Development of small-molecule Trypanosoma brucei N-myristoyltransferase inhibitors: Discovery and optimisation of a novel binding mode

Article abstract of DOI:10.1002/cmdc.201500301

The enzyme N-myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high-throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X-ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X-ray crystallography/structure-based design approach, the benzomorpholinone series was further optimised, increasing activity against T. brucei NMT by >1000-fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T. brucei. HATs off to diversity! Screening a diverse library against Trypanosoma brucei N-myristoyltransferase (NMT) identified hits based on thiazolidinone and benzomorpholine scaffolds. X-ray crystallography of these compounds bound to Leishmania major NMT revealed novel active site binding conformations. Using the structural information, the benzomorpholine scaffold was optimised to a blood-brain barrier penetrant compound with activity against TbNMT of <0.002 μm.

Full text of DOI:10.1002/cmdc.201500301

DOI: 10.1002/cmdc.201500301
Full Papers
Very Important Paper
Development of Small-Molecule Trypanosoma brucei
N-Myristoyltransferase Inhibitors: Discovery and
Optimisation of a Novel Binding Mode
Daniel Spinks, Victoria Smith, Stephen Thompson, David A. Robinson, Torsten Luksch,
Alasdair Smith, Leah S. Torrie, Stuart McElroy, Laste Stojanovski, Suzanne Norval,
Iain T. Collie, Irene Hallyburton, Bhavya Rao, Stephen Brand, Ruth Brenk, Julie A. Frearson,
Kevin D. Read, Paul G. Wyatt, and Ian H. Gilbert*[a]
The enzyme N-myristoyltransferase (NMT) from Trypanosoma
brucei has been validated both chemically and biologically as
a potential drug target for human African trypanosomiasis. We
previously reported the development of some very potent
compounds based around a pyrazole sulfonamide series, de-
rived from a high-throughput screen. Herein we describe work
around thiazolidinone and benzomorpholine scaffolds that
were also identified in the screen. An X-ray crystal structure of
the thiazolidinone hit in Leishmania major NMT showed the
compound bound in the previously reported active site, utilis-
ing a novel binding mode. This provides potential for further
optimisation. The benzomorpholinone was also found to bind
in a similar region. Using an X-ray crystallography/structure-
based design approach, the benzomorpholinone series was
further optimised, increasing activity against T. brucei NMT by
>1000-fold. A series of trypanocidal compounds were identi-
fied with suitable in vitro DMPK properties, including CNS ex-
posure for further development. Further work is required to in-
crease selectivity over the human NMT isoform and activity
against T. brucei.
Introduction
Human African trypanosomiasis (HAT), or African sleeping sick-
ness, is endemic in sub-Saharan Africa, claiming the lives of
about 10000 people every year.[1] The disease burden in this
area is substantial, with approximately 60 million people at risk
of infection. HAT is caused by two subspecies of the protozoan
parasite Trypanosoma brucei gambiense and T. brucei rhoden-
siense, which are transmitted to the human host by the bite of
an infected tsetse fly. If left untreated the disease is often fatal.
There is a clinical need for more effective drug therapies, be-
cause current treatments are unsatisfactory due to toxicity,
treatment failures, and inappropriate dosing regimens for
a rural African setting.[1–4]
Figure 1. DDD85646, the previously published T. brucei NMT inhibitor.
The enzyme N-myristoyltransferase (NMT) is one of only
a few genetically and chemically validated drug targets in ki-
netoplastids, with the NMT inhibitor DDD85646 (Figure 1)
being shown to act on target and to cure the mouse model of
stage-1 (non-CNS) T. brucei infection.[5–8] Functionally the NMT
enzyme is ubiquitous and is responsible for catalysing the co-
translational transfer of myristate from myristoyl-CoA to the N-
terminal glycine residue of the target protein. Herein we de-
scribe the discovery and optimisation of novel T. brucei NMT in-
hibitor scaffolds identified by high-throughput screening, with
appropriate physicochemical properties for oral bioavailability
and CNS penetration.
[a] D. Spinks, V. Smith, Dr. S. Thompson, Dr. D. A. Robinson, Dr. T. Luksch,
A. Smith, Dr. L. S. Torrie, Dr. S. McElroy, L. Stojanovski, S. Norval, I. T. Collie,
I. Hallyburton, B. Rao, Dr. S. Brand, Dr. R. Brenk, Prof. J. A. Frearson,
Dr. K. D. Read, Prof. P. G. Wyatt, Prof. I. H. Gilbert
Drug Discovery Unit, College of Life Sciences
University of Dundee, Sir James Black Centre, Dundee, DD1 5EH (UK)
E-mail: i.h.gilbert@dundee.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500301.
Results and Discussion
Hit-to-lead chemistry
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This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
In our initial programme to discover inhibitors of TbNMT,
a high-throughput screen of our diversity set was carried out.
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In addition to the pyrazole sulfonamide series previously re-
ported,[8–10] several other hits were identified. These were also
investigated to determine if they offer opportunities to devel-
op selective and blood–brain barrier penetrant TbNMT inhibi-
tors. Two hits around a thiazolidinone and benzomorpholinone
were identified and validated as hits (Figure 2).
Scheme 1. Thiazolidinone synthesis. Reagents and conditions: a) 1. aldehyde
(2 equiv), amine (1 equiv), THF, 08C, 5 min; 2. thioacetic acid (3 equiv), 08C,
10 min; b) polymer-supported carbodiimide (1.3 equiv), 208C, 16 h, 33–74%.
In parallel to a re-evaluation of the original diversity screen
data, a virtual screening exercise was carried out, the basis of
which was known interactions between the pyrazole sulfona-
mide DDD85646 and the enzyme (PDB code: 2WSA; Figure 3).
Screening our in-house database of commercially available
compounds using this pharmacophore led to the discovery of
three different compound classes (data not shown), including
examples containing the thiazoldinone scaffold (Figure 3B).
1–13 (Table 1) in yields of 33–74%. Work was initially carried
out without X-ray co-crystal structures; when this structural in-
formation was established using the Leishmania major enzyme,
key early-stage molecules were also co-crystallised with the
enzyme. As we discussed in a previous publication,[10] the
L. major NMT shows high sequence homology to both the
T. brucei and human NMTs. This has been an excellent system
to improve the activity of inhibitors, but given the similarities
and lack of high-resolution structures of the T. brucei enzyme,
it has much less use in predicting selectivities.
The benzyl ring at R2 could be replaced with a furanyl
methyl group (compound 2). However, replacement with
a nonaromatic group (R2 =ethyl, 3) led to a complete loss of
activity. Similarly, a directly attached pyridine moiety
also led to a loss of activity. This loss is probably due
to a clash of the pyridine moiety with the protein
and suboptimal filling of the hydrophobic pocket
with the methyl group. A protein–ligand structure of
6 bound to L. major NMT (LmNMT) later confirmed
this methylene-aromatic group occupies a hydropho-
bic pocket in the active site (see binding mode T1,
Figure 4A,B).
Thiazolidinone series
The initial hit expansion focused on optimising the substitu-
ents around the thiazolidinone ring. The synthetic route is
shown in Scheme 1 and consisted of condensation of the alde-
hyde with an amine and thioacetic acid to give analogues
We were keen to replace the 2-methoxyphenol
group, due to the potential for de-methylation and
oxidation to a quinone. Removal of the phenol group
from 2, as in compound 5, resulted in loss of poten-
cy, indicating the importance of this group for bind-
ing. The phenol hydrogen bond donor of com-
pounds 1 and 2 could be replaced by the indazole
isostere, as in compound 6, without loss of potency.
The complex of 6 bound to LmNMT revealed binding
mode T1 of the thiazolidinone series, (Figure 4). The
thiazolidinone ligand occupies a similar area of the
peptide binding pocket of NMT as the pyrazole sulfo-
namide ligands[8] (Figure 4B). In binding mode T1,
the thiazolidinone core of 6 packs against the side
chain of Phe90, and the carbonyl group forms a key
hydrogen bonding interaction with the side chain of
Ser330. The benzyl moiety of 6 forms p-stacking in-
teractions with the side chains of Phe88 and Phe232
(parallel and edge–face, respectively), with the
pocket enclosed by the side chains of Leu341 and
Val374. The indazole N2 lone pair forms a hydrogen
Figure 2. Thiazolidinone and benzomorpholinone hits.
bond to the backbone amide of Asp396, a key inter-
action identified in the pyrazole sulfonamide series.
Despite the ligand being prepared without chiral res-
olution, only the R enantiomer was observed bound
in the crystal structure. Compounds 1 and 2 were as-
sumed to have a similar binding mode.
Figure 3. A) Structure-based pharmacophore based on the crystal structure of DDD85646
bound to LmNMT. The key pharmacophoric interactions used were: 1) a hydrogen bond
from the pyrazole N atom to Ser330, shown in blue; 2) a p-stacking interaction between
the pyrazole and Phe90, shown in green; 3) a p-stacking interaction between the pyri-
dine and Tyr217, shown in green; 4) a hydrogen bond between the sulfonamide O atom
and His219 and Asp396. Excluded volumes are shown in purple. B) Virtual screening hit
compound 9.
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Table 1. Initial SAR data from thiazolidinone hit expansion.
Compd
R1
R2
IC50 [mm][a]
TbNMT
EC50 [mm][a]
T. brucei
LE[b]
TbNMT
Assumed
binding mode[c]
hNMT
1
22
28
>50
>50
ND
0.29
T1
2
3
7.6
>100
>100
0.33
T1
–
>100
–
4
5
>100
>100
>100
>100
ND
ND
–
–
–
–
6
20
>100
>50
0.29
T1 (5AG5)
7
8
13
>50
ND
ND
0.36
–
T2 (5AG4)
>100
>100
T2
9
31
>100
32
66
>100
>100
>100
ND
>50
>50
>50
0.35
–
T2
–
10
11
12
0.32
0.39
T2
T1
5.9
13
0.27
>100
14
0.42
T1 (5AG6)
[a] Values shown are the mean of two or more determinations; ND=not determined. [b] Ligand efficiency (LE), determined for compounds with T. brucei
NMT potency <50 mm, was calculated as 0.6ln(IC50)/(heavy atom count).[11] [c] The assumed binding mode of each analogue is classified into either of the
two specific binding modes identified by X-ray crystallography (see Figure 4); this assumption was supported by the observed SAR data and by modelling
these analogues in PyMOL.
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Figure 4. Binding mode of thiazolidinone-based ligands to LmNMT. A) Compound 6 (C atoms gold) binding to LmNMT (C atoms grey), adopting binding
mode T1. Hydrogen bonds are shown as dashed lines, and key residues are labelled. B) Overlay of the binding mode of 6 with a pyrazole sulfonamide ligand
(C atoms cyan, PDB code: 4A30).[9] C) Compound 7 (C atoms gold) binding to LmNMT (C atoms grey), adopting binding mode T2. The image was prepared
with PyMOL.
Simultaneous replacement of the R1 3-phenol-4-methoxy
groups of 1 with a 2-pyridyl unit, and truncation of the R2
benzyl group to a directly linked phenyl, resulted in compound
7 and an unexpected inversion of the binding mode from that
observed for 6, giving rise to binding mode T2 (Figure 4C).
