Synthetic Strategies towards Imidazopyridinones and 7-Azaoxindoles and their Evaluation as Antibacterial Agents

Article abstract of DOI:10.1002/ejoc.202100172

Imidazopyridinones and 7-azaoxindoles are two classes of heterocyclic compounds with only limited previous use in organic and medicinal chemistry. Various synthetic methods and routes have been evaluated to identify safe and robust chemistry to advanced i

Full text of DOI:10.1002/ejoc.202100172

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Synthetic Strategies towards Imidazopyridinones and
7-Azaoxindoles and their Evaluation as Antibacterial
Agents
Fredrik Heen Blindheim,[a] Cecilie Elisabeth Olsen,[a] Caroline Krogh Søgaard,[b]
Marit Otterlei,[b] Eirik Sundby,[c] and Bård Helge Hoff*[a]
Imidazopyridinones and 7-azaoxindoles are two classes of
heterocyclic compounds with only limited previous use in
organic and medicinal chemistry. Various synthetic methods
and routes have been evaluated to identify safe and robust
chemistry to advanced imidazopyridinone building blocks and
inhibitor structures. Preparation of the 7-azaoxindoles was
challenged by instability of the core scaffold. Key to the
successful isolation of the target structure was development of
a mild Suzuki cross-coupling in non-aqueous media. The
imidazopyridinones were potent inhibitors of the E. coli
thymidylate monophosphate kinase, while the 7-azaoxindole
showed low activity. The compounds were inactive in cell-based
studies, indicating poor cell wall penetration.
Introduction
The rise of antibiotic resistance has necessitated the develop-
ment of new antimicrobial agents with novel modes of action.[1]
One possible target for new antibiotics is thymidylate mono-
phosphate kinase (TMPK), an essential enzyme of thymidine
diphosphate and DNA synthesis.[2] Sufficient sequence differ-
ences between human- and bacterial TMPKs allows selective
inhibition,[3] and TMPK X-ray co-crystal structures are available
from a variety of organisms, making rational drug design
possible.[4] Most inhibitors targeting TMPK in prokaryotes are
based on modifications of thymine or thymidine.[4–5] The recent
years have, however, seen the development of other classes of
inhibitors, such as the metal-based complexes of 3-methyl-N-
hydroxybenzamidine developed by Lautre et al.[6] or the
imidazopyridinone 1 (Figure 1) developed by Choi et al.[7]
Besides this work only a few imidazopyridinones have been
evaluated for biological activity.[8]
Figure 1. Structures of the known inhibitor 1 and the target molecules 2 and
3.
We have attempted to identify new design concepts for
TMPK inhibitors by using the E. coli enzyme as model. Molecular
docking based on the structure 1 indicated that increased
affinity for TMPK could be obtained by introducing an electron
donating substituent at the central aromatic ring (structure 2).
Further, docking could not explain the role of the N-methyl
group of 1 in binding to TMPK and a possible model compound
to investigate this was the 7-azaoxindole 3, see Figure 1.
However, a challenge with 7-azaoxindoles is that the unsub-
stituted lactam is rather acidic and prone to condensation
reactions.[9] Thus, we set out to prepare these model com-
pounds, and herein describe complications and solutions for
the chemistry, alongside their preliminary biological evaluation
towards E. coli TMPK and a panel of human kinases.
[a] F. H. Blindheim, C. E. Olsen, B. H. Hoff
Department of Chemistry
Norwegian University of Science and Technology (NTNU)
7491 Trondheim, Norway
E-mail: bard.h.hoff@ntnu.no
www.ntnu.edu/chemistry/research/organic/applied#
[b] C. Krogh Søgaard, Prof. M. Otterlei
Department of Clinical and Molecular Medicine
Norwegian University of Science and Technology (NTNU)
7489 Trondheim, Norway
[c] Prof. E. Sundby
Department of Material Science
Norwegian University of Science and Technology (NTNU)
7491 Trondheim, Norway
Results and Discussion
Docking
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202100172
© 2021 The Authors. European Journal of Organic Chemistry published by
Wiley-VCH GmbH. This is an open access article under the terms of the
Creative Commons Attribution Non-Commercial License, which permits use,
distribution and reproduction in any medium, provided the original work is
properly cited and is not used for commercial purposes.
Docking of compound 1 was undertaken with two different E.
coli TMPK structures (PDB: 5TMP and 4TMK) using GLIDE in
extra precision (XP) mode with Schrödinger Maestro. This
revealed a number of hydrophilic and lipohilic interactions,
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Figure 2. Docking of compound 2 into E. coli TMPK (PDB: 5TMP).
among others what appeared as a cation-π interaction between
Arg51 and the central carboaromatic ring. Moreover, the role of
the N-methyl group in binding was unclear, as this group points
towards the solvent-exposed entrance with no obvious inter-
actions. Thus, we speculated that the cation-π interaction could
be strengthened by adding an electron donating group as in
structure 2 (Figure 2) and that the N-methyl unit could be
replaced by a carbon as seen in the 7-azaoxindole 3. Indeed,
the docking scores (Supplementary information file, Table S2)
indicate that compounds 2 and 3 were comparable to structure
1 as inhibitors of TMPK. Figure 3 shows an overlay of the three
docked structures 1–3 indicating only minimal distortion of the
core scaffolds. Compounds 1 and 3 were also subjected to
10 ns dynamics, which indicated very similar flexibility for the
two fused 5-membered rings (Supplementary information, Fig-
ure S17–S20).
Retrosynthetic analysis of compounds 1–3
A retrosynthetic analysis of target compounds 1–3 is shown in
Scheme 1. The linear synthesis used previously[7] to prepare
imidazopyridinone 1 involves disconnection to the commer-
cially available 3-phenyl-1-propylamine (fragment A), and an
advanced carboxylic imidazopyridinone (fragment B). This in
turn can be made from an organoboron reagent (fragment E)
Figure 3. An overlay of the docked structures 1–3.
Scheme 1. Retrosynthesis of the target compounds 1–3.
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and the heterocyclic building block (fragment D). We also
Our synthesis of imidazopyridinone 6 (fragment D) initially
made use of triphosgene[7] in yields up to 47% (see Scheme 3).
Due to the hazards related to triphosgene, sampling and
optimisation was not performed.
wanted to explore a more convergent synthesis, starting from
an advanced organoboron compound (fragment C) and the
heterocyclic building block D.
For the synthesis of target molecules 1 and 2, the
heterocyclic fragment D could be prepared from 2,3-diamino-5-
bromo-pyridine. Although the synthesis of these building blocks
has been reported,[7] we wanted to develop a route with higher
yields and without hazardous reagents.
Instead, to make the reaction safer, the carbocyclisation was
also tested with urea and carbonyldiimidazole (CDI) as cyclizing
agents. The use of urea yielded 6 in 37% yield, owing both to
by-product formation and incomplete conversion. However,
syntheses with 5 equiv. CDI, employing triethylamine as base at
reflux led to full conversion and the isolation of compound 6 in
82% yield. Finally, compound 6 was protected with trityl
chloride to form compound 7 in 88% yield.
Synthesis of imidazopyridinones 1 and 2
For the synthesis of target molecule 1, the more convergent
route was used. The assembly of 3-phenyl-1-propylamine (frag-
ment A) and 3-boronobenzoic acid (fragment E) was done
through an amide coupling with 1-[bis(dimethylamino)
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-
fluoro-phosphate (HATU) and DIPEA, see Scheme 4.
This gave boronic acid 8 in a variable 22–70% yield,
depending on the number of purification steps. The subsequent
Suzuki coupling with heteroaryl 7 (fragment D) with Pd(dppf)Cl2
gave the protected analogue 9 in 81% yield, and after
deprotection with trifluoroacetic acid (TFA) in CH2Cl2 compound
1 was obtained in 58% yield.
The aryl boronic ester 10 (see Scheme 5) needed to prepare
2 was made by a previously reported protocol[10] (see Support-
ing Information). Coupling of 10 to fragment D was first
attempted using the unprotected building block 6 (Scheme 5)
and a range of palladium catalysts. However, all attempts
resulted in low conversion, exhibited low yields of the biaryl 11,
and high amounts of methyl 3-hydroxy-4-methoxybenzoate as
the main by-product. Instead the trityl derivative 7 was used as
coupling partner, whereupon the Suzuki reaction gave a 78%
isolated yield of 12.
Two strategies were explored for attachment of 3-phenyl-1-
propylamine to fragment B, see Scheme 6. The first method
employed hydrolysis of 12 with HCl to the free acid 13.
