Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A novel antimycobacterial class targeting mycobacterial respiration

Article abstract of DOI:10.1021/acs.jmedchem.5b01542

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Full text of DOI:10.1021/acs.jmedchem.5b01542

Article
pubs.acs.org/jmc
Pyrrolo[3,4‑c]pyridine-1,3(2H)‑diones: A Novel Antimycobacterial
Class Targeting Mycobacterial Respiration
Renier van der Westhuyzen,† Susan Winks,† Colin R. Wilson,† Grant A. Boyle,† Richard K. Gessner,†
Candice Soares de Melo,† Dale Taylor,‡ Carmen de Kock,‡ Mathew Njoroge,§ Christel Brunschwig,§
Nina Lawrence,† Srinivasa P.S. Rao,○ Frederick Sirgel,∥ Paul van Helden,∥ Ronnett Seldon,†
Atica Moosa,⊥ Digby F. Warner,⊥,# Luca Arista,∇ Ujjini H. Manjunatha,○ Paul W. Smith,○
Leslie J. Street,† and Kelly Chibale*,†,§,#
†Drug Discovery and Development Centre (H3D), Department of Chemistry, University of Cape Town, Rondebosch 7701, South
Africa
‡Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
§South African Medical Research Council Drug Discovery and Development Research Unit, Department of Chemistry, University of
Cape Town, Rondebosch 7701, South Africa
∥DST/NRF Centre of Excellence in Biomedical TB Research, SA MRC Centre for TB Research, Division of Molecular Biology and
Human Genetics, Faculty of Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa
⊥MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Rondebosch
7701, South Africa
#Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
∇Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
○Novartis Institute for Tropical Diseases (NITD), 10 Biopolis Road, Chromos no. 05-01, Singapore 138670, Singapore
S
* Supporting Information
ABSTRACT: High-throughput screening of a library of small
polar molecules against Mycobacterium tuberculosis led to the
identification of a phthalimide-containing ester hit compound
(1), which was optimized for metabolic stability by replacing
the ester moiety with a methyl oxadiazole bioisostere. A route
utilizing polymer-supported reagents was designed and
executed to explore structure−activity relationships with
respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from
these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-
susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of
the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations
indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M.
tuberculosis.
diarylquinoline, Bedaquiline,4 and the nitroimidazole, Delam-
INTRODUCTION
■
anid.5
Mycobacterium tuberculosis (Mtb) is the causative agent of the
infectious disease tuberculosis (TB). Although most TB deaths
A high-throughput phenotypic screening effort at the
Novartis Institute for Tropical Diseases (NITD) identified
are preventable, the mortality rate owing to Mtb infections
remains unacceptably high.1 Furthermore, the six-month
compounds, which lie in the conventional oral drug space (Mw
300−500 Da and clogP 1−4.5), but multiple lead optimization
treatment duration using the 40-year-old first-line drugs,
isoniazid (INH) and rifampicin (RIF), often results in poor
efforts to improve antimycobacterial potency led to advanced
compounds with high lipophilicity and increased molecular
patient adherence, which can lead to cases of drug-resistant
weight.6 Generally lipophilic molecules pose a challenge to
formulation as well as other aspects of clinical development.
Interestingly, many of the classical TB specific drugs like
TB.2 The WHO estimates that, in 2013, about half a million
people developed multidrug-resistant TB (MDR-TB), which is
defined as resistance to at least INH and RIF.1,3 Therefore, new
drugs which can shorten the duration of treatment are urgently
needed. Some progress has been made with the recent approval
of two new drugs for the treatment of MDR-TB patients: the
Received: October 2, 2015
© XXXX American Chemical Society
A
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isoniazid, pyrazinamide, ethionamide, p-aminosalicylic acid, and
cycloserine occupy a unique chemical space in terms of
molecular weight (<250 Da) and lipophilicity (clogP < 2.5)
(Figure 1). To probe this distinctive chemical space, NITD
Table 1. SAR for Ester Bioisosteres and in Vitro ADMET
Data
Figure 1. Plots of clogP vs molecular weight. Most TB drugs lie
outside of conventional oral drug space as many are small and polar.
assembled a library of ∼6000 leadlike7 compounds with Mw
150−350 Da, clogP −1 to 3.5, and with no obvious reactive
groups, and carried out a proof-of-concept phenotypic screen
against M. tuberculosis. Compound 1 (Figure 2) with a Mw 339
and clogP 3.3 was selected as a representative example for
subsequent hit validation through resynthesis, retesting, and
ADME profiling.
a
14-day Alamar Blue readout against H37RvMa in GAST/Fe medium.
b
c
FaSSIF kinetic solubility assay at pH 6.5. Percentage remaining at 30
d
min assessed in human, rat and mouse liver microsomes. Cytotoxicity
against kidney epithelial cells extracted from an African green monkey
e
(VERO). Cytotoxicity against Chinese hamster ovarian cells (CHO).
f
Rifampicin positive control. n.d., not determined.
initial medicinal chemistry strategy was to improve the
metabolic stability of compound 1 while maintaining good in
vitro potency by identifying bioisosteric replacements for the
labile ester functionality. Once a suitable ester group
replacement was identified, we envisaged that initial structure-
activity relationship (SAR) studies would focus on iterative
changes to the benzyl substituent and exploration of the
phthalimide core. Here, we present results of the SAR analyses
along with our efforts toward elucidation of the target of this
antimycobacterial series through mechanism of action studies.
Figure 2. HTS hit compound 1 from screening a focused library.
cLogP and TPSA calculated by ChemDraw Professional 15.
Although compound 1 had good in vitro potency against
Mtb, the presence of the ester functionality raised concerns
regarding metabolic instability owing to hydrolysis of the ester
group, both in vitro in liver microsomes and in vivo. Ester
group hydrolysis was confirmed by showing that less than 20%
of the compound remained after incubation with human, rat,
and mouse liver microsomes after 30 min (Table 1). The
carboxylic acid was identified as the main metabolite in mouse
liver microsomes (MLM) and rat liver microsomes (RLM). In
the presence of human S9 fraction, only 35% remained after 40
min. These observations led to the conclusion that compound
1 was not expected to perform well in vivo. Therefore, the
RESULTS AND DISCUSSION
■
Chemistry. Hit compound 1 was prepared by acid-catalyzed
condensation of ethyl acetamidocyanoacetate and benzylmalei-
mide (Scheme 1).8 Similarly, reaction of N-(dicyanomethyl)-
acetamide with benzylmaleimide gave nitrile 2a. Hydrolysis of
the ethyl ester 1 under acid conditions gave acid 2b. Primary,
secondary, and tertiary amides were prepared from 2b by
utilizing amide coupling reagents EDC, HOBt, and the
appropriate amine in dichloromethane. These conditions and
appropriate alcohols were also used for the synthesis of esters
(2c−e), which were explored to determine the influence of the
B
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a
Scheme 1. Synthesis of Ester Bioisosteres
a
Reagents and conditions: (a) cat. TFA, DCE, reflux, 3 d; (b) 1, aq HCI, dioxane, reflux; (c) EDC, HOBt, amine, DCM, rt; (d) 1, CH3C(
NOH)NH2, NaH, dioxane, 60 °C; (e) (1) 1, CF3C(NOH)NH2, NaH, 65 °C, dioxane, (2) PS-BEMP, dioxane, 75 °C; (f) CH3CONHNH2, T3P,
NEt3, EtOAc, reflux; (g) TsCI, NEt3, CHCI3, 65 °C; (h) EDC, HOBt, ROH, DCM.
size of the ester group on Mtb activity. The 1,2,4-oxadiazoles 2k
and 2m were prepared by reacting 1 with the appropriate
amidoxime in sodium hydride in dioxane at 60 °C. 1,3,4-
Oxadiazole 2j was prepared in two steps from 2b by coupling
acetyl hydrazide in the presence of propylphosphonic
anhydride (T3P) and then cyclization with tosyl chloride
(TsCl) in refluxing chloroform.
reaction is enzyme-mediated and not a result of chemical
instability. Replacement of the ester functionality with a nitrile
also abolished Mtb activity but did improve metabolic stability.
Next, we turned our attention to five-membered heterocycles,
in particular oxadiazoles, because these are well-known
bioisosteres for ester groups.12 The 1,3,4-oxadiazole compound
2j was shown to be inactive, whereas the 1,2,4-oxadiazole (2k)
had significant Mtb activity and improved metabolic stability
over ester 1. Metabolite identification in the presence of mouse
liver microsomes and in mice revealed that the metabolic
pathway for compound 2k was through oxidative ring opening
of the oxadiazole ring, most likely mediated by oxidation of the
methyl group leading to metabolites 2b, 2f, and 2l (Supporting
Information Figure S1). This prompted us to investigate
blocking oxidation on the methyl group by replacement with a
trifluoromethyl group (2m). However, this modification
resulted in loss of Mtb activity. All compounds tested in this
series were not toxic to VERO cells up to 100 μM (IC50 > 100
μM).
