Toward More Drug Like Inhibitors of Trypanosome Alternative Oxidase

Article abstract of DOI:10.1021/acsinfecdis.7b00218

New tools are required to ensure the adequate control of the neglected tropical disease human African trypanosomiasis. Annual reports of infection have recently fallen to fewer than 5000 cases per year; however, current therapies are hard to administer an

Full text of DOI:10.1021/acsinfecdis.7b00218

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Article
Towards more drug like inhibitors of trypanosome alternative oxidase
Ryan West, Thomas Cunningham, Lewis E. Pennicott, Srinivasa P S Rao, and Simon E Ward
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00218 • Publication Date (Web): 21 Jan 2018
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Towards more drug like inhibitors of trypanosome alternative oxi-
dase
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Ryan A. West , Thomas Cunningham , Lewis E. Pennicott , Srinivasa P. S. Rao , Simon E.
Ward* a, c
a Sussex Drug Discovery Centre, University of Sussex, Brighton, BN1 9QJ, United Kingdom.
b Novartis Institutes for Tropical Diseases, 5300 Chiron Way, California, 94608-2916, USA.
c Medicines Discovery Institute, Park Place, Cardiff University, CF10 3AT, United Kingdom, Wards10@cardiff.ac.uk.
New tools are required to ensure the adequate control of the neglected tropical disease human African trypanosomiasis.
Annual reports of infection have recently fallen to fewer than 5000 cases per year; however, current therapies are hard to
administer and have safety concerns, hence are far from ideal. Trypanosome alternative oxidase is an exciting target for
controlling the infection; it is unique to the parasite and inhibition of this enzyme with the natural product ascofuranone
has shown to clear in vivo infections. We report the synthesis and associated structure activity relationships of inhibitors
based upon this natural product with correlation to T. b. brucei growth inhibition in an attempt to generate molecules
that possess improved physicochemical properties and potential for use as new treatments for human African trypanoso-
miasis.
Keywords: Medicinal chemistry, drug design, multipa-
rameter optimisation, human African trypanosomiasis.
and associated economic output of animals; the cost of
delivering trypanocidal agents to control ‘Nagana’ or Afri-
can animal trypanosomiasis (AAT) is estimated to be 140
million USD per year.12
Human African trypanosomiasis (HAT), otherwise known
as African sleeping sickness, is a neglected tropical dis-
ease caused by human infection by the protozoan para-
sites T. b. gambiense or T. b. rhodesiense, transmitted by
tsetse flies in sub-Saharan Africa.1 Without treatment,
HAT is invariably fatal;2 the drugs that are currently ap-
proved for treatment of both the haemolymphatic (stage
1) and encenphalitic (stage 2) phases of the disease have
either toxic side effects or require complex administration
procedures.3 Resistant strains of the parasites have been
observed in the clinic and there is a requirement for new
treatment options to guarantee the successful control of
the disease.4,5 In 2009 it was estimated that occurrences of
the disease had fallen below 20 000 incidences a year and
that 65 million people are at risk of transmission.1
Current treatments for HAT are far from ideal. Stage 1 of
the disease is treated with suramin for T. b. rhodesiense or
pentamidine for T. b. gambiense, both compounds display
undesired toxic side effects in the clinic.13 Resistance
against pentamidine has also been observed and linked to
mutations in the P2 amino purine and other trypanosome
uptake transporters.5 Both suramin and pentamidine are
unable to permeate into the CNS to sufficient levels, and
thus are ineffective against stage 2 infections.14 The or-
gano-arsenide melarsoprol and the more recent combina-
tion therapy of nifurtimox and eflornithine (NECT) are
currently used to treat infections of T. b. gambiense and T.
b. rhodesiense respectively. Both treatments have their
limitations. Melarsoprol shows significant toxic effects in
those treated, causing encephalopathy in 5-10% of people
treated with the drug.15 Incidences of resistance to me-
larsoprol are becoming more commonplace, again linked
to mutations in the P2 amino purine uptake transporters;
associated failure rates in the clinic have been reported to
be between 20%-30% in the early 2000’s.5 NECT requires
the administration of eflornithine via slow intravenous
infusion of 200 mg/kg every 12 hours for 7 days as it is
only trypanostatic, thus requiring trypanocidal action by
the co-administered nifurtimox and innate host clearance
mechanisms.16
The trypanosome, first discovered by Sir David Bruce in
1894,6 undergoes diverse lifecycle adaptations conferring
viability in both the tsetse fly vector and mammalian
host.7,8 A predominant feature of Trypanosoma brucei is
that it is capable of successfully evading host immune
responses, predominantly due to its capacity to diversify
the surface antigens that it presents to its host;9 this abil-
ity has been a major factor for preventing the develop-
ment of an efficacious vaccine against the pathogen.10 T.
b. gambiense is responsible for >95% of cases of HAT and
is a zoonotic disease, residing in a variety of domestic
animals and livestock.11 Trypanosome infection in cattle
and other livestock also has a large burden on the health
With the limitations of the currently approved medicines
for treating HAT and the increasing reports of resistance
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to these therapies, it is vital to discover new and im- A major contribution to the poor multi-parameter opti-
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proved treatments against the pathogen. An ideal target
for drug discovery for HAT is the trypanosome alternative
oxidase (TAO).17 TAO is the sole terminal oxidase enzyme
in the aerobic respiratory pathway for the long slender
blood stage form of Trypanosoma brucei subspecies. The
enzyme utilises oxygen and ubiquinol as substrates for
the efficient generation of cellular ATP as the parasite
does not express regular cytochrome respiratory com-
plexes.18 TAO is a di-iron (non-heme) oxidoreductase that
is located in the inner-mitochondrial membrane of the
trypanosome.19 As TAO is not found in mammalian sys-
tems, there is a unique opportunity to attain compounds
with enhanced selectivity and circumvent on-target tox-
icity.19 It has recently been reported that the pentamidine
and melarsoprol drug resistant parasites have an in-
creased sensitivity to inhibition of TAO, as these parasites
have a reduced capacity to efflux cellular glycerol that is
produced as the result of anaerobic respiration.20 The
crystal structure of TAO has been reported, allowing a
structure based design approach to new molecules.21 Na-
nomolar inhibition of TAO has been reported with the
natural product ascofuranone (AF (1)) and its analogues
in validated biochemical assays,22 demonstrating correla-
tion in ex vivo growth inhibition23 and efficacy in in vivo
clearance models.24
misation (MPO)26 predicted CNS-penetration score for AF
(1) and colletochlorin B (CCB (2)) is attributed to its high
lipophilicity. Clearly the major contributor to the high
lipophilicity of CCB (2) is from the geranyl ‘lipophilic tail’
(figure 2). Compounds with a cLogP above 5 are common-
ly observed to be P-gp substrates, and tend to have a high
metabolic liability. We herein report the synthesis and
SAR investigation of analogues focused on reducing the
lipophilicity of the tail region of structural analogues of
AF (1) and CCB (2), with an aim to improve physicochem-
ical properties and increase the predicted CNS penetra-
tion MPO score.
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Figure 2. Per atom contribution to lipophilicity of asco-
furanone (AF) (1) and colletochlorin B (CCB) (2)
Figure 1. Summary of previous work23
Results and Discussion
Our previous work identified robust synthetic routes to
close analogues of AF (1) and structure activity relation-
ships of the synthesized molecules, highlighting the re-
quirement of both the chloro and methyl substituents on
the aromatic ring for high potency (figure 1).23 With these
routes in place we set out to further explore structurally
similar analogues with the aim to reduce lipophilicity,
and improve the drug like properties of these molecules.
Our aim of reducing lipophilicity was to specifically im-
prove aqueous solubility, decrease metabolism and im-
prove the potential to achieve efficacious CNS concentra-
tions, as new compounds for HAT must be effective
against both stages of the disease.
The biochemical assessment of the bicyclic 1,2-isoxazole
intermediate prepared to access the aldehyde replace-
ment with the nitrile analogue of CCB (2), had shown
unexpectedly high potency for inhibiting TAO. Previous
reports had postulated that a hydrogen bond donor was
needed at this position for potent inhibition of TAO.22
There are many hurdles preventing permeability of organ-
ic drug molecules to the brain. The blood brain barrier
provides significant protection to xenobiotics; tight junc-
tions around capillaries prevent permeation and the ex-
pression of ABC-transporters actively transport solubil-
ised organic molecules out and away from the barrier and
the brain.25 Compounds that traverse readily into the CNS
tend to have high passive permeability, have low efflux
from ABC-transporters like P-glyco-protein (P-gp) and
have low metabolic turnover in human liver microsomes,
providing the basis for the generation of a predictive scor-
ing tool for CNS penetration.26
Figure 3. Isoxazole Kemp elimination
To confirm this result, a synthetic route to the mono
phenolic compound (11) was devised (scheme 1), to rule
out the activity arising from an in situ formation of nitrile
compound via a Kemp elimination (figure 3).27
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Scheme 1. Mono-phenol analogue synthesis
ACS Infectious Diseases
Selective bromination of the phenolic aldehyde was
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achieved by treatment with trimethylbenzylammonium
tribromide at 0 °C; non-selective and over bromination
was observed using N-bromosuccinimide.28 The aldehyde
(6) could be successfully converted to the nitrile (7) by
oxime formation with hydroxylamine hydrochloride and
subsequent in situ dehydration. Protection of the phenol
(8) to the anisole (9) with dimethylsulfate was required to
facilitate a high yielding Suzuki-Miyaura cross-coupling
using tri-n-octylborane as the coupling partner that was
formed in situ by reacting 1-octene with a solution of 1M
borane in THF.29 Final deprotection of the anisol (10) to
the free phenol using trimethylsilyl chloride and sodium
iodide provided the monophenol analogue (11) for bio-
chemical and ex vivo assessment (table 1).30
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Reaction conditions: a) trimethylphenylammonium tribro-
mide, CH2Cl2 / MeOH rt, 3 h, 77%, b) NH2OH·HCl, EtOH,
reflux, 16 h, 90%, c) NCS, Et3N, MeCN, 0°C – rt, 16 h, 70%, d)
dimethyl sulfate, acetone, rt, 16 h, 98%, e) i) BH3·THF, 1-
octene, 0°C, 20 min, ii) toluene, H2O, K3PO4, RuPhos,
Pd(OAc)2, 50°C, 16 h, 90% f) TMS-Cl, NaI, MeCN, 80°C, 16 h,
65%.