Compounds that adopted binding mode T2 show the R1 2-pyr-
idyl subunit forming a hydrogen bonding interaction with the
side chain of Ser330, and the thiazolidinone carbonyl group
forming a hydrogen bonding interaction with the side chain of
Asn376. The X-ray crystal structure also revealed the R2 sub-
stituent to be located in the hydrophobic peptide binding
groove, lying in a similar plane to the aryl group in the pyra-
zole sulfonamide series (Figure 4). In binding mode T2, and in
contrast to 6, the S enantiomer was bound in the active site. It
is unclear why compound 7 displayed selectivity for TbNMT
over hNMT (Table 1). However, this observed selectivity was
lost upon removal of the (R2-phenyl)-3-chloro substituent, seen
with 9, although this is partly due to the overall diminished
potency observed for this compound. Substitution at the ortho
(11) or meta (12) positions of the R2 phenyl group appeared to
be preferred over para substitution (10) which may be due to
a clash of this substituent with the side chain of Tyr217.
teraction and a significant improvement in potency (20-fold,
IC50: 0.27 mm) and a slight improvement in ligand efficiency to
0.42 (Figure 5 and Table 1).
The thiazolidinone series, in particular compound 13, pres-
ents a good starting point for further optimisation. It should
be possible to gain additional affinity by accessing an addition-
al local binding interaction or filling a local hydrophobic
pocket by using a structure-based design approach. Replace-
ment of the phenol subunit with a bioisostere has proved ach-
ievable; this substituent could be further optimised.[12] Most
compounds were not sufficiently potent against the enzyme to
give significant activity in the parasite assays. Compound 13,
the most potent compound, showed a 50-fold decrease in ac-
tivity in going from enzyme to cell. However, further work is
required to derive compounds with greater enzyme and para-
site activity.
During our exploration of the structure–activity relationship
(SAR) around the thiazolidinone scaffold, the 2-pyridylmethy-
lene subunit of 12 was identified as the most ligand efficient
R2 substituent (LE=0.39; Table 1). Modelling of 12 into the
binding sites of 6 and 7 could explain the efficiency of binding.
Assuming 12 adopted binding mode T1, there was no clear
ligand–protein interaction with the His219 residue; however,
we postulated a hydrogen bonding interaction between the
ligand and residue Asn376, with the R2 2-pyridyl nitrogen atom
as the hydrogen bond acceptor. Compound 13 was synthes-
ised; it is a hybrid of compounds 12 and 1, with the addition
of 4-hydro-3-methoxy to create an additional hydrogen bond
with His219, seeking to afford a significant improvement in po-
tency. X-ray crystallography confirmed 13 as achieving this in-
Figure 5. Binding of 13 to LmNMT. Hydrogen bonds are shown as dashed
lines, and key residues are labelled.
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Figure 6. Binding mode of benzomorpholinone ligands. A) Binding mode of 14 (C atoms gold) to LmNMT. B) Similarity in core binding of the benzomorpholi-
none of 14 (C atoms gold) and the thiazolidinone of 13 (C atoms blue). C) Binding mode of 14 (C atoms gold) compared with the pyrazole sulfonamide
ligand DDD85646 (C atoms cyan, PDB code: 2WSA).[8] The image was prepared using PyMOL.
Benzomorpholinone series
TbNMT IC50 potencies around the 10 mm level. The labile ester
The hit compound 14 (Figure 2) has a low molecular weight
(Mr =235 Da), good ligand efficiency (LE=0.42), and provided
an excellent starting point for further optimisation (Table 2 and
Figure 6). The initial optimisation focus for the benzomorpholi-
none series was to identify replacements for the potentially hy-
drolysable ester functional group of 14.
functional group was replaced by a pyrrolidine amide (com-
pound 17) without loss of potency, although the dimethylami-
no amide (compound 18) showed a fivefold decrease in poten-
cy. It was also found that C1-linked amides 19, 20, and 21 lost
all potency. Although the reason for these significant potency
losses is unclear, the decrease in lipophilicity for these particu-
lar compounds may be partly responsible for the lower poten-
cies observed.
C1-linked heterocyclic isosteres for the ester compound 14
were tolerated, although there was a requirement for a heter-
oatom in the a position, presumably to act as a hydrogen
bond acceptor (HBA) with the side chain of Asn376. The vector
and relative hydrogen bond potential of the lone pair on the
HBA atom, coupled with the orientation of the alkyl substitu-
ent appeared critical drivers for potency. The heterocyclic iso-
stere with the highest potency was the methyl-isoxazole 23
(TbNMT IC50: 2.9 mm). This compound displayed a fourfold im-
provement in TbNMT potency over 14. The thiazole (24), pyra-
zole (32), and oxadiazole (27) heterocyclic analogues lost sig-
nificant potency relative to compound 23. In the case of 24
and 32, we hypothesised from our binding models that the
methyl group is suboptimally positioned, and for oxadiazole
27 the oxygen is a relatively weak HBA moiety. This explana-
tion was further supported by the recovery in potency ob-
served for the isomeric oxadiazole 28 (TbNMT IC50: 7.0 mm). In
this oxadiazole isomer 28, the nitrogen atom presumably acts
as the HBA motif, and is predicted to have a similar HBA po-
tential as the isoxazole nitrogen atom in 23.[13,14] There was no
potency gain observed upon adding further lipophilic bulk to
the methyl group in the b position of the heterocycle, for ex-
ample homologation to isobutyl analogue in 29 or 30. Further-
more, for both compounds this substitution decreased the LE
significantly.
Synthetic route to the benzomorpholinone series
Chloroacetylchloride was reacted with the appropriately substi-
tuted 2-aminophenol to give the NH benzomorpholinone in-
termediate (Scheme 2). This was then alkylated with potassium
carbonate as the base, with heating in DMF. Workup and purifi-
cation yielded compounds 14–38.
Compound 14 was successfully co-crystallised with LmNMT.
The complex shows the ligand bound in a an orientation simi-
lar to that of the thiazolidinone ligand 13, creating a very simi-
lar interaction pattern (Figure 6). The benzomorpholinone
packs against the side chain of Phe90 with the carbonyl form-
ing a hydrogen bond to the side chain of Ser330 (Figure 6A).
The carbonyl group of the ester moiety forms a hydrogen
bond interaction with the side chain of Asn376, occupying
a similar position to the pyridyl group of compound 13 (Fig-
ure 6B).
On the fragment level, a one- (C1, 14) or two- (C2, 16)
carbon linker to the ester was equally well tolerated, with
Scheme 2. Benzomorpholinone synthesis. Reagents and conditions: a) 2-
chloroacetyl chloride (1 equiv), 2-aminophenol (1 equiv), K2CO3 (1 equiv),
DMF, 808C, 3 h; b) K2CO3 (1.4 equiv), DMF, 708C, 18 h; overall yield: 30–70%
(two steps).
The 2-pyridyl analogue 25 retained the potency of the ester
14, but was found to be nearly 10-fold less potent than 23.
This was not predicted by the HBA potential. The reason for
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ther potency (relative to 23) was lost when the
methyl group was added at a suboptimal position (a
to the heteroatom) as in compound 31, and all po-
tency was lost with the 4-pyridyl isomer 26, presuma-
bly due to loss of the hydrogen bond with Asn376.
Table 2. Initial SAR data from benzomorpholinone hit expansion.
Compd
R
IC50 [mm][a]
TbNMT
LE[b]
TbNMT
hNMT
Benzomorpholinone hit-to-lead strategy
14
15
16
CH2COOEt
CH(CH3)COOEt
CH2CH2COOEt
12
>100
13
>100
>100
>100
0.42
–
0.38
Compound 23 showed similar potency and LE as
those of the initial hit 14, and lacks the metabolically
and chemically labile ester functional group. This
scaffold was developed further in a hit-to-lead cam-
paign to investigate other substitution vectors using
a rational structure-based design approach based on
X-ray crystallography data. This approach used the
binding mode overlay of the pyrazole sulfonamide
inhibitor DDD85646 with 14 (Figure 6). From our pre-
vious work on the pyrazole sulfonamide series, ap-
pending a basic moiety to reach the C-terminal car-
boxylic acid gave an approximate 1000-fold increase
in potency.[8–10] Using this hypothesis, cross-over com-
pounds were designed to target nanomolar efficacy
in the parasite growth assay, good pharmacokinetic
parameters, and selectivity for parasite efficacy (rela-
tive to hNMT enzyme inhibition) to identify com-
pounds suitable for evaluation in an animal model of
HAT. The selectivity ratio of T. brucei cell versus hNMT
enzyme potency was used as a prediction for the in
vivo therapeutic window in the pyrazole sulfonamide
series, and formed the basis of ranking and selecting
a back-up series for lead optimisation (Table 3).[10]
17
11
>100
0.34
18
19
20
CH2CH2CON(CH3)2
CH2CON(CH3)2
CH2CONHCH3
50
>100
>100
>100
>100
>100
–
–
–
21
>100
ND
–
22
23
24
>100
>100
0.31
0.43
2.9
24
25
26
27
44
22
>100
>100
>100
>100
0.33
0.38
–
>100
>100
–
Hit-to-lead discussion
Investigation of simple halogen and methyl group
substitution around the core benzomorpholinone
scaffold indicated substitution with a halogen (Cl or
Br), as in 36 and 37, is tolerated at the 7-position
(Table 3). The 8-bromo analogue 38 showed similar
potency to 7-bromo compound 37. This implies that
the 7- or 8-positions are suitable vectors for exten-
sion of 23 toward the C terminus, in agreement with
the X-ray crystal structure (Figure 6).
28
29
7.0
>100
>100
0.40
0.32
18
9
30
30
0.34
Extended analogues, postulated to achieve the in-
teraction with the C-terminal residue in the active
site, were designed and synthesised. Data are listed
in Table 3. These analogues were designed to interact
31
32
41
80
69
0.31
–
>100
directly, or through
a water-mediated hydrogen
bond, with the C-terminal carboxylate group to
obtain potency gains as observed for the previously
reported pyrazole sulfonamide series.[8,9] Crystal struc-
ture analysis of 44 in complex with LmNMT con-
firmed that this interaction was achieved (Figure 7).
[a] Values shown are the mean of two or more determinations; ND=not determined.
[b] Ligand efficiency (LE), determined for compounds with T. brucei NMT potency
<50 mm, was calculated as 0.6ln(IC50)/(heavy atom count).[11]
the loss in potency might be that the methyl group contrib-
utes favourably to binding, or alternatively this heterocycle
does not achieve an optimised hydrogen bonding interaction
with the protein due to an unfavourable HBA orientation. Fur-
The benzomorpholinone core of 44 retained the binding
mode of the original hit 14, with the methyl isoxazole packing
against Phe232 and the nitrogen lone pair forming a hydrogen
bond to Asn376. The substituent at the 7-position of the ben-
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with the protein chain, but hy-
drogen bonded to a highly coor-
dinated water molecule, an in-
teraction observed in previous
series of NMT inhibitors.[8,9] The
potencies of the extended ana-
logues 39–44 all indicate this in-
teraction has been achieved,
with all six analogues showing
>100-fold improvement in po-
tency at TbNMT.
Table 3. Benzomorpholinone series hit to lead optimisation.
Compd
R
IC50 [mm][a]
EC50 [mm][a]
Selectivity
ratio
LE[b]
TbNMT
TbNMT
hNMT
T. brucei
MRC5
33
34
35
36
37
38
H
2-CH3
6-Chloro
7-Chloro
7-Bromo
8-Bromo
2.9
>100
>100
>100
>100
>100
>100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
–
–
–
–
–
–
0.43
–
–
0.37
0.37
0.37
Notably,
potencies
of
>100
>100
7.1
6.4
11
<0.002 mm were at the limit of
the enzyme assay’s sensitivity;
compounds at this end of the
potency range are probably sub-
nanomolar inhibitors. In previous
works[8–10] we reported that
there is a good correlation be-
tween activity against the para-
site and inhibition of TbNMT, so
we reported a selectivity mea-
sure using the EC50 value against
the parasite in predicting selec-
tivity.[10] Relative selectivities cal-
culated as (hNMT IC50)/(T. brucei
cell assay EC50) were used to
compare compounds.[10] Whilst it
is unknown if inhibition of mam-
malian NMT is dose limiting or
whether the compounds have
off-target effects, this “selectivi-
ty” value turned out to be better
at predicting the “therapeutic
ratio” in mouse models of infec-
tion than a simple ratio of cellu-
lar activities. For example, 44
was found to be >10-fold more
potent in the parasite growth
assay than the other five extend-
ed compounds. Unfortunately,
the hNMT potency of 44 was
correspondingly increased as
well (hNMT IC50: 0.04 mm), so al-
though the cellular selectivity
(MRC5 cell/T. brucei cell) was
promising at 1400-fold, the more
informative hNMT IC50/T. brucei
cell assay EC50 (in vivo predictive)
selectivity index was only sixfold.
