The Discovery and Development of Thienopyrimidines as Inhibitors of Helicobacter pylori That Act through Inhibition of the Respiratory Complex i

Article abstract of DOI:10.1021/acsinfecdis.0c00300

The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughp

Full text of DOI:10.1021/acsinfecdis.0c00300

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The Discovery and Development of Thienopyrimidines as Inhibitors
of Helicobacter pylori That Act through Inhibition of the Respiratory
Complex I
Alex K. Mugengana,∇ Nicole A. Vita,∇ Autumn Brown Gandt,∇ Kevin Moran, George Agyapong,
Lalit K. Sharma, Elizabeth C. Griffith, Jiuyu Liu, Lei Yang, Ekaterina Gavrish, Kirk E. Hevener,
Michael D. LaFleur, and Richard E. Lee*
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ABSTRACT: The successful treatment of Helicobacter pylori
infections is becoming increasingly difficult due to the rise of
resistance against current broad spectrum triple therapy regimens.
In the search for narrow-spectrum agents against H. pylori, a high-
throughput screen identified two structurally related thienopyr-
imidine compounds that selectively inhibited H. pylori over
commensal members of the gut microbiota. To develop the
structure−activity relationship (SAR) of the thienopyrimidines
against H. pylori, this study employed four series of modifications
in which systematic substitution to the thienopyrimidine core was
explored and ultimately side-chain elements optimized from the
two original hits were merged into lead compounds. During the development of this series, the mode of action studies identified
H. pylori’s respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential
for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB−NuoD binding interface was generated to help rationalize
the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against
H. pylori, improved safety indices, and a good overall pharmacokinetic profile with the exception of high protein binding and poor
solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no
efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase
solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The
thienopyrimidine series developed in this study demonstrates that NuoB−NuoD of the respiratory complex I can be targeted for
development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future
advancement of these studies.
KEYWORDS: Helicobacter pylori, thienopyridine, NuoD, complex I inhibitor
In a previous effort to discover novel chemical matter
selective against H. pylori, a high-throughput screening
protocol was developed to address the microaerophilic
environment required to grow H. pylori.6 Hits found in the
high-throughput screening campaign were then screened
against Staphylococcus aureus to remove promiscuous com-
pounds and prioritize selective compounds. From this
approach, we previously reported Hpi1 (Figure 1A), a selective
anti-H. pylori compound with excellent in vitro potency
Helicobacter pylori is a major causative agent of gastritis, peptic
ulcers, and gastric cancer. According to the Centers for Disease
Control and Prevention (CDC), more than 500,000 cases of
H. pylori infections are diagnosed annually in the United States.
A triple therapy, consisting of a proton pump inhibitor and the
broad-spectrum antibiotics clarithromycin and amoxicillin, is
the recommended treatment against H. pylori infection.1
However, the rate of successful treatment with the use of the
triple therapy regimen has dropped below 80% due to the rise
of antibiotic resistance.2−5 Additionally, the intensive use of
the broad-spectrum antibiotics for the treatment of H. pylori
infections perturbs the gut microbiome, leading to side effects
such as diarrhea and drug resistance in commensal pathogens.1
Given the rise of H. pylori resistance, new drugs are needed for
the treatment of H. pylori infections.
Special Issue: Gut Pathogens
Received: May 11, 2020
Published: January 20, 2021
https://dx.doi.org/10.1021/acsinfecdis.0c00300
© 2021 American Chemical Society
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Figure 1. (A) Structure of Hpi1, a selective anti-H. pylori compound that emerged as a positive hit from a high-throughput screen against H. pylori.6
(B, C) Structures of thienopyrimidines hits 1 and 2 that were identified using a similar screening approach with the H. pylori IC50 of 1.55 and 1.72
μM, respectively. (D) Areas substituted in this study to improve potency, drug-like properties, and cytotoxicity.
a
Scheme 1. Synthesis of Thienopyrimidine Derivatives
a
Reagents and conditions: (i) sulfur, morpholine, ethanol, 70 °C, microwave, 20 min, 90%; (ii) formamide, reflux, 18 h, 80%; (iii) POCl3, N,N-
dimethylaniline, reflux, 14 h, 90%; (iv) ethanol, 150 °C, microwave, 1 h, 80−93%.
(H. pylori minimum inhibitory concentration (MIC) of 0.002
μg/mL) compared to clarithromycin (0.008 μg/mL).6 This
protocol was subsequently applied to a larger library of
219,197 compounds, which yielded 2027 compounds as
positive hits. The hit compounds were clustered and
subsequently prioritized on the basis of their solubility and
their chemical tractability. The second screen identified two
thienopyrimidine compounds, 1 and 2 (Figure 1B,C), with an
H. pylori 50% inhibitory concentration (IC50) of 1.55 and 1.72
μM, respectively. Their activities were confirmed through
resynthesis and testing.
Herein, we report the structure−activity relationship (SAR)
developed from the modification of thienopyrimidines 1 and 2
against H. pylori, which explored substitutions around the
thienopyrimidine core and merged optimized side-chain
elements from the two hits. Hit to lead development was
based on H. pylori activity, cytotoxicity, and in vivo and ex vivo
efficacy using a mouse model of infection. During these
studies, NADH/ubiquinone oxidoreductase (complex I)
subunit NuoD was identified as a putative target of the
thienopyrimidine series by analyzing resistant mutants using
whole-genome sequencing and performing genetic trans-
formation and complementation of resistance. To help
rationalize the SAR, explore the mechanism of action, and
guide further development of the series, a homology model of
the H. pylori NuoB−NuoD binding interface was generated.
These studies produced tractable thienopyrimidine leads with
a defined mechanism of action suitable for further optimization
and validated the NuoB−NuoD interface as a target for the
development of novel narrow spectrum agents against
H. pylori.
RESULTS AND DISCUSSION
■
Generation of Optimized Thienopyrimidines. Four
series of new compounds were synthesized to develop the SAR
around the two thienopyrimidine hits as outlined in Schemes
1−3.7−10 Initial SAR efforts focused on examining the N-alkyl
hydroxyl moiety of hit compound 1, series-1 (Table 1), and
the phenethylamine motif of compound 2, series-2 (Table 2).
Side chains based on lead compounds from each series were
then merged into compounds for series-3 (Table 3). Finally,
series-4 explored the differing substitution patterns observed
for compounds 1 and 2 at C5 and C6 of the thienopyrimidine
ring in combination with an optimized phenylglycinol 4-
position side chain from series-3 (Table 4).
To generate compounds in series-1−3 (Scheme 1),
phenylacetaldehyde 3 was reacted with ethyl 3-cyanopropa-
noate 4 to give phenylthiophene 5. Heating 5 in excess
formamide formed pyrimidone 6. Chlorination and dehydra-
tion of the pyrimidone with phosphoryl chloride afforded
chlorothienopyrimidine 7, which was coupled with a variety of
primary amines and a secondary amine to give targeted 4-
position substituted H. pylori inhibitors (8−30). Commercially
available amine starting materials with hydroxyl and aromatic
moieties, similar to the moieties identified within the hit
compounds, were used to synthesize analogs. These amines
were selected on the basis of their structural resemblance to
the amine substituents of the hit compounds and refined as
data from synthesized compounds developed the SAR of each
series.
Compounds in each series were evaluated for their activity
against H. pylori and cytotoxicity (Tables 1−4). Antimicrobial
activity was measured using IC50 assays against H. pylori type
strains ATCC 43504 and SS1, a mouse adapted strain used in
the mouse model of infection. Activity was also monitored
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a
Table 1. Structure-Activity Relationship of Series-1
a
All pIC50 (−log IC50) data are the mean of at least 3 measurements. Error measured in SEM.
a
Table 2. Structure-Activity Relationship of Series-2
a
All pIC50 (−log IC50) data are the mean of at least 3 measurements. Error measured in SEM.
against a target-based resistant mutant H. pylori SS1 NuoD
A402P to ensure the developing anti-H. pylori SAR remained
on target. Poor solubility of these 4 compound series
sometimes resulted in large standard deviations; therefore,
the comparison of the IC50 between the two strains served to
guide the development of the series, leading to successful
identification of lead compounds 25 and 26 with a >69-fold
increase in potency for both strains. The IC50 data is presented
as pIC50, the negative log of the molar IC50 value to better
follow the emerging SAR.
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a
Table 3. Structure-Activity Relationship of Series-3
a
All pIC50 (−log IC50) data are the mean of at least 3 measurements. Error measured in SEM.
a
Table 4. Structure-Activity Relationship of Series-4 with Substitution at the 5- and 6-Positions of the Thienopyrimidine Core
a
All pIC50 (−log IC50) data are the mean of at least 3 measurements. Error measured in SEM.