Table 1. Biological assay assessment of isoxazole and mono-phenol analogues
†
*
#
#
Structure
TAO pIC50
T. b. b. pEC50
HepG2 pCC50
5.1 ± 0.1
2
8.5 ± 0.3
8.4 ± 0.1
4
3
8.4 ± 0.2
8.3 ± 0.4
8.9 ± 0.3
7.0 ± 0.1
4.5 ± 0.1
6.4 ± 0.1
7.0 ± 0.2
7.6 ± 0.1
5.1 ± 0.2
< 4.3
4.9 ± 0.1
5.3 ± 0.2
5.1 ± 0.1
4.7 ± 0.1
< 4.3
11
46
10
†: Negative log concentration and standard deviation of compounds required for 50% inhibition of trypanosome alternative oxi-
dase; *: Negative log concentration and standard deviation of compounds required for 50% growth inhibition of T. b. brucei Lis-
ter427; #: Negative log concentration and standard deviation of compounds required for 50% cytotoxicity of HepG2 cell line. (n ≥
2 in all assays)
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Interestingly, the monophenol analogue (11) retained high Scheme 2. Amide tail linker synthesis
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potency against TAO that corresponded to the most po-
tent inhibition of T. b. brucei growth for a compound not
containing the benzaldehyde functionality present in AF
and CCB. The removal of one of the flanking substituents
ortho to the brominated coupling position facilitated its
reactivity and thus exploration of this tail. This synthetic
route was adopted for further diversification of this tail
region. The des-methyl starting material was accessible
from commercially available sources and was used to ini-
tially investigate tail modifications.
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The introduction of polarity into the lipophilic tail was
devised to reduce cLogP and improve upon the predicted
CNS penetration MPO score (>4 required for high pre-
dicted CNS exposure). The introduction of amide func-
tionality in alternate positions in the chain were planned
to mimic the n-octyl (C8H17) or geranyl chain. The inclu-
sion of the amide functionality had the largest predicted
improvement to both cLogP and MPO score (Row C –
table 2). 1-Alkene esters were converted to trialkylboranes
using the methodology previously employed to access the
n-octyl analogues (scheme 2). These were then coupled
with the brominated intermediate (14). The esters were
converted to the desired amides by either in situ hydroly-
sis under the demethylation conditions, with subsequent
HATU amide coupling with the required alkylamine, or a
direct aminolysis of the ester by using 1,5,7-
triazabicyclo[4.4.0]dec-5-ene (TBD) and the desired al-
kylamine with subsequent anisole deprotection with
TMSCl / NaI to provide the amide chain compounds for
biological assessment (table 3).31
Reaction conditions: a) NBS, MeCN, rt, 16 h, 85%, b) dime-
thyl sulfate, acetone, reflux, 3 h, 90%, c) i) BH3·THF, 1-alkene
ester, 0 °C, 20 min, ii) toluene, H2O, K3PO4, RuPhos,
Pd(OAc)2, 16 h, 50°C, 90%, d) TMSCl, NaI, MeCN, 80 °C, 16 h,
35-85%. e) HATU, Et3N, R1’R2’NH, MeCN, rt, 3 h, 60-90%, f)
TBD, R1’R2’NH, MeCN, rt, 1 – 16 h 70-85%.
The acrylic ester could not be successfully coupled under
these conditions; we postulate that the required trialkyl-
borane may not form as the alkene is too electron defi-
cient. The desired ester was synthesized by a Heck-
Mizoroki cross-coupling and subsequent reduction of the
double-bond, before chlorination and amide formation
with TBD (scheme 3).
Scheme 3. Heck cross-coupled amide tail linker syn-
thesis
Table 2. cLogP and predicted MPO scores for planned
analogues.
Row
Structure
cLogP MPO Score26
A
Reaction conditions: a) ethyl acrylate, SingaCycle™, K2CO3,
NMP, 100 °C, 16 h, 30%, b) triethylsilane, Pd/C, EtOH, rt, 16
h, 78%, c) SO2Cl2, Et2O, rt, 96 h, 57%, d) TBD, n-butylamine,
MeCN, 16h, 39%.
6.2
6.3
3.5
4.6
4.9
3.5
3.8
5.5
4.8
4.2
(4)
B
(11)
A selection of ether chain analogues were also planned
and synthesised (D – table 2), these were expected to be
more chemically similar to that of the aliphatic chain with
a smaller amount of polarity introduced in comparison to
the amide. These compounds were less chemically tracta-
ble; in our hands the anisole de-protection to the phenol
proved challenging. The deprotection conditions that we
explored led to the cleavage of the desired ether function-
ality present in the chain to the terminal alcohol. To miti-
gate this synthetic hurdle, alternative protecting groups
were investigated. Bulkier silyl protecting groups hin-
dered the cross-coupling and failed to provide the desired
ether tail analogues. Reaction with the phenolic ester (27)
provided some cross-coupled product, the stability of this
protecting group perhaps unsurprisingly, proved to be
labile under the reaction coupling conditions. The de-
acetylated products (29 to 31) were observed and isolated
from the reactions however significant proportions of the
C
D
E
(43)
cLogP calculated from ChemAxon Marvin Sketch, calculated
MPO as described by Wager et al.26
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Assessment of these less lipophilic analogues showed a
starting material ester (28) saponified before cross-
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7
coupling providing a major by-product of the deacetylat-
ed phenol (14) thus resulting in lower isolated yields
(scheme 4).
large decrease in potency compared to the n-octyl ana-
logues, highlighting that even minor increases in polarity
in this part of the lipophilic tail was not tolerated. The
most promising analogues included polarity at the termi-
nal of the chain (22 and 23); however, disappointingly,
these compounds resulted in no measurable inhibition of
T. b. brucei growth (table 3).
Scheme 4. Ether tail linker synthesis
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Reaction conditions: a) Ac2O, pyridine, DCM, rt, 16 h, 74%,
b) i) BH3·THF, 1-alkene ether, 0°C, 20 min, ii) toluene, H2O,
K3PO4, RuPhos, Pd(OAc)2, 16 h, 50 °C, 7-24%.
Table 3. Biological assessment of polar tail analogues
†
#
R3
R5
CH3
H
cLogP
6.3
TAO pIC50
8.9 ± 0.3
7.0 ± 0.1
T. b. b. pEC50*
7.6 ± 0.1
5.1 ± 0.2
<4.3
11
46
27
5.8
CH3
3.5
5.5 ± 0.2
19
20
21
H
H
H
3.0
2.9
3.0
<4.5
-
-
-
4.6 ± 0.1
4.6 ± 0.1
22
23
H
H
3.4
3.6
5.1 ± 0.1
<4.3
<4.3
5.4 ± 0.2
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30
31
H
H
H
4.1
4.1
4.1
4.8 ± 0.1
5.6 ± 0.1
5.2 ± 0.1
4.7 ± 0.1
4.6 ± 0.1
<4.3
†: Negative log concentration and standard deviation of compounds required for 50% inhibition of trypanosome alternative oxi-
dase; * Negative log concentration and standard deviation of compounds required for 50% growth inhibition of T. b. brucei Lis-
:
ter427. (n ≥ 2 in all assays performed).
This data suggested that a lipophilic tail was required for
the potent inhibition of TAO. To further probe this ob-
servation; analogues were prepared to assess the length of
tail needed for potent inhibition of TAO and whether
total lipophilicity could be decreased by reduction of the
carbon chain length (E – table 2). Previous reports had
shown large decreases in potency against TAO at carbon
chain length below 3. The C1-7 analogues (39 to 45) were
prepared in a similar fashion to that of the n-octyl ana-
logue (11); C1-4 analogues were however prepared directly
using the alkyl boronic acid or trimeric alkyl boroxines as
the volatile alkene hydrocarbons were not accessible
(scheme 5).
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Scheme 5. Carbon chain length analogue synthesis
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Reaction conditions: a) i) BH3·THF, 1-alkene, 0 °C, 20 min, ii)
toluene, H2O, K3PO4, RuPhos, Pd(OAc)2, 16 h, 50 °C, 65-90%,
or 1-boronic acid/boroxine, K3PO4, RuPhos, Pd(OAc)2, 16 h,
80 °C b) TMSCl, NaI, MeCN, 80 °C, 16 h, 35-85%.
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Table 4. Biological assessment of carbon chain length analogues
†
*
#
R3
cLogP
6.3
TAO pIC50
8.9 ± 0.3
8.5 ± 0.1
8.2 ± 0.1
7.8 ± 0.1
7.7 ± 0.1
7.1 ± 0.1
T. b. b. pEC50
7.6 ± 0.1
6.6 ± 0.2
6.1 ± 0.1
11
n-C8H17
n-C7H15
n-C6H13
n-C5H11
n-C4H9
n-C3H7
n-CH3
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39
5.8
5.4
4.9
4.5
6.0 ± 0.1
5.3 ± 0.1
4.1
4.7 ± 0.1
4.7 ± 0.1
3.2
6.2 ± 0.1
†: Negative log concentration and standard deviation of compounds required for 50% inhibition of trypanosome alternative oxi-
dase; * Negative log concentration and standard deviation of compounds required for 50% growth inhibition of T. b. brucei Lis-
:
ter427. (n ≥ 2 for all assays performed).
Interestingly, the reduced carbon chain length analogues
showed good potency against TAO (table 4), until a nota-
ble decrease in enzymatic inhibition was observed at
compounds with C3 or below, corroborating previous
reports.22 What was more evident was the decrease ob-
served in efficacy against T. b. brucei growth inhibition as
chain length and cLogP decreased. Compounds showed a
distinct drop off in potency against T. b. brucei for each
carbon reduction. To interrogate this data further correla-
tions of cLogP and biochemical and cellular potency were
carried out (figure 4).
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Figure 4. Correlations of cLogP vs TAO inhibition pIC50 (left) and cLogP vs T. b. b. pIC50 growth inhibition (right). LipE is
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lipophilic ligand efficiency (pIC50 – cLogP)32.
Biochemical potency for these compounds track to a lipo-
philic ligand efficiency of 3. Cellular potency, however
correlates to a lipophilic ligand efficiency of 1, relating to a
100-fold decrease in potency from the biochemical activity
to the cellular assay. The 2 log decrease in cellular poten-
cy observed here could be due a combination of reasons
that we are currently investigating, it is of interest that
CCB (2) shows a smaller decrease in potency from the
biochemical to cellular assay. The permeability of the
compounds though the cell and mitochondrial mem-
branes of the trypanosome may reduce as the lipophilicity
decreases. The compounds could be substrates for efflux
transport, that may reduce the intracellular concentra-
tion. The TAO inhibition kinetics of the compounds may
be altered with the reduction in carbon chain length that
could result in faster dissociation, resulting in reduced
efficacy on target. The compounds may not locate to the
desired cell compartment as lipophilicity may be required
for the inhibitors to be retained in the inner mitochondri-
al membrane where the enzyme is located. Colletochlorin
B could be acting on an unknown complimentary anti-
parasitic target. On-going studies to better understand
these data will aid future analogue design and synthesis
to identify more drug like inhibitors of TAO.
identify potent inhibitors of TAO that possess improved
physicochemical properties than that of the natural prod-
ucts from which they are derived. Our exploration in this
area has previously shown the requirement for the methyl
and chloro substituents on the aromatic ring for high po-
tency inhibition of TAO. Our efforts reported here for
modulating the lipophilicity of this molecule by the in-
troduction of polar functionality in the tail region of this
chemotype has proved challenging. Polar functionality in
this region of the molecule was shown to not be tolerated
with large decreases in potency against TAO observed.