Compound 40 had the highest
hNMT IC50/T. brucei cell assay
EC50 selectivity index of 13-fold,
but unfortunately this com-
39
40
41
42
0.008
<0.002
<0.002
0.009
0.7
1.0
0.9
24
0.41
>50
2
13
5
0.40
>0.38
>0.40
0.38
0.08
0.19
0.35
24
19
>50
69
43
44
<0.002
<0.002
0.9
0.15
22
10
6
6
>0.40
>0.40
0.04
0.007
[a] Values shown are the mean of two or more determinations; ND=not determined. [b] Ligand efficiency (LE),
determined for compounds with T. brucei NMT potency <50 mm, was calculated as 0.6ln(IC50)/(heavy atom
count) for TbNMT.[11] [c] Selectivity ratio: hNMT (EC50)/T. brucei cell (EC50).
Figure 7. Crystallographically determined binding mode of 44 bound to LmNMT. A) Orientation of 44 (C atoms
gold) bound to LmNMT. Hydrogen bonds are shown as dashed lines. B) Comparison of 44 with the pyrazole
sulfonamide compound DDD85646 (C atoms cyan, PDB code: 2WSA). The image was prepared using PyMOL.
zomorpholinone extended down the peptide binding groove
presenting the methyl-piperidine group toward the C-terminal
carboxylate. The piperidine nitrogen did not interact directly
pound had a poorer T. brucei cellular efficacy (EC50: 80 nm) and
was not sufficiently potent to progress into an in vivo efficacy
study.
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Pharmacokinetic and physicochemical properties of the
benzomorpholinone series
Conclusions
Compounds around two different cores, thiazolidinones and
benzomorpholinones, were validated as hits for TbNMT. The
benzomorpholinones originated from HTS, and the thiazolidi-
nones were discovered by HTS and virtual screening in parallel.
Interestingly, the thiazolidinone compounds displayed two dis-
tinct binding modes with different enantiomers bound
(Figure 4). One of the binding
The lead compounds showed a good balance of physicochemi-
cal properties (Table 4), which were within acceptable lead op-
timisation limits. Lipophilicity was clearly higher for the N-
methyl analogues, although the clogD values were still accept-
able. The mouse hepatic microsomal intrinsic clearance was
modes was very similar to that
Table 4. In vitro pharmacokinetic and physicochemical properties.
adopted by the benzomorpholi-
nones (Figures 6 and 7). All bind-
ing modes of the described
compounds differ from the bind-
ing modes of the previously de-
scribed pyrazole sulfonamide
compounds (Figures 4B and 7B),
or a number of structures of
Compd
R
Mr [Da]
clogP/clogD[a]
PSA [Š2][a]
68
CLint (m)[b]
PPB [%][b]
ND
Leishmania NMT with various li-
gands that were published sub-
sequent to the work reported
herein.[15,16] This enabled us to
explore new vectors and interac-
tions when attempting to opti-
mise the affinities of the hit
compounds.
39
383
3.8/1.2
2.0
40
41
42
412
397
399
3.1/1.0
4.0/1.7
2.7/1.0
62
59
68
ND
10
ND
82
The thiazolidinone hit 1,
which displayed relatively weak
TbNMT potency with no selectiv-
ity over hNMT, was expanded
into a hit series of compounds
using a one-pot, two-step reac-
tion. The indazole isostere 6 for
the methoxyphenol was quickly
identified as being equipotent
and more selective (displaying
no measurable inhibition of
hNMT). The discovery of a bind-
ing mode switch for the thiazoli-
7.0
ND
43
44
385
397
2.8/0.6
4.0/1.7
77
59
ND
12
ND
94
[a] Polar surface area, calculated partition (clogP) and distribution (clogD) coefficients calculated using StarDrop
from Optibrium. [b] CLint (m) [mLminÀ1 (g liver)À1] and plasma protein binding (PPB) were determined using the
protocol described in the Experimental Section; ND=not determined.
high for N-methyl analogues, most probably driven by N-de-
methylation to the secondary amines. The secondary amines
(e.g., 39) were inherently more metabolically stable and still
showed enzyme and T. brucei cellular activity in the nanomolar
range (Table 3).
dinone in compound 7 and the highly ligand efficient lead 13
provides a potential starting point for lead optimisation
chemistry, although cellular efficacy was a potential issue for
this series (best in class T. brucei EC50: 6.3 mm). Further optimi-
sation of 13 should be possible with a structure-based design
approach to target additional interactions in the binding site.
Alternatively, following on from SAR observations with the pyr-
azole sulfonamide and benzomorpholinone series, a hydrogen
bonding/salt bridge ligand–protein interaction with the C-ter-
minal carboxylate group could be targeted. This should lead to
nanomolar TbNMT potencies (as previously observed), which
Interestingly, we observed that the vector of substitution
from the benzomorpholinone core had a substantial effect on
the plasma protein binding (PPB) of these compounds and
their ability to cross the blood–brain barrier in in vivo studies,
where the 8-substituted compound had a much higher brain-
to-blood ratio (B/B=27) than the 7-substituted compound (B/
B=0.12). The latter property is essential for progression into
lead optimisation and for candidates suitable for clinical trials,
as the parasite has already invaded the CNS before most pa-
tients present for treatment.
should afford potent parasite cell activities at T. brucei.[8,9]
A
suitable group could be designed, with correct vector and
linker to achieve this interaction, as seen in the benzomorpho-
linone series hit-to-lead strategy. However, this strategy is not
without risk, because data appear to suggest that targeting
the C-terminal residue for a ligand–protein interaction may
also result in correspondingly potent hNMT inhibition, as ob-
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served with the benzomorpholinone series (Table 3). This could
lead to potential issues with selectivity and toxicity (through
mammalian NMT inhibition). The thiazolidinone scaffold also
offers the opportunity to explore previously unexplored vec-
tors, with the possibility of identifying new protein–ligand in-
teractions which could deliver selective potency at TbNMT
(over hNMT).
Experimental Section
Chemistry
Chemicals and solvents were purchased from Aldrich Chemical Co.,
Fluka, ABCR, VWR, Acros, Fisher Chemicals, and Alfa Aesar, and
were used as received unless otherwise stated. Air- and moisture-
sensitive reactions were carried out under an inert atmosphere of
argon in oven-dried glassware. Analytical thin-layer chromatogra-
phy (TLC) was performed on pre-coated TLC plates (layer 0.20 mm
silica gel 60 with fluorescent indicator UV254, Merck). Developed
plates were air dried and analyzed under a UV lamp (l 254/
365 nm). Flash column chromatography was performed using pre-
packed silica gel cartridges (230–400 mesh, 40–63 mm, SiliCycle)
(unless otherwise stated) using a Teledyne ISCO Combiflash Com-
panion or Combiflash Retrieve. 1H and 13C NMR spectra were re-
corded on a Bruker Avance II 500 spectrometer (1H at 500.1 MHz,
13C at 125.8 MHz), or a Bruker DPX300 spectrometer (1H at
300.1 MHz). Chemical shifts (d) are expressed in ppm recorded
using the residual solvent as internal reference in all cases. Signal
splitting patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), pentet (p), multiplet (m), broad (br), or a combination
thereof. Coupling constants (J) are quoted to the nearest 0.1 Hz.
LC–MS analyses were performed with either an Agilent HPLC 1100
series instrument connected to a Bruker Daltonics MicrOTOF or an
Agilent Technologies 1200 series HPLC connected to an Agilent
Technologies 6130 quadrupole spectrometer, where both instru-
ments were connected to an Agilent diode array detector. LC–MS
On the benzomorpholinone series, we started with the low-
molecular-weight, ligand-efficient hit compound 14. We identi-
fied several bioisosteric replacements of the chemically labile
ester functionality, two of which additionally displayed im-
proved TbNMT potency and good ligand efficiency. A struc-
ture-based drug design approach was instigated, using 23 as
the parent template for substitution. Vectors for attaining sub-
stitution toward the protein’s C terminus were identified by X-
ray crystallography and from initial SAR investigations around
the core scaffold. Six compounds, 39–44, were described
which appear to have achieved this additional ligand–protein
interaction, and which showed the expected increase in poten-
cy at TbNMT. Within this series we developed TbNMT enzyme
potency from the 10 mm level to <0.002 mm. The enzyme and
cellular potencies correlated directly, indicating an on-target
effect of killing the parasites in culture. Due to high structural
homology between the human and T. brucei NMT enzyme or-
thologues, improvements in potency toward the sub-10 nm
levels at TbNMT were often accompanied by a corresponding
improvement in hNMT inhibition (Table 3). Compound 42 gave
some indication that TbNMT/hNMT selectivity may be achieva-
ble within this series, although 42 itself had insufficient cellular
potency to progress further and into in vivo efficacy studies.
In compounds from these two series there was a 30- to 50-
fold decrease in activity going from TbNMT enzyme to T. brucei
cell efficacy, which further narrowed selectivity over mammali-
an NMT inhibition; this may have the potential to drive toxicity.
Compounds from the benzomorpholinone series afforded
potent antiparasitic cell activities (T. brucei EC50: 0.007 mm for
44); they display promising in vitro DMPK profiles, showing
this series has the potential to be optimised further to deliver
orally active antiparasitic compounds. The 8-substituted benzo-
morpholinone series in particular shows good potential for
stage-2 in vivo efficacy, although improvements in selectivity
would be needed to extend the therapeutic window and to
allow higher dose levels in order to maximise the chances of
curing stage-2 infected mice. It is unclear at this time how to
rationally achieve improved selectivity to the required levels,
although this could be possible by using currently undiscov-
ered selectivity pockets and protein secondary structure differ-
ences between T. brucei and human NMT.
chromatographic separations were conducted with
a Waters
Xbridge C18 column, 50 mm”2.1 mm, 3.5 mm particle size; mobile
phase: H2O/MeCN+0.1% HCOOH, or H2O/MeCN+0.1% NH3;
linear gradient from 80:20 to 5:95 over 3.5 min and then held for
1.5 min; flow rate: 0.5 mLminÀ1. All tested compounds had a mea-
sured purity of 95% (by TLC and UV) as determined by this analyti-
cal LC–MS system. High-resolution electrospray MS measurements
were performed on a Bruker Daltonics MicrOTOF mass spectrome-
ter. Microwave-assisted chemistry was performed using a Biotage
initiator microwave synthesiser.
General procedure for the synthesis of thiazolidinones: To
a cooled solution of aldehyde (2 mmol) in THF (5 mL) at 08C, was
added amine (1 mmol) and the reaction was stirred for 5 min. Thio-
acetic acid (3 mmol) was then added, and stirring continued for
10 min.
Polymer-supported
carbodiimide
(1.33 mmol,
1.33 mmolgÀ1 loading) was then added, the reaction warmed to
RT, and stirring continued for 16 h. The reaction was filtered, the
resin washed (THF, MeOH) and the filtrate concentrated in vacuo.
The resultant crude residue was taken up in CH2Cl2, washed (10%
citric acid (aq), water, saturated sodium bicarbonate (aq), brine),
dried (MgSO4) and concentrated in vacuo. The resultant crude resi-
due was purified by column chromatography (0–100% EtOAc/
hexane) to give the title compound.