Disappointingly, the following HATU mediated coupling with 3-
phenyl-1-propylamine, yielded only 3% of product. Rather than
optimizing this amide coupling we instead went for a direct
amination of the methyl ester 12. Traditionally this has been
achieved with trimethyl aluminum,[11] but due to its highly
The logical steps from commercially available 2,3-diamino-5-
bromopyridine to the key imidazopyridinone fragment D,
involves a regioselective methylation and a carbocyclisation.
The methylation protocols tested to arrive at 5-bromo-N3-
methylpyridine-2,3-diamine (5) are shown in Scheme 2.
Compound 5 was first synthesized via the N3-Cbz-protected
derivative 4. Initial reactions with benzyl chloroformate (Cbz-Cl)
3
°
were conducted at 0 C, but low selectivity towards N -
protection was observed. The best conditions identified was a
°
reaction time of 30 min at À 98 C, quenching with excess water
and then slowly increasing the temperature. However, the high
selectivity comes with the price of mediocre conversion and an
isolated yield of 46–49% in 3–15 gram scales.
Alternative methylation strategies were also explored. The
use of N-benzyloxy-carbonyloxy)succinimide (Cbz-OSu) required
a much higher reaction temperature and stronger base to
proceed and resulted in low selectivity, while methylation with
methyl iodide did not lead to product formation under the
conditions attempted. Next, reductive cleavage of the Cbz
derivative 4 with LiAlH4 gave the methylated aminopyridine 5
in 82% isolated yield. Finally, we tested a two-step one-pot
protocol, in which the Cbz derivative 4 was directly reduced by
LiAlH4 without intermediate purification. This process per-
formed on par with the original protocol on a small scale but
had issues with Cbz-precipitation/mass transfer and aggregates
of alumina salts during work-up when scaling to grams.
Although simple, more development work is needed to harvest
any benefit.
Scheme 2. Preparation of 5 from 2,3-diamino-5-bromopyridine.
Scheme 3. Synthesis of key building block 7 for fragment A.
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Scheme 4. Synthesis of target molecule 1.
achieved in 400 mg scale using 3 equiv. of both coupling agent
and amine. The intermediate 14 was not isolated but directly
deprotected using TFA, leading to an overall yield of 82%, with
the target molecule 2 isolated as its TFA-salt.
Overall, both synthetic routes from precursor 17 to the end
products 1 and 2 worked satisfactory, and the choice of method
will depend on the task at hand. The three-step approach
employing DABAL-Me3 to form the amide, provided higher yield
than the more convergent method employing Suzuki cross-
coupling with the boronic acid 8. It must however be
emphasized that these final steps have not been optimised.
Synthesis of 7-azaoxindole 3
The 7-azaoxindole 3 was first planned prepared as described in
Scheme 1.
Although there was some precedence for the cross-coupling
Scheme 5. Two strategies for merging fragment D with fragment E.
between
5-bromo-1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-one
(16) and aryl boronic acids,[13] the initial Suzuki reactions of 16
were slow, proceeded with low conversions and only trace
amounts of product 19a were formed. The lack of reactivity
was assumed to be caused by coordination of the substrate to
palladium. To circumvent this issue, a protection strategy was
pyrophoric nature, bis(trimethyl-aluminum)-1,4-diazabi-cyclo
[2.2.2]octane adduct (DABAL-Me3), which can release trimethyl
aluminum in situ, was used.[12] Successful amide formation was
Scheme 6. The end-game in preparation of the target structure 2.
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Scheme 7. First route to the target structure 3 and the structure of the model compound 18c.
sought. Insertion of SEM-, trityl- and acetyl N-protections were
unsuccessful in our hands (multiple products, data not shown),
while reaction of 16 with Boc2O gave the double N,O-diBoc
derivative 17 in 81% yield (Scheme 7).
The Boc-protecting group is usually stable in basic aqueous
media, but the initially tested Suzuki cross-couplings with 17
and 3-methoxycarbonylphenylboronic acid, catalyzed by
acidic conditions were too harsh giving by-products assumed
originating from lactam ring opening and side reactions on the
α-carbon. In the search for milder methods, we found that
lipase B from Candida antarctica mediated the hydrolysis, but at
a sluggish pace, giving the acid 19b in 19% yield after 5 days.
Unfortunately, coupling of the carboxylic acid 19b with 3-
phenyl-1-propylamine using HATU failed to give the target
°
Pd2(dba)3 and PCy3 in 1,4-dioxane/H2O (1:1) at 100 C, revealed
structure 3 and instead formed a complex mixture of
extensive degradation of both substrate and product. However,
it was found that decomposition was far less pronounced in
THF/t-BuOH. After tuning of the reaction conditions on a model
components (not shown). Fortunately, we knew that the 7-
azaoxindole scaffold tolerated mild Suzuki cross-coupling
conditions and we instead performed a reaction between 17
and 8, which gave precursor 20 in 47% yield (Scheme 8). Finally,
acid hydrolysis enabled the isolation of the target 3 in 46%
yield.
Although compound 3 was successfully prepared, carrying
the unstable azaoxoindole through three steps is challenging.
Thus, in an alternative approach, the plan was to introduce the
labile γ-lactam at the end. As our major concern was the
oxidation step, we first evaluated the chemistry from 23 to 19b.
The synthesis is outlined in Scheme 9.
°
reaction giving 18c, the use of XPhos Pd G2 and XPhos at 60 C
appeared as the best solution (see Supporting Information). It
should be noted that the model compound 18c (Scheme 8)
was unstable during silica-gel column chromatography, but
purification by reversed phase silica (RP) gave 75% yield. With a
decent outcome for the model reaction, we proceeded with
more relevant boronic acids. Ideally, coupling with 3-carbox-
yphenyl boronic acid would lead to a shorter overall route, but
the reactivity was too low compared to the rate of degradation.
However, coupling with 3-methoxycarbonylphenyl boronic acid,
lead to complete conversion in two hours. Purification of the
biaryl 18a by silica-gel column chromatography resulted in 68%
yield (Scheme 7).
Oxidation of 7-azaindols has previously been described
using a two-step procedure containing double-bromination at
C-3 followed by zinc reduction.[9a] In our hands the aqueous
bromination was carried out at room temperature, and after
one hour full conversion was observed by 1H NMR spectro-
scopy. However, quenching of the reaction mixture with either
Na2S2O5, Na2S2O3 or Na2SO3 led to decomposition, and we
therefore proceeded with a simple extraction as work up.
Attempts at purifying the dibrominated intermediate 24 also
led to severe decomposition on both NP and RP columns.
Instead, the following reduction with Zn powder in AcOH was
performed on the crude material which gave the acid 19b in
41% yield over two steps The process described above was
then tested in the oxidation of the more advanced derivative
26. To prepare the 7-azaindole 26, the protected intermediate
22 was first coupled with 3-phenyl-1-propylamine using HATU
giving the amide 25 in 58% yield (Scheme 10). This was
followed by deprotection with Cs2CO3 in MeOH/THF giving the
precursor 26 in 55% yield. However, the final oxidation failed at
Boc-deprotection of 7-azaoxindole 18a yielded 19a in 47%
yield. In the subsequent ester hydrolysis, conventional basic or
1
the bromination stage. H NMR analysis indicated that multiple
Scheme 8. Final steps towards the target 3.
sites were brominated and treatment with Zn powder did not
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Scheme 9. Synthesis of 19b with oxidation of the 7-azaindole 23.
Table 1. Evaluation of compound 1–3 as E.coli TMPK inhibitors in
enzymatic and in antimicrobial assays.
Enzymatic assay TMPK
Antimicrobial activity, MIC
E. coli
[μg/Ml]
Compound Inhibition [%][a] IC50 [μM][b] E. coli[c]
S. aureus[d]
1
2
3
61
69
9
1.4
5.6
83
>256
>256
>256
>256
>256
>256
[a] Measured at 8.3 μM. [b] See Supporting Information for IC50 curves. [c] E.
coli–MG1655. [d] S. aureus – ATCC29213.
Scheme 10. Preparation of the advanced intermediate 26 and attempted
oxidation.
are potent inhibitors towards the E. coli TMPK variant (IC50
curves are shown in Supporting Information). In contrast to that
indicated by molecular docking, efficient binding was not seen
for compound 3. Obviously, our docking procedures on this
rather flexible protein must be improved. We have attempted
to grow co-crystal structures of E. coli TMPK and compounds
1–3, but these efforts have not yielded crystals of sufficient
quality.
reduce the complexity of the mixture. Although the strategy
might be applicable for other more electron deficient aromatic
amines, we decided to abandon this route for the preparation
of 3.
Overall synthesis of the 7-azaoxindole 3 proved challenging
mainly due to the instability of the core structure. Among
others, the typically mild HATU amidation protocol could not
be applied. However, the 7-azaoxindole in protected form
tolerated Suzuki cross-coupling to yield advanced intermedi-
ates, providing a route for future design of new 7-azaoxindoles.
