, J = 7.6
Hz), 8.67 (1H, d, J = 8.1 Hz); LC-MS m/z 360.0 [M + H+].
8-(Morpholine-4-carbonyl)indolo[2,1-b]quinazoline-6,12-
dione (1y) (ref 45): yellow solid (60%); 1H NMR (300 MHz,
CDCl3) δ 3.63 (8H, s), 7.73−7.78 (1H, m), 7.90−7.94 (2H, m), 7.97
(2H, d, J = 3.6 Hz), 8.34 (1H, d, J = 7.7 Hz), 8.53 (1H, dd, J = 7.9, 0.7
Hz); LC-MS m/z 362.1 [M + H+].
General Procedure for the Synthesis of A-Ring-Aza
Tryptanthrins (4). To a solution of 2-[1H-benzotriazole-1-yl]-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; 1 mmol),
N-methylmorpholine (1.8 mmol), and 4- or 2-aminonicotinic acid or
3-aminoisonicotinic acid (1 mmol) in DMF (3 mL) was added a
solution of isatin (0.9 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU; 2 mmol) in DMF (3 mL) over 10 min at rt. After 20 h, the
reaction mixture was quenched with 1 N citric acid aqueous solution
(10 mL). The reaction mixture was filtered, and the filtrate was
extracted with CHCl3 (3×). The organic layers were dried with
MgSO4, filtered, and concentrated. The reaction mixture was purified
via column chromatography (eluted with 5% MeOH in CH2Cl2) to
give 4- or 2- or 3-aza tryptanthrin, respectively.
tert-Butyl 4-(6,12-dioxo-6,12-dihydroindolo[2,1-b]-4-azaqui-
nazolin-9-yl)piperazine-1-carboxylate (4h). To a solution of 4g
(260 mg, 0.92 mmol) in NMP (5 mL) was added tert-butyl piperazine-
1-carboxylate (210 mg, 1.11 mmol) at rt. The red reaction mixture was
stirred at 90 °C for 2 h and was concentrated and dissolved in MeOH.
To the reaction mixture was added ether to precipitate solids, which
were filtered and dried to give the title compound as a red solid (320
mg, 80%): 1H NMR (300 MHz, CDCl3) δ 1.49 (9H, s), 3.63 (8H, d, J
= 7.8 Hz), 6.61 (1H, dd, J = 8.7, 1.3 Hz), 7.57 (1H, dd, J = 7.7, 4.6
Hz), 7.69 (1H, d, J = 8.8 Hz), 7.91 (1H, d, J = 1.2 Hz), 8.66 (1H, dd, J
= 7.6, 1.4 Hz), 9.06−9.07 (1H, m); 13C NMR (75 MHz, CDCl3) δ
28.3, 46.6, 80.5, 100.7, 110.6, 111.7, 119.1, 124.1, 127.8, 136.3, 148.4,
149.1, 154.5, 156.3, 156.7, 157.6, 158.5, 177.3; HRESIMS m/z
433.1746 [M+] (calcd for C23H23N5O4, 433.1750); anal. C 63.73, H
5.35, N 16.16%, calcd for C23H23N5O4, C 63.93, H 5.29, N 16.02%.
9-Morpholinoindolo[2,1-b]-4-azaquinazoline-6,12-dione
(4i). Using morpholine instead of tert-butyl piperazine-1-carboxylate,
the procedure for 4h was adopted to give the title compound as a red
1
solid (73%): H NMR (300 MHz, TFA-d) δ 4.45−4.74 (8H, m),
7.49−7.62 (1H, m), 8.40−8.53 (1H, m), 8.64−8.86 (2H, m), 9.67−
9.78 (1H, m), 10.00−10.11 (1H, m); HRESIMS m/z 334.1066 [M+]
(calcd for C18H14N4O3, 334.1068); anal. C 64.66, H 4.22, N 16.76%,
calcd for C18H14N4O3, C 64.66, H 4.17, N 16.48%.