General procedure for the synthesis of benzomorpholinones:
Appropriate 2-chloroacetyl chloride (21.3 mmol) was added drop-
wise to a mixture of appropriate 2-aminophenol (21.3 mmol) and
K2CO3 (21.3 mmol) in DMF (40 mL). The reaction mixture was
heated at 808C for 3 h. The solvent was then removed in vacuo,
the residue was partitioned with CH2Cl2 and water, and the organ-
ics concentrated. The solid obtained was triturated and filtered
with Et2O to give substituted-2H-benzo[b][1,4]oxazin-3(4H)-one.
Starting from the singleton HTS hits 1 and 14, and the virtu-
al screening hit 9, we have described the development of two
potent TbNMT enzyme inhibitor series, which provide good
starting points for drug discovery programmes for the treat-
ment of stage-1 and stage-2 HAT. These provide novel scaffolds
and give opportunities for exploring new vectors.
Appropriately
substituted-2H-benzo[b][1,4]oxazin-3(4H)-one
(0.67 mmol) was taken into DMF (5 mL). Alkyl halide (0.74 mmol)
and anhydrous K2CO3 (1.01 mmol) were added and stirred at 608C
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overnight. The reaction mixture was concentrated in vacuo, the
residue partitioned between CH2Cl2 and saturated aqueous
NaHCO3, the organics dried (MgSO4) and concentrated to dryness.
Purification by column chromatography (0–5% MeOH/CH2Cl2) gave
the title compounds as solids.
aldehyde (214 mg, 2 mmol). Compound 11 was obtained as an off-
white solid (290 mg, 50%): H NMR (500 MHz, [D6]DMSO): d=8.58
(brs, 1H), 7.78–7.82 (m, 1H), 7.53–7.59 (m, 2H), 7.31–7.38 (m, 2H),
7.25 (m, 1H), 7.05 (brs, 1H), 6.14 (brs, 1H), 4.09 (dd, J=15.6,
1.4 Hz, 1H), 3.82 ppm (d, J=15.5 Hz, 1H); LC–MS: m/z=291 [M+
H].
1
3-(4-Chlorophenyl)-2-(pyridin-2-yl)thiazolidin-4-one (10): Pre-
pared using 4-chloroaniline (382 mg, 4 mmol) and pyridine-2-car-
baldehyde (214 mg, 2 mmol). Compound 10 was obtained as
a beige solid (250 mg, 43%): 1H NMR (500 MHz, [D6]DMSO): d=
8.49 (m, 1H), 7.58 (m, 1H), 7.13–7.20 (m, 6H), 6.02 (d, J=1.3 Hz,
1H), 4.01 (dd, J=16, 1.3 Hz, 1H), 3.73 ppm (d, J=16 Hz, 1H); LC–
MS: m/z=291 [M+H].
Thiazolidinone series compounds
3-Benzyl-2-(4-hydroxy-3-methoxyphenyl)thiazolidin-4-one
(1):
Prepared using benzylamine (0.11 mL, 1 mmol) and vanillin
(304 mg, 2 mmol). Compound 1 was obtained as an off-white solid
(233 mg, 74%): 1H NMR (500 MHz, [D6]DMSO): d=9.23 (s, 1H),
7.33–7.24 (m, 3H), 7.11–7.07 (m, 2H), 6.81 (d, J=2.0 Hz, 1H), 6.77–
6.70 (m, 2H), 5.48 (d, J=1.6 Hz, 1H), 4.76 (d, J=15.3 Hz, 1H), 3.93
(dd, J=1.6 and 15.5 Hz, 1H), 3.78–3.67 ppm (m, 5H); LC–MS: m/z=
316 [M+H].
2-Phenyl-3-(pyridin-2-ylmethyl)thiazolidin-4-one (12): A solution
of 2-pyridylmethylamine (412 mL, 4 mmol) and benzaldehyde
(305 mL, 3 mmol) in EtOH (12 mL) was heated in a microwave at
1008C for 10 min. Thioacetic acid (2 mmol) was added to the reac-
tion, which was heated for a further 10 min at 1008C. Zinc chloride
(2 mmol) was then added and the reaction mixture was heated at
high absorbance setting (in microwave) at 1008C for 15 min,
before being cooled and transferred into a separating funnel.
EtOAc (30 mL) and 1n sodium hydroxide (30 mL) was added to
the separating funnel, which was then shaken. The organic layer
was collected and washed with brine (2”30 mL), then dried with
MgSO4. The solvent was removed in vacuo and the crude mixture
was purified by column chromatography (0–6% MeOH/CH2Cl2) to
yield compound 12 as an off-white solid (216 mg, 40%): 1H NMR
(500 MHz, [D6]DMSO): d=8.5 (m, 1H), 7.76 (m, 1H), 7.32–7.40 (m,
5H), 7.29 (m, 1H), 7.20 (d J=8 Hz, 1H), 5.79 (d, J=1.7 Hz, 1H), 4.87
(d, J=16, 1H), 3.97 (dd, J=15.4, 1.8 Hz, 1H), 3.76–3.82 ppm (m,
2H); LC–MS: m/z=271 [M+H].
3-(Furan-2-ylmethyl)-2-(4-hydroxy-3-methoxyphenyl)thiazolidin-
4-one (2): Prepared with furfurylamine (0.088 mL, 1 mmol) and va-
nillin (304 mg, 2 mmol). Compound 2 was obtained as an off-white
1
solid (202 mg, 66%): H NMR (500 MHz, [D6]DMSO): d=9.23 (s, 1H),
7.58 (dd, J=0.9 and 1.8 Hz, 1H), 6.85 (d, J=1.7 Hz, 1H), 6.75–6.71
(m, 2H), 6.38 (dd, J=1.8 and 3.5 Hz, 1H), 6.15 (dd, J=0.8 and
3.3 Hz, 1H), 5.51 (d, J=1.6 Hz, 1H), 4.74 (d, J=15.3 Hz, 1H), 3.93
(dd, J=1.6 and 15.5 Hz, 1H), 3.77–3.67 ppm (m, 5H); LC–MS: m/z=
306 [M+H].
3-Ethyl-2-(4-hydroxy-3-methoxyphenyl)thiazolidin-4-one (3): Pre-
pared using 2m ethylamine in THF (0.5 mL, 1 mmol) and vanillin
(304 mg, 2 mmol). Compound 3 was obtained as an off-white solid
1
(155 mg, 61%): H NMR (500 MHz, [D6]DMSO): d=9.22 (s, 1H), 6.94
(d, J=2.0 Hz, 1H), 6.81 (dd, J=2.1 and 8.2 Hz, 1H), 6.77 (d, J=
8.0 Hz, 1H), 5.74 (d, J=1.7 Hz, 1H), 3.82 (dd, J=1.6 and 15.5 Hz,
1H), 3.77 (s, 3H), 3.64 (d, J=15.2 Hz, 1H), 3.44 (m, 1H), 2.67 (m,
1H), 0.91 ppm (t, J=7.2 Hz, 3H); LC–MS: m/z=254 [M+H].
2-(4-Hydroxy-3-methoxyphenyl)-3-(pyridin-2-ylmethyl)thiazoli-
din-4-one (13): Prepared as for 12, using 2-pyridylmethylamine
(412 mL, 4 mmol) and 4-hydroxy-3-methoxybenzaldehyde (304 mg,
2 mmol) in EtOH (12 mL). Purification by column chromatography
(0–6% MeOH/CH2Cl2) gave compound 13 as an off-white solid
(142 mg, 22%): 1H NMR (500 MHz, [D6]DMSO): d=9.21 (brs, 1H),
8.49 (m, 1H), 7.73–7.76 (m, 1H), 7.26–7.29 (m, 1H), 7.19 (d J=8 Hz,
1H), 6.86 (brs, 1H), 6.72 (m, 2H), 5.70 (d, J=1.7 Hz, 1H), 4.79 (d,
J=16, 1H), 3.76–3.94 (m, 3H), 3.72 ppm (s, 3H); LC–MS: m/z=317
[M+H].
2-(4-Hydroxy-3-methoxyphenyl)-3-(pyridin-2-yl)thiazolidin-4-one
(4): Prepared using 2-aminopyridine (94 mg, 1 mmol) and vanillin
(304 mg, 2 mmol). Compound 4 was obtained as an off-white solid
1
(200 mg, 66%): H NMR (500 MHz, [D6]DMSO): d=9.04 (s, 1H), 8.31
(m, 1H), 7.91–7.86 (m, 1H), 7.85–7.81 (m, 1H), 7.18–7.13 (m, 1H),
6.92 (d, J=2.0 Hz, 1H), 6.75 (s, 1H), 6.70 (dd, J=2.0 and 8.3 Hz,
1H), 6.62 (d, J=8.2 Hz, 1H), 4.08 (dd, J=1.1 and 16.0 Hz, 1H), 3.87
(d, J=16.0 Hz, 1H), 3.71 ppm (s, 3H); LC–MS: m/z=303 [M+H].
Compounds 7, 8 and 9 were purchased from ChemDiv.
3-(Furan-2-ylmethyl)-2-(3-methoxyphenyl)thiazolidin-4-one (5):
Prepared using Furfurylamine (0.088 mL, 1 mmol) and 3-methoxy-
benzaldehyde (272 mg, 2 mmol). Compound 5 was obtained as
Benzomorpholinone series compounds
1
a clear colourless oil (93 mg, 33%): H NMR (500 MHz, [D6]DMSO):
Ethyl 2-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)acetate (14): 2H-
benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), ethyl bromoa-
cetate (124 mg, 0.74 mmol) and anhydrous K2CO3 (139 mg,
1.01 mmol) were taken up in DMF (5 mL) and stirred at 608C over-
night. The reaction mixture was concentrated in vacuo, the residue
partitioned with CH2Cl2 and saturated aqueous NaHCO3. The organ-
ic layer was dried (MgSO4) and concentrated to dryness. Purifica-
tion by column chromatography (0–5% MeOH/CH2Cl2) gave com-
pound 14 as a white solid (132 mg, 84%): 1H NMR (500 MHz,
[D6]DMSO): d=7.03–7.09 (m, 4H), 4.73 (s, 2H), 4.71 (s, 2H), 4.16 (q,
J=7.2 Hz, 2H), 1.21 ppm (t, J=7.2 Hz, 3H); LC–MS: m/z=236 [M+
H].
d=7.58 (dd, J=0.9 and 1.8 Hz, 1H), 7.38 (t, J=7.8 Hz, 1H), 6.95–
6.86 (m, 3H), 6.38 (dd, J=1.8 and 3.5 Hz, 1H), 6.17 (dd, J=0.8 and
3.3 Hz, 1H), 5.58 (d, J=1.6 Hz, 1H), 4.80 (s, 0.5H), 4.77 (s, 0.5H),
3.94 (d, J=1.6 Hz, 0.5H), 3.91 (d, J=1.6 Hz, 0.5H), 3.70–3.60 ppm
(m, 5H); LC–MS: m/z=290 [M+H].
3-Benzyl-2-(1H-indazol-6-yl)thiazolidin-4-one (6): Prepared using
benzylamine (0.11 mL, 1 mmol) and indazole-5-carbaldehyde
(292 mg, 2 mmol). Compound 6 was obtained as an off-white solid
(155 mg, 50%): 1H NMR (500 MHz, [D6]DMSO): d=13.15 (s, 1H),
8.07 (m, 1H), 7.68 (m, 1H), 7.35–7.25 (m, 4H), 7.10 (m, 1H), 7.33 (m,
1H), 5.68 (d, J=1.4 Hz, 1H), 4.84 (d, J=15.4 Hz, 1H), 4.01 (dd, J=
1.5 and 15.7 Hz, 1H), 3.87 (d, J=15.8 Hz, 1H), 3.71 ppm (s, 3H);
LC–MS: m/z=303 [M+H].