The second route employing a late stage oxidation/reduction
sequence is highly applicable if the molecule in question is not
prone to over- bromination.
Antimicrobial activity testing in culture revealed all three
compounds to have no activity towards both E. coli and S.
aureus, likely due to poor cell wall penetration. Although certain
design concepts can be applied to improve cell wall
penetration,[15] these are difficult to apply on inhibitors for
TMPK without proper structural information of binding mode.
To reveal if the imidazopyridinone 2 and the 7-azaoxindole
3 could have any off-targets or alternative applications, they
were assayed towards a panel of 60 human kinases at 500 nM
test concentration. Overall, these two model compounds
possessed low ability to inhibit these selected kinases, with the
highest activity seen towards the colony simulated factor 1
receptor (CSF1R) with 17 and 18% inhibition, respectively. Thus,
further development as CSF1R inhibitors is a potential applica-
tion of both compound classes. Interestingly, in contrast to that
seen in the TMPK assay, compounds 2 and 3 have rather similar
potency towards the human kinases. Figure 4 shows the 15
most inhibited kinases ranked by the inhibition towards
compound 2. All data can be found in the Supporting
Information file.
Biological evaluation of compounds 1–3
Compound 1 has previously been found active towards TMPK
from Pseudomonas aeruginosa.[7] We initially confirmed that
compound 1 was also an active E. coli TMPK inhibitor. Although
the sequence similarity as analysed by BLAST[14] was only 44%,
the two enzymes have very similar folding.[4] The potency of
compounds 1–3 towards E. coli TMPK was determined in an
enzymatic fluorescence assay. Single-point inhibition measure-
ments at 8.3 μM revealed the imidazopyridinones 1 and 2 to be
equipotent, while the 7-azaoxindole 3 showed low inhibition
(Table 1). Further, IC50 measurements confirmed that 1 and 2
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mass determination in positive and negative mode was performed
on a “Synapt G2-S” Q-TOF instrument from WatersTM. Samples
were ionized using an ASAP probe (APCI). No chromatographic
separation was done prior to the mass analysis. Calculated exact
mass and spectra processing was done by WatersTM Software
(Masslynx V4.1 SCN871). NMR spectra were recorded using the
Bruker DPX 400 MHz and 600 MHz Avance III HD NMR spectrom-
eters. Chemical shifts (δ) are recorded in parts per million relative to
TMS (δH=0.00, δC=0.0) or [D6]DMSO (δH=2.50, δC=39.5), and
coupling constants (J) are measured in hertz (Hz).
Figure 4. Evaluation of compound 2 and 3 towards a panel of kinases.
Synthesis of 4[16]
A solution of 2,3-diamino-5-bromopyridine (15.0 g, 79.9 mmol), dry
°
THF (190 mL) and dry pyridine (22.5 mL) was stirred at À 98 C for
Conclusion
10 minutes before the dropwise addition of benzyl chloroformate
(14.8 mL, 102 mmol). The reaction was then stirred for a further 30
minutes, quenched with H2O (10 mL) and stirred for an additional
Synthetic routes for preparing three potential bacterial thymi-
dylate monophosphate kinase inhibitors have been evaluated.
The key building block for the imidazopyridinone based
inhibitors were made in 4 steps. Three of the transformations
were carefully evaluated and the yield and safety were
improved by substituting triphosgene with 1,1’-carbonyldiimi-
dazole in the key carbo-cyclisation step. A modular approach
°
15 minutes at À 98 C. The reaction mixture was subsequently
concentrated to approx. 50% of the original volume in vacuo, and
EtOAc (200 mL) and brine (100 mL) were added. The resulting
emulsion was split in two, and the bottom phase extracted with
EtOAc (100 mL) and brine (50 mL). The resulting organic phase was
added to the top phase, which was then extracted with EtOAc
(100 mL) and brine (50 mL). This resulted in phase separation. The
combined water-phases were extracted with EtOAc (6×100 mL)
and the combined organic phases washed with brine (100 mL),
dried over anhydrous Na2SO4, filtered, concentrated in vacuo and
washed with toluene (5×2 mL), yielding a dark brown solid. The
crude material was then immobilized on Celite, split in three and
purified by flash chromatography on three columns (Rf =0.27,
EtOAc/n-pentane 1:1). Impure product fractions were combined
and recrystallized from MeCN (15 mL), resulting in a total of 11.7 g
employing
bis(trimethylaluminum)-1,4-diazabicyclo-[2.2.2]
octane in the amide forming reaction proved highly efficient.
Synthesis of the 7-azaoxindole was challenged by instability of
the core scaffold. This was solved by proper selection of
protection group and development of a mild Suzuki cross-
coupling in non-aqueous media, allowing for the isolation of
target 3 by a 3-step route. An alternative strategy employing
late stage oxidation with molecular bromine was successfully
executed on model compounds but failed when performed on
a more electron rich substrate. The imidazopyridinones were
potent inhibitors of the E. coli TMPK in enzymatic assays.
However, in contrast to that indicated by molecular docking,
the 7-azaoxindole possessed low activity. Obviously, the
docking procedures on these flexible proteins must be
developed. Unfortunately, none of the compounds showed
activity towards cultures of E. coli or S. aureus, probably due to
poor cell membrane permeability.
°
(36.4 mmol, 46%) of a brown solid, mp. 152–153 C (lit. [16] 157–
158 C). 1H NMR (400 MHz, [D6]DMSO) δ=8.98 (s, 1H), 7.87 (br s,
°
1H), 7.79 (d, J=2.3 Hz, 1H), 7.32–7.45 (m, 5H), 6.09 (s, 2H), 5.16 (s,
2H). 1H NMR and 13C NMR were in accordance with the
literature.[7,16]
Synthesis of 5[16]
LiAlH4 (1.17 g, 30.8 mmol) was added to a solution of benzyl-(2-
amino-5-bromopyridin-3-yl)-carbamate (4) (2.50 g, 7.77 mmol) in
diethyl ether (150 mL) cooled at 0 C under an N2-atmosphere.
Following stirring at 0 C for 15 minutes and at RT for 5 hours, the
°
°
°
mixture was cooled to 0 C and H2O (1.6 mL), aqueous NaOH (10%,
1.6 mL) and more H2O (3.6 mL) were added sequentially. The
solution was then filtered to remove LiAlH4 residues and the filtrate
concentrated in vacuo. The resulting brown solid was immobilized
on Celite and purified by flash chromatography (Rf =0.30, EtOAc/n-
Experimental Section
Reagents and general methods
pentane 3:1), yielding 1.28 g (6.36 mmol, 82%) of the desired
1
°
°
product as a white solid, mp. 136–137 C (lit. [16] 137–138 C). H
All reagents and solvents used were purchased from Merch, VWR or
Alpha Aesar and used without further purification. Reactions
sensitive to moisture or oxygen were conducted under an N2
atmosphere using oven-dried glassware and solvents dried over
molecular sieves for 24 hours, or collected from an MBraun SPS-800
solvent purifier. HPLC was performed on an Agilent 1100 series
instrument with an Agilent Poroshell C18 (4.6×100 mm) column
with 2.7 μm pore size. Agilent Chemstation was used as software.
Purity analysis and reaction monitoring was done starting with a 5-
minute isocratic elution with H2O/MeCN (9:1) followed by a 30-
minute linear gradient ending at H2O/MeCN (0:100) at 0.8 mL/min
flow. Hydrolytic reactions were analyzed with a 2-minute isocratic
elution with H2O/MeCN (9:1) followed by a 13-minute linear
gradient ending at H2O/MeCN (0:100) at 0.5 mL/min flow. Accurate
NMR (400 MHz, [D6]DMSO) δ=7.28 (d, J=1.9 Hz, 1H), 6.55–6.56 (m,
1
1H), 5.65 (s, 2H), 5.22 (m, 1H), 2.69 (d, J=4.9 Hz, 3H), H NMR and
13C NMR were in accordance with the literature.[7,16]
Synthesis of 6[7]
5-Bromo-N3-methylpyridine-2,3-diamine (5) (503 mg, 2.50 mmol)
and carbonyldiimidazole (2.00 g, 12.4 mmol) were flushed with N2
three times. THF (10 mL) and NEt3 (1.7 mL) were added, and then
refluxed for 30 min. The solvent was removed in vacuo, EtOAc
(50 mL) and H2O (25 mL) were added and the pH in the aqueous
phase adjusted to 3 with HCl (2 M) and NaHCO3 soln. (sat.). The
phases were separated, and the aqueous phase extracted with
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EtOAc (2×50 mL). The combined organic layers were washed with
Synthesis of 9
brine (25 mL), dried over anhydrous Na2SO4, filtered and concen-
trated in vacuo. The resulting crude was immobilized on Celite and
purified by flash chromatography (Rf =CHCl3/acetone 5:1), yielding
463 mg (2.03 mmol, 82%) of the desired product as a yellow solid.