The reduction in the carbon chain length of the tail re-
sulted in identifying analogues that retained high inhibi-
tory activity of TAO, however ex vivo efficacy against T. b.
brucei growth was diminished. We will continue to try to
understand this disconnect and use this generated data
for future compound design towards more drug like in-
hibitors of TAO.
Materials and Methods
Experimental Details. ChemAxon Calculator Plugins
were used for structure property prediction and calcula-
tion (cLogP), Marvin 15.5.4, 2015, ChemAxon
(http://www.chemaxon.com). All commercial reagents
were purchased from Sigma-Aldrich, Alfa Aesar, Apollo
Scientific, Fluorochem or Tokyo Chemical Industry and of
the highest available purity. Unless otherwise stated,
chemicals were used as supplied without further purifica-
tion. Anhydrous solvents were purchased from Acros
(AcroSeal™) or Sigma-Aldrich (SureSeal™) and were
stored under nitrogen. 40-60 petrol ether refers to the
fraction with a boiling point between 40 °C and 60 °C.
Anhydrous solvents and reagents were used as purchased.
Thin layer chromatography (TLC) was carried out using
glass plates pre-coated with Merck silica gel 60 F254.
Melting points were determined using an OptiMelt appa-
ratus and are uncorrected. Proton nuclear magnetic reso-
We also noticed positive correlation between cLogP and
both biochemical and cellular activity (figure 4). Exam-
ples of potency correlating to high lipophilicity have pre-
viously been observed with other membrane proteins and
enzymes requiring lipophilic substrates.33,34
Conclusions
HAT continues to provide a challenge for the develop-
ment of new therapies; cases continue to be reported with
resistance more frequently observed. Discoveries of new
brain penetrant T. b. gambiense and T. b. rhodesiense
trypanocides are vital for ensuring the adequate control of
HAT in the future. Our work in this area has been to
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Page 8 of 29
nance spectra were recorded at 500 MHz on a Varian well, and the plates were incubated for 48 hours at 37 °C
1
2
3
4
5
6
7
8
VNMRS 500 MHz spectrometer (at 30 °C), using residual
isotopic solvent (CHCl3, δ = 7.27 ppm, DMSO δ = 2.50
ppm, MeOH δ = 3.31 ppm) as an internal reference. Chem-
ical shifts are quoted in parts per million (ppm). Coupling
constants (J) are recorded in Hertz (Hz). Carbon nuclear
magnetic resonance spectra were recorded at 125 MHz on
a Varian 500 MHz spectrometer and are proton decou-
pled, using residual isotopic solvent (CHCl3, δ = 77.00
ppm, DMSO δ = 39.52 ppm, MeOH δ = 49.00 ppm) as an
internal reference. Proton and carbon spectra assign-
ments are supported by DEPT editing. High resolution
mass spectrometry (HRMS) data (ESI) was recorded on
Bruker Daltonics, Apex III, ESI source: Apollo ESI with
methanol as spray solvent. Only molecular ions, fractions
from molecular ions and other major peaks are reported
as mass/charge (m/z) ratios. LCMS data was recorded on
a Waters 2695 HPLC using a Waters 2487 UV detector
and a Thermo LCQ ESI-MS. Samples were eluted through
a Phenomenex Lunar 3 μm C18 50 mm × 4.6 mm column,
using water and acetonitrile acidified by 0.1% formic acid
at 1 ml/min and detected at 254 nm. The gradient em-
ployed was a 7 min method 30-90% MeCN over a 5 min
gradient, held at 90% MeCN for 1 min, then re-
equilibrated to 30% MeCN over 1 min. All experiments
were carried out under an inert atmosphere of N2 unless
otherwise stated.
in 5% CO2 incubators. Parasite numbers in individual
plate wells were determined through quantification of
intracellular ATP amount. The CellTiter-Glo luminescent
cell viability reagent (Promega) was added to plate wells,
and ATP-dependent luminescence signal was measured
on Tecan M1000 plate Reader after 30 min incubation.
Suramin an anti-trypanosomal drug was used positive
control and DMSO was used as negative control. pIC50
values were calculated using Graph Pad Prism software by
plotting the luminescence values in sigmoidal dose re-
sponse curves. Suramin was used as a positive control in
screening. (pIC50 6.7 ± 0.1)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hep-G2 Cytotoxicity Assay. Human hepatocellular car-
cinoma (HepG2) cells were obtained from ATCC and
grown in RPMI media. 25 µL of 1.6 x 104 cells / mL were
dispensed into sterile 384 well Griener clear plates and
incubated at 37 °C in 5% CO2 incubator for 24 h. Once the
cells adhered, 125 nL of 10-point, 3-fold serially diluted
compounds in DMSO were transferred on to cells. After
incubating for additional 96 h at 37 °C in 5% CO2 incuba-
tor, cells were added with CCK-8 reagent to each well.
Plates were further incubated for 3 h followed by absorb-
ance reading at 450 nM using Envision reader. Absorb-
ance values were used for determination of cytotoxic con-
centration (pCC50) required to inhibit 50% growth.
Purmycin was used as positive control in screening (pCC50
6.3 ± 0.1).
TAO Absorbance Assay. 1-Ubiquinol turnover was
measured by recording the increase in absorbance at 278
nm (Greiner 96-well UV star flat bottom plates with BMG
PHERAstar FS photo spectrometer) to monitor the in-
crease of 1-ubiquinone concentration kinetically over 6
min with purified rTAO (3 nM). A final concentration of
15 µM 1-ubiquinol was used under the following condi-
tions: 50 mM Tris-HCl; 0.05% (w/v) C10E8; pH 7.4; 25 °C).
The sigmoidal curve of the inhibition was observed by 10-
point 3-fold serial dilution of test compounds to generate
IC50 values.
Synthesis
5-Chloro-7-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-1,2-
benzoxazol-6-ol (3)
To a solution of triphenylphosphine (42 mg, 0.16 mmol)
in dichloromethane (2 mL) was added 2,3-dichloro-5,6-
dicyano-p-benzoquinone (37 mg, 0.16 mmol). The dark
reaction mixture was stirred at room temperature for 1
minute during which time the colour faded. 4-Chloro-2-
2E)-3,7-dimethylocta-2,6-dien-1-yl]-6-[(hydroxyimino)m
ethyl]benzene-1,3-diol (35 mg, 0.10 mmol) in dichloro-
methane (0.5 mL) was added to the reaction mixture in 1
portion and this was stirred for 1 minute before being
concentrated under reduced pressure. Purification by
flash column chromatography (10 g silica), eluting with a
gradient of petrol ether : ethyl acetate (100 : 0) to (90 : 10),
gave the title compound as a white solid (24 mg, 72%). 1H
NMR (500 MHz, Chloroform-d) δ 8.60 (s, 1H), 6.10 (s, 1H),
5.42 – 5.32 (m, 1H), 5.10 – 5.01 (m, 1H), 3.66 (d, J = 7.3 Hz,
2H), 2.58 (s, 3H), 2.11 – 2.05 (m, 2H), 2.05 – 1.98 (m, 2H),
1.84 (d, J = 1.2 Hz, 3H), 1.64 (d, J = 1.4 Hz, 3H), 1.57 (d, J =
1.2 Hz, 3H).
Growth inhibition assays. Bloodstream form Trypano-
soma brucei brucei Lister 427 parasites were continuously
passaged in HMI-9 medium formulated from IMDM me-
dium (Invitrogen), 10% heat-inactivated fetal bovine se-
rum, 10% Serum Plus medium supplement (SAFC Biosci-
ences), 1 mM hypoxanthine (Sigma-Aldrich), 50 µM bath-
ocuproine disulfonic acid (Sigma-Aldrich), 1.5 mM cyste-
ine (Sigma-Aldrich), 1 mM pyruvic acid (Sigma-Aldrich),
39 µg / mL thymidine (Sigma-Aldrich), and 14 µL / L beta-
mercapthoethanol (Sigma-Aldrich); all concentrations of
added components refer to that in complete HMI-9 me-
dium. The parasites were cultured in 10 mL of HMI-9 me-
dium in T75 CELL-STAR tissue culture flasks at 37 °C / 5%
CO2.
2-Methyl-4-hydroxy-5-bromo-benzaldehyde (6)
To a solution of 2-methyl-4-hydroxy-benzaldehyde (10 g,
73.45 mmol) in CH2Cl2 (100 ml) and methanol (50 ml) was
added a solution of phenyltrimethylammonium tribro-
mide (30.07 g, 77.12 mmol) in CH2Cl2 : methanol (1:1, 100
ml) at -5 °C. The reaction was warmed to ambient tem-
perature and stirred for 16 h. The reaction was concen-
trated under reduced pressure and the residue was added
1 M HCl (aq) (200 mL) to precipitate a white solid, the
To determine growth inhibitory potency of compounds
against T. b. brucei bloodstream form parasites, 200 nL of
10-point, 3-fold serially diluted compounds in DMSO were
transferred to the wells of white, solid bottom 384-well
plates (Greiner Bio-One) by either Echo 555 acoustic liq-
uid handling system or Mosquito. Then, 104 of T. b. brucei
parasites in 40 µL of HMI-9 medium were added to each
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Page 9 of 29
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solid was collected by filtration and dried under reduced
(C), 116.3 (C), 116.0 (C), 111.0 (C), 61.0 (CH3), 19.2 (CH3).
LRMS EI+ 261 m/z.
1
2
pressure to give the title compound as a cream precipitate
(14.67 g, 94 %): m.p. 160–165 °C, (methanol / water). IR
3-Bromo-5-chloro-4-hydroxybenzonitrile (13)
1
(neat, vmax) cm-1, 3063, 1384, 1310, 1136. H NMR (500MHz,
3
4
5
6
7
8
9
N-Bromosuccinimide (0.30 g, 1.71 mmol) was added to a
solution of 3-chloro-4-hydroxybenzonitrile (0.25g, 1.63
mmol) in acetonitrile (5 mL). To the reaction mixture was
added water (15 mL), precipitating a white solid, this was
collected by filtration and dried under reduced pressure
to provide the title compound as a white solid (0.36 g,
84%). m.p. 166-169 °C. IR (neat, vmax), cm-1 3411, 2229, 1471,
D6-DMSO) δ 11.35 (1H, br s), 9.95 (1H, s), 7.91 (1H, s), 6.83
(1H, s), 2.49 (3H, s). 13C NMR (126MHz, D6-DMSO) δ 190.8
(CH), 159.2 (C), 142.3 (C), 137.1 (CH), 128.0 (C), 119.1 (CH),
107.4 (C), 19.1 (CH3). HRMS (ESI-) m/z [M-H]- calculated
for C8H6BrO2 212.9557, found 212.9550.