N,N-Dimethyl-2-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)aceta-
mide (19): 2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol),
dimethylcarbamic chloride (76 mL, 0.74 mmol) and anhydrous
K2CO3 (139 mg, 1.01 mmol) were treated as described for the syn-
3-(2-Chlorophenyl)-2-(pyridin-2-yl)thiazolidin-4-one (11): Pre-
pared using 3-chloroaniline (382 mg, 4 mmol) and pyridine-2-carb-
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thesis of 14. After workup a solid was obtained, trituration with
Et2O gave compound 19 as a white solid (122 mg, 78%): H NMR
0.91 mmol) and dimethylamine (1.64 mmol), to give compound 18
as an off-white solid (110 mg, 49%): H NMR (500 MHz, [D6]DMSO):
1
1
(500 MHz, CDCl3): d=6.99–7.02 (m, 3H), 6.76–7.79 (m, 1H), 4.70 (s,
2H), 4.69 (s, 2H), 3.15 (s, 3H), 3.02 (s, 3H); LC–MS: m/z=235 [M+
H].
d=7.22 (m, 1H), 7.06 (brs, 1H), 7.02 (brs, 2H), 4.63 (brs, 2H), 4.09
(m, 2H), 2.92 (brs, 3H), 2.82 (brs, 3H), 2.63 ppm (brs, 2H); LC–MS:
m/z=249 [M+H].
4-(Benzo[d]oxazol-2-ylmethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(22): 2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), 2-
(chloromethyl)benzo[d]oxazole (124 mg, 0.74 mmol) and anhy-
drous K2CO3 (139 mg, 1.01 mmol) were treated as described for the
synthesis of 14 to give compound 22 as a white solid (148 mg,
79%): 1H NMR (500 MHz, CDCl3): d=7.70–7.74 (m, 1H), 7.50–7.54
(m, 1H), 7.33–7.37 (m, 2H), 7.10–7.13 (m, 1H), 7.02–7.04 (m, 2H),
6.98–7.02 (m, 1H), 5.42 (s, 2H), 4.78 ppm (s, 2H); LC–MS: m/z=281
[M+H].
4-((5-Methylisoxazol-3-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (23): 2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), 3-
(chloromethyl)-5-methylisoxazole (97 mg, 0.74 mmol) and anhy-
drous K2CO3 (139 mg, 1.01 mmol) were treated as described for the
synthesis of 14. A solid was obtained by trituration with Et2O to
give compound 23 as a white solid (162 mg, 99%): 1H NMR
(500 MHz, CDCl3): d=7.21–7.26 (m, 1H), 6.98–7.03 (m, 3H), 5.98–
5.99 (m, 1H), 5.14 (s, 2H), 4.68 (s, 2H), 2.38–2.39 ppm (m, 3H); LC–
MS: m/z=245 [M+H].
Ethyl 3-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoate (16):
2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), ethyl 3-bro-
mopropanoate (134 mg, 0.74 mmol) and anhydrous K2CO3
(139 mg, 1.01 mmol) were treated as described for the synthesis of
4-((2-Methylthiazol-4-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (24): 2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), 4-
(chloromethyl)-2-methylthiazole
hydrochloride
(136 mg,
0.74 mmol) and anhydrous K2CO3 (200 mg, 1.45 mmol) were treat-
1
14 to give compound 16 as a white solid (152 mg, 91%): H NMR
ed as described for the synthesis of 14 to give compound 24 as an
1
(500 MHz, CDCl3): d=7.04–7.06 (m, 2H), 7.01–7.03 (m, 2H), 4.61 (s,
2H), 4.23–4.27 (m, 2H), 4.15 (q, J=7.2 Hz, 2H), 2.68–2.72 (m, 2H),
1.26 ppm (t, J=7.2 Hz, 3H); LC–MS: m/z=250 [M+H].
off-white solid (136 mg, 78%): H NMR (300 MHz, CDCl3): d=7.18–
7.24 (m, 1H), 6.99–7.03 (m, 3H), 6.95–6.97 (m, 1H), 5.23 (s, 2H),
4.69 (s, 2H), 2.74 ppm (s, 3H); LC–MS: m/z=261 [M+H].
4-(3-Oxo-3-(pyrrolidin-1-yl)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (17): Step 1: Ethyl 3-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)-
propanoate (16) (2 g, 8.03 mmol) was taken up in THF (100 mL),
treated with 2n aqueous KOH (100 mL), and the reaction was
stirred at RT for 1 h. The reaction was then heated at 608C, and
stirring continued for 2 h. The solvent was removed in vacuo and
the aqueous phase was acidified to pH 2 using concentrated HCl.
The aqueous phase was then extracted with EtOAc (2”50 mL), and
the combined extracts were dried in vacuo to give key intermedi-
ate 3-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoic acid (1.7 g,
96%): 1H NMR (500 MHz, [D6]DMSO): d=12.4 (brs, 1H), 7.25 (m,
1H), 7.07 (m, 1H), 7.02 (m, 2H), 4.62 (s, 2H), 4.14 (q, J=7.2 Hz, 2H),
2.54 ppm (m, 2H); LC–MS: m/z=221 [M+H].
N-Methyl-2-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)acetamide
(20): Step 1: 2H-benzo[b][1,4]oxazin-3(4H)-one (1.0 g, 6.7 mmol),
ethyl bromoacetate (818 mL, 7.4 mmol) and anhydrous K2CO3
(1.39 g, 10.1 mmol) were taken up in DMF (30 mL) and stirred at
508C for 3 h. The reaction mixture was concentrated in vacuo, the
residue partitioned with CH2Cl2 and saturated aqueous NaHCO3.
The organic layer was collected, dried (MgSO4) and concentrated
to dryness to give intermediate ethyl 2-(3-oxo-2H-benzo[b]
[1,4]oxazin-4(3H)-yl)acetate as a clear gum (1.34 g, 5.7 mmol, 85%):
1H NMR (500 MHz, CDCl3): d=7.00–7.04 (m, 3H), 6.75–6.79 (m, 1H),
4.69 (s, 2H), 4.66 (s, 2H), 4.26 (q, J=7.2 Hz, 2H), 1.29 ppm (t, J=
7.2 Hz, 3H).
Step 2:
Ethyl
2-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)acetate
Step 2: 3-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoic acid
(200 mg, 0.91 mmol) was taken into CH2Cl2 (5 mL), added carbonyl-
diimidazole (162 mg, 1 mmol). Stirred for 15 min then added
1.2 equiv amine (pyrrolidine, 77 mg, 1.09 mmol). Stirred for 18 h at
RT. Transferred reaction mixture to a separating funnel, added
CH2Cl2 (30 mL) and washed the organic layer with 1n HCl (20 mL),
2N NaHCO3 (20 mL) and then brine (30 mL). Dried organic layer
with MgSO4, and removed the solvent in vacuo. Purification by
column chromatography (0–6% MeOH/CH2Cl2) yielded compound
17 as a white solid (80 mg, 32%): 1H NMR (500 MHz, [D6]DMSO):
d=7.24 (m, 1H), 7.06 (m, 1H), 7.01 (m, 2H), 4.63 (m, 2H), 4.11 (m,
2H), 3.34 (m, 2H), 3.28 (m, 2H), 2.57 (m, 2H), 1.84 (m, 2H),
1.75 ppm (m, 2H); LC–MS: m/z=275 [M+H].
(1.34 g, 5.7 mmol) was taken up in THF (20 mL), KOH (640 mg,
11.4 mmol) in water (5 mL) was added and the reaction mixture
stirred at 608C for 3 h. Solvent was removed in vacuo, the residue
acidified to pH 3–4 by addition of 1m HCl, then extracted into
EtOAc. The organic layer was collected, then dried (MgSO4) and
concentrated to dryness to give intermediate 2-(3-oxo-2H-benzo[b]
[1,4]oxazin-4(3H)-yl)acetic acid as a white solid (1.1 g, 5.3 mmol,
93%): 1H NMR (300 MHz, [D6]DMSO): d=13.11 (s, 1H), 7.02–7.08
(m, 4H), 4.71 (s, 2H), 4.65 ppm (s, 2H).
Step 3:
2-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)acetic
acid
(200 mg, 0.96 mmol) was taken up in CH2Cl2 (10 mL), 2m methyl-
amine in THF (966 mL, 1.92 mmol) and carbonyldiimidazole
(172 mg, 1.06 mmol) was added and the reaction mixture was
stirred at RT for 18 h. The reaction mixture was then diluted with
further CH2Cl2 (50 mL) and partitioned with saturated aqueous
NaHCO3. The organic layer was collected, dried (MgSO4) and con-
centrated to dryness. The resulting solid was filtered off and tritu-
rated with Et2O to give compound 20 as an off-white solid (97 mg,
46%): 1H NMR (300 MHz, [D6]DMSO): d=7.16–7.19 (m, 1H), 7.05–
7.08 (m, 2H), 7.00–7.04 (m, 1H) 6.18 (brs, 1H), 4.70 (s, 2H) 4.54 (s,
2H), 2.82 ppm (d, J=4.9 Hz, 3H); LC–MS: m/z=221 [M+H].
4-(2-Oxo-2-(pyrrolidin-1-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (21): The carboxylic acid intermediate was prepared from 14,
as in step 1 for the synthesis of 17. The free acid was then taken
into CH2Cl2 and reacted with carbonyldiimidazole and pyrrolidine
as in step 2 for the synthesis of 17. Workup and purification as de-
scribed for the preparation of 17 afforded compound 21 as
a white solid (51%): 1H NMR (500 MHz, [D6]DMSO): d=6.95–7.03
(m, 4H), 4.68 (m, 4H), 3.58 (m, 2H), 3.32 (m, 2H), 1.95 (m, 2H),
1.81 ppm (m, 2H); LC–MS: m/z=261 [M+H].
4-(Pyridin-2-ylmethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(25):
N,N-Dimethyl-3-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propan-
amide (18): Prepared as described for the synthesis of 17 using 3-
(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoic acid (200 mg,
2H-benzo[b][1,4]oxazin-3(4H)-one (50 mg, 0.34 mmol), 2-(chlorome-
thyl)pyridine hydrochloride (60 mg, 0.37 mmol) and anhydrous
K2CO3 (69 mg, 0.50 mmol) were treated as described for the syn-
ChemMedChem 2015, 10, 1821 – 1836
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thesis of 14 and purified by column chromatography (EtOAc/
Hexane) to give compound 25 as a white solid (36 mg, 44%):
1H NMR (500 MHz, CDCl3): d=8.58–8.60 (m, 1H), 7.64 (td, J=7.7,
1.8 Hz, 1H), 7.23–7.26 (m, 1H), 7.19–7.22 (m, 1H), 7.03–7.06 (m,
1H), 6.97–7.02 (m, 2H), 6.91–6.95 (m, 1H), 5.28 (s, 2H), 4.75 ppm (s,
2H); LC–MS: m/z=241 [M+H].
to
a
solution of (1-methyl-1H-pyrazol-3-yl)methanol (100 mg,
0.89 mmol) in CHCl3 and heated at 608C for 2 h. The solvent was
removed in vacuo and the resulting crude 3-(chloromethyl)-1-
methyl-1H-pyrazole hydrochloride material used without purifica-
tion in the next step: LC–MS: m/z=131.1 [M+H].