1H NMR (400 MHz, [D6]DMSO) δ=11.72 (s, 1H), 7.99 (d, J=2.0 Hz,
A mixture of 6-bromo-1-methyl-3-trityl-1,3-dihydro-2H-imidazo[4,5-
b]pyridin-2-one (7) (201 mg, 0.428 mmol), (3-((3-phenylpropyl)
carbamoyl)phenyl)boronic acid (8) (156 mg, 0.552 mmol),
PdCl2(dppf) (16 mg, 0.022 mmol) and K2CO3 (176 mg, 1.273 mmol)
under N2 were added 1,4-dioxane (1.5 mL) and water (0.5 mL) and
1
1H), 7.73 (d, J=2.0 Hz, 1H), 3.27 (s, 3H). H NMR and 13C NMR were
°
in accordance with literature.[7]
was agitated at 100 C for 3 hours. The mixture was cooled to
ambient temperature and was added water (25 mL) and EtOAc
(25 mL), before the aqueous layer was extracted with EtOAc (3×
15 mL). The combined organic layer was dried over Na2SO4, filtered
through a celite pad and concentrated in vacuo. The crude product
was purified by flash chromatography (Silica, Rf =0.33, gradient
100% CH2Cl2 to 1:9 EtOAc/CH2Cl2) to afford 9 in 81% yield (217 mg,
Synthesis of 7[7]
Trityl chloride (1.45 g, 5.20 mmol) was added to a solution of 6-
bromo-1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one
(6)
1
(1.04 g, 4.57 mmol), NEt3 (2 mL) in CH2Cl2 (30 mL) and stirred
overnight. The solvent was removed in vacuo, CH2Cl2 (50 mL) and
H2O (25 mL) was added and the phases separated. The aqueous
phase was extracted with CH2Cl2 (3×50 mL) and H2O (25 mL). The
combined organic layers were washed with brine (25 mL) before
drying with anhydrous Na2SO4, filtration and concentration in vacuo
yielded the crude product as a yellow solid. The solid was purified
0.345 mmol) as a white waxy solid; H NMR (600 MHz, [D6]DMSO)
δ=8.53 (t, J=5.6 Hz, 1H), 8.16 (d, J=2.1 Hz, 1H), 8.10 (t, J=1.9 Hz,
1H), 7.83–7.79 (m, 3H), 7.54–7.50 (m, 7H), 7.29–7.22 (m, 10H), 7.19–
7.14 (m, 4H), 3.33 (s, 3H), 3.31–3.28 (m, 2H), 2.65–2.63 (m, 2H), 1.87–
1.82 (m, 2H). 13C NMR (150 MHz, [D6]DMSO) δ=165.9, 153.3, 143.0
(3C), 142.9, 141.7, 137.4, 137.0, 135.3, 129.1, 129.01, 128.98, 128.5
(6C), 128.30 (2C), 128.28 (2C), 127.3 (6C), 126.5, 126.3 (3C), 125.7,
125.4, 124.9, 111.8, 73.8, 39.0, 32.7, 30.9, 26.9;
by flash chromatography (EtOAc/n-pentane 2:9–1:1), resulting in
1
°
1.93 g (4.02 mmol, 88%) of a yellow solid, mp. 219–220 C. H NMR
(600 MHz, [D6]DMSO) δ=8.01–8.03 (m, 1H), 7.96 (d, J=2.2 Hz, 1H),
7.92 (d, J=1.9 Hz, 1H), 7.52–7.54 (m, 6H), 7.23–7.27 (m, 7H), 7.17–
7.19 (m, 3H), 6.98 (d, J=8.7 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.34 (s,
3H); 13C NMR (150 MHz, [D6]DMSO) δ=152.90, 142.68, 142.02 (3C),
138.40, 128.47 (6C), 127.28 (6C), 126.34, 126.29 (3C), 116.08, 112.31,
73.84, 26.90; IR (neat, cmÀ 1) ν=705 (s), 1136 (m), 1367 (s), 1467 (m),
1737 (s); HRMS (TOF ASAP+): m/z calcd. for C26H21N3O79Br: 470.0868
[M+H]+, found: 470.0863. 1H NMR, 13C NMR and MS spectra were in
accordance with literature.[7]
Synthesis of 1[7]
To a stirring solution of 3-(1-methyl-2-oxo-3-trityl-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)-N-(3-phenylpropyl)benzamide
(9)
(172 mg, 0.274 mmol) in CH2Cl2 (5 mL) TFA (1 mL) was added. After
1 hour, the solution was concentrated under reduced pressure, and
residual TFA was removed by co-evaporation with MeOH (3×
15 mL). A portion (56%, 103 mg) of the crude material was added
brine (25 mL) and EtOAc (25 mL) before the aqueous phase was
extracted with additional EtOAc (3×25 mL) and the combined
organic phase was dried over Na2SO4, filtered and dried in vacuo.
The crude material was purified by flash chromatography (silica,
Rf =0.16, gradient: 100% EtOAc to 1:9 MeOH/EtOAc) to afford 1 in
Synthesis of 8
To a solution of 3-boronobenzoic acid (504 mg, 3.04 mmol) and
HATU (1.37 g, 3.61 mmol) in dry CH2Cl2 (40 mL), DIPEA (0.67 mL,
3.85 mmol) was added. The solution was stirred at ambient
temperature for 30 minutes before 3-phenylpropan-1-amine
(435 μL, 3.06 mmol) was added, and the reaction was stirred for
20 hours. Full conversion was observed by TLC (Rf =0.41, CH2Cl2/
MeOH 1:9), and water (50 mL) was added. The mixture was
extracted with CH2Cl2 (3×25 mL) before the combined organic layer
was washed with brine (25 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was
purified by flash chromatography (silica, Rf =0.41, CH2Cl2/MeOH
gradient 0:100 to 1:9) before the obtained oil was added CH2Cl2
(6 mL) and n-pentane (20 mL). The formed solids were filtered off
°
58% yield (35 mg, 0.091 mmol) as a white solid; mp 183.0–184.1 C;
1H NMR (600 MHz, [D6]DMSO) δ=11.65 (s, 1H), 8.60 (t, J=5.6 Hz,
1H), 8.30 (d, J=2.0 Hz, 1H), 8.14 (t, J=1.8 Hz, 1H), 7.87–7.83 (m, 2H),
7.81 (d, J=2.0 Hz, 1H), 7.57 (t, J=7.7 Hz, 1H), 7.31–7.24 (m, 4H),
7.20–7.17 (m, 1H), 3.38 (s, 3H), 3.34–3.31 (m, 2H), 2.66 (t, J=7.7 Hz,
2H), 1.89–1.84 (m, 2H); 13C NMR (150 MHz, [D6]DMSO) δ=166.0,
154.1, 143.3, 141.8, 138.3, 138.1, 135.4, 129.2, 128.98, 128.96, 128.30
(2C), 128.27 (2C), 126.2, 125.7, 125.5, 125.2, 112.2, 39.0, 32.7, 30.9,
26.5; HRMS (ASAP-TOF) m/z calcd. for C23H23N4O2: 387.1821, [M+
H]+, found 387.1822. 1H NMR and 13C NMR were in accordance with
literature.[7]
and
a portion of the solids (84%) were purified by flash
chromatography (C-18 silica, Rf =0.24, H2O/MeOH gradient 1:1 to
1:2) before the obtained solids were dissolved in CH2Cl2 (6 mL) and
were dropwise added Et2O (4 mL). The formed solids were filtered
off and were stirred in refluxing H2O (150 mL) for 30 min, before the
mixture was cooled to ambient temperature and the solids were
filtered off. This afforded 212 mg (0.749 mmol) of (3-((3-phenyl-
propyl)carbamoyl)phenyl)boronic acid (8) as a white powder, which
Synthesis of 12
6-Bromo-1-methyl-3-trityl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-
one (7) (196 mg, 0.417 mmol), methyl 4-methoxy-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate
0.634 mmol), K2CO3 (173 mg, 1.25 mmol) and Pd(dppf)Cl2 (17.9 mg,
24.4 μmol) were dissolved in degassed 1,4-dioxane (1.5 mL) and
(10)
(185 mg,
°
1
H2O (0.5 mL) under an N2 atmosphere, heated to 100 C and stirred
corresponds to a yield of 29%. H NMR (600 MHz, [D6]DMSO) δ=
for 2.5 hours. The mixture was then cooled, filtered through a Celite
pad and concentrated in vacuo. The resulting brown solid was
distributed between EtOAc (10 mL) and H2O (10 mL). The aqueous
phase was extracted with EtOAc (6×10 mL) and the combined
organic layers washed with H2O (10 mL) and brine (10 mL), dried
over anhydrous Na2SO4, filtered and concentrated in vacuo to yield
the crude product as a brown oil. Immobilisation on Celite and
purification by flash chromatography (Rf =0.19, EtOAc/toluene
1:10) yielded 213 mg (0.324 mmol, 78%) of a yellow solid, decomp.