With the 1,2,4-oxadiazole identified as a more metabolically
stable ester bioisostere, we next explored changes to the N-
benzyl substituent in order to improve potency and aqueous
solubility. For rapid SAR exploration, a parallel synthesis route
was designed that would enable preparation of various
analogues with minimum purification (Scheme 2). Cyclization
of maleimide and ethyl acetamidocyanoacetate gave N-
phthalimide, compound 3, which could be transformed into
the versatile precursor 4 by reacting N′-hydroxyacetimidamide
with 3 under basic conditions. Alkylation of 4 with a variety of
benzyl bromides and chlorides proceeded smoothly in the
presence of polymer-supported 2-tert-butylimino-2-diethylami-
no-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine on polystyr-
ene (PS-BEMP), in dioxane, at 75 °C. After the reaction was
In Vitro Antimycobacterial Activity. Compounds pre-
pared in this manner were tested against the fully virulent Mtb
H37RvMa9 in GAST/Fe medium using the standard broth
micro dilution method10 and minimum inhibitory concen-
tration (MIC) values determined at 14 days (Table 1). Kinetic
solubility of the test compounds was determined in fasted state
simulated intestinal fluid (FaSSIF) medium, and metabolic
stability of selected compounds in human, rat, and mouse liver
microsomes was assessed by incubating for 30 min and
measuring the percentage of compound remaining.11 As shown
in Table 1, the methyl (2c), ethyl (1), propyl (2d), and
cyclopropyl (2e) esters all had good in vitro activity against Mtb
H37RvMa. The primary (2f), secondary (2g), tertiary (2i), and
hydroxamic (2h) amides were not active at the highest
concentration tested (MIC90 > 160 μM). The carboxylic acid
2b maintained some Mtb activity, albeit at least 10-fold lower
than the original hit compound 1. As expected, 2b had
improved microsomal metabolic stability in microsomes relative
to 1 and exhibited significantly improved aqueous solubility, as
did the inactive amide analogues. The improved metabolic
stability of 2b suggests that ester hydrolysis is the main route of
metabolism. The ester 1 was also hydrolyzed into 2b in mouse
plasma, in Mtb supernatant (data not shown), and in mouse
liver microsomes even in the absence of NADPH, indicating
contribution by non-CYP450 enzymes. However, no hydrolysis
occurred in a control containing buffer only, indicating that the
C
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a
Scheme 2. Synthesis of Methyl Oxadiazole Analogues
Table 2. SAR and Solubility Data for Changes Made to the
Phthalimide Group
a
Reagents and conditions: (a) cat. TFA, dichloroethane reflux, 3 d; (b)
CH3C(NOH)NH2, NaH, dioxane, 60 °C; (c) (1) RBr or RCI, PS-
BEMP, dioxane, 24 h, 75 °C, (2) PS-PhSH, 75 °C.
complete, thiophenol on polystyrene (PS-PhSH) was added to
scavenge any remaining benzyl bromide or chloride. Removal
of the resins by filtration gave the final products, which were
often >95% pure. Remaining impurities consisted of unreacted
benzyl bromide or 4 which could be easily removed by flash
chromatography.
We initially explored the effect of adding small halogens to
the benzyl ring. Benzyl analogues with para and meta halogens
(F, Cl, Br, and CF3) were favored over ortho substituted
analogues (Table 2). Methoxy and trifluoromethoxy groups
also showed good to excellent anti-Mtb activity. α-Methylbenzyl
analogue 5l also maintained good anti-Mtb activity with some
improvement in solubility probably resulting from disrupting
the planarity of the molecule. Replacement of the benzyl group
with a 4-pyridylmethyl (5s) was investigated to improve
solubility but resulted in a loss of activity with an increase in
solubility. A series of compounds with larger changes in the
para position was prepared to explore potential new
interactions and/or to improve the solubility of this series in
aqueous media. Analogues with nitrile (5u), carboxamide (5v),
thiotrifluoromethyl (5w), hydroxymethyl (5x), and N,N-
dimethylamino (5y) functionalities all showed a significant
loss in activity. Methylsulfone analogue 5t showed good Mtb
potency but no improvement in solubility over 2k. Hetero-
cycles in the para position, e.g., pyridyl, pyrazole, and
pyrrolidinone, were mainly detrimental to activity. However,
the p-fluorophenyl ether derivative 5ab maintained good
activity and exhibited some improvement in solubility relative
to the benzyl analogue 2k. The increase in lipophilicity of this
compound compared to other analogues might have con-
tributed to the improved activity. Finally, saturated cyclic
heterocycles such as the piperidinylmethyl analogue 5ad and
tetrahydropyranmethyl 5ae resulted in loss of activity while
improving solubility.
a
14-day Alamar Blue readout against H37RvMa in GAST/Fe medium.
FaSSIF kinetic solubility assay at pH 6.5. Rifampicin positive control.
b
c
n.d., not determined.
Scheme 3. Synthesis of Analogues with Changes to the
Core
a
We also investigated some changes to the pyrrolo[3,4-
c]pyridine-1,3(2H)-dione scaffold of 1 (Scheme 3). These
changes, which included removing the benzylic methylene,
compound 6,8 and the methyl group on the pyrrolo[3,4-
c]pyridine-1,3(2H)-dione core, compound 7, both led to a
significant loss in activity (Table 3). Reduction of the carbonyls
on the phthalimide moiety was also explored. Reduction using
NaBH4 gave a mixture of monohydroxyl regio-isomers, which
were separated by preparative HPLC. The 1-hydroxy
compound 8a resulted in a 4-fold drop in activity, while the
3-hydroxy isomer 8b retained the potency of the original hit
compound 1. Furthermore, both of these compounds showed a
a
Reagents and conditions: (a) cat. TFA, DCE, reflux, 3 d; (b) NaBH4,
DCM, MeOH, 0 °C.
significant improvement in aqueous solubility and microsomal
stability and were not toxic to VERO cells.
Further in Vitro Biological Profiling of Selected
Compounds. Having identified numerous compounds with
D
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activity against the full clinical panel (Table 4) in 7H9 medium.
This result established the activity of this series against clinical
Mtb strains from diverse phylogenetic origins and, moreover,
suggested that the apparent inactivity against the laboratory
strain, H37RvMa, in 7H9 medium was likely a consequence of
extended adaption to in vitro medium over decades of research
use.9
Table 3. SAR of Changes Made to the Heterocyclic Scaffold
and in Vitro ADMET Data
In Vitro and in Vivo Pharmacokinetic Studies. In vitro
clearance indicated that there was insignificant turnover for
compounds 5h and 5k in human, rat, and mouse microsomes
(Table 5). However, compound 5h was more stable across all
three species, more stable in the presence of human S9 fraction,
and had moderate stability in human plasma. The measured log
D was similar for these analogues and within an acceptable
space for oral drugs, while compound 5h showed high
permeability in a PAMPA assay and was highly protein
bound. On the basis of these results, compound 5h was
selected for progression into mouse PK studies.
With the excellent in vitro microsomal stability and moderate
solubility observed for 5h, a mouse PK study was carried out at
5 mg/kg iv and 20 mg/kg po (Table 6). When dosed iv
compound 5h showed a high clearance and volume of
distribution and had poor plasma exposure (AUC 8 μM·min
and Cmax = 0.033 μM respectively at 20 mg/kg) when dosed
orally. As the compound showed high in vivo clearance leading
to poor blood exposures, it was coadministered with amino-
benzotriazole (ABT), which is a known nonspecific cytochrome
P450 inhibitor, and is used frequently to reduce the CYP-
mediated metabolism of compounds.17 Dosing iv with ABT
indicated that the clearance improved to moderate (170.78−
94.45 mL/min/kg). Dosing orally with ABT showed an
increased exposure, resulting in a Cmax 2-fold above the
MIC90 with a 10-fold higher AUC compared to non-ABT
exposure. The disconnect between in vitro and in vivo
clearance suggests that clearance is through a non CYP-
mediated process, most probably through hydrolysis of the
oxadiazole by amidases and esterases present in the plasma.