5-Bromo-4-hydroxy-2-methyl-benzonitrile (7)
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A solution of 2-methyl-4-hydroxy-5-bromo-benzaldehyde
(20.00 g, 93.0 mmol), in acetonitrile (300 mL) was added
hydroxylamine hydrochloride (6.92 g, 99.5 mmol). The
reaction mixture was heated to reflux and stirred for 16
hours. The reaction mixture was cooled to 50 °C and add-
ed further hydroxylamine hydrochloride (1.5 g, 21.5
mmol). The reaction mixture was heated to reflux for a
further 3 hours. This was collected by filtration and dried
under vacuum to give 13.08 g of an off-white solid. Analy-
sis showed a good purity product. The filtrate was con-
centrated under vacuum. The residue was triturated in
MeCN : water (1:2) and sonicated, the solid was collected
by filtration and dried under vacuum, to provide a further
6.67 g (19.75, quant.). m.p. 194-199 °C (MeCN / water). IR
(neat, νmax) cm-1 3203, 2232 (CN), 1595, 1402, 1386, 1260,
1327, 1294, 1245, 1204, 1156. H NMR (500MHz, CDCl3) δ
7.75 (s, 1H), 7.64 (s, 1H) 6.37 (s, 1H). 13C NMR (126 MHz,
CDCl3) δ 152.8 (C), 135.0 (C), 132.6 (CH), 121.7 (C), 116.3 (C),
110.9 (C), 106.1 (C). HRMS (ESI-) m/z [M-H]- calcd for
C7H2BrClNO 229.9014, found 231.8984.
3-Bromo-5-chloro-4-methoxybenzonitrile (14)
Dimethyl sulfate (0.15 mL, 1.61 mmol) was added to a sus-
pension of potassium carbonate (0.22 g, 1.61 mmol) and 3-
bromo-5-chloro-4-hydroxy-benzonitrile (0.30 g, 1.29
mmol) in acetone (10 mL). The reaction mixture was
heated to reflux for 30 min, cooled to RT and added wa-
ter. The resulting precipitate was collected by filtration
and dried under reduced pressure to provide the title
compound as a white solid (0.29 g, 90%). m.p. 115-116 °C,
1
IR (neat, vmax), cm-1 2236 (CN), 1539, 1470, 1419, 1272. H
1
1221. H NMR (500MHz, D6-DMSO) - δ 11.38 (1H, br s),
NMR (500MHz, CDCl3) δ 7.78 (s, 1H), 7.66 (s, 1H), 3.97 (s,
3H). 13C NMR (126 MHz, CDCl3) δ 157.6 (C), 135.4 (CH),
133.2 (CH), 130.4 (C), 119.5 (C), 116.0 (C), 109.9 (C), 61.0
(CH3). HRMS (ESI-) m/z [M-H]- calcd for C8H5BrClNO
243.9170, found 243.9165.
7.90 (1H, s), 6.92 (1H, s), 2.49 (3H, s). 13C NMR (126MHz,
D6-DMSO) δ 158.6 (C), 143.4 (C), 137.1 (CH), 117.9 (CH),
117.7 (C), 107.3 (C), 103.8 (C), 19.1 (CH3). HRMS (ESI-) m/z
[M-H]- calculated for C8H5BrNO 209.9560, found
209.9554.
(2-Bromo-6-chloro-4-cyano-phenyl) acetate (28)
5-Bromo-3-chloro-4-hydroxy-2-methyl-benzonitrile (8)
3-Bromo-5-chloro-4-hydroxy-benzonitrile (500 mg, 2.15
mmol) was dissolved in dichloromethane (10 mL), before
pyridine (0.26 mL, 3.23 mmol) and acetic anhydride (0.24
mL, 2.58 mmol) were added. The reaction mixture was
stirred at room temperature for 12 h. The reaction mixture
was taken up in water (10 mL) and the organic compo-
nents were washed with water (2 × 10 mL). The chlorinat-
ed layer was separated, washed with brine (1 × 10 mL),
dried over MgSO4, filtered and concentrated in vacuo, to
give the title compound as a granular white solid (462 mg,
74%). m.p. 116-119 °C; IR (neat, vmax, cm-1) 3088, 2238, 1774;
1H NMR (500 MHz, CDCl3) δ 7.83 (s, 1H), 7.72 (s, 1H), 2.44
(s, 3H); 13C (126 MHz, CDCl3) δ 166.3 (C), 149.3 (C), 135.0
(CH), 132.7 (CH), 130.3 (C), 119.3 (C), 115.7 (C), 112.2 (C),
20.3 (CH3); LRMS m/z (EI+) 275.
Triethylamine (0.83 mL, 5.97 mmol) was added to a solu-
tion of 5-bromo-2-methyl-4-hydroxybenzonitrile (1.15 g,
5.42 mmol) in acetonitrile (50 mL). 1-chloropyrorolidine-
2,5-dione (1.52 g, 11.38 mmol) was added portionwise to
the stirring solution. To the reaction mixture was added
water (50 mL), precipitating a pale-yellow solid that was
collected by filtration to provide the title compound (1.11
g, 83%). m.p. 166-169 °C, IR (neat, vmax), cm-1 3239, 2236
1
(CN), 1581, 1468, 1384, 1294, 1224, 1147. H NMR (500MHz,
CDCl3) δ 7.70 (s, 1H), 6.39 (s, 1H), 2.58 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 152.5 (C), 140.9 (C), 134.4 (CH), 122.1
(CH), 116.4 (C), 107.5 (CH), 106.9 (C), 19.0 (CH3). HRMS
(ESI-) m/z [M-H]- calcd for C8H4BrClNO 243.9170, found
243.9165.
5-Bromo-3-chloro-4-methoxy-2-methyl-benzonitrile (9)
General protocol for alkene cross-couplings
Dimethyl sulfate (0.85 mL, 8.95 mmol) was added to a
suspension of 5-bromo-3-chloro-4-hydroxy-benzonitrile
(2.10 g, 8.52 mmol), and potassium carbonate (1.41 g, 10.22
mmol) in acetone (80 mL). The reaction mixture was
heated to reflux for 3 hours, the reaction was cooled to RT
and added water (160 mL) precipitating a white solid that
was collected by vacuum filtration to give the title com-
pound (2.07 g, 93%). m.p. 119-120 °C, IR (neat, vmax), cm-1
1-Alkene (2.54 mmol) was dropwise added to an ice
cooled solution of 1M borane in THF (0.91 mmol). This
solution was warmed to room temperature and stirred for
15 min. The solution was quenched with the addition of
water (0.25 mL), and diluted with toluene (2.5 mL). Potas-
sium phosphate tribasic (0.43 g, 2.03 mmol), aromatic
bromide (1.01 mmol), palladium(II) acetate (0.01 g, 0.025)
mmol
and
2-dicyclohexylphosphino-2’-6’-
1
2229 (CN), 1457, 1372, 1275, 1048. H NMR (500 MHz,
diisopropoxybiphenyl (0.02 g, 0.05 mmol) were added.
The reaction mixture sealed and heated 100°C for 16
CDCl3) δ 7.74 (s, 1H), 3.94 (s, 3H), 2.58 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 157.4 (C), 141.7 (C), 134.9 (CH), 131.3
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Page 10 of 29
hours. The reaction mixture was partitioned between wa- (CH2), 29.80 (CH2), 28.8 (CH2), 24.6 (CH2). LRMS EI+ 295
1
2
3
4
5
6
7
8
ter (10 mL) and EtOAc (20 mL). The organic layer was
separated, dried over MgSO4, filtered and concentrated
under vacuum. The residue was purified by flash silica
chromatography, eluting with petroleum ether to 10-50%
EtOAc followed by purification by preparative HPLC 50%
MeCN in water to 100% MeCN to provide the title com-
pounds (11-90%).
m/z.
Methyl
phenyl)heptanoate (18)
IR (neat, vmax), cm-1 2933, 2232 (CN), 1734, 1472, 1276. H
NMR (500 MHz, CDCl3) δ 7.52 (s, 1H), 7.38 (s, 1H), 3.88 (s,
3H), 3.66 (s, 3H), 2.64 (m, 2H), 2.31 (m, 2H), 1.66-1.55 (m,
7-(3-chloro-5-cyano-2-methoxy-
1
13
4H), 1.36 (m, 4H). C NMR (126 MHz, CDCl3) δ 174.3 (C),
3-Chloro-4-methoxy-2-methyl-5-octyl-benzonitrile (10)
158.4 (C), 139.6 (C), 132.6 (CH), 132.0 (CH), 129.1 (C), 117.9
(C), 108.6 (C), 61.3 (CH3), 51.7 (CH3), 34.1 (CH2), 30.14
(CH2), 30.08 (CH2), 29.2 (CH2), 29.0 (CH2), 24.9 (CH2).
HRMS (ESI+) m/z [M+Na]+ calcd for C16H20ClNNaO3
332.1024, found 332.1019.
9
IR (neat, vmax) cm-1 2913, 2849, 2226 (CN), 1468, 1291, 1074.
1H NMR (500 MHz, CDCl3) δ 7.36 (s, 1H), 3.87 (s, 3H), 2.62
(t, J = 7.9 Hz, 2H), 2.57 (s, 3H), 1.61-1.55 (m, 2H), 1.36-1.27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(m,
10H),
0.89
(t,
J
=
6.8
Hz,
3H).
13C NMR (126 MHz, CDCl3) δ 158.2 (C), 139.4 (C), 136.3 (C),
132.0 (CH), 129.7 (C), 117.7 (C), 109.4 (C), 60.9 (CH3), 31.8
(CH2), 30.2 (CH2), 29.7 (CH2), 29.4 (CH2), 29.3 (CH2), 29.1
3-(3-Butoxypropyl)-5-chloro-4-hydroxy-benzonitrile (29)
IR (neat, vmax, cm-1) 2923, 2236, 1H NMR (500 MHz, CDCl3)
7.51 (s, 1H), 7.31 (s, 1H), 3.50 (t, J = 6.6 Hz, 2H), 3.43 (t, J =
5.9 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H), 1.90 (p, J = 6.4 Hz,
2H), 1.62( p, J = 7.1 Hz, 2H), 1.40 (h, J = 7.5 Hz, 2H), 0.95 (t,
J = 7.5 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 154.9 (C),
133.0 (CH), 131.5 (CH), 130.4 (C), 121.8 (C), 118.2 (C), 104.0
(C), 71.1 (CH2), 68.5 (CH2), 31.5 (CH2), 29.0 (CH2), 26.4
(CH2), 19.3 (CH2), 13.9 (CH3); HRMS (ESI-) m/z [M-H]-
calcd for C14H17ClNO2 266.0953, found 266.0953.
(CH2),
22.6
(CH2),
18.8
(CH3),
14.0
(CH3).
LRMS EI+ 293 m/z.
5-Chloro-4-methoxy-5-octylbenzonitrile (46 intermedi-
ate)
IR (neat, vmax) cm-1 2925, 2854, 2232 (CN), 1470, 1426, 1275.