8.42 (t, J=5.5 Hz, 1H), 8.24 (br s, 1H), 8.15 (s, 2H), 7.90–7.89 (m, 1H),
7.84–7.83 (m, 1H), 7.41 (t, J=7.5 Hz, 1H), 7.30–7.27 (m, 2H), 7.24–
7.23 (m, 2H), 7.19–7.16 (m, 1H), 3.29–3.26 (m, 2H), 2.65–2.62 (m, 2H),
1.86–1.81 (m, 2H); 13C NMR (150 MHz, [D6]DMSO) δ=166.8, 141.8,
136.5, 134.0, 133.0, 128.6, 128.29, 128.26, 127.2, 125.7, 38.9, 32.6,
30.9.
1
3
°
point: 215 C. H NMR (600 MHz, CDCl3) δ=8.01 (dd, JHÀ H =8.7 Hz,
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4JHÀ H =2.3 Hz, 1H), 7.96 (d, J=2.2 Hz, 1H), 7.92 (d, J=1.8 Hz, 1H),
0,340 mmol) in THF (2 mL). After 2 h the solution was filtered
through celite and concentrated under reduced pressure before
EtOAc (25 mL) and brine (25 mL) was added. The aqueous layer was
extracted with EtOAc (2×25 mL), before the combined organic
layer was washed with brine (2×25 mL), dried over Na2SO4, and
dried under reduced pressure. The crude product was purified by
flash chromatography (silica, Rf =0.20, Et2O/n-pentane, 3:7). This
gave 18a in 68% yield (109 mg, 0.233 mmol) as a light-brown oil;
1H NMR (400 MHz, CDCl3) δ=8.67 (d, J=2.2 Hz, 1H), 8.28–8.27 (m,
1H), 8.06–8.04 (m, 1H), 8.00 (d, J=2.2 Hz, 1H), 7.80–7.78 (m, 1H),
7.54 (t, J=7.7 Hz, 1H), 6.27 (s, 1H), 1.68 (s, 9H), 1.58 (s, 9H); 13C NMR
(100 MHz, CDCl3) δ: 167.0, 150.0, 147.5, 145.2, 143.5, 143.1, 139.1,
131.8, 131.7, 131.1, 129.2, 128.7, 128.6, 126.9, 120.1, 93.6, 85.5, 85.1,
52.4, 28.2, 27.8; HRMS (ASAP-TOF+) m/z calcd. for C25H29N2O7
469.1975 [M+H]+, found 469.1977.
7.53–7.54 (m, 6H), 7.23–7.26 (m, 10H), 7.17–7.19 (m, 3H), 6.98 (d, J=
8.5 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.33 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ=165.7, 160.1, 154.2, 143.1, 142.9 (3C), 140.1, 132.3, 131.0,
129.2 (6C), 127.4, 127.3 (6C), 126.5 (3C), 126.6, 124.4, 122.9, 113.5,
111.6, 74.71, 55.8, 52.0, 26.9; IR (neat, cmÀ 1) ν=739 (s), 1141 (m),
1238 (m), 1263 (m), 1367 (s), 1450 (m), 1460 (m), 1707 (s), 1723 (s);
HRMS (TOF ASAP+): m/z calcd. for C35H30N3O4: 556.2236 [M+H]+,
found: 556.2238.
Synthesis of 2
3-Phenyl-1-propylamine (310 μL, 2.18 mmol) was added to a
solution of DABAL-Me3 (557 mg, 2.17 mmol) in dry THF (8 mL) and
°
stirred at 40 C under an N2 atmosphere for 1 hour. Methyl 4-
methoxy-3-(1-methyl-2-oxo-3-trityl-2,3-dihydro-1H-imidazo[4,5-b]
pyridin-6-yl)-benzoate (12) (385 mg, 0.693 mmol) in dry THF (8 mL)
was added, and the reaction refluxed for a further 4 hours. The
reaction was then concentrated in vacuo, re-dissolved in CH2Cl2
Preparation of 19a
To a mixture of tert-butyl 2-((tert-butoxycarbonyl)oxy)-5-(3-(meth-
oxycarbonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (18a)
in THF (0.10 M, 1 mL, 0.10 mmol), HCl (37%, 1 mL) was added. The
reaction was left for 15 min before NaHCO3 (sat., 20 mL) and EtOAc
(10 mL) was added. The aqueous layer was extracted with EtOAc
(2×10 mL), and the combined organic layers were washed with
brine (10 mL), dried over Na2SO4, and concentrated under reduced
pressure. The crude product was adsorbed on celite and purified by
flash chromatography (C18 silica, Rf =0.18, EtOAc/n-hexane 3:2).
°
(10 mL) and TFA (2 mL) and stirred for 1.5 hours at 22 C to remove
the protective group. Concentration in vacuo and co-evaporation
with CH2Cl2 (2×10 mL) yielded the crude product as a dark yellow
solid. Immobilisation on Celite and gradient flash chromatography
(EtOAc/MeOH 100:0–9:1) yielded 366 mg (0.567 mmol, 98% purity,
82%) of the TFA-salt of the desired product as a light brown solid,
mp. 119–122 C (decomp. on melting). 1H NMR (600 MHz, [D6]
°
DMSO) δ=11.58 (s, 1H), 8.43 (t, J=5.6 Hz, 1H), 8.03 (d, J=1.8 Hz,
1H), 7.89 (dd, 3JH,H =8.5 Hz, 4JH,H =2.3 Hz, 1H), 7.85 (d, J=2.2 Hz, 1H),
7.55 (d, J=1.8 Hz, 1H), 7.15–7.28 (m, 6H), 3.83 (s, 3H), 1.83 (quintet,
J=7.3 Hz, 2H), 2.62 (t, J=7.7 Hz, 2H), 3.32 (s, 3H), 3.28 (q, J=6.8 Hz,
2H); 13C NMR (150 MHz, [D6]DMSO) δ=165.5, 158.4, 154.1, 142.7,
141.8, 140.2, 129.6, 128.5, 128.27–128.30 (4C), 127.1, 126.75, 125.7,
This gave 19a in 47% yield (12.5 mg, 0.047 mmol); mp 239.8–
1
°
242.4 C (decomp.); H NMR (400 MHz, [D6]DMSO) δ=11.13 (s, 1H),
8.39 (d, J=2.0 Hz, 1H), 8.153–8.145 (m, 1H), 7.95–7.91 (m, 3H), 7.62
(t, J=7.8 Hz, 1H), 3.89 (s, 3H), 3.63 (s, 2H); 13C NMR (100 MHz, [D6]
DMSO) δ=175.9, 166.1, 158.2, 144.4, 138.2, 131.1, 130.44, 130.40,
129.6, 128.8, 127.9, 129.8, 120.9, 52.3, 35.4; IR (neat, cmÀ 1): 3092,
1714, 1284, 1242, 1228, 763, 571; HRMS (ASAP-TOF+) m/z calcd. for
C15H13N2O3, 269.0926 [M+H]+, found 269.0927.
126.67, 124.7, 114.7, 111.2, 55.9, 38.9, 32.7, 31.0, 26.4; IR (neat, cmÀ 1
)
ν=657 (m), 699 (m), 1019 (s), 1047 (s), 1604 (m), 1633 (m), 1686 (br
s), 2936 (br s); HRMS (TOF ASAP+): m/z calcd. for C24H25N4O3:
417.1927 [M+H]+, found: 417.1921.