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In series-1, variations to the propyl alcohol motif of
compound 1 were evaluated (Table 1). Compound 8 was
generated first to investigate the effect of N-methylation of the
amine group attached at the 4-position of the thienopyrimidine
found in hit compound 1. Compound 8 did not improve the
H. pylori pIC50 values and increased cytotoxicity. Therefore,
the free secondary aromatic was retained for the remainder of
series-1. To examine the length of the spacer contribution
between the hydroxyl group and the amine moiety in 1,
compound 9 was synthesized with a shorter ethyl motif as a
spacer and compound 15 was synthesized with a longer spacer
that included an additional methylene unit. Neither compound
consistently improved the potency compared with 1.
To investigate the potential for branching between the
amine moiety and the hydroxyl group of 1, methyl branched
regio-isomers 10 and 11 were synthesized (Table 1).
Compound 10 demonstrated a 20-fold greater potency against
H. pylori SS1 than 1, and compound 11 was 4-fold more
potent than 1. The IC50 in ATCC 43504 confirmed this with a
37-fold increase in potency for compound 10 and only a
negligible less than 2-fold increase for 11. This suggested there
may be a preference for the modification at the amine α-carbon
over the β-carbon. The methylation at this amine α-carbon
creates a chiral center; therefore, the contribution of each
isomer in 10 was determined through the synthesis of
enantiomers 12 (S) and 13 (R). The R enantiomer 13 was
found to be the active isomer, demonstrating 56-fold increased
activity over 1 in SS1, while the S enantiomer 12 did not
improve the activity over compound 1. To examine the
contribution of the hydroxyl group in 10, the O-methylated
analog 14 was synthesized. Overall, compound 14 demon-
strated less potency when compared to 10. The activity of 14
against ATCC 43504 was 29-fold less potent than 10.
Therefore, the free hydroxyl group was retained in the
remainder of this series. The importance of this hydroxyl
group was later highlighted in the modeling studies of NuoD in
complex with an active ligand (Figure 2).
To determine if a further substitution could be tolerated on
the α- and β-carbons of 10 and 11, regio-isomeric compounds
16 and 17 with an additional methylene unit were generated,
respectively. The pIC50 values of 16 and 17 were very similar
to the parent compounds 10 and 11, in both SS1 and ATCC
43504, indicating branching can be accommodated at this
position. However, compound 18, with a larger isopropyl
substitution over the ethyl motif found in 16, showed
decreased potency. Overall, within compound series-1, clear
SAR was observed, consistent with the analogs engaging a
defined molecular target. However, increased potency did
come with an increase in cytotoxicity for this series. Important
to the SAR, and carried forward into the design of series-3
compounds, was a 2-carbon linker between the amine moiety
and the hydroxy group, branching at the amine α-carbon over
the β-carbon and retaining the terminal hydroxy group.
Series-2 was designed to evaluate the substitution of the
phenyl ring in the phenylethyl side chain of compound 2. Four
analogs with varying phenyl substitutions, 19, 20, 21, and 22
(Table 2), were synthesized on the thienopyridine core.
Compound 19 displayed improved potency (11-fold against
ATCC43504) over compound 2 with a pIC50 of 6.8 0.26
(IC50 0.16 μM) and improved cytotoxicity. 19 is the direct
phenyl-thienopyrimidine hybrid of 2 with the 3,4-dimethox-
yphenethyl side chain. Substitution of the dimethoxyphenyl
motif with dimethylphenyl, compound 20, decreased com-
Figure 2. (a) Homology model of the NuoD and NuoB subunits of
complex I. (b) Snapshot of lead compound 26 (gold) in the average
conformation over a 250 ns molecular dynamic simulation in the wild-
type protein. The 250 ns MD simulation shows that compound 26 is
a hydrogen bond donor to the carbonyl oxygen at T395 and accepts a
hydrogen bond from the hydroxyl side chain of T400 labeled with
green dotted lines, respectively. Compound 26 methoxybenzyl group
π-stacks with Y376 illustrated with blue dotted lines. (c) A ligand
interaction diagram between compound 26 and the protein. Pink lines
show hydrogen bonds, and the green lines show π−π interactions. (d)
Analysis of compound 26 hydrogen bond interactions with the
respective residues over the entire 250 ns simulation.
parative potency and increased cytotoxicity. Three and four
substituted pyridyl analogs 21 and 22 were designed to
increase metabolic stability and solubility but also showed
substantially decreased potency.
The SAR progression revealed series leads 16 and 19 from
hits 1 and 2, respectively. We hypothesized that merging both
substituents at the 4-position of phenyl-thienopyrimidine could
produce analogs with increased potency. Thus, we synthesized
compound 24 (Table 3), which demonstrated activity similar
to 16 and 19 against ATCC 43504 but weaker activity against
the SS1 strain. Series-3 was designed to evaluate the
modifications of hybrid analogs based on 24 (Table 3). First,
compound 25 with a shorter spacer between the aryl motif and
the amine was generated, which demonstrated high potency
with pIC50 7.7
0.070 (IC50 0.021 μM). To evaluate the
contribution of each enantiomer, compounds 26 and 27 were
synthesized. Although the difference in magnitude is less, like
enantiomers 12 and 13, compound 26 pIC50 ATCC43504 8.0
0.13 and SS1 8.2
0.093 (0.010 μM and 0.0068 μM,
respectively), retains the activity of 25, while the S enantiomer
27 decreases activity consistently in both strains ATCC43504
7.3
0.032 and SS1 7.0
0.13 (0.053 μM and 0.11 μM
respectively). Unexpectedly, the cytotoxicity of 25 was less
than each individual isomer (26 and 27), which was
reproducible upon resynthesis and testing. The reason for
this observation is unclear at this time. Unsubstituted 28
retained the reasonable potency of the series-3 compounds
with an pIC50 7.6
0.17 (IC50 of 0.023 μM). Fluorinated
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a
Scheme 2. Synthesis of Amine Derivative 31
a
Reagents and conditions: (i) di-tert-butyl dicarbonate, Et3N, DCM, RT, 2 h; (ii) phthalimide, DIAD, Ph3P, DCM, RT, 4 h; (iii) 4 M HCl in
dioxane, DCM, methanol, RT, 4 h; (iv) Et3N, reflux, 6 h; (v) NH2NH2, methanol, RT, 6 h.
a
Scheme 3. Synthesis of Thienopyrimidine Derivatives with Modifications at Positions 5 and 6
a
Reagents and conditions: (i) Et3N, 150 °C, microwave, 1 h, 89−93%.
compounds 29 and 30 were synthesized in efforts to increase
metabolic stability. Fluorination in these compounds was
tolerated as both retained similar IC50 values to 26, which may
be useful in the future development of this series.
yielded the amine 35, which was coupled to chlorothienopyr-
imidine 7 to afford the intermediate 36. In the final step, the
deprotection of the phthalimide group of 36 afforded the
targeted thienopyrimidine 31. The substitution of the hydroxyl
group for a primary amine resulted in a large increase (>79-
fold for both SS1 and ATCC 43504) in the H. pylori IC50,
indicating that the amine replacement was not tolerated and
the hydroxyl group is required to maintain the low nM potency
observed in the other compounds in series-3. This concurs
with the modeling data for 26 (Figure 2), showing the
hydroxyl group acting as a hydrogen bond acceptor to the
T400 residue.
The final compound synthesized in series-3 was compound
31, the primary amine analog of 30. It was generated to explore
the potential of amine replacement, as primary amines have
been shown to enhance the uptake of drugs into Gram-
negative bacteria.11 Compound 31 was synthesized as
described in Scheme 2, starting from amino alcohol
intermediate 32, Boc-protection yielded alcohol 33.12,13
Substitution of the hydroxyl group of 33 with a phthalimide
group was carried out under Mitsunobu conditions to give the
intermediate 34. Next, the removal of the Boc protecting group
Series-3 confirmed our hypothesis that the lead side chains
from series-1 and series-2 could be combined, producing leads
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with low nM anti-H. pylori activity. Like compounds 12 and 13
in series-1, the R enantiomer was found to be more potent but
again led to an increase in cytotoxicity. Substitution of the
phenyl ring was tolerated, which suggests a modification of the
phenyl ring may be possible for the development of future
compounds with improved metabolic stability. Unfortunately,
the replacement of the hydroxyl with the primary amine was
not tolerated.
Compounds in series-4 explored the differing substitution
patterns observed for compounds 1 and 2 at C5 and C6 of the
thienopyrimidine ring, in combination with the phenylglycinol
4-position side chain from series-3 compound 28. Compounds
37−40 were synthesized as described in Scheme 3, and their
activities were compared to compound 28 with the simplified
phenylglycinol substitution (Table 4). Commercially available
chlorothienopyrimidines (41−44) were used in the coupling
reaction with the optimized phenylglycinol side chain to yield
the final compounds (37−40). Moving the phenyl ring to the
5-position (39 and 40) substantially reduced anti-H. pylori
potency. The dimethyl analog 37 with a similar substitution
pattern of compound 2 was not as active as compound 28,
instead having similar activity to compound 2. Compound 38
with a similar phenyl substitution pattern to series-3
compounds and an additional methyl substitution, demon-
strated somewhat decreased activity compared to 28. These
data suggested the phenyl ring at the 6-position is preferred,
and the 5-position can be a hydrogen or methyl but not a
larger phenyl group, possibly due to steric clashes in the
NuoB−NouD menaquinone binding tunnel targeted by these
inhibitors.