9-(Piperidin-1-yl)indolo[2,1-b]-4-azaquinazoline-6,12-dione
(4j). Using piperidine instead of tert-butyl piperazine-1-carboxylate, the
procedure for 4h was adopted to give the title compound as a yellow
1
solid (71%): H NMR (300 MHz, CDCl3) δ 1.75 (6H, s), 3.62 (4H,
s), 6.68 (1H, dd, J = 9.0, 2.2 Hz), 7.55 (1H, dd, J = 7.8, 4.5 Hz), 7.71
(1H, d, J = 8.9 Hz), 8.00 (1H, d, J = 2.2 Hz), 8.69 (1H, dd, J = 7.8, 1.9
Hz), 9.06 (1H, dd, J = 4.9, 1.9 Hz); HRESIMS m/z 332.1251 [M+]
(calcd for C19H16N4O2, 332.1273); anal. C 68.34, H 5.06, N 16.39%,
calcd for C19H16N4O2, C 68.66, H 4.85, N 16.86%.
8-(Trifluoromethoxy)indolo[2,1-b]-2-azaquinazoline-6,12-
1
dione (4a): yellow solid (30%); H NMR (300 MHz, DMSO-d6) δ
7.92 (2H, d, J = 5.1 Hz), 7.96 (1H, d, J = 8.2 Hz), 8.56 (1H, d, J = 8.6
Hz), 9.03 (1H, d, J = 5.4 Hz), 9.50 (1H, s); 13C NMR (75 MHz,
DMSO-d6) δ 117.9, 118.0, 118.3, 118.3, 118.7, 121.7, 122.6, 123.6,
130.3, 144.2, 146.5, 148.5, 149.8, 151.9, 154.9, 157.1, 180.7; LC-MS
m/z 334.1 [M + H+].
8-Bromoindolo[2,1-b]-4-azaquinazoline-6,12-dione (4k): yel-
1
low solid (65%); H NMR (300 MHz, DMSO-d6) δ 7.91 (1H, d, J =
5.4 Hz), 8.06−8.12 (2H, m), 8.39 (1H, d, J = 8.4 Hz), 9.02 (1H, d, J =
5.4 Hz), 9.48 (1H, s); HRESIMS m/z 326.9645 [M+] (calcd for
C14H6BrN3O2, 326.9643).
8-Bromoindolo[2,1-b]-2-azaquinazoline-6,12-dione (4b): red
1
solid (55%); H NMR (300 MHz, DMSO-d6) δ 7.92 (1H, d, J = 5.4
9-Chloroindolo[2,1-b]-2-azaquinazoline-6,12-dione (4l): yel-
Hz), 8.07−8.13 (2H, m), 8.39 (1H, d, J = 8.8 Hz), 9.02 (1H, dd, J =
5.4, 0.9 Hz), 9.49 (1H, s); HRESIMS m/z 326.9643 [M+] (calcd for
C14H6BrN3O2, 326.9629).
8-Chloroindolo[2,1-b]-4-azaquinazoline-6,12-dione (4c): yel-
low solid (59%); 1H NMR (300 MHz, CDCl3) δ 7.63 (1H, dd, J = 7.9,
4.5 Hz), 7.76 (1H, dd, J = 8.5, 2.2 Hz), 7.89 (1H, d, J = 2.1 Hz), 8.57
(1H, d, J = 8.5 Hz), 8.76 (1H, dd, J = 7.9, 1.9 Hz), 9.11 (1H, dd, J =
4.5, 1.9 Hz); HRESIMS m/z 283.0149 [M+] (calcd for C14H6ClN3O2,
283.0149); anal. C 59.28, H 2.13, N 14.81%, calcd for C14H6ClN3O2,
C 59.18, H 2.31, N 14.80%.