This is consistent with the moderate half-life found in human
plasma (t1/2 = 60 min) and the minor reduction in clearance
when codosing the compound with ABT in mice.
a
14-day Alamar Blue readout against H37RvMa in GAST/Fe medium.
b
c
FaSSIF kinetic solubility assay at pH 6.5. Percentage remaining at 30
d
min assessed in human, rat and mouse liver microsomes. Cytotoxicity
against kidney epithelial cells extracted from an African green monkey
(VERO). Rifampicin positive control. n.d., not determined.
e
submicromolar MIC90 values against Mtb H37RvMa, and
having made improvements to in vitro metabolic stability in
some cases (e.g., compound 5h), we profiled these compounds
in additional microbiological assays in vitro. Specifically,
compounds 1, 5h, and 5k were tested against a recombinant
Mtb H37RvMa strain expressing green fluorescent protein
(Mtb::gfp)13 (Table 4).14 Compounds 1, 5h, and 5k were
tested in GAST/Fe medium using this Mtb::gfp strain, and the
dose−response curve confirmed the potent MIC90 values. In
contrast, the same compounds were completely inactive when
MIC90 values were determined following 14-days incubation in
standard Middlebrook 7H9 medium supplemented with ADC
(bovine albumin fraction V), an observation reminiscent of
known inhibitors of the mycobacterial cytochrome bc1 complex
possibly suggesting pyrrolo[3,4-c]pyridine-1,3(2H)-diones tar-
get the respiratory pathway.15
To determine the potential utility of this scaffold in a clinical
setting, compounds 1, 5h, and 5j were tested for activity against
four drug-susceptible clinical Mtb isolates, selected from a
culture collection in the Department of Molecular Biology and
Human Genetics, Stellenbosch University, South Africa (Table
4). The identity of the isolates was determined by IS6110
restriction fragment length polymorphism (RFLP) and their
phylogenetic lineages assigned by spoligotyping.16 The
spoligotype patterns indicated that the strains SAWC 3906,
SAWC 2371, SAWC 3385, and SAWC 3933 belong to the
atypical Beijing, Haarlem, Cas 1/Delhi, and X families,
respectively. Pyrrolo[3,4-c]pyridine-1,3(2H)-diones retained
Target Identification Studies. To identify the molecular
target of pyrrolo[3,4-c]pyridine-1,3(2H)-diones and gain in-
sight into the mechanism of action, we attempted to raise
spontaneous resistant mutants (SRM) in H37RvMa. We were
unable to obtain SRMs of compounds from this series even
when plating on solid media containing a range of
concentrations from 2-fold to 200-fold the MIC90 value
determined in the liquid GAST/Fe assay. During routine hit
triaging, we observed that the compounds were hyperactive
against a cytochrome bd oxidase deletion mutant (ΔcydKO)
(Table 7).15 In addition, a derivative ΔcydKO deletion mutant
Table 4. MIC90 Values (μM) of Selected Compounds against M. tuberculosis Strains
reference strain
SAWC clinical isolates (7H9)
a
a
compd
H37RvMa (GAST/Fe)
Mtb::gfp (GAST/Fe)
Mtb::gfp (7H9)
2371 Haarlem
3385 Cas1/Delhi
3906 Atyp-Beijing
3933 LCC
1
<0.156
<0.156
<0.156
0.39
0.132
0.065
n.d.
>20
>20
<0.08
0.6
<0.08
0.6
<0.08
0.15
0.075
n.d.
n.d.
0.15
0.15
n.d.
n.d.
5h
5j
5k
n.d.
0.3
0.075
n.d.
0.052
0.0032
>20
n.d.
n.d.
b
RIF
0.009
0.0016
n.d.
n.d.
a
b
GFP readout. Rifampicin positive control, SAWC, South Africa Western Cape, n.d. not determined.
E
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Table 5. Properties of Two Leading Compounds 5h and 5k
VERO IC
HLM/RLM/MLM CLint
human S9 (% remaining
at 40 min)
plasma stability
t1/2 (min)
log D
pH 7.4
PAMPA pH 6.5
log Papp (cm/s)
human PPB
(% bound)
a 50
b
compd
(μM)
(mL/min/mg protein)
5h
5k
>100
>100
<11.6/<11.6/<11.6
15.4/32.4/65.7
97
93
62
3.34
3.64
−5.0
−6.1
96.7
n.d
>360
a
b
Cytotoxicity against kidney epithelial cells extracted from an African green monkey (VERO). HLM = human liver microsomes, RLM = rat liver
microsomes, MLM = mouse liver microsomes. PPB = plasma protein binding, n.d., not determined.
indicate high clearance and low plasma exposure (Cmax = 0.033
Table 6. Pharmacokinetic Parameters for Compound 5h in
C57/BL6 Mice Following Intravenous and Oral
Administration with and without ABT
μM) in contrast to good metabolic stability in liver microsomes.
Co-dosing of the compound with ABT gave an improved
clearance resulting in a circulation concentration above the
MIC90 for 6 h when dosed at 20 mg/kg. Future studies will
focus on improving exposure of the compounds in plasma by
further modifications based on the 3-hydroxyl analogue 8b,
which has good in vitro Mtb activity and improved aqueous
solubility.
a
compd 5h without
ABT
compd 5h with ABT
parameters
dose (mg/kg)
IV
oral
20
2.19
IV
oral
20
3.39
5
5
apparent t1/2 (h)
CLtotal (mL/min/kg)
Vd (L/kg)
7.01
170.78
104.24
0.622
0.17
76
7.31
94.45
53.10
1.526
0.17
−
−
0.033
−
−
0.167
0.5
80
26
EXPERIMENTAL SECTION
■
Cmax (μM)
General Methods. Purification of compounds were carried out by
either column chromatography on silica gel 60 (Fluka), particle size
0.063−0.2 mm (70−230 mesh), as the stationary phase, or by Waters
preparative HPLC using X-bridge C18 5 μm column (4.6 mm × 150
mm). Mobile phase B: 0.4% acetic acid, 10 mM ammonium acetate in
a 9:1 ratio of HPLC grade methanol and type 1 water. Mobile phase A:
0.4% acetic acid in 10 mM ammonium acetate in HPLC grade (type 1)
water; flow rate = 15.00 mL/min; detector, photodiode array (PDA).
All target compounds and intermediates were characterized by 1H
NMR and LC-MS. NMR spectra were recorded on either a Varian
Mercury-300 (1H 300.1 MHz, 13C 75.5 MHz) or Bruker-400 (1H
400.2 MHz, 13C 100.6 MHz) instrument using CDCl3, CD3OD, and
DMSO-d6 as solvents. Liquid chromatograph with mass spectrometer
(LC-MS) analysis was performed using an Agilent 1260 Infinity binary
pump, Agilent 1260 Infinity diode array detector (DAD), Agilent 1290
Infinity column compartment, Agilent 1260 Infinity standard
autosampler, and a Agilent 6120 quadrupole (single) mass
spectrometer, equipped with APCI and ESI multimode ionization
source. Purities were determined by Agilent LC-MS using a Kinetex
Core C18 2.6 μm column (50 mm × 3 mm). Mobile phase B: 0.4%
acetic acid, 10 mM ammonium acetate in a 9:1 ratio of HPLC grade
methanol, and type 1 water. Mobile phase A: 0.4% acetic acid in 10
mM ammonium acetate in HPLC grade (type 1) water, with flow rate
= 0.9 mL/min; detector, diode array (DAD), and all compounds were
confirmed to have ≥95% purity.
Tmax (h)
1
8
2
AUC0−∞ (min·μM)
oral bioavailability (%)
159
−
−
a
Values are the mean from three animals. Dash indicates that the value
was not measured or was not relevant.