Thienopyrimidines Inhibit H. pylori Respiratory
Complex I. To determine the target of the thienopyrimidines,
one resistant H. pylori mutant was successfully isolated by serial
passaging (five times) on agar medium containing increasing
concentrations of compound 1 and two additional sponta-
neous first-step mutants were obtained by plating on
compound 28. Whole genome sequencing indicated mutations
in NuoD with no other mutations present in all three strains
compared to the isogenic wild-type. The serial passage mutant
had multiple nonsynonymous mutations in nuoD, while the
two first-step resistant mutants had single nucleotide poly-
morphisms in nuoD causing an amino acid change, either
A402P or T400I (Figure 3). Seven additional resistant mutants
were generated using compound 28, all with a nonsynonymous
single-nucleotide polymorphism in nuoD, which resulted in
amino acid mutations T400I or A402P. Further, the A402P
mutation generated by compound 28 was transformed into a
drug susceptible strain of H. pylori for site directed resistance
studies to confirm that resistance was caused by non-
synonymous mutations in nuoD. IC50 studies with this strain
demonstrated resistance was transferable with the A402P
mutation alone.
Figure 3. Sequence alignment of the wild-type nuoD and two H. pylori
mutants. Resistant mutants were generated by exposing H. pylori to
compound 1 or 28 on agar plates. Whole genome sequencing was
performed, and single nucleotide polymorphism analysis identified
two spontaneous mutants, which had nonsynonymous point
mutations in nuoD. The resulting amino acid changes were threonine
to isoleucine (T400I) and alanine to proline (A402P). nuoD encodes
for the subunit D of NADH/quinone reductase also known as
complex I, a membrane protein that catalyzes the first step of
oxidative phosphorylation. Complex I is the putative target of the
thienopyrimidine series.
I.14−17 These results suggest that complex I, the protein that
catalyzes the first step of oxidative phosphorylation, is the
putative target of thienopyrimidines.18,19 Interestingly, NuoD
is also the putative target of a previously described series of
benzimidazole anti-H. pylori agents, with resistance conferring
mutations found at NuoD: G398S, F404S, and V407M (Figure
S2).20,21 This group further determined that, while nuoD is
essential in H. pylori, it is dispensable in other bacteria,
suggesting that targeting NuoD may spare the microbiome. In
accordance with this, we found that compounds 25, 26, 28, 29,
30, and 31 had limited to no activity (MIC ≥ 16 μg/mL)
against a panel of representative Gram-negative and Gram-
positive species, including commensals (Supplementary data,
Table S3).
Recent advances in the structural characterization of
complex I, one of the largest membrane protein complexes
known, have now enabled the computational analysis of
complex I inhibitors.22 The complete structure of NADH/
ubiquinone oxidoreductase (complex I) from Thermus
thermophilus was determined by Baradaran et al.; they modeled
it in decylubiquinone, showing the key interactions with Y87
and H38, which correspond to Y85 and H36 in H. pylori,
respectively.18 A protein sequence alignment showed that the
NuoB and NuoD subunits of H. pylori’s complex I have a high
degree of sequence similarity with T. thermophilus’ complex I
subunits Nqo6 (63% identity) and Nqo4 (43% identity)
(Figure S2). As discussed above, we have shown that
thienopyrimidine resistance-conferring mutations occur at
T400 and A402, positions which fall within the menaqui-
none-binding pocket (Figures 3 and S3).23−25 The location of
T400I and A402P mutations indicate that the thienopyr-
imidine series bind in the menaquinone active site. In
comparing the published benzimidazole resistance mutations
and the two mutations found above, we hypothesize that the
menaquinone active site is a target for H. pylori therapy.20
In order to further characterize interactions of the
thienopyrimidine series of inhibitors with H. pylori’s complex
I, a model of the wild-type protein (subunits NuoD and
NuoB) was built using T. thermophilus’ complex I as a
template. Inhibitor binding interactions were modeled by
computationally docking 26 into the predicted active site,
which showed hydrogen binding to T400. In order to
determine stability of the docked pose of 26, a 250 ns
To validate that all the compounds in the thienopyrimidine
series were on target, each analog was tested against the
mutant H. pylori strain SS1 NuoD A402P (Tables 1−4, Figure
S1). All of the compounds tested demonstrated significantly
decreased anti-H. pylori activity against the NuoD mutant
strain, with the exception of compounds 8, 21, 22, 39, and 40,
which did not demonstrate statistical significance, indicating
that the mechanism of the action is largely consistent
throughout the compound series (Supplementary data, Figure
S1). The gene nuoD encodes for the NuoD subunit of NADH/
quinone oxidoreductase, also known as respiratory complex
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molecular dynamic simulation was run using AMBERMD.
Simulation trajectory analysis revealed key ligand−receptor
hydrogen bonding interactions with T400 and T395 with π-
stacking interactions to Y376, all located within the
menaquinone binding pocket (Figure 2b). Additional analysis
revealed that the protein−ligand interaction stabilizes at 50 ns
and has a stable energy profile (Figure S4), concluding that the
menaquinone pocket is the active site of 26.
To determine the extent of potential selectivity, 26 was also
docked into a model of the A402P mutated NuoD subunit
using the same methods as the wild-type. A weaker docking
score of −4.4, as opposed to −11.5 in the wild-type, was
observed, suggesting a significantly reduced affinity of 26 for
the mutated subunit.
Pharmacological Properties of anti-H. pylori Thieno-
pyrimidines. To examine if the lead thienopyrimidine 26 was
suitable for the advancement into animal testing, the
pharmacological properties of its racemate 25 were determined
for reasons of cost. The in vitro ADME and in vivo
pharmacokinetic properties of 25 were determined.26−28 The
full results of these studies are provided in Tables S1 and S2,
respectively. Compound 25 showed good microsomal stability
(t1/2 = 3.8 h) and permeability (Ave Pe = 239.5 × 10−6 cm/s)
but has low solubility (1 μM) and was highly bound to human
plasma protein (HPPB % = 99). 25 showed good in vivo
exposure (AUC = 31 907.71 h·ng/mL, Table S2) at a 50 mg/
kg intraperitoneal (IP) dose with a lower exposure found upon
oral (PO) dosing in mice. The total drug concentration of 25
in the plasma was above its IC50 value for more than 8 h by IP
dosing. With good plasma exposure, which we believe is
required to treat deep seated ulcerative H. pylori infections, we
proceeded to test 25 in the H. pylori murine infection
model.29,30 Further details of this study are provided in Figure
S5.31 Compared to the metronidazole control, 25 did not show
efficacy at 50 mg/kg (PO), 50 mg/kg (IP), or 25 mg/kg
(IP).31
An ex vivo H. pylori infection model was used to investigate
the lack of efficacy of compound 25 in the in vivo infection
model. Mice were infected with an H. pylori SS1 strain, as
described previously; however, instead, the infected stomach
tissues were dissected and placed in sterile growth media
containing the active compound.29,30 These experiments were
designed to test the effectiveness of compound 25 independent
of drug metabolism and excretion. Compound 25 was found to
be highly effective in the ex vivo model with colony counts
below the limit of detection for 6 of the 10 samples (Figure 4).
These data suggest that insufficient levels of compound 25 are
present at the site of infection in the in vivo model and that
optimization of the pharmacokinetic properties of the
thienopyrimidines series is likely required to demonstrate in
vivo efficacy.
Figure 4. Effect of compound 25 against H. pylori in an ex vivo
infection model. Ten mice were infected with H. pylori strain SS1.
After 3 days, stomachs were harvested and bisected laterally. One-half
of each stomach was placed in a 12-well plate containing either
Brucella-FBS medium or Brucella-FBS medium with 0.6 μg/mL
(1.6 μM) of compound 25. Plates were incubated for 24 h, and tissues
were washed, homogenized, diluted, and plated for colony counts.
from compounds 1 and 2 on the C4-position of the
thienopyrimidine core could be combined to generate more
potent compounds. Branching at the amine α-carbon was
found to be preferred over the β-carbon with the R enantiomer
having greater potency at this position (13 and 26). Retention
of the hydroxyl group on the amine moiety from compound 1
was required to maintain potency (14 and 31), which was
shown in modeling studies to form key hydrogen bonding with
resistance residue T400 in the NuoD binding site. Fluorination
of the phenyl ring in series-3 was tolerated (29 and 30), which
may be helpful in future development to increase the metabolic
stability of these compounds. Finally, the substitution of the
C5- and C6-position of the thienopyrimidine ring suggested
the phenyl ring at C6 is preferred. This was supported in
modeling studies indicating the accommodation for larger
groups at C6 but steric hindrance in the binding site for a large
substitution at C5.
Utilizing compound 28, resistance generation, whole-
genome sequencing, and resistance transfer studies identified
NADH/ubiquinone oxidoreductase (complex I) subunit
NuoD as the putative target of the thienopyrimidine series.