8-(Trifluoromethoxy)indolo[2,1-b]-3-azaquinazoline-6,12-
dione (4d): yellow solid (15%); 1H NMR (300 MHz, CDCl3) δ 7.66
(1H, dd, J = 8.7, 2.5 Hz), 7.79 (1H, d, J = 1.3 Hz), 8.22 (1H, d, J = 5.1
Hz), 8.69 (1H, d, J = 8.7 Hz), 8.92 (1H, d, J = 5.1 Hz), 9.41 (1H, s);
13C NMR (75 MHz, CDCl3) δ 117.9, 119.5, 119.6, 123.1, 128.9, 130.7,
1
low solid (34%); H NMR (300 MHz, DMSO-d6) δ 7.92 (1H, d, J =
7.6 Hz), 8.07−8.13 (2H, m), 8.39 (1H, d, J = 8.3 Hz), 9.01−9.03 (1H,
m), 9.49 (1H, s); HRESIMS m/z 283.0142 [M+] (calcd for
C14H6ClN3O2, 283.0149).
tert-Butyl 4-(6,12-dioxo-6,12-dihydroindolo[2,1-b]-2-azaqui-
nazolin-9-yl)piperazine-1-carboxylate (4m). Using 4l instead of
4g, the procedure for 4h was adopted to give the title compound as a
1
red solid (70%): H NMR (300 MHz, CDCl3) δ 1.50 (9H, s), 3.65
(8H, s), 6.70 (1H, d, J = 7.2 Hz), 7.77 (1H, d, J = 8.8 Hz), 7.84 (1H, d,
J = 5.5 Hz), 8.04 (1H, d, J = 1.7 Hz), 8.97 (1H, d, J = 5.5 Hz), 9.61
(1H, s); 13C NMR (75 MHz, CDCl3) δ 28.3, 46.7, 80.6, 101.0, 110.7,
111.4, 118.2, 122.7, 128.1, 148.8, 150.7, 152.4, 154.4, 154.8, 157.0,
157.7, 177.3; HRESIMS m/z 433.1750 [M+] (calcd for C23H23N5O4,
433.1750).
140.9, 143.7, 145.4, 148.2, 150.4, 153.0, 156.6, 180.5; HRESIMS m/z
9-Morpholinoindolo[2,1-b]-2-azaquinazoline-6,12-dione
(4n). Using 4l instead of 4g, the procedure for 4i was adopted to give
the title compound as a red solid (71%); 1H NMR (300 MHz, CDCl3)
δ 3.60 (4H, t, J = 4.3 Hz), 3.89 (4H, t, J = 4.3 Hz), 6.73 (1H, d, J = 8.7
Hz), 7.79 (1H, d, J = 8.2 Hz), 7.84 (1H, d, J = 5.0 Hz), 8.08 (1H, s),
8.97 (1H, d, J = 5.5 Hz), 9.61 (1H, s); HRESIMS m/z 334.1067 [M+]
(calcd for C18H14N4O3, 334.1066).
333.0361 [M+] (calcd for C15H6F3N3O3, 333.0361).
8-Nitroindolo[2,1-b]-4-azaquinazoline-6,12-dione (4e): yel-
1
low solid (29%); H NMR (300 MHz, DMSO-d6) δ 7.80 (1H, dd, J
= 7.9, 4.6 Hz), 8.60 (1H, d, J = 2.3 Hz), 8.67 (1H, s), 8.75 (1H, dd, J =
3.5, 2.1 Hz), 8.77 (1H, d, J = 8.7 Hz), 9.12 (1H, dd, J = 4.6, 1.9 Hz);
HRESIMS m/z 294.0385 [M+] (calcd for C14H6N4O4, 294.0389).
L
dx.doi.org/10.1021/np3007167 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
9-(Piperidin-1-yl)indolo[2,1-b]-2-azaquinazoline-6,12-dione
(4o). Using 4l instead of 4g, the procedure for 4j was adopted to give
the title compound as a red solid (64%): 1H NMR (300 MHz, CDCl3)
δ 1.76 (6H, s), 3.65 (4H, s), 6.70 (1H, dd, J = 9.0, 2.3 Hz), 7.74 (1H,
d, J = 9.0 Hz), 7.83 (1H, d, J = 5.3 Hz), 8.04 (1H, d, J = 2.2 Hz), 8.95
(1H, d, J = 5.5 Hz), 9.61 (1H, s); HRESIMS m/z 332.1273 [M+]
(calcd for C19H16N4O2, 332.1273).
C15H9ClN2O, 268.0403); anal. C 67.29, H 3.25, N 10.18%, calcd for
C15H9ClN2O, C 67.05, H 3.38, N 10.43%.