Enzymatic hydrolysis of methyl ester 19a to 19b
Synthesis of 17
To a mixture of lipase B from Candida antarctica (39.6 mg, 807 U/g)
in phosphate buffer (0.100 M, 9.1 mL, pH 7.0), a solution of methyl
3-(2-oxo-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoate (19a)
(20.6 mg, 0.077 mmol) in 1,4-dioxane (0.9 mL) was added. The
5-Bromo-1,3-dihydro-2H-pyrrolo[2,3-b]pyridine-2-one (16) (1.00 g,
4.72 mmol), di-tert-butyl decarbonate (5.14 g, 23.55 mmol) and NEt3
(4.00 mL, 28.70 mmol) was dissolved in THF (11 mL) and heated to
reflux. After 24 h, the mixture was concentrated under reduced
pressure, before EtOAc (50 mL) was added. The organic phase was
washed with water (50 mL), and the aqueous phase was extracted
with EtOAc (2×50 mL) before the combined organic layer was
washed with brine (50 mL), dried over Na2SO4 and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (silica, Rf =0.53, Et2O/n-pentane, 5:95,). This gave
°
mixture was placed in an orbital shaker at 37 C and 200 rpm for
5 days, and incomplete conversion was observed by TLC (Rf =0.13,
CH2Cl2/MeOH 9:1). The reaction mixture was saturated with NH4SO4
and extracted with EtOAc (3×10 mL) before the combined organic
layer was washed with (NH4)2SO4-solution (sat., 10 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure. The
crude product was immobilized on celite and purified by flash
chromatography (silica, grad. EtOAc 100%– EtOAc/MeOH 9:1). This
gave 19b in 19% yield (3.8 mg, 0.015 mmol) as a light pink solid; 1H
NMR (400 MHz, [D6]DMSO) δ=11.11 (s, 1H), 8.38 (d, J=2.1 Hz, 1H),
8.14 (br t, J=1.5 Hz, 1H), 7.93–7.88 (m, 3H), 7.59 (t, J=7.7 Hz, 1H),
3.62 (s, 1H); 13C NMR (100 MHz, [D6]DMSO) δ=175.9, 167.2, 158.1,
144.4, 138.0, 131.7, 130.7, 130.4, 129.4, 129.0, 128.1, 127.0, 120.9,
35.4; HRMS (TOF ASAPÀ ) m/z calcd. for C14H9N2O3: 253.0613 [MÀ H]À ,
found 253.0608.
1
17 in 81% yield (1.57 g, 3.80 mmol) as a viscous, colorless oil; H
NMR (400 MHz, CDCl3) δ=8.42 (d, J=2.2 Hz, 1H), 7.87 (d, J=2.2 Hz,
1H), 6.14 (s, 1H), 1.62 (s, 9H), 1.53 (s, 9H); 13C NMR (100 MHz, CDCl3)
δ: 149.8, 147.1, 144.9, 143.7, 143.2, 130.5, 121.5, 115.0, 92.6, 85.5,
85.2 28.0 (3C), 27.6 (3C); IR (neat, cmÀ 1) ν=697, 734, 1144, 1264,
1378, 1450, 1460, 1537, 1633, 1717, 2859, 2934, 3026, 3056, 3085,
3326; HRMS (TOF ASAP+): m/z calcd. for C17H22N2O579Br: 413.0712
[M+H]+, found 413.0704.
Synthesis of 18a
Synthesis of 20
A mixture of (3-(methoxycarbonyl)phenyl)boronic acid (122, mg,
0.680 mmol), XPhos Pd G2 (14.8 mg, 0.019 mmol), XPhos (8.6 mg,
0.018 mmol) and Cs2CO3 (340 mg, 1.05 mmol) was added t-BuOH
(2 mL) and a solution of tert-butyl 5-bromo-2-((tert-butoxycarbonyl)
To a mixture of XPhos Pd G2 (27.3 mg, 0.035 mmol), XPhos
(17.1 mg, 0.036 mmol) and Cs2CO3 (657 mg, 2.016 mmol) under N2
atmosphere, a solution of (3-((3-phenylpropyl)carbamoyl)phenyl)
boronic acid (8) (483 mg, 1.705 mmol) and tert-butyl 5-bromo-2-
((tert-butoxycarbonyl)oxy)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate
oxy)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate
(17)
(140 mg,
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(17) (273 mg, 0.661 mmol) in THF (2 mL) and t-BuOH (2 mL) was
728, 1155, 1173, 1190, 1271, 1376, 1443; HRMS (TOF ASAP+) m/z
calcd. for C13H10N2O2S79Br, 336.9646 [M+H]+, found 336.9650.
added. After 1 hour and 40 minutes, full conversion was observed
1
by H NMR and CH2Cl2 (25 mL), water (50 mL) and brine (5 ml) was
added. The aqueous phase was extracted with CH2Cl2 (2×25 mL)
and the combined organic layer was washed with brine (50 mL),
dried over Na2SO4 and filtered before it was concentrated under
reduced pressure. The crude product was purified by reverse phase
flash chromatography (C18-silica, Rf =0.36, gradient H2O/MeOH 1:4
3-(1-(Phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic
acid (22)
5-Bromo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (21) (3.01 g,
8.93 mmol), 3-boronobenzoic acid (2.21 g, 13.5 mmol), Pd(dppf)Cl2
(328 mg, 0,448 mmol) and K2CO3 (3.71 g, 26.9 mmol) were mixed
to 1:9). This gave 20 in 47% yield (179 mg, 0.313 mmol) as a pale-
1
°
yellow solid; mp 105.8–107.2 C (decomp.); H NMR (600 MHz, [D6]
with 1,4-dioxane (30.0 mL) and water (10.0 mL) under
a N2
DMSO) δ=8.76 (d, J=2.2 Hz, 1H), 8.63 (br t, J=5.6, 5.4 Hz, 1H), 8.33
(d, J=2.1 Hz, 1H), 8.21 (br s, 1H), 7.91 (br d, J=7.8 Hz, 1H), 7.88 (br
d, J=7.8 Hz, 1H), 7.59 (t, J=7.7, 7.8 Hz, 1H), 7.30–7.27 (m, 2H), 7.25–
7.23 (m, 2H), 7.19–7.16 (m, 1H), 6.56 (s, 1H), 3.34–3.30 (m, 2H), 2.66
(br t, J=7.6, 7.7 Hz, 2H), 1.87 (br quint., J=7.3, 7.4 Hz, 2H), 1.61 (s,
9H), 1.52 (s, 9H); 13C NMR (150 MHz, [D6]DMSO) δ=165.9, 149.6,
146.9, 144.1, 142.9 142.3, 141.8, 137.6, 135.4, 131.2, 129.5, 129.2,
128.31 (2C), 128.27 (2C), 126.9, 126.7, 125.7, 125.5, 119.3, 93.8, 85.3,
84.5, 39.0, 32.7, 30.9, 27.6, 27.1; IR (neat, cmÀ 1) ν=698, 1106, 1141,
1235, 1255, 1368, 1742, 1771, 3061, 3320; HRMS (ES-TOF): m/z calcd.
for C33H38N3O6: 572.2761, [M+H]+, found 572.2764.
°
atmosphere. The mixture was stirred at 80 C for 2 hours, and full
conversion was observed by TLC (Rf =0.95, MeOH/CH2Cl2 5:95). HCl
(4 M) was added to the mixture until pH=4, before it was saturated
with NaCl and extracted with EtOAc (6×20 mL). The combined
organic layers were washed with brine (20 mL), dried over Na2SO4
and filtered before the solvents were removed under reduced
pressure. The solids were re-dissolved in a minimal amount of
MeOH, and the product was precipitated by slow addition of water.
This gave 22 in 50% yield (1.68 g, 4.44 mmol) as an off-white solid;
mp 185.4–189.4 C (decomp.) ; 1H NMR (400 MHz, [D6]DMSO) δ=
°
8.69 (d, J=2.1, 1H), 8.37 (d, J=2.1 Hz, 1H), 8.20–8.19 (m, 1H), 8.15–
8.13 (m, 2H), 7.98–7.96 (m, 3H), 7.75–7.71 (m, 1H), 7.66–7.60 (m, 3H),
6.89 (d, J=3.9 Hz, 1H), the CO2H proton was not seen; 13C NMR
(100 MHz, [D6]DMSO) δ=167.1, 146.3, 143.4, 137.8, 138.5, 134.8,
131.68, 131.60, 131.1, 129.7 (2C), 129.5 (2C), 128.56, 128.53, 127.8,
127.5, 122.7, 106.4; IR (neat, cmÀ 1) ν=580, 596, 725, 1150, 1169,
1188, 1264, 1376, 1692, 3405; HRMS (TOF ASAP+) m/z calcd. for
C20H15N2O4S, 379.0753 [M+H]+, found 379.0755.