Complex I, the first protein of the electron transport chain, is
located on the inner membrane of H. pylori and catalyzes the
first step of oxidative phosphorylation. Although complex I is
conserved through prokaryotes and eukaryotes, it is uniquely
important in H. pylori as the electron transport chain is the
only pathway to form ATP. Previous research efforts have
indicated complex I and NuoD may be a good target for the
development of anti-H. pylori agents. Researchers at
AstraZeneca identified an anti-H. pylori benzimidazole series
derived from omeprazole (Supplementary data, Figure S6) that
targets complex I of the electron transport chain at the same
site as the theinopyrimidines.20,34 In these studies, knockout
mutagenesis studies demonstrated that nuoD is an essential
gene for H. pylori but not for the majority of other bacteria,
indicating that H. pylori can be targeted selectively without
perturbation of the host microbiota. Targeting oxidative
phosphorylation has been done safely in the clinic, as
exemplified by metformin, a drug used for the treatment of
diabetes32 and in the treatment of certain cancers including
relapsed/refractory acute myeloid leukemia and solid tumors.33
In summary, we report the synthesis and evaluation of four
series of thienopyridines, through which we establish: a clear
anti-H. pylori SAR at the 4-, 5-, and 6-position of the
thienopyrimidine core; identification of potent compounds,
including lead compound 26 with an IC50 of 0.0068 μM; mode
of action studies identifying H. pylori’s respiratory complex I as
the target of the thienopyrimidines; computational analysis
utilizing a NuoD homology model that supported the SAR
observed in microbiological studies; the efficacy in an ex vivo
H. pylori model. The SAR of the thienopyridines occurred
iteratively, prior to the development of the NuoD homology
model. From these data, it was found that modified side chains
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These examples, taken together with the studies described
herein, suggest that complex I is a worthwhile target in
eradicating H. pylori, which can now be exploited using
structure-based drug design. In this study, the computational
model supported the SAR of the thienopyridines developed
from the biological studies and can be used going forward in
the further development of these agents. Although in vivo
efficacy could not be obtained for the compounds reported
here, 26 is a promising chemical probe that is worth further
pharmacological optimization to decrease cytotoxicity and
improve in vivo efficacy in the H. pylori infected mouse model.
Toward these goals, ongoing efforts are focused around
substituting the thienopyrmidine core with more hydrophilic
ring systems.
of 12−60% ethyl acetate in hexanes) to afford 7 as a yellow
solid. 1H NMR (CDCl3, 500 MHz): δ 8.85 (s, 1H), 7.79−7.77
(m, 2H), 7.62 (s, 1H), 7.54−7.46 (m, 3H); MS (ESI): m/z
247.1 (M+ + H).
General Procedure for the Amination of 4-Chloro-6-
phenylthieno[2,3-d]pyrimidine. In a microwave tube,
compound 7 (1 equiv), amine (1.5 equiv), and triethylamine
(3 equiv) were dissolved in ethanol and subjected to
microwave heating for 1 h at 150 °C. The mixture was
extracted with ethyl acetate/water, and the organic phase was
washed with brine, dried (Na2SO4), and concentrated in vacuo.
The residue was purified by column chromatography (Isolera
1, Biotage, elution gradient of 20−85% ethyl acetate in
hexanes) to afford the targeted compound.
3-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-
ol (1). Following the general procedure for the amination of 4-
chloro-6-phenylthieno[2,3-d]pyrimidine, compound 1 was
METHODS
■
General Chemistry Methods. Starting materials were
purchased from Sigma-Aldrich and Enamine. Chemical
reactions were monitored by the thin-layer chromatography
(TLC) and the Waters ACQUITY-UPLC-MS-UV system.
Microwave-assisted chemical reactions were carried out in the
Biotage Initiator+. The reaction mixtures were purified using
the Biotage Flash column chromatography system with silica
cartridges acquired from Biotage Inc. The solvents for
chromatography were purchased from Sigma-Aldrich. NMR
spectra were obtained using the Bruker 400 MHz NMR
spectrometer and the Bruker AVANCE 500 MHz NMR
spectrometer. NMR analysis was carried out using the
MestReNova software. The chemical shifts and the coupling
constants (J) are reported in ppm and hertz (Hz), respectively.
The purity of the compounds was determined to be >95% by
the UPLC-MS-UV system and NMR. The optical rotation of
the chiral compounds was determined using a JASCO P-1010
Polarimeter.
1
obtained as a white solid. H NMR (CDCl3, 400 MHz): δ
8.48 (s, 1H), 7.69−7.67 (m, 1H), 7.67−7.66 (m, 1H), 7.48−
7.43 (m, 2H), 7.41−7.37 (m, 1H), 7.33 (s, 1H), 5.70 (br, 1H),
3.85 (q, J = 6.5 Hz, 2H), 3.74 (t, J = 5.9 Hz, 2H), 1.93−1.87
(m, 2H); MS (ESI): m/z 286.1 (M+ + H).
3-(Methyl(6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
propan-1-ol (8). Following the general procedure for the
amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, com-
pound 8 was obtained as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.41 (s, 1H), 7.74 (s, 1H), 7.70−7.64 (m, 2H), 7.49−
7.43 (m, 2H), 7.41−7.36 (m, 1H), 4.67 (br, 1H), 3.96 (dd, J =
6.8, 5.4 Hz, 2H), 3.61−3.60 (m, 2H), 3.51 (s, 3H), 1.96−1.90
(m, 2H); MS (ESI): m/z 301.1 (M+ + H).
2-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-
ol (9). Following the general procedure for the amination of 4-
chloro-6-phenylthieno[2,3-d]pyrimidine, compound 9 was
1
obtained as a white solid. H NMR (CDCl3, 400 MHz): δ
Ethyl 2-Amino-5-phenylthiophene-3-carboxylate (5). In a
microwave tube, ethyl 3-cyanopropanoate 4 (1.771 mL, 16.65
mmol) was added to a solution of 2-phenylacetaldehyde 3 (2 g,
16.65 mmol), sulfur (0.587 g, 18.315 mmol), and morpholine
(1.579 mL, 18.315 mmol) in ethanol (20 mL). The resulting
mixture was submitted to microwave heating for 30 min at 70
°C. After cooling, the mixture was filtered, and the filtrate was
poured in water. The precipitate was collected, dried, and
purified by flash column chromatography (Isolera 1, Biotage,
25 g size, elution gradient of 12−60% ethyl acetate in hexanes)
8.45 (d, J = 2.7 Hz, 1H), 7.72−7.60 (m, 2H), 7.43 (ddt, J =
9.5, 7.8, 1.5 Hz, 2H), 7.40−7.30 (m, 2H), 5.70 (s, 1H), 3.93
(dt, J = 5.0, 3.3 Hz, 2H), 3.82 (ddt, J = 7.2, 5.4, 3.0 Hz, 2H);
MS (ESI): m/z 273.1 (M+ + H).
2-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-
ol (10). Following the general procedure for the amination of
4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 10 was
obtained as a white solid.1H NMR (CDCl3, 400 MHz): δ 8.38
(s, 1H), 7.62−7.57 (m, 2H 1.32 (d, J = 6.8 Hz, 3H), 7.37
(ddd, J = 7.7, 6.6, 1.6 Hz, 2H), 7.31−7.25 (m, 2H), 5.24 (d, J
= 6.7 Hz, 1H), 4.42−4.40 (m, 1H), 3.81 (dd, J = 10.9, 3.1 Hz,
1H),3.68−3.64 (m, 1H); MS (ESI): m/z 286.9 (M+ + H).
1-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-2-
ol (11). Following the general procedure for the amination of
4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 11 was
1
to afford 5 as a yellow solid. H NMR (DMSO, 500 MHz): δ
7.48−7.44 (m, 4H), 7.33 (t, J = 7.7 Hz, 2H), 7.23 (s, 1H), 7.19
(t, J = 7.4 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1
Hz, 3H); MS (ESI): m/z 248.5 (M+ + H).
6-Phenylthieno[2,3-d]pyrimidin-4(3H)-one (6). Compound
5 (1.15 g, 4.65 mmol) was dissolved in an excess of formamide,
and the resulting mixture was heated for 16 h at 160 °C. The
mixture was concentrated in vacuo and purified by flash column
chromatography (Isolera 1, Biotage, 25 g size, elution gradient
of 12−60% ethyl acetate in hexanes) to afford 6 as a red solid.
1H NMR (CDCl3, 500 MHz): δ 11.05 (s, 1H), 8.58 (s, 1H),
7.65−7.60 (m, 2H), 7.45 (s, 1H), 7.43−7.38 (m, 2H), 7.33−
7.30 (m, 1H); MS (ESI): m/z 230.1 (M+ + H).