Indolo[2,1-b]quinazolin-6(12H)-one (5c): red solid (81%); H
NMR (300 MHz, CDCl3) δ 4.93 (2H, s), 6.83 (1H, d, J = 7.9 Hz),
7.04−7.09 (2H, m), 7.18−7.28 (2H, m), 7.48 (1H, dd, J = 7.5, 1.2
Hz), 7.56−7.61 (1H, m), 7.69 (1H, d, J = 7.7 Hz); LC-MS m/z 235.2
[M + H+].
1
Indolo[2,1-b]-2-azaquinazolin-6(12H)-one (5d): red solid
9-(Piperazin-1-yl)indolo[2,1-b]-2-azaquinazoline-6,12-dione
hydrochloride (4p). To a solution of 4m (100 mg, 0.23 mmol) in
NMP (5 mL) was added a 4 M solution of HCl in dioxane (0.20 mL,
0.80 mmol) at rt. The reaction mixture was stirred at rt for 24 h and
was concentrated and dissolved in MeOH. To the reaction mixture
was added ether to precipitate solids, which were filtered and dried to
1
(2%); H NMR (300 MHz, DMSO-d6) δ 5.08 (2H, s), 6.88−6.92
(1H, m), 7.17−7.23 (2H, m), 7.46−7.53 (2H, m), 7.62−7.65 (1H, m),
7.53−7.57 (1H, m); LC-MS m/z 236.1 [M + H+].
8-Chloroindolo[2,1-b]-2-azaquinazolin-6(12H)-one (5e): red
solid (12%); 1H NMR (300 MHz, CDCl3) δ 5.06 (2H, s), 6.84 (1H, d,
J = 8.4 Hz), 7.16−7.21 (1H, m), 7.45−7.48 (1H, m), 7.58−7.61 (1H,
m), 7.71 (1H, d, J = 2.1 Hz), 8.52−8.55 (1H, m); LC-MS m/z 270.0
[M + H+].
1
give the title compound as a red solid (71%): H NMR (300 MHz,
MeOH-d4) δ 3.40 (4H, t, J = 5.1 Hz), 3.83 (4H, t, J = 5.2 Hz), 7.02
(1H, dd, J = 8.9, 2.1 Hz), 7.42 (1H, d, J = 6.9 Hz), 7.72 (1H, d, J = 8.8
Hz), 7.89 (1H, d, J = 2.0 Hz), 8.46 (1H, dd, J = 6.9, 1.1 Hz), 9.06 (1H,
d, J = 0.7 Hz); HRESIMS m/z 333.1224 [M+] (calcd for
C18H16ClN5O2, 333.1226).
8-(Trichloromethoxy)indolo[2,1-b]-2-azaquinazolin-6(12H)-
1
one (5f): red solid (10%); H NMR (300 MHz, CDCl3) δ 5.04 (2H,
s), 6.95 (1H, d, J = 8.5 Hz), 7.38 (1H, d, J = 5.1 Hz), 7.53 (1H, dd, J =
8.5, 2.1 Hz), 7.63 (1H, s), 8.43 (1H, s), 8.56 (1H, d, J = 5.1 Hz); LC-
MS m/z 320.0 [M + H+].
Indolo[2,1-b]-2-azaquinazoline-6,12-dione (4q): yellow solid
(45%); 1H NMR (300 MHz, CDCl3) δ 7.47−7.49 (1H, m), 7.82−7.86
(2H, m), 7.93 (1H, d, J = 7.5 Hz), 8.60 (1H, d, J = 8.1 Hz), 9.00 (1H,
d, J = 5.4 Hz), 9.65 (1H, s); LC-MS m/z 250.1 [M + H+].
8-Chloroindolo[2,1-b]-2-azaquinazoline-6,12-dione (4r): yel-
low solid (45%); 1H NMR (300 MHz, CDCl3) δ 7.78 (1H, dd, J = 8.6,
2.1 Hz), 7.86 (1H, d, J = 5.2 Hz), 7.89 (1H, d, J = 2.0 Hz), 8.58 (1H,
d, J = 8.5 Hz), 9.02 (1H, d, J = 5.4 Hz), 9.66 (1H, s); 13C NMR (75
MHz, CDCl3) δ 29.6, 118.2, 119.3, 122.5, 123.1, 125.5, 133.9, 138.2,
144.2, 147.0, 150.9, 151.9, 155.2, 156.9; LC-MS m/z 284.16 [M + H+].