Table 7. Activity of Selected Compounds against Mutant
Mtb Strains
MIC90 (μM) with
MIC90 (μM)
MIC90 (μM) with
Ala317Thr point mutant
a
a
a
compd
H37RvMa
with ΔcydKO
1
0.078
1.25
0.019
0.039
>10
>10
>10
>10
>10
>10
2e
5h
5j
<0.156
<0.156
0.0637
0.144
<0.019
0.039
5k
8b
<0.019
0.019
a
14-day Alamar Blue readout in GAST/Fe medium.
carrying an Ala317Thr point mutation in qcrB15 was resistant to
compounds from this series (Table 7). Together with the
differential activity in GAST/Fe versus 7H9 medium against
H37RvMa (Table 4), the observed hypersensitivity of the
ΔcydKO deletion mutant to these compounds and the cross-
resistance of the ΔcydKO/QcrBA317T mutant strongly
indicated that this series of pyrrolo[3,4-c]pyridine-1,3(2H)-
diones targets the QcrB subunit of the respiratory bc1 complex
in Mtb. Recently, multiple chemically diverse scaffolds have
been described that target QcrB, the cytochrome b subunit in
the cytochrome bc1 complex, and these include the imidazo-
[1,2-α]pyridines series from which the exciting new clinical
candidate Q203 was derived.18
Ethyl 7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxylate (1). Trifluoroacetic acid (0.06
g, 0.58 mmol) was added to a mixture of 1-benzyl-1H-pyrrole-2,5-
dione (1.0 g, 5.88 mmol) and ethyl acetamidocyanoacetate (1.31 g,
7.05 mmol) in DCE. The reaction mixture was heated at reflux for 48
h. The solvent was evaporated under reduced pressure, and the crude
product was purified by column chromatography (10−20% EtOAc/
petroleum ether) to afford 1 (1.0 g, 50%). 1H NMR (400 MHz,
DMSO) δ 7.26−7.32 (m, 5H), 6.97 (s, 2H), 4.71 (s, 2H), 4.35 (q, J =
7.11 Hz, 2H), 2.58 (s, 3H), 1.32 (t, J = 7.08 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 168.28, 167.45, 165.94, 142.53, 141.46, 135.93,
134.51, 128.77, 128.63, 128.03, 125.77, 120.59, 62.16, 41.66, 20.04,
14.23. LC-MS (APCI): m/z 340.2 [M + H]+. HPLC purity 95%.
7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-pyrrolo-
[3,4-c]pyridine-6-carbonitrile (2a). To a suspension of malononitrile
(30.0 g, 454 mmol) in water (200 mL) and acetic acid (200 mL) was
added sodium nitrite (62.70 g, 908.6 mmol) at 0−5 °C over a period
of 1 h. The temperature was slowly raised to rt and stirred until
complete consumption of malononitrile. The reaction mixture was
then extracted with EtOAc (5 × 80 mL). The combined organic layers
were washed with 10% sodium bicarbonate solution (5 × 25 mL) and
brine, dried over sodium sulfate, and evaporated. The resulting crude
product was purified by flash column chromatography (20−30%
CONCLUSIONS
■
Screening of a library of compounds in novel chemical space
led to the identification of a potent hit compound, the
pyrrolo[3,4-c]pyridine-1,3(2H)-dione 1. Hit optimization of 1
gave compound 5h by replacing the ester moiety with a methyl
oxadiazole bioisostere. This compound had good in vitro
metabolic stability and retained the excellent potency of the hit
compound. Pyrrolo[3,4-c]pyridine-1,3(2H)-diones targets my-
cobacterial respiratory cytochrome bc1, a highly promising
promiscuous target in Mtb.19 Mouse PK studies with 5h
F
DOI: 10.1021/acs.jmedchem.5b01542
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
EtOAc/petroleum ether) to afford hydroxycarbonimidoyl dicyanide
(16.0 g, 37%). 1H NMR (400 MHz, DMSO) δ 14.45 (s, 1H), 7.90 (s,
1H), 7.83(s, 1H). To a cooled suspension of hydroxycarbonimidoyl
dicyanide (16.0 g, 166 mmol) and acetic anhydride (68.0 g, 666
mmol) in acetic acid (200 mL) was added zinc dust (32.69 g, 499.0
mmol) in small portions. The suspension was stirred at rt for 3 h. The
reaction mixture was poured into crushed ice, and the pH was adjusted
to 8−9 using a saturated sodium carbonate solution. The product was
extracted with EtOAc (3 × 120 mL). The combined organic layers
were washed with water (2 × 100 mL) and brine, dried over sodium
sulfate, and evaporated. The crude product was purified by flash
column chromatography (40−50% EtOAc/petroleum ether) to afford
N-(dicyanomethyl)acetamide (3.30 g, 16%). 1H NMR (400 MHz,
DMSO) δ 7.53 (s, 1H), 2.21 (s, 1H). Trifluoroacetic acid (1.48 g, 12.9
mmol) was added to a mixture of 1-benzyl-1H-pyrrole-2,5-dione (6.02
g, 32.1 mmol) and N-(dicyanomethyl)acetamide (3.30 g, 26.8 mmol)
in DCE. The reaction mixture was heated at reflux for 48 h. The
solvent was evaporated under reduced pressure, and the crude product
was purified by flash column chromatography (10−20% EtOAc/
0.482 mmol) in DMF (2 mL) was added triethylamine (0.134 μL,
0.964 mmol) and HATU (275 mg, 0.723 mmol) and stirred for 10
min. Ammonium chloride (31 mg, 0.58 mmol) was then added and
the mixture stirred at 40 °C overnight. Addition of water resulted in an
orange precipitate, which was isolated by vacuum filtration and
purified by column chromatography (30% EtOAc/hexane) to afford 2f
1
(88 mg, 59%) as a yellow solid. H NMR (300 MHz, DMSO) δ 8.09
(s, 1H), 7.70 (s, 1H), 7.23−7.39 (m, 5H), 4.70 (s, 2H), 2.59 (s, 3H).
LC-MS (ESI): m/z 311.0 [M + H]+. HPLC purity 98%.
7-Amino-2-benzyl-N,4-dimethyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxamide (2g). A solution of 2b (100
mg, 0.32 mmol), methylamine (0.32 mL of a 2 M solution in THF,
0.64 mmol), HOBt (50 mg, 0.32 mmol), and EDC (61 mg, 0.32
mmol) in DCM (3 mL) was stirred for 24 h. Water was added to the
reaction, followed by EtOAc. The organic layer was separated, washed
with brine, and dried over MgSO4. After filtration and concentration,
the product was purified by preparative HPLC to afford 2g (8 mg,
7%). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.45−7.23 (m, 5H),
4.79 (s, 2H), 2.99 (d, J = 5.2 Hz, 3H), 2.67 (s, 3H). LC-MS (ESI): m/
z 325.1 [M + H]+. HPLC purity 99%.
1
petroleum ether) to afford 2a (0.80 g, 10%). H NMR (400 MHz,
7-Amino-2-benzyl-N-hydroxy-4-methyl-1,3-dioxo-2,3-dihydro-
1H-pyrrolo[3,4-c]pyridine-6-carboxamide (2h). 2b (100 mg, 0.32
mmol) and triethylamine (0.058 mL, 0.42 mmol) were added to
anhydrous THF (3.0 mL). The solution was cooled to 0 °C in an ice
bath, and ethyl chloroformate (0.040 mL, 0.42 mmol) was added
dropwise. The mixture was stirred for 20 min at 0 °C and left stirring
for a further 2 h at rt. The mixture was filtered and then added in a
dropwise manner to a solution of triethylamine (0.045 mL, 0.32
mmol) and hydroxylamine hydrochloride (22 mg, 0.32 mmol) in
methanol (3.0 mL) and then allowed to stir for 2 h at rt. The solvents
were removed in vacuo, and the residue was dissolved in EtOAc (10
mL). The organics were washed with 0.5 M HCl (5.0 mL) and water
(10 mL), dried over Na2SO4, and filtered. The filtrate was
concentrated under reduced pressure, and the crude product was
DMSO) δ 8.15 (s, 1H), 7.75 (s, 1H), 7.2−7.36 (m, 5H), 4.72 (s, 2H),
2.61 (s, 3H). 13C NMR (400 MHz, DMSO) δ 168.70, 168.27, 167.88,
140.68, 139.88, 136.93, 136.49 129.03, 127.92, 125.63, 119.92, 19.83.
LC-MS (APCI): m/z 310.1 [M + H2O]+. HPLC purity 98%.
7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-pyrrolo-
[3,4-c]pyridine-6-carboxylic Acid (2b). To a mixture of 1 (0.500 g,
1.47 mmol) in dioxane (6 mL) was added aqueous 1 M hydrochloric
acid (12.0 mL, 12.0 mmol), and the reaction was refluxed overnight.
The organics were removed under reduced pressure and the mixture
cooled to 0 °C to give a yellow precipitate. Filtration gave 2b (366 mg,
78%) as a yellow solid. 1H NMR (300 MHz, DMSO) δ 7.40−7.21 (m,
5H), 7.03 (s, 2H), 4.72 (s, 2H), 2.60 (s, 3H). LC-MS (ESI): m/z
312.1 [M + H]+. HPLC purity 98%.
Methyl 7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxylate (2c). A solution of 2b (100
mg, 0.32 mmol), methanol (26 μL, 0.64 mmol), HOBt (50 mg, 0.32
mmol), and EDC (61 mg, 0.32 mmol) in DCM (3 mL) was stirred for
24 h. Water was added to the reaction, followed by EtOAc. The
organic layer was separated, washed with brine, and dried over MgSO4.