4-Chloro-6-phenylthieno[2,3-d]pyrimidine (7). Compound
6 (1 g, 4.38 mmol) and N,N-dimethylaniline (0.043 mL, 0.336
mmol) were dissolved in phosphoryl trichloride (5.10 mL, 54.8
mmol), and the mixture was heated for 16 h to 95 °C. The
mixture was concentrated in vacuo and purified by flash column
chromatography (Isolera 1, Biotage, 25 g size, elution gradient
1
obtained as a white solid. H NMR (DMSO, 400 MHz): δ
8.33 (s, 1H), 8.12 (s, 1H), 7.99 (t, J = 5.9 Hz, 1H), 7.69 (d, J =
7.5 Hz, 2H), 7.51 (t, J = 6.8 Hz, 2H), 7.42−7.38 (m, J = 7.5
Hz, 1H), 4.86 (d, J = 4.8 Hz, 1H), 3.94−3.89 (m, 1H), 3.54−
3.38 (m, 2H), 1.13 (d, J = 6.2 Hz, 3H); MS (ESI): m/z 268.7
(M+ + H).
(S)-2-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
propan-1-ol (12). Following the general procedure for the
amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, com-
pound 12 was obtained as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.47 (s, 1H), 7.71−7.68 (m, 2H), 7.46 (t, J = 7.5 Hz,
2H), 7.41−7.39 (m, 1H), 7.35 (s, 1H), 5.31 (s, 1H), 4.51−
4.49 (m, 1H), 3.91 (dd, J = 11.1, 3.02 Hz, 1H), 3.75 (dd, J =
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11.1, 6.2 Hz, 1H), 1.41 (d, J = 6.9 Hz, 3H); MS (ESI): m/z
286.9 (M+ + H).
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 20 was obtained as a white solid. 1H NMR
(DMSO, 400 MHz): δ 8.37 (s, 1H), 8.07 (t, J = 5.62 Hz, 1H),
8.00 (s, 1H), 7.67 (d, J = 7.52 Hz, 2H), 7.51 (t, J = 8.08 Hz,
2H), 7.40 (t, J = 7.12 Hz, 1H), 7.06−7.05 (m, 2H), 6.99−6.97
(m, 1H), 3.73−3.68 (m, 2H), 2.87 (t, J = 7.49 Hz, 2H), 2.19
(s, 3H), 2.17 (s, 3H); MS (ESI): m/z 361.21 (M+ + H).
6-Phenyl-N-(2-(pyridin-4-yl)ethyl)thieno[2,3-d]pyrimidin-
4-amine (21). Following the general procedure for the
amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, com-
pound 21 was obtained as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.56−8.47 (m, 3H), 7.65−7.63 (m, 2H), 7.44−7.41
(m, 2H), 7.38−7.36 (m, 1H), 7.21−7.18 (m, 3H), 5.26 (s,
1H), 3.98−3.93 (m, 2H), 3.05 (t, 2H, J = 7.52 Hz); MS (ESI):
m/z 333.1 (M+ + H).
6-Phenyl-N-(2-(pyridin-3-yl)ethyl)thieno[2,3-d]pyrimidin-
4-amine (22). Following the general procedure for the
amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, com-
pound 22 was obtained as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.56−8.53 (m, 3H), 7.68−7.65 (m, J = 6.88 Hz, 2H),
7.61 (d, J = 7.91 Hz, 1H), 7.45 (dd, J = 8.45, 6.60 Hz, 2H),
7.40−7.37 (m, 1H), 7.24 (s, 1H), 5.26 (s, 1H) 3.96 (q, J =
6.73, 2H), 3.08 (t, J = 6.96 Hz, 2H); MS (ESI): m/z 333.1
(M+ + H).
(R)-2-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
propan-1-ol (13). Following the general procedure for the
amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, com-
pound 13 was obtained as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.47 (s, 1H), 7.70−7.68 (m, 2H), 7.46 (t, J = 8.2 Hz,
2H), 7.41−7.35 (m, 2H), 5.33 (s, 1H), 4.52−4.48 (m, 1H),
3.93−3.88 (m, 1H), 3.78−3.73 (m, 1H), 1.41 (d, J = 6.4 Hz,
3H); MS (ESI): m/z 286.9 (M+ + H).
N-(1-Methoxypropan-2-yl)-6-phenylthieno[2,3-d]-
pyrimidin-4-amine (14). Following the general procedure for
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 14 was obtained as a white solid. 1H NMR
(DMSO, 400 MHz): δ 8.34 (s, 1H), 8.12 (s, 1H), 7.74−7.68
(m, 3H), 7.51 (t, 2H, J = 7.4 Hz), 7.43−7.38 (m, 1H), 4.60−
4.53 (m, 1H), 3.52−3.48 (m, 1H), 3.40−3.37 (m, 1H), 3.30
(s, 3H), 1.24 (d, J = 6.7 Hz, 3H); MS (ESI): m/z 300.8 (M+ +
H).
4-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-1-
ol (15). Following the general procedure for the amination of
4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 15 was
obtained as a white solid. 1H NMR (CDCl3, 400 MHz): δ 8.47
(s, 1H), 7.67−7.64 (m, 2H), 7.45−7.25 (m, 4H), 5.67 (s, 1H),
3.79 (s, 2H), 3.69 (s, 2H), 2.16 (s, 1H), 1.87−1.84 (m, 2H),
1.75−1.73 (m, 2H); MS (ESI): m/z 301.3 (M+ + H).
2-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-1-
ol (16). Following the general procedure for the amination of
4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 16 was
obtained as a white solid. 1H NMR (CDCl3, 400 MHz): δ 8.45
(s, 1H), 7.70−7.67 (m, 2H), 7.48−7.43 (m, 2H), 7.39 (t, J =
7.48 Hz, 1H), 7.35 (s, 1H), 5.34 (d, J = 7.5 Hz, 1H), 4.33−
4.26 (m, 1H), 3.93 (dd, J = 11.1, 3 Hz, 1H), 3.80 (dd, J = 11.1,
5.8 Hz, 1H), 1.88−1.70 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS
(ESI): m/z 300.9 (M+ + H).
N-(2,3-Dimethoxybenzyl)-6-phenylthieno[2,3-d]-
pyrimidin-4-amine (23). Following the general procedure for
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 23 was obtained as a white solid. 1H NMR (CDCl3,
400 MHz) δ 8.54 (s, 1H), 7.69−7.63 (m, 2H), 7.49−7.41 (m,
2H), 7.40−7.33 (m, 1H), 7.30 (d, J = 1.2 Hz, 1H), 7.11−7.00
(m, 2H), 6.94 (dd, J = 7.8, 1.8 Hz, 1H), 5.73 (s, 1H), 4.89 (d, J
= 5.6 Hz, 2H), 3.97 (d, J = 0.9 Hz, 3H), 3.92 (d, J = 1.1 Hz,
3H); MS (ESI): m/z 378.02 (M+ + H).
3-(4-Methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-
4-yl)amino)propan-1-ol (24). Following the general proce-
dure for the amination of 4-chloro-6-phenylthieno[2,3-d]-
1-((6-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-2-
ol (17). Following the general procedure for the amination of
4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 17 was
1
pyrimidine, compound 24 was obtained as a white solid. H
NMR (CDCl3, 500 MHz): δ 8.38 (s, 1H), 7.55 (d, J = 7.65 Hz,
2H), 7.36 (t, J = 7.54 Hz, 2H), 7.30 (t, J = 7.7 Hz, 1H), 7.14−
7.11 (m, 3H), 6.80 (d, J = 8.2 Hz, 2H), 5.39 (d, J = 6.25 Hz,
1H), 4.45−4.39 (m, 1H), 3.82 (d, J = 10.8 Hz, 1H), 3.72−3.66
(m, 4H), 2.97−2.80 (m, 2H); MS (ESI): m/z 391.9 (M+ + H).
2-(4-Methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-
4-yl)amino)ethan-1-ol (25). Following the general procedure
for the amination of 4-chloro-6-phenylthieno[2,3-d]-
1
obtained as a white solid. H NMR (DMSO, 400 MHz): δ
8.33 (s, 1H), 8.12 (s, 1H), 7.96 (t, J = 5.73 Hz, 1H), 7.70−
7.67 (m, 2H), 7.51 (dd, J = 8.41, 7.01 Hz, 2H), 7.42−7.38 (m,
1H), 4.83 (d, J = 5.24 Hz, 1H), 3.70−3.62 (m, 1H), 3.61−3.55
(m, 1H), 3.44−3.37 (m, 1H), 1.58−1.48 (m, 1H), 1.42−1.31
(m, 1H), 0.93 (t, J = 7.4 Hz, 3H); MS (ESI): m/z 300.9 (M+ +
H).
1
3-Methyl-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
butan-1-ol (18). Following the general procedure for the
amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, com-
pound 18 was obtained as a white solid. 1H NMR (CDCl3, 400
MHz): δ 8.35 (s, 1H), 7.62−7.59 (m, 2H), 7.37 (t, J = 7.2 Hz,
2H), 7.32−7.25 (m, 2H), 5.30 (d, J = 7.7 Hz, 1H), 4.11−4.05
(m, 1H), 3.87−3.75 (m, 2H), 3.48 (m, 1H), 2.10−2.01 (m,
1H), 1.20−0.98 (m, 6H); MS (ESI): m/z 315.01 (M+ + H).