Indolo[2,1-b]-4-azaquinazoline-6,12-dione (4s): yellow solid
(45%); 1H NMR (300 MHz, CDCl3) δ 7.47 (1H, t, J = 7.5 Hz), 7.62
(1H, dd, J = 7.9, 4.6 Hz), 7.81 (1H, dt, J = 7.8, 1.0 Hz), 7.94 (1H, d, J
= 7.7 Hz), 8.60 (1H, d, J = 8.1 Hz), 8.76 (1H, dd, J = 7.9, 1.8 Hz), 9.10
(1H, dd, J = 4.5, 1.8 Hz); 13C NMR (75 MHz, DMSO-d6) δ 116.9,
119.1, 122.1, 124.7, 124.9, 127.2, 136.3, 137.9, 145.7, 147.6, 156.1,
157.2, 158.1, 182.3; LC-MS m/z 250.15 [M + H+].
8-(Trifluoromethoxy)indolo[2,1-b]-4-azaquinazoline-6,12-
dione (4t): yellow solid (40%); 1H NMR (300 MHz, CDCl3) δ 7.61−
7.66 (2H, m), 7.77 (1H, s), 8.66 (1H, d, J = 8.7 Hz), 8.76 (1H, dd, J =
7.9, 1.8 Hz), 9.10 (1H, dd, J = 4.5, 1.9 Hz); 13C NMR (75 MHz,
DMSO-d6) δ 117.9, 118.3, 118.6, 118.9, 121.7, 123.9, 124.9, 130.2,
136.4, 144.1, 146.5, 147.7, 156.3, 157.0, 158.0, 181.0; LC-MS m/z
334.0 [M + H+].
General Procedure for the Synthesis of C-11-Deoxy
Tryptanthrins (5). Step 1: To a solution of tryptanthrin (1 mmol)
in ether (3 mL) was added LiAlH4 (3 mmol) at 0 °C. The reaction
mixture was refluxed overnight. To the reaction mixture was added
H2O (10 mL), and the solution extracted with EtOAc (2×). The
organic phase was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The organic layers were dried with MgSO4,
filtered, and concentrated. The reaction mixture was purified via
column chromatography (eluted with 5% MeOH in CH2Cl2) to give a
reduced tryptanthrin. Step 2: To a solution of the reduced tryptanthrin
(1 mmol) in CH2Cl2 (3 mL) was added MnO2 (2 mmol) at rt. The
reaction mixture was stirred at rt for 48 h. The reaction mixture was
filtered and washed with CH2Cl2. The organic layers were dried with
MgSO4, filtered, and concentrated. The reaction mixture was purified
via column chromatography (eluted with 5% MeOH in CH2Cl2) to
give the title compound.
Indolo[2,1-b]-4-azaquinazolin-6(12H)-one (5g): red solid
1
(6%); H NMR (300 MHz, CDCl3) δ 5.06 (2H, s), 6.87 (1H, d, J
= 8.0 Hz), 7.12−7.18 (2H, m), 7.46 (1H, d, J = 7.3 Hz), 7.63 (1H, t, J
= 7.7 Hz), 7.75 (1H, d, J = 7.5 Hz), 8.52 (1H, d, J = 5.0 Hz); LC-MS
m/z 235.9 [M + H+].
8-Chloroindolo[2,1-b]-4-azaquinazolin-6(12H)-one (5h): red
solid (9%); 1H NMR (300 MHz, CDCl3) δ 5.06 (2H, s), 6.84 (1H, d,
J = 8.4 Hz), 7.16−7.21 (1H, m), 7.45−7.48 (1H, m), 7.58−7.61 (1H,
m), 7.71−7.73 (1H, m), 8.52−8.55 (1H, m); LC-MS m/z 269.9 [M +
H+].