After filtration and concentration, the product was purified by
recrystallization from MeOH/DCM to give 2c (60 mg, 57%) as a
yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.46−7.27 (m, 5H), 6.92
(br s, 2H), 4.80 (s, 2H), 4.00 (s, 3H), 2.76 (s, 3H). LC-MS (ESI): m/z
326.0 [M + H]+. HPLC purity 98%.
1
purified by preparative HPLC which afforded 2h (46 mg, 44%). H
NMR (300 MHz, CDCl3) δ 9.97 (br s, 1H), 7.47−7.21 (m, 5H), 4.80
(s, 1H), 4.79 (s, 2H). LC-MS (ESI): m/z 327.0 [M + H]+. HPLC
purity 95%.
7-Amino-2-benzyl-N,N,4-trimethyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxamide (2i). A solution of 2b (100 mg,
0.32 mmol), dimethylamine hydrochloride (52 mg, 0.64 mmol), HOBt
(50 mg, 0.32 mmol), and EDC (61 mg, 0.32 mmol) in DCM (3 mL)
was stirred for 24 h. Water was added to the reaction, followed by
EtOAc. The organic layer was separated, washed with brine, and dried
over MgSO4. After filtration and concentration, the product was
purified by preparative HPLC to afford 2i (9 mg, 8%). 1H NMR (400
MHz, CDCl3) δ 7.46−7.41 (m, 2H), 7.38−7.27 (m, 3H), 6.05 (s, 2H),
4.82 (s, 2H), 3.17 (s, 3H), 3.15 (s, 3H), 2.73 (s, 3H). LC-MS (ESI):
m/z 339.1 [M + H]+. HPLC purity 99%.
7-Amino-2-benzyl-4-methyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-
1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (2j). To a mixture of 2b
(100 mg, 0.32 mmol) and acethydrazide (25 mg, 0.32 mmol) in
EtOAc (10 mL) was added triethylamine (97 mg, 0.130 mL, 0.96
mmol). To the mixture was added T3P (0.20 mL of a 50% solution in
EtOAc, 0.32 mmol) and the reaction refluxed overnight at 80 °C. The
reaction was diluted with EtOAc (10 mL) and washed with water (10
mL), followed by saturated NaHCO3 (10 mL). After drying the
organics over Na2SO4, the solvent was reduced in vacuo to give the
crude product which was purified by recrystallization from methanol.
The product was taken into the next step without further purification.
To a stirred suspension of the hydrazide (100 mg, 0.270 mmol) in
DCM (10 mL) was added triethylamine (137 mg, 1.36 mmol)
followed by tosyl chloride (156 mg, 0.820 mmol). The mixture was
heated to 65 °C for 3 h, followed by removal of the solvent in vacuo.
The residue was taken up in EtOAc (10 mL) and washed with brine
(10 mL), dried (MgSO4), and concentrated in vacuo. The crude
product was purified by preparative HPLC to afford 2j (7 mg, 7%). 1H
NMR (400 MHz, CDCl3) δ 7.46−7.40 (m, 2H), 7.38−7.27 (m, 3H),
4.82 (s, 2H), 2.80 (s, 3H), 2.70 (s, 3H). LC-MS (ESI): m/z 350.1 [M
+ H]+. HPLC purity 99%.
Propyl 7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxylate (2d). A solution of 2b (100
mg, 0.32 mmol), 1-propanol (58 μL, 0.64 mmol), HOBt (50 mg, 0.32
mmol), and EDC (61 mg, 0.32 mmol) in DCM (3 mL) was stirred for
24 h. Water was added to the reaction, followed by EtOAc. The
organic layer was separated, washed with brine, and dried over MgSO4.
After filtration and concentration, the product was purified by
1
preparative HPLC to afford 2d (7 mg, 6%). H NMR (400 MHz,
CDCl3) δ 7.44−7.38 (m, 2H), 7.36−7.27 (m, 3H), 6.90 (br s, 2H),
4.79 (s, 2H), 4.36 (t, J = 7.0 Hz, 2H), 2.76 (s, 3H), 1.86 (dt, J = 7.5,
7.1, 2H), 1.02 (t, J = 7.4 Hz, 3H). LC-MS (ESI): m/z 354.1 [M + H]+.
HPLC purity 99%.
Cyclopropyl 7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-
1H-pyrrolo[3,4-c]pyridine-6-carboxylate (2e). A solution of 2b (100
mg, 0.32 mmol), EDC (61 mg, 0.32 mmol), and HOBt (50 mg, 0.32
mmol) was stirred in DCM (3.0 mL) for 15 min. Cyclopropanol
(0.041 mL, 0.64 mmol) was added, and the reaction was stirred for 24
h. Water (10 mL) was added to the reaction, followed by EtOAc (10
mL). The organic layer was separated, washed with brine (10 mL),
dried over Na2SO4, and concentrated in vacuo to give the crude
product which was purified by preparative HPLC to give 2e (21 mg,
19%). 1H NMR (300 MHz, DMSO) δ 7.38−7.23 (m, 5H), 6.98 (br s,
2H), 4.72 (s, 2H), 4.37−4.22 (m, 1H), 2.58 (s, 3H), 0.85−0.79 (m,
4H). LC-MS (ESI): m/z 352.1 [M + H]+. HPLC purity 95%.
7-Amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-pyrrolo-
[3,4-c]pyridine-6-carboxamide (2f). To a solution of 2b (152 mg,
G
DOI: 10.1021/acs.jmedchem.5b01542
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7-Amino-2-benzyl-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-
1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (2k). To a solution of N′-
hydroxyacetimidamide (52 mg, 0.707 mmol) in THF (10 mL) was
added NaH (27 mg, 0.707 mmol, 60% in mineral oil) and the mixture
stirred for 15 min at rt and then at 60 °C for a further 30 min. To the
mixture was added 1 (0.200 g, 0.589 mmol) and the mixture refluxed
overnight. The reaction was cooled to rt and quenched by adding
water (10 mL) and the product extracted with EtOAc (2 × 10 mL).
The combined organic layers were dried (Na2SO4), filtered, and
evaporated. The crude product was purified by preparative TLC (30%
EtOAc/hexane), followed by recrystallization from a mixture of
dichloromethane and hexane to give 2k (20 mg, 10%) as a yellow
under reduced pressure, the crude product was purified by flash
chromatography (gradient of cyclohexane/EtOAc) to give 4 (2.59 g,
1
27%). H NMR (400 MHz, DMSO) δ 11.50 (s, 1H), 7.21 (s, 2H),
2.62 (s, 3H), 2.50 (s, 3H). LC-MS (ESI): m/z 260.1 [M + H]+. HPLC
purity 98%.
General Procedure for the Synthesis of Compounds 5a−
5ae. To a solution of 4 (50 mg, 0.19 mmol, 1.1 equiv) in dioxane (4.0
mL) was added BEMP on polystyrene (262 mg, 0.576 mmol, labeling
2.2 mmol/g). The reaction was placed on an Activo-PLS organic
synthesizer and shaken for 15 min at rt, and then the appropriate
chloride or bromide (0.18 mmol, 1.0 equiv) was added and shaking
continued for 24 h at 75 °C. To the mixture was added polymer
supported thiophenol (152 mg, 1.1 eq, labeling 1.3 mmol/g) and the
mixture stirred for 18 h at 75 °C. After filtration on a VacMaster-20
system, the filtrate was concentrated to give the final product.
Compounds that were not sufficiently pure were further purified by
flash chromatography (gradient of cyclohexane/EtOAc) or triturated
with methanol.
1
solid. H NMR (300 MHz, CDCl3) δ 7.48−7.27 (m, 5H), 4.82 (s,
2H), 2.81 (s, 3H), 2.54 (s, 3H). 13C NMR (400 MHz, DMSO) δ
172.17, 169.75, 169.46, 167.45, 142.11, 139.40, 129.42, 126.98, 121.90,
19.83, 11.80. LC-MS (ESI): m/z 350.1 [M + H]+. HPLC purity 98%.
7-Amino-2-benzyl-N-cyano-4-methyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxamide (2l). To a solution of 2b
(0.150 g, 0.482 mmol) in a mixture of DMF (2 mL) and acetonitrile
(2 mL), were added EDC (0.110 g, 0.578 mmol), HOBt (0.089 g, 0.58
mmol), and cyanamide (0.020 g, 0.48 mmol). After stirring for 30 min,
DiPEA (0.170 mL, 0.964 mmol) was added and the reaction mixture
was stirred at rt overnight. The crude reaction mixture was
concentrated in vacuo and purified by preparative HPLC to give 2l
7-Amino-2-(4-fluorobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5a). Yield 5a: (28
mg, 40%). 1H NMR (400 MHz, CDCl3) δ 7.49−7.37 (m, 2H), 7.08−
6.95 (m, 2H), 4.78 (s, 2H), 2.81 (s, 3H), 2.54 (s, 3H). LC-MS (ESI):
m/z 368.1 [M + H]+. HPLC purity 95%.