N-(3,4-Dimethoxyphenethyl)-6-phenylthieno[2,3-d]-
pyrimidin-4-amine (19). Following the general procedure for
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 19 was obtained as a white solid. 1H NMR (CDCl3,
400 MHz): δ 8.54 (s, 1H), 7.65 (d, J = 7 Hz, 2H), 7.45 (t, J =
7.04 Hz, 2H), 7.40−7.36 (m, 1H), 7.20 (s, 1H), 6.88−6.78
(m, 3H), 5.28 (br, 1H), 3.95−3.90 (m, 5H), 3.87 (s, 3H), 2.99
(t, J = 7.04 Hz, 2H); MS (ESI): m/z 392.01 (M+ + H).
N-(3,4-Dimethylphenethyl)-6-phenylthieno[2,3-d]-
pyrimidin-4-amine (20). Following the general procedure for
pyrimidine, compound 25 was obtained as a white solid. H
NMR (DMSO, 400 MHz): δ 8.28 (s, 1H), 8.21 (s, 1H), 8.01
(d, J = 8.08 Hz, 1H), 7.75−7.67 (m, 2H), 7.55−7.47 (m, 2H),
7.44−7.33 (m, 3H), 6.95−6.84 (m, 2H), 5.48−5.33 (m, 1H),
4.89 (t, J = 5.69 Hz, 1H), 3.91−3.63 (m, 5H); MS (ESI): m/z
378.1 (M+ + H).
(R)-2-(4-Methoxyphenyl)-2-((6-phenylthieno[2,3-d]-
pyrimidin-4-yl)amino)ethan-1-ol (26). Following the general
procedure for the amination of 4-chloro-6-phenylthieno[2,3-
d]pyrimidine, compound 26 was obtained as a white solid. 1H
NMR (DMSO, 400 MHz): δ 8.28 (s, 1H), 8.24 (s, 1H), 8.13
(d, J = 8.2 Hz, 1H), 7.73−7.70 (m, 2H), 7.54−7.50 (m, J =
8.12 Hz, 2H), 7.43−7.39 (m, 1H), 7.37−7.34 (m, 2H), 6.89
(d, J = 8.6 Hz, 2H), 5.43−5.38 (m, 1H), 4.99 (t, J = 6.16 Hz,
1H), 3.79−3.69 (m, 5H); MS (ESI): m/z 378.1 (M+ + H).
(S)-2-(4-Methoxyphenyl)-2-((6-phenylthieno[2,3-d]-
pyrimidin-4-yl)amino)ethan-1-ol (27). Following the general
procedure for the amination of 4-chloro-6-phenylthieno[2,3-
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d]pyrimidine, compound 27 was obtained as a white solid. 1H
NMR (DMSO, 400 MHz): δ 8.28 (s, 1H), 8.24 (s, 1H), 8.14
(d, J = 7.76 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.52 (t, J = 7.72
Hz, 2H), 7.43−7.35 (m, 3H), 6.89 (d, J = 9 Hz, 2H), 5.41 (q, J
= 7.88 Hz, 1H), 4.99 (t, J = 6.04 Hz, 1H), 3.79−3.69 (m, 5H);
MS (ESI): m/z 378.1 (M+ + H).
(R)-2-Phenyl-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)-
amino)ethan-1-ol (28). Following the general procedure for
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 28 was obtained as a white solid. 1H NMR (CDCl3,
400 MHz): δ 8.46 (s, 1H), 7.69 (d, J = 6.88 Hz, 2H), 7.47−
7.34 (m, 9H), 5.85 (s, 1H), 5.50−5.47 (m, 1H), 4.17−4.09
(m, 2H); MS (ESI): m/z 348.9 (M+ + H).
(R)-2-(2-Fluoro-4-methoxyphenyl)-2-((6-phenylthieno-
[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (29). Following the
general procedure for the amination of 4-chloro-6-
phenylthieno[2,3-d]pyrimidine, compound 29 was obtained
as a yellow solid. 1H NMR (CDCl3, 400 MHz): δ 8.45 (s, 1H),
7.67 (d, J = 6.16 Hz, 2H), 7.45−7.29 (m, 5H), 6.70 (s, 2H),
6.02 (s, 1H), 5.70 (s, 1H), 4.14−4.05 (m, 2H), 3.81 (s, 3H);
MS (ESI): m/z 396.01 (M+ + H).
(R)-2-(3-Fluoro-4-methoxyphenyl)-2-((6-phenylthieno-
[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (30). Following the
general procedure for the amination of 4-chloro-6-
phenylthieno[2,3-d]pyrimidine, compound 30 was obtained
as a yellow solid. 1H NMR (CDCl3, 400 MHz): δ 8.42 (s, 1H),
7.67−7.65 (m, 2H), 7.54 (s, 1H), 7.45−7.35 (m, 3H), 7.22−
7.16 (m, 2H), 6.98 (t, J = 8.6 Hz, 1H), 6.31 (s, 1H), 5.45−5.41
(m, 1H), 4.14−4.06 (m, 2H), 3.90 (s, 3H); MS (ESI): m/z
397.3 (M+ + H).
(R)-2-((5,6-Dimethylthieno[2,3-d]pyrimidin-4-yl)amino)-
2-phenylethan-1-ol (37). Following the general procedure for
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 37 was obtained as a white solid. 1H NMR (CDCl3,
400 MHz): δ 8.35(s, 1H), 7.44−7.28 (m, 5H), 6.28 (s, 1H),
5.47−5.43 (m, 1H), 4.07 (d, J = 4.58 Hz, 2H), 2.51 (s, 3H),
2.46 (s, 3H); MS (ESI): m/z 301.00 (M+ + H).
(R)-2-((5-Methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)-
amino)-2-phenylethan-1-ol (38). Following the general
procedure for the amination of 4-chloro-6-phenylthieno[2,3-
d]pyrimidine, compound 38 was obtained as a white solid. 1H
NMR (CDCl3, 400 MHz): δ 8.48 (s, 1H), 7.48−7.36 (m, 9H),
7.11−7.07 (m, 1H), 5.89 (d, J = 6.84 Hz, 1H), 5.49 (q, J =
5.44 Hz, 1H), 4.17−4.09 (m, 2H), 1.28 (s, 3H); MS (ESI): m/
z 361.9 (M+ + H).
fluoro-4-methoxyphenyl)-2-hydroxyethanaminium chloride 32
(0.267 g, 1.208 mmol) and triethylamine (0.506 mL, 3.63
mmol) in dichloromethane (4 mL). The reaction mixture was
stirred for 2 h at room temperature. The mixture was
concentrated and purified by flash column chromatography
(Isolera 1, Biotage, 10 g size, elution gradient of 12−80% ethyl
acetate in hexanes). Pure fractions were evaporated to afford
33 as a white solid. 1H NMR (CDCl3, 500 MHz): δ 7.08−7.03
(m, 2H), 6.96 (t, J = 8.33 Hz, 1H), 5.21 (d, J = 6.55 Hz, 1H),
4.73 (s, 1H), 3.90−3.81 (m, 5H), 2.11 (s, 1H), 1.46 (s, 9H);
MS (ESI): m/z 284.3 (M+ + H).
tert-Butyl (R)-(2-(1,3-Dioxoisoindolin-2-yl)-1-(3-fluoro-4-
methoxyphenyl)ethyl)carbamate (34). Diisopropyl azodicar-
boxylate (0.282 mL, 1.458 mmol) was added dropwise at room
temperature to a solution of 33 (0.320 g, 1.122 mmol),
phthalimide (0.215 g, 1.458 mmol), and Ph3P (0.382 g, 1.458
mmol) in THF (3.8 mL). The resulting mixture was stirred for
16 h at room temperature. The mixture was extracted with
ethyl acetate/water, and the organic phase was washed with
brine, dried (Na2SO4), and evaporated in vacuo. The residue
was purified by flash column chromatography (Isolera 1,
Biotage, 10 g size, elution gradient of 12−60% ethyl acetate in
hexanes) to afford 34 as a yellow solid. 1H NMR (CDCl3, 500
MHz): δ 7.80 (m, 2H), 7.66 (m, 2H), 7.04 (m, 2H), 6.87 (m,
1H), 5.28 (d, J = 8.1 Hz, 1H), 4.94−4.92 (m, 1H), 3.89−3.81
(m, 5H), 1.20 (s, 9H); MS (ESI): m/z 415.7 (M+ + H).
(R)-2-(2-Amino-2-(3-fluoro-4-methoxyphenyl)ethyl)-
isoindoline-1,3-dione (35). HCl (4 M in 1,4-dioxane, 0.9 mL,
3.6 mmol) was added to a solution of 34 (0.372 g, 0.897
mmol) in a mixture of DCM (2.5 mL) and methanol (1 mL),
and the resulting mixture was stirred for 16 h at room
temperature. The mixture was subjected to acid−base
extraction with ethyl acetate/water, and the organic phase
was washed with brine, dried (Na2SO4), and concentrated in
vacuo. The resulting residue 35 (yellow oil) was used in the
1
next step without further purification. H NMR (MeOD, 500
MHz): δ 7.88−7.78 (m, 4H), 7.20 (dd, J = 12.4, 2.20 Hz, 1H),
7.08 (dd, J = 8.65, 2.14 Hz, 1H), 7.00(t, J = 8.49 Hz, 1H), 4.25
(t, J = 7.27 Hz, 1H), 3.88−3.84 (m, 5H); MS (ESI): m/z
315.5 (M+ + H).