8-(Trifluoromethoxy)indolo[2,1-b]-4-azaquinazolin-6(12H)-
1
one (5i): red solid (10%); H NMR (300 MHz, CDCl3) δ 5.07 (2H,
s), 6.89−6.93 (1H, m), 7.17−7.22 (1H, m), 7.46−7.52 (2H, m), 7.62−
7.64 (1H, m), 8.54−8.56 (1H, m); LC-MS m/z 320.0 [M + H+].
1,2,3,4-Ttetrahydroindolo[2,1-b]quinazoline-6,12-dione (6).
A solution of A-ring-saturated isatoic anhydride28 (403 mg, 2.41
mmol), isatin (425 mg, 2.89 mmol), and Et3N (930 mg, 9.19 mmol) in
toluene (20 mL) was refluxed for 16 h. The reaction mixture was
quenched with H2O and then extracted with EtOAc (2×). The organic
layers were dried with MgSO4, filtered, and concentrated. The reaction
mixture was purified via column chromatography (eluted with EtOAc/
hexanes) to give the title compound as a yellow solid (85.1 mg, 14%):
1H NMR (300 MHz, CDCl3) δ 1.76−1.88 (4H, m), 2.63−2.68 (2H,
m), 2.76−2.80 (2H, m), 7.39 (1H, dt, J = 7.5, 0.7 Hz), 7.72 (1H, dt, J
= 7.8, 1.3 Hz), 7.83 (1H, dd, J = 7.5, 0.8 Hz), 8.51 (1H, d, J = 8.0 Hz);
13C NMR (75 MHz, CDCl3) δ 21.3, 21.9, 22.9, 31.5, 117.9, 121.3,
125.4, 127.4, 128.5, 137.9, 144.7, 146.0, 158.4, 159.4, 182.7; 13C NMR
(75 MHz, DMSO-d6) δ 20.9, 21.5, 22.5, 31.0, 116.9, 121.7, 124.8,
126.5, 127.1, 137.4, 145.2, 145.6, 157.9, 158.4, 182.4; LC-MS m/z
253.1 [M + H+].
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +82-42-860-7541. Fax: +82-42-860-7348. E-mail: pkim@
krict.re.kr.
Notes
The authors declare no competing financial interest.
8-(Trifluoromethoxy)indolo[2,1-b]quinazolin-6(12H)-one
1
(5a): red solid (79%); H NMR (300 MHz, CDCl3) δ 5.01 (2H, s),
ACKNOWLEDGMENTS
■
6.90 (1H, d, J = 8.5 Hz), 7.13 (1H, dd, J = 6.8, 1.0 Hz), 7.28−7.35
(2H, m), 7.49 (1H, dd, J = 8.5, 2.4 Hz), 7.55 (1H, dd, J = 7.4, 1.3 Hz),
7.62 (1H, d, J = 1.3 Hz); HRESIMS m/z 318.0603 [M+] (calcd for
C16H9F3N2O2, 318.0616).
8-Chloroindolo[2,1-b]quinazolin-6(12H)-one (5b): red solid
(78%); 1H NMR (300 MHz, CDCl3) δ 4.98 (2H, s), 6.83 (1H, d, J =
8.3 Hz), 7.12 (1H, d, J = 6.9 Hz), 7.24−7.35 (2H, m), 7.52−7.59 (2H,
m), 7.69 (1H, d, J = 1.8 Hz); HRESIMS m/z 268.0428 [M+] (calcd for
We are very grateful to Prof. Lester A. Mitscher for sharing
background information on tryptanthrins. Without his
encouragement, this work would never have begun. The
authors also would like to thank Dr. S.-H. Lee at KRICT for
synthetic help and Dr. C. B. Cooper at TB Alliance for helpful
discussions. This research was supported in part by KRICT and
Brain Korea 21.
M
dx.doi.org/10.1021/np3007167 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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DEDICATION
■
Dedicated to Dr. Lester A. Mitscher, of the University of
Kansas, for his pioneering work in the discovery of bioactive
natural products and their derivatives.
(34) Li, J.; Miao, S.; Xie, Y.; Wang, J.; Cao. W.; Bi. L.; Wang, S.
Chromatographia 2012, October 2, 1−6.
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