7-Amino-2-(4-chlorobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
1
(12 mg, 7%). H NMR (300 MHz, DMSO) δ 7.37−7.23 (m, 5H),
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5b). Yield 5b: (45
1
7.10 (br s, 2H), 4.72 (s, 2H), 2.58 (s, 3H). LC-MS (ESI): m/z 336.1
mg, 64%). H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.4 Hz, 2H),
[M + H]+. HPLC purity 97%.
7.30 (d, J = 8.4 Hz, 2H), 4.78 (s, 2H), 2.81 (s, 3H), 2.54 (s, 3H). LC-
MS (ESI): m/z 384.1 [M + H]+. HPLC purity 95%.
7-Amino-2-benzyl-4-methyl-6-(3-(trifluoromethyl)-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (2m). To a solu-
tion of (E)-2,2,2-trifluoro-N′-hydroxyacetimidamide (91 mg, 0.707
mmol) in anhydrous dioxane (3 mL) at −10 °C was added sodium
hydride (71 mg, 1.77 mmol, 60% in mineral oil). The mixture was
stirred for 15 min at rt followed by heating for 30 min at 65 °C. Ethyl
7-amino-2-benzyl-4-methyl-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]-
pyridine-6-carboxylate (1) (200 mg, 0.589 mmol) was then added in
one portion and the mixture stirred overnight at 65 °C. After cooling
to rt, the reaction was quenched by adding satd NH4Cl (1 mL) and
stirring for 15 min. The mixture was transferred to a separatory funnel,
water (20 mL) added, and the product extracted with EtOAc (2 × 20
mL). After drying over Na2SO4, filtration and concentration under
reduced pressure the crude product was taken up in dioxane (2 mL)
and BEMP on polystyrene (234 mg, 0.514 mmol, labeling 2.2 mol/g)
was added. The mixture was heated at 75 °C for 2 h. LC-MS of the
reaction indicated that all material had been converted to the desired
product. The solution was filtered and the solvent removed in vacuo to
yield a yellow solid. Purification by preparative HPLC gave 2m (10
mg, 17%) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 7.44−7.47
(m, 2H), 7.31−7.40 (m, 3H), 4.86 (s, 2H), 2.85 (s, 3H). LC-MS
(ESI): m/z 404.1 [M + H]+. HPLC purity 99%.
Ethyl 7-Amino-4-methyl-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-
c]pyridine-6-carboxylate (3). Trifluoroacetic acid (0.060 g, 0.60
mmol) was added to a mixture of 1H-pyrrole-2,5-dione (6.0 g, 6.2
mmol) and ethyl acetamidocyanoacetate (10.6 g, 6.2 mmol) in DCE.
The reaction mixture was heated at reflux for 48 h. The solvent was
evaporated under reduced pressure, and the crude product was
triturated with methanol followed by diethyl ether to afford 3 (5.0 g,
32%) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 11.43 (s, 1H),
6.91 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.56 (s, 3H), 1.33 (t, J = 7.1 Hz,
3H). LC-MS (ESI): m/z 250.1 [M + H]+. HPLC purity 95%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-1H-pyrrolo-
[3,4-c]pyridine-1,3(2H)-dione (4). To a solution of N′-hydroxyaceti-
midamide (2.85 g, 38.5 mmol) in anhydrous dioxane (160 mL) at −10
°C was added sodium hydride (2.57 g, 64.2 mmol, 60% in mineral oil).
The mixture was stirred for 15 min at rt followed by heating for 30
min at 65 °C. The ester 3 (8.00 g, 32.1 mmol) was then added in one
portion and the mixture stirred overnight at 65 °C. After cooling to rt,
the reaction was quenched by adding satd NH4Cl (100 mL) and
stirring for 15 min. The mixture was transferred to a separatory funnel,
water (400 mL) added, and the product was extracted with EtOAc (2
× 400 mL). After drying over MgSO4, filtration, and concentration
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(4-
(trifluoromethyl)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
1
(5c). Yield 5c: (64 mg, 83%). H NMR (400 MHz, CDCl3) δ 7.63−
7.48 (m, 4H), 4.87 (s, 2H), 2.82 (s, 3H), 2.54 (s, 3H). LC-MS (ESI):
m/z 418.2 [M + H]+. HPLC purity 95%.
7-Amino-2-(4-methoxybenzyl)-4-methyl-6-(3-methyl-1,2,4-oxa-
diazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5d). Yield 5d:
1
(47 mg, 66%). H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.6 Hz,
2H), 6.86 (d, J = 8.6 Hz, 2H), 4.75 (s, 2H), 3.78 (s, 3H), 2.81 (s, 3H),
2.54 (s, 3H). LC-MS (ESI): m/z 380.1 [M + H]+. HPLC purity 95%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(4-
(trifluoromethoxy)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
(5e). Yield 5e: (40 mg, 50%). 1H NMR (400 MHz, CDCl3) δ 7.47 (d,
J = 8.6 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 4.81 (s, 2H), 2.81 (s, 3H),
2.54 (s, 3H). LC-MS (ESI): m/z 434.1 [M + H]+. HPLC purity 95%.
7-Amino-2-(4-bromobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5f). Yield 5f: (73
1
mg, 96%). H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.4 Hz, 2H),
7.31 (d, J = 8.4 Hz, 2H), 4.76 (s, 2H), 4.66 (s, 1H), 2.81 (s, 3H), 2.54
(s, 3H). LC-MS (ESI): m/z 430.0 [M + H]+. HPLC purity 99%.
7-Amino-2-(3-fluorobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5g). Yield 5g: (29
mg, 43%). 1H NMR (400 MHz, CDCl3) δ 7.34−7.01 (m, 4H), 4.81 (s,
2H), 2.82 (s, 3H), 2.54 (s, 3H). LC-MS (ESI): m/z 368.1 [M + H]+;
HPLC purity 95%.
7-Amino-2-(3-chlorobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5h). Yield 5h: (43
mg, 61%). 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 7.35−7.19 (m,
3H), 4.76 (s, 2H), 2.80 (s, 3H), 2.52 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 171.91, 167.96, 167.24, 167.14, 143.92, 139.15, 137.70,
134.67, 130.88, 130.09, 128.74, 128.36, 126.83, 125.03, 120.21, 41.13,
19.94, 11.74. LC-MS (ESI): m/z 384.0 [M + H]+. HPLC purity 95%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(3-
(trifluoromethyl)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
1
(5i). Yield 5i: (26 mg, 34%). H NMR (400 MHz, CDCl3) δ 7.69 (s,
1H), 7.63 (d, J = 8.3 Hz, 1H), 7.56 (t, J = 6.6 Hz, 1H), 7.47 (t, J = 7.8
Hz, 1H), 4.87 (s, 2H), 2.82 (s, 3H), 2.55 (s, 3H). LC-MS (ESI): m/z
418.1 [M + H]+. HPLC purity 96%.
7-Amino-2-(3-methoxybenzyl)-4-methyl-6-(3-methyl-1,2,4-oxa-
diazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5j). Yield 5j:
1
(50 mg, 70%). H NMR (400 MHz, CDCl3) δ 7.26 (dd, J = 8.6, 7.2
Hz, 2H), 7.01 (d, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.83 (d, J = 8.3 Hz,
H
DOI: 10.1021/acs.jmedchem.5b01542
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 4.79 (s, 2H), 3.80 (s, 3H), 2.81 (s, 3H), 2.54 (s, 3H). LC-MS
(ESI): m/z 380.1 [M + H]+. HPLC purity 95%.
(s, 3H), 2.54 (s, 3H). LC-MS (ESI): m/z 450.3 [M + H]+. HPLC
purity 99%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(3-
7-Amino-2-(4-(hydroxymethyl)benzyl)-4-methyl-6-(3-methyl-
(trifluoromethoxy)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5x).
1
1
(5k). Yield 5k: (35 mg, 43%). H NMR (400 MHz, CDCl3) δ 7.40−
Yield 5x: (51 mg, 74%). H NMR (400 MHz, CDCl3) δ 7.43 (d, J
7.32 (m, 2H), 7.29 (s, 1H), 7.16 (s, 1H), 4.82 (s, 2H), 2.82 (s, 3H),
2.54 (s, 3H). LC-MS (ESI): m/z 434.1 [M + H]+. HPLC purity 95%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(1-phe-
nylethyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5l). Yield 5l: (21
= 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 4.82 (s, 2H), 4.67 (s, 2H),
2.81 (s, 3H), 2.54 (s, 3H), 1.64 (br s, 1H). LC-MS (ESI): m/z 380.3
[M + H]+. HPLC purity 98%.