(R)-2-(2-(3-Fluoro-4-methoxyphenyl)-2-((6-phenylthieno-
[2,3-d]pyrimidin-4-yl)amino)ethyl)isoindoline-1,3-dione
(36). Compound 36 was synthesized following the general
procedure for amination of 4-chloro-6-phenylthieno[2,3-d]-
1
pyrimidine. H NMR (CDCl3, 500 MHz): δ 8.31 (s, 1H),
(R)-2-((6-Methyl-5-phenylthieno[2,3-d]pyrimidin-4-yl)-
amino)-2-phenylethan-1-ol (39). Following the general
procedure for the amination of 4-chloro-6-phenylthieno[2,3-
d]pyrimidine, compound 39 was obtained as a white solid. 1H
NMR (CDCl3, 400 MHz): δ 8.42 (s, 1H), 7.57 (t, J = 7.63 Hz,
1H), 7.45−7.41 (m, 2H), 7.27−7.21 (m, 5H), 6.90−6.88 (m,
2H), 5.33 (d, J = 5.6 Hz, 1H), 5.20−5.16 (m, 1H), 3.78−3.69
(m, 2H), 2.33 (s, 3H); MS (ESI): m/z 361.9 (M+ + H).
(R)-2-Phenyl-2-((5-phenylthieno[2,3-d]pyrimidin-4-yl)-
amino)ethan-1-ol (40). Following the general procedure for
the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine,
compound 40 was obtained as a white solid. 1H NMR (CDCl3,
400 MHz): δ 8.45 (s, 1H), 7.69−7.67 (m, 2H), 7.47−7.35 (m,
9H), 5.96−5.94 (m, 1H), 5.51−5.47 (m, 1H), 4.17−4.10 (m,
2H); MS (ESI): m/z 348.1 (M+ + H).
7.88−7.85 (m, 2H), 7.75−7.73 (m, 4H), 7.54 (s, 1H), 7.49 (t,
J = 7.55, 1H), 7.42 (t, J = 7.35 Hz, 1H), 7.25−7.22 (m, 2H),
6.99−6.95 (m, 1H), 6.91 (s, 1H), 5.66−5.62 (m, 1H), 4.24−
4.12 (m, 2H), 3.89 (s, 3H); MS (ESI): m/z 525.5 (M+ + H).
(R)-1-(3-Fluoro-4-methoxyphenyl)-N1-(6-phenylthieno-
[2,3-d]pyrimidin-4-yl)ethane-1,2-diamine (31). Compound
36 (0.203 g, 0.393 mmol) was dissolved in methanol, and a
80% hydrazine hydrate solution (0.073 mL,1.2 mmol) was
added dropwise to the solution at 0 °C. The reaction mixture
was stirred for 16 h at room temperature. The mixture was
extracted with ethyl acetate/water, and the organic phase was
washed with brine, dried (Na2SO4), and evaporated in vacuo.
The residue was purified by flash column chromatography
(Isolera 1, Biotage, 10 g size, elution gradient of 2−20%
1
methanol in ethyl acetate) to afford 31 as a yellow solid. H
tert-Butyl (R)-(1-(3-Fluoro-4-methoxyphenyl)-2-
hydroxyethyl)carbamate (33). Di-tert-butyl dicarbonate
(0.290 g, 1.329 mmol) was added to a solution of (R)-1-(3-
NMR (DMSO, 500 MHz): δ 8.27 (s, 1H), 8.15 (s, 1H), 7.75
(d, 2H, J = 7.6 Hz), 7.54 (t, 2H, J = 7.6 Hz), 7.44 (t, 1H, J =
7.6 Hz), 7.26 (dd, J = 12.5, 2 Hz, 1H), 7.20 (d, 1H, J = 7.6
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Hz), 7.12 (t, J = 8.6 Hz, 1H), 5.28−5.25 (m, 1H), 3.80 (s,
3H), 3.00−2.91 (m, 2H), 1.25−1.18 (m, 2H); MS (ESI): m/z
396.4 (M+ + H).
method, H. pylori ATCC 43504 was serially passaged onto
plates containing increasingly higher concentrations of 1 until
the strain was able to grow at 8× the MIC of the wild-type.
The DNA from this resistant mutant was then purified and
transformed into strain HM-274 to confirm stable genetically
transferable resistance prior to sequencing. Genomic DNA was
purified from the three resistant mutants as well as HM-274
wild-type using ZR Fungal/Bacterial DNA MiniPrep (Zymo
Research) according to the manufacturer’s instructions. Whole
genome sequencing was performed by Tufts Medical Center.
Genome annotation was accomplished using RAST, and single
nucleotide polymorphism (SNP) identification was performed
with CLC Workbench. The two spontaneous resistant mutants
had SNPs in nuoD, which caused one amino acid change,
either A402P or T400I. The serial passage mutant had multiple
nonsynonymous mutations in nuoD. There were no other
mutations shared by all three strains and not present in the
isogenic background strain.
Seven additional spontaneous resistant mutants were
subsequently isolated using the same methodology. Genomic
DNA was purified from each, and nuoD was amplified by PCR.
The PCR products were sequenced and compared to wild-type
nuoD. All seven had mutations in nuoD resulting in an amino
acid change, either A402P or T400I.
In order to ensure that no secondary mutations were causing
thienopyrimidine resistance, a wild-type copy of nuoD was
modified to contain the A402P mutation using the Q5 Site
Directed Mutagenesis kit (New England Biolabs) according to
the manufacturer’s instructions. The mutation was confirmed
by PCR and sequencing, and then, wild-type SS1 was
transformed with this plasmid and selected for on plates
containing 28. The A402P mutation in nuoD conferred
resistance in strain SS1. Several colonies were chosen, and
the mutation was confirmed by PCR and sequencing. An SS1
isolate with the NuoD A402P mutation was used in further
testing to confirm on target activity of the thienopyrimidine
series (see above).
Ex Vivo Efficacy Study. This model was adapted from the
standard mouse model of H. pylori infection using SS1. Briefly,
6 to 8 week-old female C57BL/6 (specific-pathogen-free) mice
were obtained from Charles River Laboratories. Five mice were
housed per cage and allowed to acclimate for at least 72 h.
Animals were inoculated by oral gavage, three times within 1
week with 0.2 mL of an H. pylori SS1 suspension (∼109 CFU/
dose). The infection was allowed to stabilize for 1 to 2 weeks
after the final gavage. Mice were euthanized, and the stomachs
were excised, bisected laterally into equal halves along the long
curvature, and washed three times with sterile PBS. Each half
was added to the well of a 12 well plate containing Brucella
broth supplemented with 10% FBS and Skirrow’s with or
without 100× the MIC of compound 25. Each half from
individual stomachs was assigned to a control and treatment
group. After 24 h, the stomach halves were washed, weighed,
homogenized, serially diluted, and plated on selective medium.
These plates were incubated for 7 days, and then, colonies
were counted to determine the H. pylori burden. Data was log
transformed, and statistical significance was determined using a
Mann−Whitney test with GraphPad Prism software. A P-value
less than 0.05 was considered significant. The study was
repeated to ensure reproducibility. Animal use complied with
the Animal Welfare Act, the Guide for the Care and Use of
Laboratory Animals, and the Office of Laboratory Animal
Welfare.
H. pylori IC50 Dose Responses. H. pylori strains ATCC
43504, SS1, or SS1 NuoD A402P were grown overnight in
Brucella broth containing Skirrow’s selective medium supple-
ment and 10% fetal bovine serum (FBS) or brain heart
infusion broth containing Skirrow’s and 10% FBS. Cultures
were grown in vented 25 cm2 tissue culture flasks at 37 °C and
10% CO2 with rotation at 50 rpm. 2-Fold serial dilutions of test
and control compounds were performed in DMSO or water as
appropriate, and 1 μL was transferred to a 96 well plate. 99 μL
of a 1:10 dilution of an overnight culture of H. pylori in
Brucella/Skirrow’s/10% FBS was added to each well. Plates
were incubated for 18−20 h, and 5 μL of 3 mM resazurin was
added to each well. Plates were incubated for an additional 3−
5 h, and resazurin reduction was measured using a BioTek
Synergy Mx (BioTek, Winooski, VT) with an excitation
wavelength of 540 nm and emission read at 590 nm. Percent
inhibition compared to growth controls was calculated, and the
50% inhibitory concentration (IC50) was determined using
XLFit software (IDBS, London, United Kingdom). pIC50, the
negative log of the IC50 value when converted to molar units,
was calculated on the basis of the individual IC50 values along
with the standard error of the mean (SEM). All pIC50 data
with 4 or more independent replicates were analyzed for
outliers using Grubbs’s test with α set to 0.05 (GraphPad
Prism). A t test was used to compare the replicate pIC50 values
for SS1 wild-type and NuoD A402P mutant (GraphPad
Prism). P-values < 0.05 were considered significant.