1H NMR (500 MHz, CDCl3) δ 7.53 (s, 1H), 7.40 (s, 1H), 3.90
(s, 3H), 2.66 (t,J= 7.9 Hz, 2H), 1.60 (m, 2H), 1.40 – 1.26 (m,
10H), 0.90 (t, J= 6.8 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
158.2 (C), 139.7 (C), 132.4 (CH), 131.7 (CH), 128.9 (C), 117.7
(C), 108.5 (C), 61.1 (CH3), 31.8 (CH2), 30.2 (CH2), 30.0
(CH2), 29.4 (CH2), 29.3 (CH2), 29.1 (CH2), 22.6 (CH2), 14.0
(CH3). HRMS (ESI-) m/z [M-H]- calcd for C15H19ClNO
264.1161, found 264.1153.
3-Chloro-5-(5-ethoxypentyl)-4-hydroxy-benzonitrile (30)
1H (500 MHz, CDCl3) δ 7.49 (s, 1H), 7.33 (s, 1H), 3.47 (q, J =
7.1 Hz, 2H), 3.41 (t, J = 6.3 Hz, 2H), 2.66 (t, J = 7.8 Hz, 2H),
1.62 (p, J = 8 Hz, 4H), 1.40 (p, J = 7.8 Hz, 2H), 1.20 (t, J =
13
7.0 Hz, 3H). C (126 MHz, CDCl3) δ 153.6 (C), 132.6 (CH),
130.4, (CH), 120.5 (C), 118.1 (C), 104.1 (C), 88.2 (C), 70.5
(CH2), 66.2 (CH2), 30.2 (CH2), 29.4 (CH2), 28.7 (CH2), 26.0
(CH2), 15.1 (CH3); HRMS (ESI-) m/z [M-H]- calcd for
C14H17ClNO2 266.0953, found 266.0942.
Methyl 4-(3-chloro-5-cyano-2-methoxy-phenyl)butanoate
(15)
IR (neat, vmax), cm-1 2952, 2231 (CN), 1732, 1427, 1276, 993,
1
877. H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 2.1 Hz, 1H),
3-Chloro-4-hydroxy-5-(6-methoxyhexyl)benzonitrile (31)
IR (neat, νmax, cm-1); 2932, 2860, 2233 (CN), 1174, 1116; H
NMR (500 MHz, CDCl3) δ 7.49 (s, 1H), 7.33 (s, 1H), 3.55 (s,
3H) 3.36 (t, J = 6.6 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H), 1.57 (p,
J = 7.4 Hz, 4H), 1.37 (m, 4H); C (126 MHz, CDCl3) δ 153.3
(C), 132.7 (CH), 130.3, (CH), 120.4 (C), 110.0 (C), 100.2 (C),
88.3 (C), 72.8 (C), 58.6 (C), 30.1 (CH2), 29.5 (CH2), 29.1
(CH2), 28.9 (CH2), 25.9 (CH3); HRMS (ESI-) m/z [M-H]-,
calcd for C14H17ClNO2 266.0953, found 266.0941.
7.40 (d, J = 2.1 Hz, 1H), 3.90 (s, 3H), 3.68 (s, 3H), 2.70 (t, J =
7.7 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.92 (m, 2H). 13C NMR
(126 MHz, CDCl3) δ 158.2 (C), 139.7 (C), 132.4 (CH), 131.7
(CH), 128.9 (C), 117.7 (C), 108.5 (C), 61.1 (CH3), 31.8 (CH2),
30.2 (CH2), 30.0 (CH2), 29.4 (CH2), 29.3 (CH2), 29.1 (CH2),
22.6 (CH2), 14.0 (CH3). LRMS EI+ 267 m/z.
1
13
Methyl
5-(3-chloro-5-cyano-2-methoxy-
phenyl)pentanoate (16)
1
IR (neat, vmax), cm-1 2951, 2233 (CN), 1733, 1472, 1275. H
NMR (500 MHz, CDCl3) δ 7.53 (s, 1H), 7.38 (s, 1H), 3.89 (s,
3H), 3.67 (s, 3H), 2.67 (m, 2H), 2.35 (m, 2H), 1.65 (m, 4H).
13C NMR (126 MHz, CDCl3) δ 173.8 (C), 158.2 (C), 139.9 (C),
132.4 (C), 132.0 (C), 128.9 (C), 117.6 (C), 108.5 (C), 61.1
(CH3), 51.6 (CH3), 33.7 (CH2), 29.7 (CH2), 29.5 (CH2), 24.6
(CH2). LRMS EI+ 281 m/z.
3-Chloro-4-methoxy-2-methyl-5-pentyl-benzonitrile (36)
1H NMR - (500MHz, CDCl3) δ 7.35 (s, 1H), 3.85 (s, 3H),
2.60 (t, J = 7.8 Hz, 2H), 2.55 (s, 3H), 1.57 (t, J = 7.5 Hz, 2H),
1.41-1.18 (m, 4H), 0.89 (t, J = 6.7 Hz, 3H). 13C NMR - (126
MHz, CDCl3) δ 158.1 (C), 139.4 (C), 136.3 (C), 132.0 (CH),
129.6 (C), 117.8 (C), 109.3 (C), 60.9 (CH3), 31.5 (CH2), 29.9
(CH2), 29.7 (CH2), 22.4 (CH2), 18.8 (CH3), 13.9 (CH3).
Methyl 6-(3-chloro-5-cyano-2-methoxy-phenyl)hexanoate
(17)
3-Chloro-5-hexyl-4-methoxy-2-methyl-benzonitrile (37)
1H NMR - (500MHz, CDCl3) δ 7.35 (s, 1H), 3.85 (s, 3H),
2.60 (t, J = 7.6 Hz, 2H), 2.54 (s, 3H), 1.61-1.48 (m, 2H), 1.41-
1.17 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H). 13C NMR - (126 MHz,
CDCl3) δ 158.1 (C), 139.3 (C), 136.3 (C), 132.0 (CH), 129.6
(C), 117.7 (C), 109.3 (C), 60.9 (CH3), 31.6 (CH2), 30.1 (CH2),
29.7 (CH2), 29.1 (CH2) 22.5 (CH2), 18.8 (CH3), 14.0 (CH3).
1
IR (neat, vmax), cm-1 2948, 2232 (CN), 1733, 1472, 1275. H
NMR (500 MHz, CDCl3) δ 7.53 (s, 1H), 7.38 (s, 1H), 3.89 (s,
3H), 3.67 (s, 3H), 2.65 (m, 2H), 2.32 (m, 2H), 1.70-1.57 (m,
13
4H), 1.39 (m, 2H). C NMR (126 MHz, CDCl3) δ 174.0 (C),
158.2 (C), 139.2 (C), 132.4 (CH), 131.9 (CH), 128.9 (C), 117.7
(C), 108.5 (C), 61.1 (CH3), 51.5 (CH3), 33.8 (CH2), 29.82
3-Chloro-5-heptyl-4-methoxy-2-methyl-benzonitrile (38)
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1H NMR - (500MHz, CDCl3) δ 7.35 (s, 1H), 3.85 (s, 3H),
Chloro(trimethyl)silane (0.26 mL, 2.0 mmol) was added
1
2
3
4
5
6
7
8
2.60 (t, J = 7.9 Hz, 2H), 2.54 (s, 3H), 1.61-1.50 (m, 2H), 1.38-
1.11 (m, 8H), 0.87 (t, J = 6.7 Hz, 3H). C NMR - (126 MHz,
CDCl3) δ 158.1 (C), 139.3 (C), 136.3 (C), 132.0 (CH), 129.6
(C), 117.7 (C), 109.3 (C), 60.9 (CH3), 31.7 (CH2), 30.8 (CH2),
29.7 (CH2), 29.3 (CH2) 29.0 (CH2), 22.6 (CH2), 18.8 (CH3)
14.1 (CH3).
to a suspension of sodium iodide (0.31 g, 2.0 mmol) and
anisole (0.51 mmol) in acetonitrile (2 mL). The reaction
mixture was heated to reflux for 4-96 hours. The cooled
reaction mixture was partitioned between 0.1 M sodium
thiosulfate (10 mL) and ethyl acetate (2 x 20 mL). The
organic layers were separated, combined dried over
MgSO4, filtered and concentrated under vacuum. The
residue was purified by flash silica chromatography, elut-
ing with petroleum ether to 30% ethyl acetate to provide
the phenolic compounds (28-80%).
13
General protocol for alkylboronic acid Suzuki-
Miyaura cross-coupling.
9
Dicyclohexyl-[2-(2,6-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
diisopropoxyphenyl)phenyl]phosphane (9 mg, 0.02
mmol) and palladium(II) acetate (2 mg, 0.01 mmol) was
added to a degassed suspension of 5-bromo-3-chloro-4-
methoxy-2-methyl-benzonitrile (100 mg, 0.38 mmol), bo-
3-Chloro-4-hydroxy-2-methyl-5-octyl-benzonitrile (11)
IR (neat, vmax) cm-1 3360, 2928, 2850, 2222 (CN), 1601, 1473,
1461, 1163. 1H NMR (500 MHz, CDCl3) δ 7.31 (s, 1H), 6.08 (s,
1H), 2.64 (t J = 7.7 Hz, 2H), 2.56 (s, 3H), 1.59 (m, 2H), 1.45-
1.20 (m, 10H) 0.91 (t J = 7.2 Hz, 3H). 13C NMR (126MHz,
CDCl3) δ 153.0 (C), 138.2 (C), 132.2 (CH), 128.9 (C), 121.0
(C), 118.0 (C), 110.0 (C), 31.8 (CH2), 29.9 (CH2), 29.3 (CH2),
29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 22.6 (CH2), 18.7 (CH2),
14.0 (CH3). HRMS (ESI-) m/z [M-H]- calculated for
C16H21ClNO 278.1317, found 278.1303.
ronic
acid or trialkylboroxine (0.58 mmol) and tripo-
tassium phosphate (244 mg, 1.15 mmol) in toluene (2 mL).
The reaction mixture was sealed, degassed, put under a
nitrogen atmosphere and heated to 100°C for 16-72 h. The
reaction mixture was filtered through a pad of celite, the
filtrate was partitioned between ethylacetate and water,
the organics were separated, washed with brine, dried
over MgSO4, filtered and concentrated under vacuum.
The residue was purified by flash chromatography eluting
with petroleum ether to 10%-25% ethylacetate in petrole-
um ether. Material was further purified by reverse phase
flash chromatography, eluting with water to MeOH if
required to provide the cross-coupled analogues de-
scribed (36-67%).
4-(3-Chloro-5-cyano-2-hydroxy-phenyl)butanoic acid (19
a)
IR (neat, vmax), cm-1 3345, 2569, 2228 (CN), 1691, 1474, 1253.
1H NMR (500 MHz, CDCl3) δ 12.06 (br s, 1H), 10.43 (br s,
1H), 7.81 (d, J = 2.0 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 2.61 (t, J
= 7.5 Hz, 2H), 2.19 (t, J = 7.5 Hz, 2H), 1.73 (quintet, J = 7.5
13
Hz, 2H). C NMR (126 MHz, CDCl3) δ 174.6 (C), 155.6 (C),
3-Chloro-4-methoxy-2,5-dimethyl-benzonitrile (32)
133.1 (CH), 132.4 (C), 131.9 (CH), 121.6 (C), 118.7 (C), 102.8
(C), 33.6 (CH2), 29.5 (CH2), 24.5 (CH2). HRMS (ESI-) m/z
[M-H]- calcd for C11H9ClNO3 238.0276, found 238.0266.