Step 2: 2H-benzo[b][1,4]oxazin-3(4H)-one (111 mg, 0.74 mmol),
crude 3-(chloromethyl)-1-methyl-1H-pyrazole hydrochloride (as-
sumed 0.89 mmol) and anhydrous K2CO3 (300 mg, 2.17 mmol) were
treated as described for the synthesis of 14 to give compound 32
4-(Pyridin-4-ylmethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(26):
2H-benzo[b][1,4]oxazin-3(4H)-one (50 mg, 0.34 mmol), 4-(chlorome-
thyl)pyridine hydrobromide (93 mg, 0.37 mmol) and anhydrous
K2CO3 (69 mg, 0.50 mmol) were treated as described for the syn-
thesis of 14 and purified by column chromatography (0–20%
EtOAc/Hexane) to give compound 26 as a white solid (45 mg,
55%): 1H NMR (500 MHz, CDCl3): d=8.56–8.59 (m, 2H), 7.16–7.19
(m, 2H), 7.05 (dd, J=8.0, 1.8 Hz, 1H), 7.01 (td, J=7.6, 1.4 Hz, 1H),
6.91–6.95 (m, 1H), 6.75 (dd, J=8.1, 1.3 Hz, 1H), 5.17 (s, 2H),
4.76 ppm (s, 2H); LC–MS: m/z=241 [M+H].
1
as a gum (170 mg, 94% over two steps): H NMR (500 MHz, CDCl3):
d=7.30–7.33 (m, 1H), 7.26–7.27 (m, 1H), 6.96–7.01 (m, 3H), 6.17
(d, J=2.2 Hz, 1H), 5.12 (s, 2H), 4.67 (s, 2H), 3.88 ppm (s, 3H); LC–
MS: m/z=244 [M+H].
Ethyl 2-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoate (15):
2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), ethyl 2-bro-
mopropanoate (96 mL, 0.74 mmol) and anhydrous K2CO3 (139 mg,
1.01 mmol) were treated as described for the synthesis of 14 then
purified by column chromatography (0–20% EtOAc/hexane) to
give compound 15 as an off-white solid (100 mg, 60%): 1H NMR
(300 MHz, CDCl3): d=6.99–7.05 (m, 3H), 7.83–7.86 (m, 1H), 5.36 (q,
J=7.1 Hz, 1H), 4.62 (q, J=16.5 Hz, 2H), 4.18–4.25 (m, 2H), 1.64 (d,
J=7.1 Hz, 3H), 1.20 ppm (t, J=7.1 Hz, 3H); LC–MS: m/z=250 [M+
H].
4-((3-Methyl-1,2,4-oxadiazol-5-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one
(27):
2H-benzo[b][1,4]oxazin-3(4H)-one
(100 mg, 0.67 mmol), 5-(chloromethyl)-3-methyl-1,2,4-oxadiazole
(98 mg, 0.74 mmol) and anhydrous K2CO3 (139 mg, 1.01 mmol)
were treated as described for the synthesis of 14 to give com-
pound 27 as an off-white solid (54 mg 33%): 1H NMR (500 MHz,
CDCl3): d=6.99–7.08 (m, 3H), 6.86–6.89 (m, 1H, 5.33 (s, 2H), 4.74
(s, 2H), 2.40 ppm (s, 3H); LC–MS: m/z=246 [M+H].
8-Bromo-4-((5-methylisoxazol-3-yl)methyl)-2H-benzo[b]
4-((5-Methyl-1,2,4-oxadiazol-3-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one (38): Step 1: 2-chloroacetyl chloride (1.7 mL,
21.3 mmol) was added dropwise to a mixture of 2-amino-6-bromo-
phenol (4 g, 21.3 mmol) and K2CO3 (2.94 g, 21.3 mmol) in DMF
(40 mL). The reaction mixture was heated at 808C for 3 h then the
solvent removed in vacuo. The residue was partitioned between
CH2Cl2 and water, the organic layer was collected, dried with
MgSO4 and the solvent was then removed in vacuo. The resulting
solid was filtered off and triturated with Et2O to give intermediate
8-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one as an off-white solid
(1.87 g, 39%): 1H NMR (500 MHz, CDCl3): d=8.67 (brs, 1H), 7.23
(dd, J=8.1, 1.5 Hz, 1H) 6.86 (t, J=8.0 Hz, 1H), 6.79 (dd, J=7.9,
1.5 Hz, 1H), 4.74 ppm (s, 2H); LC–MS: m/z=226/228 [M+H].
[1,4]oxazin-3(4H)-one
(28):
2H-benzo[b][1,4]oxazin-3(4H)-one
(100 mg, 0.67 mmol), 3-(chloromethyl)-5-methyl-1,2,4-oxadiazole
(98 mg, 0.74 mmol) and anhydrous K2CO3 (139 mg, 1.01 mmol)
were treated as described for the synthesis of 14 to give com-
pound 28 as a white solid (135 mg, 82%): 1H NMR (300 MHz,
CDCl3): d=6.99–7.04 (m, 4H), 5.24 (s, 2H), 4.72 (s, 2H), 2.58 ppm (s,
3H); LC–MS: m/z=246.0 [M+H].
4-((5-Isobutyl-1,2,4-oxadiazol-3-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one
(29):
2H-benzo[b][1,4]oxazin-3(4H)-one
(100 mg, 0.67 mmol), 3-(chloromethyl)-5-isobutyl-1,2,4-oxadiazole
(129 mg, 0.74 mmol) and anhydrous K2CO3 (139 mg, 1.01 mmol)
were treated as described for the synthesis of 14 to give com-
pound 29 as a white solid (162 mg, 84%): 1H NMR (500 MHz,
CDCl3): d=6.98–7.03 (m, 4H), 5.25 (s, 2H), 4.72 (s, 2H), 2.75 (d, J=
7.2 Hz, 2H), 2.19 (hept, J=6.8 Hz, 1H), 0.98 ppm (d, J=6.7 Hz, 6H);
LC–MS: m/z=288 [M+H].
Step 2:
0.88 mmol),
8-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one
(200 mg,
(128 mg,
3-(chloromethyl)-5-methylisoxazole
0.97 mmol) and anhydrous K2CO3 (183 mg, 1.33 mmol) were treat-
ed as described for the synthesis of 14 to give compound 38 as
a pale-yellow solid (252 mg, 89%): 1H NMR (500 MHz, CDCl3): d=
7.24 (dd, J=8.2, 1.4 Hz, 1H), 7.20 (dd, J=8.2, 1.3 Hz, 1H), 6.89 (t,
J=8.2 Hz, 1H), 5.98 (d, J=0.8 Hz, 1H), 5.13 (s, 2H), 4.77 (s, 2H),
2.39 ppm (d, J=0.8 Hz, 3H); LC–MS: m/z=322/324 [M+H].
4-((5-Isobutylisoxazol-3-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (30): 2H-benzo[b][1,4]oxazin-3(4H)-one (100 mg, 0.67 mmol), 3-
(chloromethyl)-5-isobutylisoxazole (128 mg, 0.74 mmol) and anhy-
drous K2CO3 (139 mg, 1.01 mmol) were treated as described for the
synthesis of 14 to give compound 30 as an off-white solid
(165 mg, 86%): 1H NMR (500 MHz, CDCl3): d=7.21–7.25 (m, 1H),
6.98–7.03 (m, 3H), 5.97 (s, 1H), 5.15 (s, 2H), 4.68 (s, 2H), 2.58 (d, J=
7.1 Hz, 2H), 2.01 (hept, J=6.8 Hz, 1H), 0.93 ppm (d, J=6.6 Hz, 6H);
LC–MS: m/z=287 [M+H].
2-Methyl-4-((5-methylisoxazol-3-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one (34): Step 1: 2-chloropropanoyl chloride
(4.45 mL, 46 mmol) was added dropwise to a mixture of 2-amino-
phenol (5 g, 46 mmol) and K2CO3 (6.33 g, 46 mmol) in DMF (50 mL)
at 08C. The reaction mixture was heated at 608C for 2 h then the
solvent removed in vacuo. The residue was partitioned between
CH2Cl2 and water, the organic layer was collected, dried with
MgSO4 and the solvent was then removed in vacuo. The resulting
solid was filtered and triturated with Et2O to give intermediate 2-
4-((6-Methylpyridin-2-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (31): 2H-benzo[b][1,4]oxazin-3(4H)-one (50 mg, 0.34 mmol), 2-
(bromomethyl)-6-methylpyridine (69 mg, 0.37 mmol) and anhy-
drous K2CO3 (69 mg, 0.50 mmol) were treated as described for the
synthesis of 14 to give compound 31 as a white solid (71 mg,
82%): 1H NMR (500 MHz, CDCl3): d=7.50 (t, J=7.7 Hz, 1H), 7.04–
7.06 (m, 1H), 6.95–7.02 (m, 4H), 6.91–6.95 (m, 1H), 5.24 (s, 2H),
4.74 (s, 2H), 2.58 ppm (s, 3H); LC–MS: m/z=255 [M+H].
methyl-2H-benzo[b][1,4]oxazin-3(4H)-one as
a colourless gum
(4.25 g, 57%): 1H NMR (500 MHz, CDCl3): d=8.69 (brs, 1H), 6.95–
7.02 (m, 3H) 6.82–6.86 (m, 1H), 4.68 (q, J=6.9 Hz, 1H), 1.60 ppm
(d, J=6.8 Hz, 3H); LC–MS: m/z=164 [M+H].
Step 2:
2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one
(100 mg,
4-((1-Methyl-1H-pyrazol-3-yl)methyl)-2H-benzo[b][1,4]oxazin-
3(4H)-one (32): Step 1: Thionyl chloride (0.5 mL, excess) was added
0.61 mmol), 3-(chloromethyl)-5-methylisoxazole (89 mg, 0.68 mmol)
and anhydrous K2CO3 (127 mg, 0.92 mmol) were treated as de-
ChemMedChem 2015, 10, 1821 – 1836
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scribed for the synthesis of 14, then purified by column chroma-
tography (0–20% EtOAc/hexane) to give compound 34 as an off-
white solid (140 mg, 89%): H NMR (500 MHz, CDCl3): d=7.18–7.21
(m, 1H), 6.98–7.02 (m, 3H), 5.95–5.96 (m, 1H), 5.18 (d, J=15.7 Hz,
1H), 5.08 (d, J=15.7 Hz, 1H), 4.69 (q, J=6.8 Hz, 1H), 2.38 (d, J=
0.9 Hz, 3H), 1.60 ppm (d, J=6.8 Hz, 3H); LC–MS: m/z=259 [M+H].
Step 2: tert-butyl 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-
dihydro-2H-benzo[b][1,4]oxazin-8-yl)butyl)piperidine-1-carboxylate
was taken up in CH2Cl2 (10 mL), trifluoroacetic acid (1 mL, excess)
was added dropwise and the reaction mixture was stirred at RT for
3 h. The solvent was removed in vacuo, the residue was taken into
CH2Cl2 (30 mL) then partitioned between CH2Cl2 and saturated
aqueous NaHCO3. The organic layer was collected, dried (MgSO4)
and concentrated in vacuo. The solid obtained was filtered off and
triturated with Et2O to give compound 39 as a white solid (61 mg,
17% over two steps): 1H NMR (500 MHz, [D6]DMSO): d=6.98 (dd,
J=7.8, 1.9 Hz, 1H), 6.94 (t, J=7.6 Hz, 1H), 6.91 (dd, J=7.2, 1.9 Hz,
1H), 6.14 (s, 1H), 5.13 (s, 2H), 4.74 (s, 2H), 2.87–2.94 (m, 2H), 2.55–
2.60 (m, 2H), 2.37 (s, 3H), 1.75–1.83 (m, 2H), 1.54–1.62 (m, 2H),
1.47–1.54 (m, 2H), 1.27–1.34 (m, 2H), 1.13–1.25 (m, 3H), 0.91–
1.10 ppm (m, 2H); LC–MS: m/z=384 [M+H].