Synthesis of target structure 3
A solution of tert-butyl 2-((tert-butoxycarbonyl)oxy)-5-(3-((3-phenyl-
propyl)carbamoyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate
(20) (100 mg, 0.175 mmol) in 1,4-dioxane (4 mL) was added HCl
(37%, 2 mL), and was stirred at ambient temperature. The reaction
was monitored by reverse phase TLC (C18-silica, Rf =0.57, MeCN/
H2O 7:3), and full conversion was observed after 1 hour. A solution
of KHCO3 (sat., 20 mL) was added to the reaction which was further
stirred for 20 minutes. The mixture was then extracted with CH2Cl2
(7×25 mL) before the combined organic layer was washed with
brine (25 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was recrystallized from a
3-(1H-Pyrrolo[2,3-b]pyridin-5-yl)benzoic acid (23)
To
a stirring mixture of 3-(1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]
pyridin-5-yl)benzoic acid (22) (821 mg, 2.17 mmol) in MeOH (9 mL)
and THF (9 mL), Cs2CO3 (2.12 g, 6.51 mmol) was added. The reaction
was left at ambient temperature overnight, before full conversion
was observed by TLC (Rf =0.17, MeOH/EtOAc 1:9). The mixture was
added EtOAc (10 mL) under stirring before it was added HCl (0.1 M)
until pH=4 and was extracted with a mixture of MeOH and EtOAc
(1:9, 3×50 mL). The combined organic layer was washed with
(NH4)2SO4 (30%, 30 mL), dried over Na2SO4 and filtered before the
solvents were evaporated under reduced pressure. The crude
product was stirred in i-PrOH (10 mL) for 15 minutes and filtered.
minimal amount of EtOAc, this gave 3 in 46% yield (30 mg,
1
°
0.081 mmol) as a white solid; mp 190.5–196.3 C (decomp.); H NMR
(400 MHz, CDCl3) δ=10.03 (s, 1H), 8.32 (s, 1H), 7.88 (s, 1H), 7.67 (s,
1H), 7.62 (s, 1H), 7.68–7.58 (m, 3H), 7.45 (t, J=7.7 Hz, 1H), 7.30–7.15
(m, 5H), 6.54 (m, 1H), 3.58 (s, 2H), 3.56–3.51 (m, 2H), 2.76–2.72 (m,
2H), 2.04–1.97 (m, 2H); 13C NMR (100 MHz, CDCl3) δ=175.6, 167.3,
157.2, 144.9, 141.6, 138.2, 135.7, 131.4, 131.1, 129.6, 129.4, 128.7
(2C), 128.5 (2C), 126.2, 126.1, 125.6, 120.4, 40.2, 35.7, 33.7, 31.2; IR
(neat, cmÀ 1) ν=569, 696, 745, 761, 809, 1228, 1600, 1628, 1650,
1705, 3061, 3312, 3439; HRMS (ASAP-TOF) m/z calcd. for C23H22N3O2:
372.1712, [M+H]+, found 372.1712.
This gave 23 in 53% yield (273 mg, 1.15 mmol) as a light brown
1
°
solid; mp 256.2–257.5 C (decomp.); H NMR (400 MHz, [D6]DMSO)
δ=11.75 (s, 1H), 8.53 (d, J=2.1 Hz, 1H), 8.26 (d, J=2.0 Hz, 1H),
8.22–8.21 (m, 1H), 7.98–7.96 (m, 1H), 7.94–7.92 (m, 1H), 7.61 (t, J=
7.8 Hz, 1H), 7.54–7.52 (br t, J=3.1, 2.7 Hz, 1 H), 6.53–6.52 (dd, J=
1.8, 1.6 Hz, 1H), the CO2H proton was not seen; 13C NMR (100 MHz,
[D6]DMSO) δ=167.4, 148.2, 141.4, 139.5, 131.7, 131.3, 129.4, 127.7,
127.5, 127.3, 127.2, 126.3, 119.8, 100.3; IR (neat, cmÀ 1) ν: 534, 726,
754, 1263, 1281, 1304, 1670, 3318; HRMS (TOF ASAP-) m/z calcd.
C14H9N2O2, 237.0664, [MÀ H]À , found 237.0660.
5-Bromo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (21)[17]
5-Bromo-1H-pyrrolo[2,3-b]pyridine (2.13 g, 10.8 mmol) was dis-
°
solved in DMF (20 mL) and cooled to 0 C, before NaH (283 mg,
10.8 mmol) was added in two portions over 5 minutes. After
addition, the mixture was heated to ambient temperature, and
PhSO2Cl (1.65 mL, 12.93 mmol) was added dropwise over 5 minutes.
After 1.5 hours full conversion was observed by TLC (Rf =0.48,
EtOAc/n-pentane 1:3), and water (60 mL) was added. After stirring
for an additional 20 minutes, the solids were filtered and washed
with water (2×15 mL), before they were air-dried for 30 minutes.
3-(3,3-Dibromo-2-oxo-2,3-dihydro-1H-pyrrolo[2,3-b]
pyridin-5-yl)benzoic acid (24)
To a stirring solution of 3-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid
(23) (214 mg, 0.898 mmol) in t-BuOH (3.2 mL) and water (3.2 mL),
Br2 (115 μL, 2.25 mmol) was added. The reaction was placed in the
dark and let stir for 1 hour, after which full conversion was observed
by 1H NMR. The reaction mixture was added water (15 mL) and
MeOH (3 mL), before it was extracted with EtOAc (4×20 mL). The
combined organic layer was washed with brine (40 mL), dried over
Na2SO4, filtered, and the solvents were evaporated under reduced
pressure. This gave crude 24 in 44% yield (169 mg, 0.396 mmol) as
This gave 21 in 92% yield (3.35 g, 9.93 mmol) as a white solid; mp
1
°
148.6–150.3 C; H NMR (600 MHz, CDCl3) δ=8.43 (d, J=2.1 Hz 1H),
8.16 (d, J=7.6 Hz, 2H), 7.95 (d, J=2.1 Hz, 1H), 7.73 (d, J=4.0 Hz,
1H), 7.59–7.67 (m, 1H), 7.50–7.47 (m, 2H), 6.53 (d, J=4.0 Hz); 13C
NMR (150 MHz, CDCl3) δ=145.59, 145.55, 138.1, 134.4, 131.9 (2C),
129.2 (2C), 128.1, 128.0, 124.5, 115.4, 104.8; IR (neat, cmÀ 1) ν=590,
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1
a red solid; analysis of the crude material H NMR (400 MHz, [D6]
N-(3-Phenylpropyl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)
DMSO) δ=13.15 (br s, 1H). 12.14 (s, 1H), 8.58 (d, J=2.2 Hz, 1H), 8.40
(d, J=2.2 Hz, 1H), 8.21 (br t, J=1.6, 1.5 Hz, 1H), 7.99 (m, 2H), 7.63 (t,
J=7.8 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO) δ=170.2, 167.1,
151.9, 148.7, 136.7, 132.0, 131.7, 131.3, 131.2, 129.5, 128.8, 127.4,
126.1, 44.3; IR (neat, cmÀ 1) ν=614, 735, 760, 1277, 1290, 1602, 1666,
1750, 2853, 2923, 3017, 3078 ;HRMS (TOF ASAP+) m/z calcd.
C14H9N2O379Br2, 410.8980 [M+H]+, found 410.8976.
benzamide (26)
To a stirring solution of N-(3-phenylpropyl)-3-(1-(phenylsulfonyl)-
1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (25) (0.53 g, 1.069 mmol) in
MeOH (10.0 mL) and THF (10.0 mL), Cs2CO3 (1.02 g, 3.13 mmol) was
added. The mixture was left overnight, and full conversion was
observed by TLC (Rf =0.49 MeOH/EtOAc 1:9). The solvents were
removed under reduced pressure before the solids were dissolved
in CH2Cl2 (20 mL) and water (40 mL) was added. The mixture was
extracted with CH2Cl2 (3×20 mL) before the combined organic layer
was washed with KHCO3 (sat., 20 mL) and brine (20 mL), and dried
over Na2SO4, filtered and concentrated under reduced pressure. The
crude product was purified through hot filtration with CH2Cl2 (3×
3-(2-Oxo-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic
acid (19b)
To a mixture of 3-(3,3-dibromo-2-oxo-2,3-dihydro-1H-pyrrolo[2,3-b]
pyridin-5-yl)benzoic acid (24) (133 mg, 0.323 mmol) in AcOH (3 mL),
zinc powder (213 mg, 3.258 mmol) was added. The mixture was
stirred overnight, and full conversion was observed by 1H NMR
before water (125 mL) was added. The mixture was extracted with
EtOAc (3×50 mL) before additional water (25 mL) and EtOAc
(25 mL) was added. The combined organic layer was washed with
brine (2×25 mL), dried over Na2SO4, filtered, and concentrated
under reduced pressure. This gave 19b in 93% yield (76 mg,
10 mL) which gave 26 as a white solid in 55% yield (207 mg,
0.582 mmol); mp 199.8–204.1 C (decomp.); H NMR (400 MHz, [D6]
1
°
DMSO) δ=11.77 (s, 1H), 8.64–8.60 (m, 2H), 8.29 (d, J=2.0 Hz, 1H),
8.19 (br s, 1H), 7.89–7.83 (m, 2H), 7.59–7.53 (m, 2H), 7.31–7.23 (m,
4H), 7.20–7.16 (m, 1H), 6.53 (dd, J=3.2, 1.8 Hz, 1H), 3.34–3.31 (m,
2H (overlaps with H2O signal)), 2.68–2.64 (m, 2H), 1.91–1.84 (m, 2H);
13C NMR (100 MHz, [D6]DMSO) δ=166.1, 148.2, 141.8, 141.6, 139.1,
135.3 129.4, 129.0, 128.33 (2C), 128.29 (2C), 127.6, 127.1, 126.3,
125.9, 125.7, 125.4, 119.7, 100.2, 39.0, 32.7, 30.9; IR (neat, cmÀ 1) ν:
698, 754, 893, 1265, 1579, 1630, 3321; HRMS (TOF ASAP+) m/z
calcd. C23H22N3O, 356.1763 [M+H]+, found 356.1767.