7-Amino-2-(4-(dimethylamino)benzyl)-4-methyl-6-(3-methyl-
1
mg, 32%). H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 7.6 Hz, 2H),
1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5y).
1
7.36 (t, J = 7.5 Hz, 2H), 7.31−7.26 (m, 1H), 5.54 (q, J = 7.3 Hz, 1H),
2.80 (s, 3H), 2.54 (s, 3H), 1.93 (d, J = 7.3 Hz, 3H). LC-MS (ESI): m/
z 364.1 [M + H]+. HPLC purity 99%.
Yield 5y: (18 mg, 24%). H NMR (400 MHz, CDCl3) δ 7.33 (d, J
= 8.7 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H), 4.72 (s, 2H), 2.92 (s, 6H),
2.80 (s, 3H), 2.54 (s, 3H). LC-MS (ESI): m/z 393.4 [M + H]+. HPLC
purity 95%.
7-Amino-2-(2-fluorobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5m). Yield 5m:
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(4-(2-oxo-
1
(57 mg, 89%). H NMR (400 MHz, CDCl3) δ 7.43−7.35 (m, 1H),
pyrrolidin-1-yl)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5z).
1
7.32−7.26 (m, 1H), 7.13−7.06 (m, 2H), 4.90 (s, 2H), 2.82 (s, 3H),
2.54 (s, 3H). LC-MS (ESI): m/z 368.3 [M + H]+. HPLC purity 99%.
7-Amino-2-(2-chlorobenzyl)-4-methyl-6-(3-methyl-1,2,4-oxadia-
zol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5n). Yield 5n: (62
Yield 5z: (36 mg, 47%). H NMR (400 MHz, CDCl3) δ 7.58 (d, J =
8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 4.79 (s, 2H), 3.83 (t, J = 7.0 Hz,
2H), 2.81 (s, 3H), 2.59 (t, J = 8.1 Hz, 2H), 2.54 (s, 3H), 2.22−2.07
(m, 2H). LC-MS (ESI): m/z 433.4 [M + H]+. HPLC purity 99%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(4-(pyri-
din-4-yl)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5aa).
Yield 5aa: (43 mg, 55%). 1H NMR (400 MHz, CDCl3) δ 8.68−
8.62 (m, 2H), 7.68−7.41 (m, 6H), 4.88 (s, 2H), 2.82 (s, 3H), 2.54 (s,
3H). LC-MS (ESI): m/z 427.3 [M + H]+. HPLC purity 97%.
7-Amino-2-(4-(4-fluorophenoxy)benzyl)-4-methyl-6-(3-methyl-
1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5ab).
Yield 5ab: (44 mg, 53%). 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J =
8.6 Hz, 2H), 7.10−6.85 (m, 6H), 4.78 (s, 2H), 2.81 (s, 3H), 2.54 (s,
3H). LC-MS (ESI): m/z 460.2 [M + H]+. HPLC purity 99%.
2-(4-(1H-Pyrazol-1-yl)benzyl)-7-amino-4-methyl-6-(3-methyl-
1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5ac).
Yield 5ac: (73 mg, 97%). 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J =
2.5 Hz, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.52
(d, J = 8.6 Hz, 2H), 6.45 (t, J = 2.1 Hz, 1H), 4.84 (s, 2H), 4.74 (s,
1H), 2.82 (s, 3H), 2.54 (s, 3H). LC-MS (ESI): m/z 416.2 [M + H]+.
HPLC purity 98%.
1
mg, 91%). H NMR (400 MHz, CDCl3) δ 7.41 (dd, J = 14.0, 8.2 Hz,
1H), 7.34−7.21 (m, 3H), 5.00 (s, 2H), 2.85 (s, 3H), 2.57 (s, 3H). LC-
MS (ESI): m/z 384.3 [M + H]+. HPLC purity 98%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(2-
(trifluoromethyl)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
(5o). Yield 5o: (53 mg, 72%). 1H NMR (400 MHz, CDCl3) δ 7.71 (d,
J = 7.7 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.22
(d, J = 7.7 Hz, 1H), 5.07 (s, 2H), 2.84 (s, 3H), 2.55 (s, 3H). LC-MS
(ESI): m/z 418.3 [M + H]+. HPLC purity 99%.
7-Amino-2-(2-methoxybenzyl)-4-methyl-6-(3-methyl-1,2,4-oxa-
diazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5p). Yield 5p:
1
(46 mg, 93%). H NMR (400 MHz, CDCl3) δ 7.32−7.15 (m, 2H),
6.97−6.82 (m, 2H), 4.88 (s, 2H), 3.86 (s, 3H), 2.82 (s, 3H), 2.54 (s,
3H). LC-MS (ESI): m/z 380.3 [M + H]+. HPLC purity 99%.
7-Amino-2-(4-(difluoromethoxy)benzyl)-4-methyl-6-(3-methyl-
1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5q).
1
Yield 5q: (68 mg, 91%). H NMR (400 MHz, CDCl3) δ 7.44 (d, J
= 8.6 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.48 (t, J = 73.7 Hz, 1H), 4.80
(s, 2H), 4.77 (s, 1H), 2.81 (s, 3H), 2.54 (s, 3H). LC-MS (ESI): m/z
416.1 [M + H]+. HPLC purity 98%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(piperi-
din-4-ylmethyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5ad). tert-
Butyl 4-((7-amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-1,3-
dioxo-1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)methyl)piperidine-1-
7-Amino-2-((5-chloropyridin-2-yl)methyl)-4-methyl-6-(3-methyl-
1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5r).
1
carboxylate yield (Boc-5ad): Yield Boc-5ad: (88 mg, 99%). H NMR
1
Yield 5r: (52 mg, 70%). H NMR (400 MHz, CDCl3) δ 8.48 (d, J
(400 MHz, CDCl3) δ 4.12 (m, 2H), 3.56 (d, J = 7.1 Hz, 2H), 2.82 (s,
3H), 2.67 (t, J = 11.4 Hz, 2H), 2.54 (s, 3H), 2.02−1.86 (m, 1H), 1.65
(d, J = 12.6 Hz, 2H), 1.48−1.40 (m, 11H). LC-MS (ESI): m/z 457.5
[M + H]+. HPLC purity 99%. To a solution of tert-butyl 4-((7-amino-
4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-1,3-dioxo-1,3-dihydro-2H-
pyrrolo[3,4-c]pyridin-2-yl)methyl)piperidine-1-carboxylate (Boc-5ad)
(88 mg, 0.20 mmol) in DCM (2.0 mL) was added trifluoroacetic acid
(2 mL) and the mixture stirred for 1 h at rt. The reaction mixture was
concentrated and dissolved in DCM (30 mL). The organics were
washed with satd NaHCO3 (3 × 20 mL). Filtration and concentration
gave 5ad (54 mg, 75%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
δ 3.56 (d, J = 7.1 Hz, 2H), 3.11 (d, J = 12.3 Hz, 2H), 2.82 (s, 3H),
2.65−2.50 (m, 5H), 2.01−1.82 (m, 1H), 1.68 (d, J = 12.7 Hz, 2H),
1.26−1.17 (m, 2H). LC-MS (ESI): m/z 357.4 [M + H]+. HPLC purity
98%.
= 2.3 Hz, 1H), 7.66 (dd, J = 8.3, 2.5 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H),
4.95 (s, 2H), 2.82 (s, 3H), 2.55 (s, 3H). LC-MS (ESI): m/z 385.1 [M
+ H]+. HPLC purity 98%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(pyridin-
4-ylmethyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione (5s). Yield 5s:
1
(34 mg, 54%). H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 5.3 Hz,
2H), 7.30 (d, J = 5.5 Hz, 2H), 4.82 (s, 2H), 2.82 (s, 3H), 2.55 (s, 3H).
LC-MS (ESI): m/z 351.1 [M + H]+. HPLC purity 99%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(4-
(methylsulfonyl)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
(5t). Yield 5t: (45 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J
= 8.1 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 4.90 (s, 2H), 3.02 (s, 3H),
2.82 (s, 3H), 2.55 (s, 3H). LC-MS (ESI): m/z 428.1 [M + H]+. HPLC
purity 95%.
4-((7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-1,3-
dioxo-1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)methyl)benzonitrile
(5u). Yield 5u: (60 mg, 89%). 1H NMR (400 MHz, CDCl3) δ 7.64 (d,
J = 8.1 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H), 4.85 (s, 2H), 2.82 (s, 3H),
2.55 (s, 3H). LC-MS (ESI): m/z 375.1 [M + H]+. HPLC purity 99%.