1H NMR - (500MHz, CDCl3) δ 7.35 (s, 1H), 3.84 (s, 3H),
2.55 (s, 3H), 2.29 (s, 3H). 13C NMR - (126 MHz, CDCl3) δ
158.3 (C), 139.5 (C), 132.9 (CH), 131.5 (C), 129.6 (C), 117.6
(C), 109.2 (C), 60.2 (CH3), 18.8 (CH3), 16.0 (CH3).
5-(3-Chloro-5-cyano-2-hydroxy-phenyl)pentanoic acid (20
a)
IR (neat, vmax), cm-1 3093, 2867, 2243 (CN), 1723, 1471, 1105.
1H NMR (500 MHz, CDCl3) δ 11.98 (br s, 1H), 10.40 (br s,
1H), 7.78 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 2.59 (t,
J = 7.2 Hz, 2H), 2.20 (m, 2H), 1.49 (m, 4H). 13C NMR (126
MHz, CDCl3) δ 174.8 (C), 155.6 (C), 133.8 (C), 133.0 (C),
131.8 (C), 121.5 (C), 118.8 (C), 102.7 (C), 33.9 (CH2), 29.8
(CH2), 28.7 (CH2), 24.5 (CH2). HRMS (ESI-) m/z [M-H]-
calcd for C12H11ClNO3 252.0433, found 252.0423.
3-Chloro-5-ethyl-4-methoxy-2-methyl-benzonitrile (33)
1H NMR - (500MHz, CDCl3) δ 7.37 (s, 1H), 3.86 (s, 3H),
13
2.75-2.60 (m, 2H), 2.56 (s, 3H), 1.22 (t, J = 7.5 Hz, 3H). C
NMR - (126 MHz, CDCl3) δ 158.0 (C), 139.4 (C), 137.5 (C),
131.4 (CH), 129.6 (C), 117.8 (C), 109.5 (C), 60.9 (CH3), 22.3
(CH2), 18.8 (CH3), 14.4 (CH3).
3-Chloro-4-methoxy-2-methyl-5-propyl-benzonitrile (34)
1H NMR - (500MHz, CDCl3) δ 7.35 (s, 1H), 3.86 (s, 3H),
2.60 (t, J = 6.7 Hz, 2H), 2.55 (s, 3H), 1.66-1.56 (m, 2H), 0.96
(t, J = 7.4 Hz, 3H). 13C NMR - (126 MHz, CDCl3) δ 158.2 (C),
139.4 (C), 136.0 (C), 132.1 (CH), 129.7 (C), 117.8 (C), 109.3
(C), 60.9 (CH3), 31.7 (CH2), 23.3 (CH2), 18.9 (CH3), 13.9
(CH3).
6-(3-chloro-5-cyano-2-hydroxy-phenyl)-N-methyl-
hexanamide (21 b)
IR (neat, vmax), cm-1 3395, 2941, 2226 (CN), 1638, 1559,
1291, 1271. 1H NMR (500 MHz, D6-DMSO) δ 10.37 (br s, 1H,
OH-7), 7.76 (s, 1H, CH-6), 7.67 (br s, 1H, NH-17), 7.51 (s,
1H, CH-4), 2.56 (t, J = 7.14 Hz, 2H, CH2-11), 2.52 (m, 3H,
CH3-19), 2.01 (t, J = 7.2 Hz, CH2-15), 1.47 (m, 4H, CH2-
12/14), 1.22 (m, 2H, CH2-13). 13C NMR (126 MHz, D6-
DMSO) δ 172.9 (C16), 155.6 (C2), 133.2 (C3), 133.0 (CH-4),
131.7 (CH-6), 121.5 (C1), 118.8 (C9), 102.6 (C5), 35.6 (CH2-
15), 29.9 (CH2-11), 29.0 (CH2-12), 28.8 (CH2-13), 25.8
(CH3-19), 25.5 (CH2-14). HRMS-ESI m/z [M+H]+ calcd for
C14H17ClN2NaO2 303.0871, found 303.0865.
5-Butyl-3-chloro-4-methoxy-2-methyl-benzonitrile (35)
1H NMR - (500MHz, CDCl3) δ 7.35 (s, 1H), 3.86 (s, 3H),
2.62 (t, J = 7.7 Hz, 2H), 2.55 (s, 3H), 1.59-1.51 (m, 2H), 1.41-
1.31 (m, 2H), 0.93 (t, 3H). 13C NMR - (126 MHz, CDCl3) δ
158.1 (C), 139.4 (C), 136.3 (C), 132.1 (CH), 129.7 (C), 117.8
(C), 109.3 (C), 60.9 (CH3), 32.3 (CH2), 31.7 (CH2), 22.5
(CH2), 18.8 (CH3), 13.8 (CH3).
General protocol for demethylation with trimethylsilyl
iodide.
7-(3-Chloro-5-cyano-2-hydroxy-phenyl)-N-methyl-
heptanamide (22 b)
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1H NMR - (500MHz, CDCl3) δ 7.57 (d, J = 2.0 Hz, 1H), 7.40
(CH2), 22.6 (CH2), 18.8 (CH3), 14.1 (CH3). LRMS, (ESI-) [M-
H]- 249.9 m/z.
1
2
3
4
5
6
7
8
(d, J = 2.0 Hz, 1H), 2.69 (s, 3H), 2.66 (t, J = 7.4 Hz, 2H),
2.16 (t, J = 7.6 Hz, 2H), 1.70-1.50 (m, 4H), 1.44-1.22 (m, 4H).
13C NMR - (126 MHz, CDCl3) δ 172.4 (C), 155.2 (C),132.9
(C), 132.1 (CH), 130.8 (CH), 121.0 (C), 117.8 (C), 102.8 (C).
LRMS, (ESI+)[M+H]+ 295.1 m/z.
3-Chloro-5-heptyl-4-hydroxy-2-methyl-benzonitrile (45)
1H NMR - (500MHz, CDCl3) δ 7.30 (s, 1H), 6.11 (br s, 1H),
2.61 (t, J = 7.5, 2H), 2.54 (s, 3H), 1.61-1.53 (m, 2H), 1.35-1.22
(m, 8H), 0.88 (t, J = 6.2 Hz, 3H). 13C NMR - (126 MHz,
CDCl3) δ 153.1 (C), 138.2 (C), 132.2 (CH), 128.9 (C), 121.0 (C),
118.1 (C), 105.1 (C), 31.7 (CH2), 30.0 (CH2), 29.2 (CH2) 29.09
(CH2), 29.06 (CH2), 22.6 (CH2), 18.8 (CH3), 14.1 (CH3).
LRMS, (ESI-), [M-H]- 264.0 m/z.
7-(3-Chloro-5-cyano-2-hydroxy-phenyl)-N,N-dimethyl-
heptanamide (23 b)
1H NMR - (500MHz, CDCl3) δ 7.48 (s, 1H) 7.31 (s, 1H), 6.93
9
(br s, 1H), 3.00 (s, 3H), 2.96 (s, 3H), 2.69-2.59 (m, 2H),
13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.34-2.24 (m, 2H), 1.67-1.52 (m, 4H), 1.42-1.29 (m, 4H). C
5-Chloro-4-hydrox-5-octylbenzonitrile (46)
NMR - (126 MHz, CDCl3) δ 173.2 (C), 153.7 (C), 132.6 (CH),
132.0 (C), 130.5 (CH), 120.8 (C), 118.2 (C), 104.1 (C), 37.3
(CH3), 35.5 (CH3), 33.1 (CH2), 29.9 (CH2), 28.83 (CH2),
28.80 (CH2), 28.75 (CH2), 24.6 (CH2). LRMS, (ESI+)
[M+H]+ 309.1 m/z.
m.p. 60.1 – 61.8 °C. IR (neat, vmax) cm-1 3362, 2927, 2849,
2230 (CN), 1595, 1471, 1316, 1241, 1168. H NMR (500 MHz,
1
CDCl3) δ 7.49 (s, 1H), 7.34 (s, 1H), 6.07 (s, 1H), 2.66 (m,
2H), 1.59 (m, 2H), 1.49-1.17 (m, 10H), 0.88 (m, 3H). 13C
NMR (126 MHz, CDCl3) δ 153.4 (C), 132.9 (C), 132.2 (CH),
130.4 (CH), 120.5 (C), 118.2 (CH), 104.6 (C), 32.0 (CH2), 30.3
(CH2), 29.5 (CH2), 29.5 (CH2), 29.4 (CH2), 29.2 (CH2), 22.8
(CH2), 14.3 (CH3). HRMS (ESI-) m/z [M-H]- calcd for
C15H19ClNO 264.1161, found 264.1151.
3-Chloro-4-hydroxy-2,5-dimethyl-benzonitrile (39)
1H NMR - (500MHz, CDCl3) δ 7.29 (s, 1H), 6.21 (br s, 1H),
2.53 (s, 3H), 2.25 (s, 3H). 13C NMR - (126 MHz, CDCl3) δ
153.4 (C), 138.3 (C), 132.8 (CH), 124.2 (C), 120.8 (C), 118.0
(C), 104.9 (C), 18.7 (CH3), 15.9 (CH3). LRMS, (ESI-) [M-H]-
179.9 m/z.
General protocol for HATU amide coupling.
Triethylamine (0.07 mL, 0.49 mmol) was added to a solu-
tion of carboxylic acid (0.2 mmol), HATU (0.08 g, 0.22
mmol), and amine (0.3 mmol) in acetonitrile (0.5 mL).
The reaction was stirred at RT for 16 h. The reaction mix-
ture was partitioned between ethyl acetate (5 mL) and
water (5 mL). The organic layer was separated, washed
with brine, dried over MgSO4, filtered and concentrated
under reduced pressure. The residue was purified by flash
silica chromatography eluting with petroleum ether to
ethyl acetate in petroleum ether, then further purified by
reverse phase chromatography eluting with water to
MeOH if necessary to provide the amides described (29-
67%).
3-Chloro-5-ethyl-4-hydroxy-2-methyl-benzonitrile (40)
1H NMR - (500MHz, CDCl3) δ 7.32 (s, 1H), 6.14 (s, 1H), 2.66
13
(q, J = 7.6 Hz, 2H), 2.54 (s, 3H), 1.21 (t, J = 7.6 Hz, 3H). C
NMR - (126 MHz, CDCl3) δ 153.0 (C), 138.3 (C), 131.4 (CH),
130.1 (C), 121.0 (C), 118.1 (C), 105.2 (C), 23.1 (CH2), 18.8
(CH3), 13.4 (CH3). LRMS, (ESI-) [M-H]- 194.0 m/z.