Cytotoxicity Assays. FaDu (ATCC HTB-43) and HepG2
(ATCC HB-8065) cell lines were routinely maintained
according to the protocols provided by the American Type
Culture Collection (ATCC). For cytotoxicity IC50 determi-
nation, 100 μL of cell suspension in Eagle’s Minimal Essential
Medium (EMEM) supplemented with 10% FBS was seeded
into black sided, tissue culture treated 96 well plates (Corning
3603) at a concentration of 3 × 105 cells/mL. Plates were
incubated for 24 h in a 37 °C, 5% CO2 incubator to allow for
cell adhesion and growth. Spent media was aspirated from the
test plates, and serial 2-fold dilutions of test compounds mixed
with fresh EMEM + 10% FBS were added to the plates at 100
μL per well. The plates were incubated for 72 h. The media
was aspirated, and FBS-free EMEM and resazurin (final
concentration of 0.15 mM) were added to the plate. Plates
were incubated for 3−5 h, and, then resazurin reduction was
measured using a BioTek Synergy Mx (BioTek, Winooski, VT)
with an excitation wavelength of 540 nm and emission read at
590 nm. Percent inhibition compared to growth controls was
calculated, and the 50% inhibitory concentration (IC50) curves
were generated using XLFit software (IDBS, London, United
Kingdom). IC50 values were converted to pIC50 values as
described above and the average and SEM were calculated.
Generation of Resistant Mutants, Sequencing, and
Target Identification. Mutants resistant to the thienopyr-
imidine series were generated in two separate ways. Strain
HM-274 was chosen for these studies, since it had been
previously sequenced and annotated. Spontaneous mutants of
HM-274 were selected by concentrating and harvesting
susceptible wild-type cultures and spreading them onto plates
containing twice the minimum inhibitory concentration
(MIC) of 28. Two stably resistant isolates were recovered
that had MICs 8-fold higher than the wild-type. In the second
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Computational Studies of H. pylori’s Complex I.
General Computational Methods. The sequence analysis of
T. thermophilus and H. pylori was done using the online Clustal
Omega tool. For residue numbering, a multiple sequence
alignment was performed with M. musculus, B. taurus,
H. sapiens, A. fumagatis, H. pylori, E. coli, and M. tuberculosis
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00300.
■
sı
Table S1: in vitro ADME profile of compound 25; Table
S2: mouse pharmacokinetic profile of compound 25;
Table S3: MIC values of select thienopyrimidines
against a panel of representative Gram-negative and
Gram-positive species; Figure S1: a comparison of pIC50
values between SS1 and SS1 A402P mutant; Figure S2:
multiple species sequence alignment of respiratory
complex I thienopyrimidine binding site; Figure S3:
docked conformations of known complex I ligands and
inhibitors; Figure S4: a complete energy profile of the
protein ligand complex; Figure S5: in vivo efficacy of
compound 25; Figure S6: structures of the optimized
benzimidazole and omeprazole (PDF)
̈
using CLC Genomic Workbench 11. Schrodinger’s Maestro
was used to build three comparative models and for docking.35
AMBERMD was used to run all simulations.36
Homology Modeling. A homology model of the subunits
NuoD and NuoB of NADH/ubiquinone oxidoreductase in
̈
Helicobacter pylori was created using Schrodinger’s Phase
program.37 The comparative model was created using a
Thermus thermophilus protein sequence as a template (PDB:
4HEA). An additional homology model was built with a
A402P mutation in the NuoD subunit using the same
template. Both models were built using energy-based methods.
The comparative models were then prepared by AMBERMD’s
Leap and then minimized using the steepest decent for 10 000
steps using AMBERMD’s PMEMD implementation.36
AUTHOR INFORMATION
Corresponding Author
■
Molecular Docking. A series of thienopyrimidines and
̈
piericidin A was docked using Schrodinger’s Induced Fit
Richard E. Lee − Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, United States; orcid.org/
0000-0002-2397-0443; Email: Richard.Lee@stjude.org
docking based upon the quinone binding channel, based on
previous Thermus thermophilus and mitochondrial studies of
ubiquinone binding (Y85, R40, V403, and N401), mutations
found from the Mills et al.20 group (G398, F404, and V407),
and a ligand in our thienopyrimidine series (T400 and
A402).14,18,19,23,33 All residue numbers are based on the
protein sequence of H. pylori. The default protocol was used
with a box size that was extended to allow docking of ligands
with up 30 Å in length and sampled each ligand conformation
using an energy window of 2.5 kcal/mol for ring conformation
sampling and an energy penalty for nonplanar amide bond
Authors
Alex K. Mugengana − Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, United States; Department of
Pharmaceutical Sciences, University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States
Nicole A. Vita − Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, United States; Department of
Pharmaceutical Sciences, University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States
Autumn Brown Gandt − Arietis Pharma, Boston,
Massachusetts 02118, United States
Kevin Moran − Arietis Pharma, Boston, Massachusetts 02118,
United States
George Agyapong − Arietis Pharma, Boston, Massachusetts
02118, United States
Lalit K. Sharma − Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, United States
Elizabeth C. Griffith − Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, United States
Jiuyu Liu − Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, United States
Lei Yang − Department of Chemical Biology and Therapeutics,
St. Jude Children’s Research Hospital, Memphis, Tennessee
38105, United States
Ekaterina Gavrish − Arietis Pharma, Boston, Massachusetts
02118, United States
Kirk E. Hevener − Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis,
Tennessee 38163, United States; orcid.org/0000-0002-
5584-3625
conformations.38 The ligands were prepared in Schrodinger
̈
using LigPrep.39 The best poses were chosen using the
induced-fit docking (IFD) score (a combination of Glide Score
and Prime energy calculations) and visual inspection.38 The
glide scores are representative ranking of ligands based upon
the specific molecular docking run based on the interactions
between ligand and protein. The thienopyrimidines were
docked using the same methods into the T400I model and
A402P model. The ligands were then compared using the IFD
score.
Molecular Simulations. AMBERMD was used for molec-
ular dynamic simulations. The chosen poses from the
molecular docking were parametrized using Leap.36 The
protein−ligand complex was solvated using TIP3P with a
12.0 Å octahedron periodic box and neutralized with Na. The
apo protein was subjected to the same solvation and
neutralization methods as the protein−ligand complex. The
parametrized structures were saved, and the protein−ligand
complex was used for simulations using GPU accelerated
PMEMD.36 The ligand−protein complex was minimized using
the steepest decent for 10 000 steps utilizing the SHAKE
algorithm. The minimized protein−ligand complex was then
heated using 2 fs time steps over 50 000 steps to reach 303 K.
The heated structure was equilibrated for 20 ns and then
further subjected to a 250 ns production run using 2 fs time
steps. After the simulation was completed, the AMBER MM-
PBSA protocol was used to calculate binding energies and ptraj
was used for analysis of protein−ligand interactions.36 All
images were created using UCSF Chimera.40
Michael D. LaFleur − Arietis Pharma, Boston, Massachusetts
02118, United States
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compound accumulation yield a broad-spectrum antibiotic. Nature
545 (7654), 299−304.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.0c00300
(12) Addie, M., Ballard, P., Buttar, D., Crafter, C., Currie, G., Davies,
B. R., Debreczeni, J., Dry, H., Dudley, P., Greenwood, R., Johnson, P.
D., Kettle, J. G., Lane, C., Lamont, G., Leach, A., Luke, R. W. A.,
Morris, J., Ogilvie, D., Page, K., Pass, M., Pearson, S., and Ruston, L.
(2013) Discovery of 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydrox-
ypropyl]-1-(7H-pyrrolo[2,3-d]pyrimidin −4-yl)piperidine-4-carboxa-
mide (AZD5363), an orally bioavailable, potent inhibitor of Akt
kinases. J. Med. Chem. 56 (5), 2059−2073.
Author Contributions
∇A.K.M., N.A.V., and A.B.G. contributed equally to this study.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(13) Sen, S. E., and Roach, S. L. (1995) A convenient two-step
procedure for the synthesis of substituted allylic amines from allylic
alcohols. Synthesis 1995 (7), 756−758.
The research reported in this publication was supported by the
National Institute of Allergy and Infectious Diseases of the
National Institutes of Health under award number AI102452
and by ALSAC, St. Jude Children’s Research Hospital. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institute
of Health or other funding agencies. We kindly thank Robin
Lee for her assistance in editing the manuscript. Molecular
graphics and analyses were performed with UCSF Chimera,
developed by the Resource for Biocomputing, Visualization,
and Informatics at the University of California, San Francisco,
with support from NIH P41-GM103311.
(14) Sazanov, L. A. (2007) Respiratory complex I: mechanistic and
structural insights provided by the crystal structure of the hydrophilic
domain. Biochemistry 46 (9), 2275−2288.
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