4-((7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-1,3-
dioxo-1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)methyl)benzamide
(5v). Yield 5v: (17 mg, 23%).1H NMR (300 MHz, DMSO) δ 7.93 (br
s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.29 (br m,
3H), 4.79 (s, 2H), 2.64 (s, 3H), 2.49 (s, 3H). LC-MS (ESI): m/z
393.1 [M + H]+. HPLC purity 96%.
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione
1
(5ae). Yield 5ae: (65 mg, 99%). H NMR (400 MHz, CDCl3) δ 3.98
(dd, J = 11.4, 3.3 Hz, 2H), 3.57 (d, J = 7.1 Hz, 2H), 3.35 (td, J = 11.7,
1.6 Hz, 2H), 2.82 (s, 3H), 2.55 (s, 3H), 2.12−1.92 (m, 1H), 1.64−1.56
(m, 2H), 1.50−1.32 (m, 2H). LC-MS (ESI): m/z 358.2 [M + H]+.
HPLC purity 97%.
Ethyl 7-Amino-4-methyl-1,3-dioxo-2-phenyl-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxylate (6). The reaction was performed
according to the procedure of Shimada et al. to yield the title
compound (36 mg, 7%).8 The 1H NMR data were in agreement with
7-Amino-4-methyl-6-(3-methyl-1,2,4-oxadiazol-5-yl)-2-(4-
((trifluoromethyl)thio)benzyl)-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-
dione (5w). Yield 5w: (73 mg, 91%). 1H NMR (400 MHz, CDCl3) δ
7.63 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 4.84 (s, 2H), 2.82
1
the reported spectra. H NMR (300 MHz, CDCl3) δ 7.57−7.37 (m,
5H), 4.51 (q, J = 7.1 Hz, 2H), 2.83 (s, 3H), 1.48 (t, J = 7.1 Hz, 3H).
LC-MS (ESI): m/z 326.0 [M + H]+. HPLC purity 95%.
I
DOI: 10.1021/acs.jmedchem.5b01542
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Ethyl 7-Amino-2-benzyl-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]-
pyridine-6-carboxylate (7). To a cooled suspension of ethyl (E)-2-
cyano-2-(hydroxyimino)acetate (0.50 g, 3.52 mmol) and acetic
anhydride (1.40 g, 14.1 mmol) in formic acid (5 mL) was added
zinc dust (0.700 g, 10.7 mmol) in small portions. The suspension was
stirred at rt for 4 days. The reaction was diluted with water (10 mL),
and the pH was adjusted to 8−9 using a saturated sodium carbonate
solution. The product was extracted with EtOAc (2 × 50 mL). The
combined organic layers were washed with water (2 × 50 mL) and
brine, dried over Na2SO4, and evaporated. Crude ethyl 2-cyano-2-
formamidoacetate (0.25 g, 46%) was taken to the next step without
further purification. LC-MS APCI: calculated for C6H8N2O3 156.14;
observed m/z [M + H]+ 157.4. Trifluoroacetic acid (0.020 g, 0.16
mmol) was added to a mixture of 1-benzyl-1H-pyrrole-2,5-dione
(0.250 g, 1.60 mmol) and ethyl 2-cyano-2-formamidoacetate (0.360 g,
1.92 mmol) in DCE. The reaction mixture was heated at reflux for 48
h. The solvent was evaporated under reduced pressure, and the crude
product was purified by flash column chromatography (10−20%
EtOAc/petroleum ether) to afford 7 (0.23 g, 44%) as a yellow solid.
1H NMR (400 MHz, DMSO) δ 8.34 (s, 1H), 7.27−7.35 (m, 5H), 7.19
ACKNOWLEDGMENTS
■
We acknowledge Helena I. M. Boshoff from the National
Institute of Allergy and Infectious Diseases (NIAID), for
technical assistance and advice with in vitro antimycobacterial
assays, and Sridevi Bashyam from Syngene International Ltd for
synthetic chemistry support. This work was supported by
grants from the Novartis Research Foundation, the Bill &
Melinda Gates Foundation, and the Strategic Health
Innovation Partnership (SHIP) unit of the South African
Medical Research Council (SAMRC). The University of Cape
Town, SAMRC, and South African Research Chairs Initiative of
the Department of Science and Technology, administered
through the South African National Research Foundation, are
gratefully acknowledged for support (K.C.).
ABBREVIATIONS USED
■
TB, tuberculosis; Mtb, Mycobacterium tuberculosis; INH,
isoniazid; RIF, rifampicin; MDR, multidrug-resistant; ADME,
absorption, distribution, metabolism and excretion; HTS, high
throughput screening; HLM, human liver microsomes; RLM,
rat liver microsomes; MLM, mouse liver microsomes; EDC, N-
(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochlor-
ide; HOBt, 1-hydroxybenzotriazole hydrate; T3P, propylphos-
phonic anhydride; TsCl, p-toluenesulfonyl chloride; FaSSIF,
fasted state simulated intestinal fluid; NADPH, reduced
nicotinamide adenine dinucleotide phosphate; PS-BEMP,
polymer-supported 2-tert-butylimino-2-diethylamino-1,3-di-
methyl-perhydro-1,3,2-diazaphosphorine; PS-PhSH, thiophenol
on polystyrene; ADMET, absorption, distribution, metabolism,
excretion, and toxicity; SAWC, South Africa Western Cape;
HPLC, high-performance liquid chromatography; LC-MS,
liquid chromatography−mass spectrometry; MIC90, lowest
concentration of drug that inhibits growth of more than 90%
of the bacterial population; RFLP, restriction fragment length
polymorphism; PAMPA, parallel artificial membrane perme-
ability assay; PPB, plasma protein binding; ABT, amino-
benzotriazole; CYP cytochrome P450 enzymes; SRM, sponta-
neous resistant mutants
(s, 2H), 4.74 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, DMSO) δ 168.41, 167.17, 165.89, 142.49,
136.72, 134.90, 129.95, 129.47, 129.01, 127.93, 127.89, 120.06, 61.81,
41.34, 14.49. LC-MS (APCI): m/z 326.2 [M + H]+. HPLC purity 97%
Methyl 7-Amino-2-benzyl-1-hydroxy-4-methyl-3-oxo-2,3-dihy-
dro-1H-pyrrolo[3,4-c]pyridine-6-carboxylate (8a) and Ethyl 7-
Amino-2-benzyl-3-hydroxy-4-methyl-1-oxo-2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine-6-carboxylate (8b). To a cooled suspension of
1 (1.0 g, 2.94 mmol) in methanol (25 mL) and DCM (15 mL) was
added sodium borohydride (0.11 g, 2.94 mmol) in small portions over
a period of 30 min and stirred for 1 h at the same temperature.
Solvents were evaporated under reduced pressure, and the crude
product was purified by preparative HPLC to afford 8a (0.075 g, 8%)
1
and 8b (0.11 g, 11%). 8a: H NMR (400 MHz, CDCl3) δ 7.37−7.45
(m, 5H), 6.91 (s, 2H), 5.62 (s, 1H), 5.10 (d, J = 14.7 Hz, 1H), 4.39 (d,
J = 14.8 Hz, 1H), 3.98 (s, 3H), 2.55 (s, 3H). 13C NMR (100 MHz,
DMSO) δ 167.09, 166.58, 159.38, 158.87, 158.18, 142.56, 140.62,
136.83, 128.98, 128.22, 127.93, 127.69, 122.46, 79.82, 52.21, 42.37,
19.91. LC-MS (APCI): m/z 328.2 [M + H]+. HPLC purity 99%. 8b:
1H NMR (400 MHz, CD3OD) δ 8.13−8.06 (m, 5H), 7.39 (s, 2H),
6.57 (s, 1H), 5.67 (d, J = 15.4 Hz, 1H), 5.11−5.17 (m, 3H), 3.21 (s,
3H), 2.13 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 167.46,
167.41, 159.86, 159.49, 159.12, 143.19, 141.39, 138.54, 129.78, 129.31,
129.12, 129.09, 129.04, 128.71, 128.49, 123.33, 80.63, 61.82, 43.19,
20.70, 15.47. LC-MS (APCI): m/z 342.2 [M + H]+. HPLC purity 99%
REFERENCES
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01542.
■
(1) Global Tuberculosis Report 2014; World Health Organisation:
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Chemistry, biology assays, in vitro ADMET assays,
metabolite identification studies, mouse pharmacokinetic
studies with 5h, references (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +27-21-6502553. Fax: +27-21-6505195. E-mail: Kelly.
Chibale@uct.ac.za.
■
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
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