3-Chloro-4-hydroxy-2-methyl-5-propyl-benzonitrile (41)
1H NMR - (500MHz, CDCl3) δ 7.29 (s, 1H), 6.21 (br s, 1H),
2.60 (t, J = 7.7 Hz, 2H), 2.53 (s, 3H), 1.62-1.56 (m, 2H), 0.94
(t, J = 7.3 Hz, 3H). 13C NMR - (126 MHz, CDCl3) δ 153.2 (C),
138.3 (C), 132.2 (CH), 128.7 (C), 121.1 (C), 118.1 (C), 105.0 (C),
31.9 (CH2), 22.2 (CH2), 18.8 (CH3), 13.7 (CH3). LRMS (ESI-)
[M-H]- 207.9 m/z
4-(3-Chloro-5-cyano-2-hydroxy-phenyl)-N-propyl-
butanamide (19 b)
5-Butyl-3-chloro-4-hydroxy-2-methyl-benzonitrile (42)
1H NMR - (500MHz, CDCl3) δ 10.73 (br s, 1H), 7.95 (br t,
1H), 7.73 (s, 1H), 7.45 (s, 1H), 2.97, (m, 2H), 2.56 (t, J = 7.4
Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.71 (m, 2H), 1.37 (m, 2H),
0.81 (t, J = 7.4 Hz, 3H). 13C NMR - (126 MHz, CDCl3) δ 172.0
(C), 155.4 (C), 132.8 (CH), 131.9 (C), 131.4 (CH), 121.2 (C),
118.3 (C), 102.12 (C), 40.3 (CH2), 34.3 (CH2), 29.1 (CH2), 25.0
(CH2), 22.3 (CH2), 11.4 (CH3). LRMS, (ESI-) [M-H]- 281.0
m/z.
1H NMR - (500MHz, CDCl3) δ 7.29 (s, 1H), 6.22 (br s, 1H),
2.61 (t, J = 7.8 Hz, 2H), 2.53 (s, 3H), 1.60-1.50 (m, 2H), 1.40-
1.29 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR - (126 MHz,
CDCl3) δ 153.2 (C), 138.2 (C), 132.1 (CH), 128.9 (C), 121.1 (C),
118.1 (C), 105.0 (C), 31.2 (CH2), 29.6 (CH2), 22.3 (CH2), 18.7
(CH3), 13.8 (CH3). LRMS (ESI-), [M-H]- 221.9 m/z.
3-Chloro-4-hydroxy-2-methyl-5-pentyl-benzonitrile (43)
1H NMR - (500MHz, CDCl3) δ 7.30 (s, 1H), 6.10 (br s, 1H),
2.62 (t, J = 7.8 Hz, 2H), 2.54 (s, 3H), 1.65-1.52 (m, 2H), 1.37-
1.27 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H). 13C NMR - (126 MHz,
CDCl3) δ 153.1 (C), 138.2 (C), 132.2 (CH), 128.9 (C), 121.0 (C),
118.1 (C), 105.1 (C), 31.4 (CH2), 29.9 (CH2), 28.8 (CH2), 22.4
(CH2), 18.8 (CH3), 14.0 (CH3). LRMS, (ESI-) [M-H]- 235.9
m/z.
5-(3-Chloro-5-cyano-2-hydroxy-phenyl)-N-ethyl-
pentanamide (20 b)
IR (neat, vmax), cm-1 3353, 2945, 2231 (CN), 1649, 1560, 1462,
1174. 1H NMR - (500MHz, D6-DMSO) δ 10.16 (br 1H), 7.76-
7.72 (br m, 2H), 7.49 (s, 1H), 3.02 (m, 2H), 2.58 (t, J = 6.3
Hz, 2H), 2.02 (t, J = 6.3 Hz, 2H), 1.45 (m, 4H), 0.96 (t, J =
7.2 Hz, 3H). 13C NMR - (126 MHz, D6-DMSO) δ 172.1 (C),
156.0 (C), 133.0 (C), 132.9 (C), 131.7 (C), 121.6 (C), 118.9 (C),
102.1 (C), 35.7 (CH2), 33.6 (CH2), 29.9 (CH2), 28.9 (CH2),
25.4 (CH2), 15.2 (CH3). HRMS (ESI+) m/z [M+Na]+ calcd
for C14H17ClN2NaO2 303.0871, found 303.0865.
3-Chloro-5-hexyl-4-hydroxy-2-methyl-benzonitrile (44)
1H NMR - (500MHz, CDCl3) δ 7.29 (s, 1H), 6.18 (br s, 1H),
2.61 (t, J = 7.8 Hz, 2H), 2.53 (s, 3H), 1.61-1.50 (m, 2H), 1.38-
1.23 (m, 6H), 0.92-0.84 (m, 3H). 13C NMR - (126 MHz,
CDCl3) δ 153.1 (C), 138.2 (C), 132.2 (CH), 128.9 (C), 121.0 (C),
118.1 (C), 105.0 (C), 31.6 (CH2), 30.0 (CH2), 29.0 (CH2), 28.9
Ethyl(2E)-3-5-cyano-2-hydroxy-4-methylphenyl)prop-2-
enoate (24)
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hours. The reaction mixture was partitioned between 1M
Potassium carbonate (0.98 g, 7.07 mmol) was added to a
1
2
3
4
5
6
7
8
solution of 5-bromo-4-hydroxy-2-methyl-benzonitrile
(0.50 g, 2.36 mmol) and ethyl acrylate (0.77 mL, 7.07 mL)
in 1-methyl-2-pyrrolidinone (10 mL). [1,3-bis(2,4,6-
trimethylphenyl)-2,3-dihydro-1H-imidazol-2-ylidene]({2-
[(dimethylamino)methyl]phenyl})palladiumylium chlo-
ride (SingaCycle®) was added to the suspension and the
reaction was sealed under nitrogen and heated at 100°C
for 48 h. The reaction mixture was cooled to RT and di-
luted with ethyl acetate (50 mL) and filtered through a
celite pad. The filtrate was washed with 1M HCl (aq) (25
mL), 0.5 M LiCl (aq) (25 mL), brine (25 mL), dried over
MgSO4 filtered and concentrated under vacuum. The res-
idue was purified by flash silica chromatography eluting
with petroleum ether to 15% ethyl acetate in petroleum
ether to provide the title compound as a white solid (0.16
g, 29%). m.p. 142.6-149.8 °C. IR (neat, vmax), cm-1 2923,
2221, 1600, 1274. 1H NMR (500 MHz, D6-DMSO) δ 11.30 (br
s, 1H) 8.07 (s, 1H), 7.73 (d, J = 16 Hz, 1H), 6.90 (s, 1H), 6.70
(d, J = 16 Hz, 1H), 4.17 (d, J = 7.2 Hz, 2H), 2.38 (s, 3H), 1.24
HCl (aq) (10 mL) and ethyl acetate (2 x 20 mL). The com-
bined organics were separated, dried over MgSO4, filtered
and concentrated under vacuum. The residue was puri-
fied by flash silica chromatography, eluting with petrole-
um ether to 25% ethyl acetate in petroleum ether to pro-
vide the title compound as a white solid (0.043 g, 75%). IR
1
(neat, vmax), cm-1 3340, 2997, 2228 (CN), 1712, 1210, 1177. H
NMR (500 MHz, CDCl3) δ 7.33 (s, 1H), 7.03 (s, 1H), 4.14 (q,
J = 7.1 Hz, 2H), 2.94 (t, J = 7.1 Hz, 2H), 2.65 (t, J = 7.1 Hz,
2H), 2.55 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 173.6 (C), 153.6 (C), 139.4 (C), 132.6 (CH),
126.7 (C), 117.9 (C), 105.3 (C), 61.1 (CH2), 33.7 (CH2), 25.3
(CH2), 18.9 (CH3), 14.2 (CH3). HRMS (ESI-) m/z [M-H]-
calcd for C13H13NO3 266.0589, found 266.0576.
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27
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General protocol for 1,5,7-triazabicyclo[4.4.0]dec-5-
ene amide formation.
Amine (0.33 mmol) was added to a solution of 1,5,7-
triazabicyclo[4.4.0]dec-5-ene (0.04 g, 0.26 mmol) and
ester (0.13 mmol) in acetonitrile (0.5 mL). The reaction
was stirred at RT for 5-40 h. The reaction mixture was
partitioned between ethyl acetate (25 mL) and water (25
mL). The organic layer was separated, washed with brine,
dried over MgSO4, filtered and concentrated under re-
duced pressure. The residue was purified by flash silica
chromatography eluting with petroleum ether to 30-50%
ethyl acetate in petroleum ether to provide the amides
described (27-80%).
13
(t, J = 7.2 Hz, 3H). C NMR (126 MHz, D6-DMSO) δ 160.9
(C), 160.6 (C), 145.4 (C), 138.2 (CH), 134.4 (CH), 120.3 (C),
119.4 (CH), 118.5 (C), 118.1 (CH), 103.19 (CN), 60.4 (CH2),
20.5 (CH3), 14.6 (CH3) HRMS (ESI-) m/z [M-H]- calcd for
C13H12NO3 230.0823, found 230.0814.
Ethyl 3-(5-cyano-2-hydroxy-4-methyl-phenyl)propanoate
(25)
Triethylsilane (0.50 g, 4.32 mmol) was added to a solution
ethyl (E)-3-(5-cyano-2-hydroxy-4-methyl-phenyl)prop-2-
enoate (0.10 g, 0.43 mmol) and 10% palladium on carbon
(4 mg, 0.04 mmol) in ethanol (5 mL). The reaction mix-
ture was stirred at RT for 16 h. Further triethylsilane (0.50
g, 4.32 mmol) was added and the reaction mixture was
stirred for a further 16 h. The nitrogen purged reaction
mixture was filtered through a pad of celite, the filtrate
was partitioned between 1M HCl (aq) (10 mL) and ethyl
acetate (2 x 20 mL). The combined organics were separat-
ed, dried over MgSO4, filtered and concentrated under
vacuum. The residue was purified by flash silica chroma-
tography, eluting with petroleum ether to 50% ethyl ace-
tate in petroleum ether to provide the title compound as a
white solid (0.08 g, 77%). IR (neat, vmax), cm-1 3496, 2927,
6-(3-Chloro-5-cyano-2-methoxy-phenyl)-N-methyl-
hexanamide (21 a)
IR (neat, vmax), cm-1 3300, 2938, 2230 (CN), 1639, 1557, 1281.
1H NMR - (500MHz, CDCl3) δ 7.52 (d, J = 2.0 Hz, 1H), 7.37
(d, J = 2.0 Hz, 1H), 5.44 (br s, 1H), 3.88 (s, 3H), 2.80 (d, J =
5.0 Hz, 3H), 2.65 (t, J = 7.6 Hz, 2H), 2.17 (t, J = 7.6 Hz, 2H),
1.68 (m, 2H), 1.59 (m, 2H), 1.37 (m, 2H). 13C NMR - (126
MHz, CDCl3) δ 173.5 (C), 158.4 (C), 139.4 (C), 132.6 (CH),
132.0 (CH), 129.1 (C), 117.8 (C), 108.6 (C), 61.3 (CH3), 36.6
(CH2), 30.1 (CH2), 30.0 (CH2), 29.1 (CH2), 26.4 (CH2), 25.5
(CH3). HRMS (ESI+) m/z [M+Na]+ calcd for
C15H19ClN2NaO2 317.1027, found 317.1019.