°
0.299 mmol) as a light yellow solid; ; mp 147.9–195.7 C (decomp.);
1H NMR (400 MHz, [D6]DMSO) δ=11.11 (s, 1H), 8.38 (d, J=2.1 Hz,
1H), 8.14 (br t, J=1.5 Hz, 1H), 7.93–7.88 (m, 3H), 7.59 (t, J=7.7 Hz,
1H), 3.62 (s, 2H), the CO2H proton is not seen; 13C NMR (100 MHz,
[D6]DMSO) δ=175.9, 167.2, 158.1, 144.4, 138.0, 131.7, 130.7, 130.4,
129.4, 129.0, 128.1, 127.0, 120.9, 35.4; IR (neat, cmÀ 1) ν= 567, 730,
753, 1211, 1242, 1279, 1463, 1684, 1723, 3157; HRMS (TOF ASAP+)
m/z calcd. C14H9N2O3, 253.0613, [M+H]+, found 253.0608.
E. coli TMPK assay
Inhibition of EcTMPK was determined by Profoldin using the E. coli
Thymidylate Kinase Assay Kit Plus (ProFoldin Catalog No.
TMK100KE), where dTMP was phosphorylated by ATP and TMPK
and the formation of ADP was measured by adding a fluorescing
dye. Single-point inhibitions were measured at 8.3 μM inhibitor
concentrations, and an ADP control assay was performed to correct
for possible ADP inhibition of the compounds. The IC50-values were
determined from a similar assay, where 2-fold dilution series from
10 mM to 0.02 mM were used.
N-(3-Phenylpropyl)-3-(1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]
pyridin-5-yl)benzamide (25)
To a solution of 3-(1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)
benzoic acid (22) (118 mg, 0.312 mmol) and HATU (147 mg,
0.387 mmol) in dry CH2Cl2, DIPEA (0.070 mL, 0.402 mmol) was
added. The solution was stirred at ambient temperature for 20
minutes before 3-phenylpropan-1-amine (0.060 mL, 0.422 mmol)
was added. The solution was left overnight, and full conversion was
observed by TLC (Rf =0.26, EtOAc/toluene 1:4). Phosphate buffer
(0.100 M, 20 mL, pH 5.8) was added and the mixture was extracted
with CH2Cl2 (3×10 mL), before the combined organic layer was
washed with brine (20 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was
dissolved in a minimal amount of DMSO and was precipitated by
MIC measurements
The MIC of compound 1–3 towards Escherichia coli (MG1655) and
Staphylococcus aureus (ATCC29213) was determined following the
standards recommended by the Clinical and Laboratory Standards
Institute (CLSI) for the broth microdilution method.[18] Briefly, the
bacterial suspension were adjusted to 0.5 McFarland standard (~1×
108 colony forming units (CFU)/mL) and diluted 1:200 in Cation-
Adjusted Mueller-Hinton Broth (CAMHB, 22.5 mg/mL Ca2+, 11 mg/
mL Mg2+). The suspension was subsequently added to polypropy-
lene microtiter plates (100 μL/well, ~5×104 CFU/well) already
prepared with different concentrations of the thymidylate kinase
inhibitors (11 μL/well, twofold serial dilutions). The plates were
dropwise addition of water. This gave 25 in 58% yield (89 mg,
1
°
0.180 mmol) as an off-white solid; mp 65–75 C (decomp.); H NMR
(400 MHz, [D6]DMSO) δ=8.73 (d, J=2.1 Hz, 1H), 8.58 (t, J=5.4 Hz,
1H), 8.38 (d, J=2.1 Hz, 1H), 8.147–8.145 (m, 2H), 8.132–8.131 (m,
1H), 7.98 (d, J=3.9 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.85 (d, J=1.6,
1H), 7.73 (bt, J=7.4, 1H), 7.65–7.63 (m, 2H), 7.57 (t, J=7.4, 1H),
7.29–7.27 (m, 2H), 7.24–7.23 (m, 2H), 7.18–7.16 (m, 1H), 6.90 (d, J=
4.0 Hz, 1H), 3.33–3.30 (m, 2H), 2.65 (t, J=7.7 Hz, 2H), 1.85 (quint.,
J=7.6, 7.3 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO) δ=165.8, 146.2,
143.5, 141.7, 137.5, 137.4, 135.4, 134.8, 131.4, 129.6 (2C), 129.1 (2C),
128.4, 128.34, 128.32 (2C), 128.2 (2C), 127.8, 127.5, 126.7, 125.7,
125.6, 122.6, 106.3, 39.0*, 32.6, 30.8; IR (neat, cmÀ 1) ν=580, 596,
751, 1153, 1188, 1375, 1603, 1639, 1721, 3314; HRMS (TOF ASAP+)
m/z calcd. C29H26N3O3S, 496.1695, [M+H]+, found 496.1692.
°
incubated at 37 C overnight for 20–24 h before inspection for
visible growth and determination of MIC values.
Kinase panel
The compounds were supplied in a 10 mM DMSO solution, and
enzymatic kinase inhibition potency was determined by Termo-
Fisher (Invitrogen) using their Z’-LYTE® assay technology,[19] at
500 nM in duplicates. ATP concentration used was in most cases
equal to Km. Otherwise the ATP concentration was 100 μM.
*Overlaps with DMSO. Detected by HSQC and HMBC.
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The X-ray crystal structures of the protein (PDB: 5TMP and 4TMK)
were prepared using the protein preparation wizard, which is part
of the Maestro software package (Maestro, v11.6.013, release 2018–
2; Schrödinger, LLC, New York, NY, USA). Bond orders and formal
charges were added for het-groups, and hydrogens were added to
all atoms in the system. Water molecules beyond 5 Å from het-
groups were removed. To alleviate steric clashes that may exist in
the original PDB structures, an all-atom constrained minimization
was carried out with the Impact Refinement module (Impref)
(Impact, v5.0; Schrödinger, LLC) using the OPLS3 force field. The
minimization was terminated when the energy converged or the
RMSD reached a maximum cutoff of 0.30 Å. The resulting protein
structures were used in the following docking study. Ligands were
drawn using ChemBioDraw (ChemBioDraw Ultra 13.0, Cambridge-
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v2.2; Schrödinger, LLC). For the computational investigation of the
receptor-inhibitor structures, the energy minimized structures of
5TMP and ligands were subsequently docked using GLIDE in XP
mode and Maestro Schrödinger.[20] The resulting docked poses
were analysed using Glide pose viewer tool. For dynamic
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points when building the model systems. Dynamic simulations
were conducted for 10 ns simulation time using Maestros Desmond
suite, the OPLS3e force field and a TIP4P solvent model. Briefly, this
was performed by putting the docked protein- ligand complex
inside a minimized solvent box and adding ions (Na+ or ClÀ ) in
order to have an electrical neutral system. Finally, NaCl was added
to a total concentration of 0.15 M, which is approximately the
physiological concentration of monovalent ions. This gave normally
a system of approximately 39 000 atoms. Molecular dynamics were
then calculated on these systems using the isothermal-isobaric
(NPT) ensemble at 300 K and 1.01325 bar. Trajectory analysis were
performed using Desmond’s Simulation Interactions Diagram tool.
All the graphical pictures were made using Maestro.
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[18] CLSI document M07-A9. Wayne, PA: Clinical and LaboratoryStandards
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[19] B. A. Pollok, B. D. Hamman, S. M. Rodems, L. R. Makings, Aurora
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Acknowledgements
The support from the Research Council of Norway to the
Norwegian NMR Platform (project number 226244/F50) and the
Trond Mohn Fundation are highly appreciated. So is the help from
the Mass Spectrometry Lab at the NV Faculty at NTNU. Roger
Aarvik is thanked for technical support.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: 7-Azaoxindole
Imidazopyridinone Suzuki cross-coupling
monophosphate kinase
·
Carbocyclization
·
Manuscript received: February 10, 2021
Revised manuscript received: April 13, 2021
Accepted manuscript online: April 13, 2021
·
·
Thymidylate
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© 2021 The Authors. European Journal of Organic Chemistry published
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