1
7-Bromo-4-((5-methylisoxazol-3-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one (37): Step 1: 2-chloroacetyl chloride
(2.33 mL, 29.3 mmol), 2-amino-5-bromophenol (5 g, 26.6 mmol)
and K2CO3 (4.41 g, 31.9 mmol) were treated as described in step 1
for the synthesis of 38 to give intermediate 7-bromo-2H-benzo[b]
[1,4]oxazin-3(4H)-one as an off-white solid. The product was puri-
fied as described in step 1 of the synthesis of compound 38
(4.90 g, 81%): 1H NMR (500 MHz, CDCl3): d=7.91 (brs, 1H), 7.15–
7.16 (m, 1H) 7.10 (dd, J=8.4, 2.1 Hz, 1H), 6.68 (d, J=8.4 Hz, 1H),
4.63 ppm (s, 2H); LC–MS: m/z=226/228 [M+H].
4-(4-(4-((5-Methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-
benzo[b][1,4]oxazin-8-yl)butyl)homopiperazine-1-methyl
(40):
Step 1: Following the methodology reported by Billotte,[17] a solu-
tion of tert-butyl 4-(but-3-en-1-yl)homopiperazine-1-carboxylate
(775 mg, 3.1 mmol) in THF (2 mL), under argon at RT, was treated
dropwise with 9-BBN (0.5m in THF, 7.7 mL, 3.9 mmol). The reaction
mixture was heated at 908C in the microwave for 30 min. The re-
sulting solution was then transferred into a stirred mixture of 38
(500 mg, 1.55 mmol) and 2m potassium phosphate solution
(1.5 mL, 3.1 mmol) in DMF (2 mL) and water (1 mL) under argon.
After bubbling argon through the reaction for 5 min at RT,
Pd(PPh3)4 (90 mg, 5 mol%) was added, the reaction vessel sealed
and heated at 908C in the microwave for 120 min. The reaction
mixture was then concentrated in vacuo, diluted with CH2Cl2 and
aqueous ammonia solution, the organic layer was washed with
brine, then dried (MgSO4) and concentrated in vacuo. Column
chromatography with 0–10% MeOH/(0.1%) NH3/CH2Cl2 eluent, fol-
lowed by freeze drying, gave intermediate tert-butyl 4-(4-(4-((5-
methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]-
oxazin-8-yl)butyl)homopiperazine-1-carboxylate (200 mg, 0.4 mmol,
26%): LC–MS: m/z=499 [M+H].
Step 2:
7-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one
(4.19 g,
19 mmol), 3-(chloromethyl)-5-methylisoxazole (2.68 g, 20 mmol)
and anhydrous K2CO3 (3.84 g, 28 mmol) were treated as described
for the synthesis of 14 to give compound 37 as a white solid
(4.60 g, 77%): 1H NMR (500 MHz, CDCl3): d=7.14–7.15 (m, 1H),
7.11–7.14 (m, 1H), 7.08–7.11 (m, 1H), 5.97–5.98 (m, 1H), 5.11 (s,
2H), 4.67 (s, 2H), 2.39 ppm (d, J=0.8 Hz, 3H); LC–MS: m/z=322/
324 [M+H].
6-Chloro-4-((5-methylisoxazol-3-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one (35): Step 1: 2-amino-4-chlorophenol
(1.44 g, 10 mmol), 2-bromoacetylbromide (3.03 g, 15 mmol) and
K2CO3 (1.66 g, 12 mmol) were treated as described in step 1 for the
synthesis of 38 to give intermediate 6-chloro-2H-benzo[b]
[1,4]oxazin-3(4H)-one (1.36 g, 74%): LC–MS: m/z=185 [M+H].
Step 2:
6-chloro-2H-benzo[b][1,4]oxazin-3(4H)-one
(53 mg,
0.29 mmol), 3-(chloromethyl)-5-methylisoxazole (42 mg, 0.32 mmol)
and anhydrous K2CO3 (60 mg, 0.44 mmol) were treated as de-
scribed for the synthesis of 14 to give compound 35 as an off-
white solid (57 mg, 71%): 1H NMR (500 MHz, [D6]DMSO): d=7.23
(m, 1H), 7.07 (m, 2H), 6.16 (brs, 1H), 5.17 (s, 2H), 4.77 (s, 2H),
2.37 ppm (s, 3H); LC–MS: m/z=280/281 [M+H].
Step 2: tert-butyl 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-
dihydro-2H-benzo[b][1,4]oxazin-8-yl)butyl)homopiperazine-1-car-
boxylate (200 mg, 0.4 mmol) was stirred overnight at RT with tri-
fluoroacetic acid (2 mL) in CH2Cl2 (10 mL). The reaction mixture
was then concentrated in vacuo, and taken up in MeOH (10 mL).
The product was purified using an SCX column, eluting with 2n
NH3/MeOH, followed by silica chromatography with 0–10% MeOH/
(0.1%) NH3/CH2Cl2 eluent to give intermediate 4-(4-(4-((5-methyli-
soxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-8-yl)-
butyl)homopiperazine (95 mg, 0.24 mmol, 60%): LC–MS: m/z=399
[M+H].
7-Chloro-4-((5-methylisoxazol-3-yl)methyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one (36): Step 1: 2-amino-5-chlorophenol
(1.44 g, 10 mmol), 2-bromoacetylbromide (3.03 g, 15 mmol) and
K2CO3 (1.66 g, 12 mmol) were treated as described in step 1 for the
synthesis of 38 to give intermediate 7-chloro-2H-benzo[b]
[1,4]oxazin-3(4H)-one (1.29 g, 70%): LC–MS: m/z=185 [M+H].
Step 2:
7-chloro-2H-benzo[b][1,4]oxazin-3(4H)-one
(35 mg,
0.24 mmol), 3-(chloromethyl)-5-methylisoxazole (32 mg, 0.24 mmol)
and anhydrous K2CO3 (40 mg, 0.37 mmol) were treated as de-
scribed for the synthesis of 14 to give compound 36 as an off-
white solid (38 mg, 57%): 1H NMR (500 MHz, [D6]DMSO): d=7.16
(m, 1H), 7.12 (m, 2H), 6.16 (brs, 1H), 5.15 (s, 2H), 4.80 (s, 2H),
2.36 ppm (s, 3H); LC–MS: m/z=280/281 [M+H].
Step 3:
4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-
(54 mg,
2H-benzo[b][1,4]oxazin-8-yl)butyl)homopiperazine
0.14 mmol) was stirred in CHCl3 with paraformaldehyde (5 equiv)
for 1 h at RT. Sodium cyanoborohydride (5 equiv) was then added
and the reaction stirred overnight at RT. A further 2 equivalents of
paraformaldehyde and sodium cyanoborohydride were added and
the reaction stirred for a further 18 h: LC–MS showed product at
m/z=413 [M+H]. The product was recovered by SCX purification,
eluting with 2n NH3/MeOH. The solvent was removed in vacuo to
give the title compound (40) as an off-white solid (50 mg,
0.12 mmol, 86%): 1H NMR (500 MHz, CDCl3): d=7.06 (dd, J=8,
1.5 Hz, 1H), 6.86–6.93 (m, 2H), 5.98 (d, J=0.8 Hz, 1H), 5.12 (s, 2H),
4.74 (s, 2H), 4.65 (s, 2H), 2.71 (m, 4H), 2.63 (m, 5H), 2.49 (t, 7.5 Hz,
2H), 2.38 (d, J=0.8 Hz, 2H), 2.37 (s, 3H), 1.82 (m, 2H), 1.54 ppm
(m, 4H); LC–MS: m/z=413 [M+H].
4-((5-Methylisoxazol-3-yl)methyl)-8-(4-(piperidin-4-yl)butyl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (39): Step 1: tert-butyl 4-(but-3-en-
1-yl)piperidine-1-carboxylate (334 mg, 1.40 mmol), 9-BBN (0.5m in
THF, 3.35 mL, 1.68 mmol) and 38 (300 mg, 0.93 mmol) were treated
as described in step 1 for the synthesis of 40. The Suzuki reaction
was carried out at 908C for 1 h, then at 110 8C for 30 min, to give
intermediate tert-butyl 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-
oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-8-yl)butyl)piperidine-1-car-
boxylate: LC–MS: m/z=484 [M+H].
ChemMedChem 2015, 10, 1821 – 1836
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4-((5-Methylisoxazol-3-yl)methyl)-8-(4-(1-methylpiperidin-4-yl)-
butyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (41): 4-(but-3-en-1-yl)-1-
methylpiperidine (107 mg, 0.70 mmol), 9-BBN (0.5m in THF,
0.86 mL, 0.93 mmol) and 38 (150 mg, 0.47 mmol) were treated as
described in step 1 for the synthesis of 40 to give compound 41
synthesis of 40. Purification by column chromatography (5–30%
EtOAc/pet ether) gave intermediate tert-butyl 4-(2-(4-((5-methyli-
soxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl)ethoxy)piperidine-1-carboxylate (184 mg, 0.39 mmol): LC–MS: m/
z=472 [M+H].
1
as an off-white solid (65 mg, 35%): H NMR (500 MHz, [D6]DMSO):
Step 3: tert-butyl 4-(2-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-
dihydro-2H-benzo[b][1,4]oxazin-7-yl)ethoxy)piperidine-1-carboxyl-
ate (40 mg, 0.085 mmol) was taken into CH2Cl2 (6 mL), trifluoroace-
tic acid (1 mL) was added and the reaction was stirred at RT for
2 h. Solvent was removed in vacuo, and the residue was dissolved
up in MeOH (10 mL) and purified on an SCX column, elution with
2n NH3/MeOH. This yielded compound 43 as an off-white solid
d=7.09 (m, 1H), 6.94 (m, 1H), 6.89 (m, 1H), 6.00 (brs, 1H), 5.14 (s,
2H), 4.67 (s, 2H), 2.84 (m, 2H), 2.62 (m, 2H), 2.40 (s, 3H), 2.27 (s,
3H), 1.86–1.91 (m, 2H), 1.53–1.70 (m, 6H), 1.35 (m, 2H), 1.21–
1.31 ppm (m, 3H); LC–MS: m/z=398 [M+H].
(R)-4-((5-Methylisoxazol-3-yl)methyl)-7-(3-((1-methylpyrrolidin-3-
yl)methoxy)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(42):
1
(25 mg, 73%): H NMR (500 MHz, [D6]DMSO): d=6.79 (m, 1H), 6.63
Step 1: (R)-tert-butyl 3-((allyloxy)methyl)pyrrolidine-1-carboxylate
(225 mg, 0.93 mmol), 9-BBN (0.5m in THF, 2.36 mL, 1.18 mmol) and
37 (200 mg, 0.62 mmol) were treated as described in step 1 for the
synthesis of 40. The Suzuki reaction was carried out at 1208C for
90 min, to give (R)-tert-butyl 3-((3-(4-((5-methylisoxazol-3-yl)meth-
yl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)propoxy)methyl)-
pyrrolidine-1-carboxylate, which was used without further purifica-
tion: LC–MS: m/z=486 [M+H].
(m, 2H), 5.89 (brs, 1H), 5.20 (brs, 2H), 4.88 (s, 2H), 4.48 (s, 2H),
3.60 (brs, 1H), 3.04–3.16 (m, 4H), 2.65 (m, 1H), 2.30 (m, 2H), 2.15
(m, 2H), 2.12 (s, 3H), 1.50 (m, 2H), 0.98 ppm (m, 2H); LC–MS: m/
z=372 [M+H].