7-(3-Chloro-5-cyano-2-methoxy-phenyl)-N-methyl-
heptanamide (22 a)
1
2225 (CN), 1716, 1199, 1250. H NMR (500 MHz, CDCl3) δ
1H NMR - (500MHz, CDCl3) δ 7.52 (d, J = 2.0 Hz, 1H), 7.37
(d, J = 2.0 Hz, 1H), 5.41 (br s, 1H), 3.88 (s, 3H), 2.80 (d, J =
5.0 Hz, 3H), 2.64 (t, J = 7.9 Hz, 2H), 2.16 (t, J = 7.6 Hz, 2H),
1.69-1.52 (m, 4H), 1.42-1.28 (m, 4H). 13C NMR - (126 MHz,
CDCl3) δ 173.5 (C), 158.2 (C), 139.4 (C), 132.4 (C), 131.8 (C),
128.9 (C), 117.7 (C), 108.4 (C), 61.1 (CH3), 36.6 (CH2), 30.0
(CH2), 29.9 (CH2), 29.1 (CH2), 29.0 (CH2), 26.3 (CH3), 25.5
(CH2). LRMS, (ESI+) [M+H]+ 309.1 m/z.
8.25 (s, 1H), 7.32 (s, 1H), 6.80 (s, 1H), 4.16 (q, J = 7.3 Hz,
2H), 2.84 (m, 2H), 2.70 (m, 2H), 2.44 (s, 3H), 1.25 (t, J = 7.3
Hz). 13C NMR (126 MHz, CDCl3) δ 175.9 (C), 158.4 (C),
142.5 (C), 135.1 (CH), 125.8 (C), 119.0 (CH), 118.6 (C), 104.3
(C), 61.8 (CH2), 35.0 (CH2), 24.0 (CH2), 20.1 (CH3), 14.0
(CH3). HRMS (ESI+) m/z [M+H]+ calcd for C13H15NNaO3
256.0944, found 256.0941.
Ethyl
3-(3-chloro-5-cyano-2-hydroxy-4-methyl-
phenyl)propanoate (26)
7-(3-Chloro-5-cyano-2-methoxy-phenyl)-N,N-dimethyl-
heptanamide (23 a)
Sulfuryl chloride (0.02 mL, 0.26 mmol) was added to a
1H NMR - (500MHz, CDCl3) δ 7.52 (s, 1), 7.37 (s, 1H), 3.88
(s, 3H), 2.99 (s, 3H), 2.94 (s, 3H), 2.64 (m, 2H), 2.30 (m,
2H), 1.70-1.53 (m, 4H), 1.43-1.32 (m, 4H). 13C NMR - (126
MHz, CDCl3) δ 173.0 (C), 158.2 (C), 139.5 (C), 132.4 (C),
131.8 (C), 128.9 (C), 117.7 (C), 108.4 (C), 61.1 (CH3, 37.3
solution
ethyl
3-(5-cyano-2-hydroxy-4-methyl-
phenyl)propanoate (0.05 g, 0.21 mmol) in diethyl ether (1
mL). The reaction mixture was stirred at RT for 96 h. Fur-
ther sulfuryl chloride (0.02 mL, 0.26 mmol) was added
and the reaction mixture was stirred for a further 16
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Bruce, D. Preliminary report on the tsetse fly disease or
nagana in Zululand. Bennet & Davis. Durban 1895, 28.
(CH3), 35.4 (CH3), 33.2 (CH2), 30.1 (CH2), 29.9 (CH2), 29.2
(CH2), 29.2 (CH2), 25.0 (CH2). LRMS, (ESI+) [M+H]+ 323.1
m/z.
1
2
3
4
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7
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Nagle, A. S.; Khare, S.; Kumar, A. B.; Supek, F.; Buchynskyy,
A.; Mathison, C. J. N.; Chennamaneni, N. K.; Pendem, N.;
Buckner, F. S.; Gelb, M. H.; et al. Recent Developments in
Drug Discovery for Leishmaniasis and Human African
Trypanosomiasis. Chem. Rev. 2014 DOI: 10.1021/cr500365f.
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Trypanosoma brucei. Journal of cell science. January 2005,
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10.1146/annurev.pa.35.040195.000521.
N-Butyl-3-(3-chloro-5-cyano-2-hydroxy-4-methyl-
phenyl)propenamide (27)
IR (neat, vmax), cm-1 3369, 2928, 2220 (CN), 1632, 1562, 1157.
1H NMR (500MHz, CDCl3) δ 10.35 (br s, 1H), 7.24 (s, 1H),
5.55 (br s, 1H), 3.26 (m, 2H), 2.91 (m, 2H), 2.59 (m, 2H),
2.54 (s, 3H), 1.45 (m, 2H), 1.28 (m, 2H), 0.90 (t, J = 7.5 Hz,
3H). 13C NMR - (126 MHz, CDCl3) δ 173.6 (C), 155.6 (C),
140.0 (C), 132.6 (CH), 127.4 (C), 123.9 (C), 118.3 (C), 104.0
(C), 39.9 (CH2), 36.3 (CH2), 31.4 (CH2), 25.0 (CH2), 19.9
(CH2), 18.9 (CH3), 13.6 (CH3). HRMS (ESI+) m/z [M+Na]+
calcd for C15H19ClN2NaO2 317.1027, found 317.1019.
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AUTHOR INFORMATION
Corresponding Author
Trop.
Dis.
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DOI:
10.1371/journal.pntd.0000333.
* Simon E. Ward, Sêr Cymru Professor in Translational Drug
Discovery & Director of Medicines Discovery Institute, Car-
diff University, Park Place, Cardiff, CF10 3AT, UK
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The trypanosome alternative oxidase: a potential drug
target? Parasitology 2016, First View, 1–9 DOI:
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Aquaglyceroporin-null trypanosomes display glycerol
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Kido, Y.; Shiba, T.; Inaoka, D. K.; Sakamoto, K.; Nara, T.;
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Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding Sources
This work was supported by the Biotechnology and Biologi-
cal Sciences Research Council [Grant Ref: BB/L017180/1]
iCASE with Novartis Institute for Tropical Diseases.
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Acknowledgment
We would like to acknowledge and thank Rima Palkar and
Nahdiyah Ghafar for carrying out the T. b. brucei growth
inhibition assays.
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ABBREVIATIONS
AAT, African animal trypanosomiasis; AF, ascofuranone;
CCB, colletochlorin B; CNS, central nervous system; HAT,
human African trypanosomiasis; LipE, lipophilic efficiency;
MPO, multi-parameter-optimisation; NECT, Nifurtimox and
Eflornithine combination therapy; TAO, trypanosome alter-
native oxidase; T. b. b., trypanosoma brucei brucei; TBD, 1,5,7-
triazabicyclo[4.4.0]dec-5-ene
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Figure 1. Summary of previous work.22
152x41mm (96 x 96 DPI)
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Figure 2. Per atom contribution to lipophilicity of ascofuranone (AF) (1) and colletochlorin B (CCB) (2)
137x214mm (96 x 96 DPI)
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Figure 3. Isoxazole Kemp elimination
87x36mm (300 x 300 DPI)
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Figure 4. Correlations of cLogP vs TAO inhibition pIC50 (left) and cLogP vs T. b. b. pIC50 growth inhibition
(right). LipE is lipophilic ligand efficiency (pIC50 – cLogP)32.
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†: Negative log concentration and standard deviation of compounds required for 50% inhibition of
trypanosome alternative oxidase; *: Negative log concentration and standard deviation of compounds
required for 50% growth inhibition of T. b. brucei Lister427; #: Negative log concentration and standard
deviation of compounds required for 50% cytotoxicity of HepG2 cell line. (n ≥ 2 in all assays).
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cLogP calculated from ChemAxon Marvin Sketch, calculated MPO as described by Wager et al.26
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†: Negative log concentration and standard deviation of compounds required for 50% inhibition of
trypanosome alternative oxidase; *: Negative log concentration and standard deviation of compounds
required for 50% growth inhibition of T. b. brucei Lister427. (n ≥ 2 in all assays performed).
281x217mm (96 x 96 DPI)
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†: Negative log concentration and standard deviation of compounds required for 50% inhibition of
trypanosome alternative oxidase; *: Negative log concentration and standard deviation of compounds
required for 50% growth inhibition of T. b. brucei Lister427. (n ≥ 2 in all assays performed).
322x149mm (96 x 96 DPI)
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Reaction conditions: a) trimethylphenylammonium tribro-mide, CH2Cl2 / MeOH rt, 3 h, 77%, b) NH2OH·HCl,
EtOH, reflux, 16 h, 90%, c) NCS, Et3N, MeCN, 0°C – rt, 16 h, 70%, d) dimethyl sulfate, acetone, rt, 16 h,
98%, e) i) BH3·THF, 1-octene, 0°C, 20 min, ii) toluene, H2O, K3PO4, RuPhos, Pd(OAc)2, 50°C, 16 h, 90% f)
TMS-Cl, NaI, MeCN, 80°C, 16 h, 65%.
119x68mm (300 x 300 DPI)
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Reaction conditions: a) NBS, MeCN, rt, 16 h, 85%, b) dime-thyl sulfate, acetone, reflux, 3 h, 90%, c) i)
BH3·THF, 1-alkene ester, 0 °C, 20 min, ii) toluene, H2O, K3PO4, RuPhos, Pd(OAc)2, 16 h, 50°C, 90%, d)
TMSCl, NaI, MeCN, 80 °C, 16 h, 35-85%. e) HATU, Et3N, R1’R2’NH, MeCN, rt, 3 h, 60-90%, f) TBD,
R1’R2’NH, MeCN, rt, 1 – 16 h 70-85%.
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Reaction conditions: a) ethyl acrylate, SingaCycle™, K2CO3, NMP, 100 °C, 16 h, 30%, b) triethylsilane,
Pd/C, EtOH, rt, 16 h, 78%, c) SO2Cl2, Et2O, rt, 96 h, 57%, d) TBD, n-butylamine, MeCN, 16h, 39%.
132x37mm (300 x 300 DPI)
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Reaction conditions: a) Ac2O, pyridine, DCM, rt, 16 h, 74%, b) i) BH3·THF, 1-alkene ether, 0°C, 20 min, ii)
toluene, H2O, K3PO4, RuPhos, Pd(OAc)2, 16 h, 50 °C, 7-24%.
124x46mm (300 x 300 DPI)
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Reaction conditions: a) i) BH3·THF, 1-alkene, 0 °C, 20 min, ii) toluene, H2O, K3PO4, RuPhos, Pd(OAc)2, 16
h, 50 °C, 65-90%, or 1-boronic acid/boroxine, K3PO4, RuPhos, Pd(OAc)2, 16 h, 80 °C b) TMSCl, NaI, MeCN,
80 °C, 16 h, 35-85%.
126x44mm (300 x 300 DPI)
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