4-((5-Methylisoxazol-3-yl)methyl)-7-(4-(1-methylpiperidin-4-yl)-
butyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (44): 4-(but-3-en-1-yl)-1-
methylpiperidine (107 mg, 0.70 mmol), 9-BBN (0.5m in THF,
0.86 mL, 0.93 mmol) and 37 (150 mg, 0.47 mmol) were treated as
described in step 1 for the synthesis of compound 40 to give com-
pound 44 as a white solid (65 mg, 35%): 1H NMR (500 MHz,
[D6]DMSO): d=7.01–7.04 (m, 1H), 6.86–6.87 (m, 1H), 6.83–6.86 (m,
1H), 6.13–6.14 (m, 1H), 5.12 (s, 2H), 4.72 (s, 2H), 2.68–2.73 (m, 2H),
2.48–2.51 (m, 2H), 2.37 (d, J=0.6 Hz, 3H), 2.12 (s, 3H), 1.74–1.80
(m, 2H), 1.54–1.60 (m, 2H), 1.24–1.31 (m, 2H), 1.17–1.23 (m, 2H),
1.04–1.14 ppm (m, 3H); LC–MS: m/z=398 [M+H].
Step 2: (R)-tert-butyl 3-((3-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-
3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)propoxy)methyl)pyrroli-
dine-1-carboxylate (assumed 0.62 mmol) was deprotected as de-
scribed for the synthesis of 39 to give (R)-4-((5-methylisoxazol-3-yl)-
methyl)-7-(3-(pyrrolidin-3-ylmethoxy)propyl)-2H-benzo[b][1,4]oxa-
zin-3(4H)-one, which was used without further purification: LC–MS:
m/z=386 [M+H].
Step 3: (R)-4-((5-methylisoxazol-3-yl)methyl)-7-(3-(pyrrolidin-3-ylme-
thoxy)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(assumed
0.62 mmol) was taken up in dry CHCl3 (5 mL), paraformaldehyde
(93 mg, 3.10 mmol) and sodium triacetoxyborohydride (657 mg,
3.10 mmol) added and the reaction mixture stirred overnight at RT.
Water (5 mL) was added, the layers separated and the organics
concentrated to dryness and purified by column chromatography
0–10% MeOH/(0.1%) NH3/CH2Cl2 eluent to give compound 42 as
a white solid (63 mg, 25% over three steps): 1H NMR (500 MHz,
[D6]DMSO): d=7.09–7.13 (m, 1H), 6.79–7–6.84 (m, 2H), 5.97–5.98
(m, 1H), 5.12 (s, 2H), 4.65 (s, 2H), 3.54–3.59 (m, 1H), 3.40–3.46 (m,
3H), 3.20–3.25 (m, 1H), 2.57–2.67 (m, 3H), 2.52 (s, 2H), 2.34–2.41
(m, 1H), 2.37–2.38 (m, 3H), 1.95–2.03 (m, 2H), 1.81–1.90 (m, 3H),
1.73–1.81 (m, 1H), 1.63–1.71 ppm (m, 1H); LC–MS: m/z=400 [M+
H].
Protein crystallography
LmNMT protein–ligand complexes were determined using methods
described previously.[9] Diffraction data were measured either in-
house using a rotating anode source (Rigaku Micromax 007)
equipped with an image plate detector (R-AXIS IV+ +) or at syn-
chrotron sources (ESRF beamline ID14–1 and Diamond beamline
I03). Coordinates for LmNMT–ligand complexes and associated dif-
fraction data have been deposited in the RCSB Protein Data Bank
(PDB) with accession codes 5AG5, 5AG4, 5AG6, 5AG7, and 5AGE for
compounds 6, 7, 13, 14, and 44 respectively.
4-((5-Methylisoxazol-3-yl)methyl)-7-(3-(piperidin-4-yloxy)propyl)-
2H-benzo[b][1,4]oxazin-3(4H)-one (43): Step 1: 4-hydroxy-N-Boc-
piperdine (3.02 g, 15 mmol) was taken into THF (150 mL) and
cooled to 08C. NaH (60% suspension on mineral oils) (900 mg,
22.5 mmol) was added slowly, and the reaction was warmed to RT
(10 min) then cooled back to 08C. Allyl bromide (2 g, 16.5 mmol)
was added and the reaction was stirred for 18 h at RT. The reaction
was diluted with EtOAc (80 mL) and transferred to separating
funnel. The organic layer was washed with water (50 mL), brine
(50 mL) then dried (MgSO4) and concentrated in vacuo. Purification
by column chromatography (5–30% EtOAc/pet ether) yielded in-
termediate tert-butyl 4-(allyloxy)piperidine-1-carboxylate (1.52 g,
72%): 1H NMR (500 MHz, CDCl3): d=5.95 (m, 1H), 5.31 (m, 1H),
5.20 (m, 1H), 4.04 (d, J=5.7, 2H), 3.80 (s, 2H), 3.52 (m, 1H), 3.10
(m, 2H), 1.85 (m, 2H), 1.54 (m, 2H), 1.48 ppm (s, 9H); LC–MS: m/
z=142 [M+H].
Virtual screening
Preparation of small-molecule database: A subset of the Drug
Discovery Unit Dundee in-house database containing 2.2 million
commercially available compounds was created using the crite-
ria:[18]
- No unwanted groups;
- No primary and secondary amides, or sulfonamides to avoid
high polar surface area;
- Number of heavy atoms between 10 and 2;
- clogP in the range of 0–4;
- Fewer than five ring systems;
- Fewer than eight rotatable bonds;
- Fewer than seven HBAs, fewer than four HBDs;
- Sum of HBAs and HBDs >1 but <9;
- No fused ring systems.
Step 2: tert-butyl 4-(allyloxy)piperidine-1-carboxylate (219 mg,
1.55 mmol), 9-BBN (0.5m in THF, 3.4 mL, 1.7 mmol) and 37
(150 mg, 0.47 mmol) were treated as described in step 1 for the
ChemMedChem 2015, 10, 1821 – 1836
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Full Papers
In total, 145127 molecules were contained in this subset. Subse-
quently, protonation states and tautomers were calculated using
in-house scripts based on OpenEye’s python toolkit (OEChem, ver-
sion 1.7.2.4, OpenEye Scientific Software Inc., Santa Fe, NM (USA),
2009 (http://www.eyesopen.com), whereas for acidic groups with
estimated pKa values between 5 and 9, both the neutral and
charged forms were stored. Corina (Molecular Networks, Germany)
was used to generate three-dimensional coordinates from smiles.
equilibrium dialysis plate. Dialysis versus isotonic buffer (150 mL)
was carried out for 5 h in a temperature-controlled incubator at
~378C (Barworld Scientific Ltd., UK) using an orbital microplate
shaker at 125 rpm (Barworld Scientific). At the end of the incuba-
tion period, aliquots of plasma or buffer were transferred to mi-
cronic tubes (Micronic B.V., the Netherlands), and the composition
in each tube was balanced with control fluid such that the volume
of buffer to plasma was the same. Sample extraction was per-
formed by the addition of 400 mL acetonitrile containing an appro-
priate internal standard. Samples were allowed to mix for 1 min
and then centrifuged at 3000 rpm in 96-well blocks for 15 min (Al-
legra X12-R, Beckman Coulter, USA). All samples were analysed by
UPLC–MS/MS on a Quattro Premier XE Mass Spectrometer (Waters
Corp., USA). The unbound fraction was determined as the ratio of
the peak area in buffer to that in plasma.
Pharmacophore search: The module UNITY in SYBYL 8.0 was ap-
plied to perform a flexible 3D pharmacophore search. The crystal
structure of LmNMT in complex with a pyrazole sulfonamide (PDB
code: 4A30) was used as receptor. Acceptor atoms, donor sites, ar-
omatic features, and exclusion volumes were selected to create
the UNITY query as described in the Results and Discussion section
(Figure 3). The UNITY database was created by uploading the mol2
file that was created by Corina. The import options “perceive chir-
ality at carbons”, “perceive chirality at nitrogen and phosphorous”,
and “perceive chirality at double bonds” were chosen; 8773 com-
pounds passed the pharmacophore filter and were subsequently
clustered using the Jarvis–Patrick algorithm of the Daylight cluster-
ing package (Daylight, Aliso Viejo, CA, USA). Representative hits
per cluster were minimised in the binding site using the Moloc
MAB force field.[19] The binding modes obtained were visually in-
spected, and a list of 200 compounds was selected for purchase.
Mouse brain penetration studies: Compounds 41 and 44 were
dosed as a bolus solution intravenously at 1 mg free base per kg
(dose volume: 5 mLkgÀ1; dose vehicle: 10% DMSO, 95% saline or
10% DMSO, 40% PEG400, 50% saline) to female NMRI mice (n=6).
At 5 and 30 min (compound 41 only) following intravenous bolus
injection of test compound, mice (n=3 per time point) were
placed under terminal anaesthesia with isofluorane. A blood
sample was taken by cardiac puncture into two volumes of dis-
tilled water and the brain removed. After suitable sample prepara-
tion, the concentration of test compound in blood and brain was
determined by UPLC–MS/MS using a Quattro Premier XE (Waters,
USA). For each mouse at each time point, the concentration in
Drug metabolism and pharmacokinetics (DMPK)
brain (nggÀ1) was divided by the concentration in blood (ngmLÀ1
)
to give a brain/blood ratio. The mean value obtained was quoted.
In vitro intrinsic clearance assay (CLint (m)): Test compound (0.5 mm)
was incubated with female CD1 mouse liver microsomes (Xenotech
LLC; 0.5 mg(mL 50 mm potassium phosphate buffer)À1, pH 7.4),
and the reaction was started by the addition of excess NADPH
(8 mg(mL 50 mm potassium phosphate buffer)À1, pH 7.4). Immedi-
ately, at time zero, then at 3, 6, 9, 15 and 30 min, an aliquot (50 mL)
of the incubation mixture was removed and mixed with acetoni-
trile (100 mL) to stop the reaction. Internal standard was added to
all samples, the samples were centrifuged (10 min, 58C, 3270 g) to
sediment precipitated protein and the plates then sealed prior to
UPLC–MS/MS analysis using a Quattro Premier XE (Waters Corp.,
USA).
Ethics. All regulated procedures on living animals were carried out
under the authority of a project license issued by the Home Office
under the Animals (Scientific Procedures) Act 1986, as amended in
2012 (and in compliance with EU Directive EU/2010/63). License
applications will have been approved by the University’s Ethical
Review Committee (ERC) before submission to the Home Office.
The ERC has a general remit to develop and oversee policy on all
aspects of the use of animals on University premises and is a sub-
committee of the University Court, its highest governing body.
Parasite and NMT assays: These were carried out as described pre-
viously.[8–10]
XLfit (IDBS, UK) was used to calculate the exponential decay and
consequently the rate constant (k) from the ratio of peak area of
test compound to internal standard at each time point. The rate of
intrinsic clearance (CLint: [mLminÀ1 (g liver)À1]) of each test com-
Acknowledgements
pound was then calculated using Equation (1), in which
V
The authors thank the Wellcome Trust (grants 077705, 094090,
and strategic award WT083481) for financial support of these
studies and OpenEye for free software licenses. The authors ac-
knowledge Dr. Paul Fyfe (University of Dundee) for supporting
the in-house X-ray facility and thank Diamond Light Source (DLS)
and the European Synchrotron Facility (ESRF) for synchrotron
beam time and staff support. The authors also thank Daniel
James for data management and Gina McKay (University of
Dundee) for performing HRMS analyses and for assistance with
performing other NMR and MS analyses.
[mL(mg protein)À1] is the incubation volume per mg protein
added, and microsomal protein yield is taken as 52.5 mg protein
per gram of liver. Verapamil (0.5 mm) was used as a positive control
to confirm acceptable assay performance.
CLint ¼ k  V  ðmicrosomal protein yieldÞ
ð1Þ
In vitro plasma protein binding experiments: In brief, a 96-well equi-
librium dialysis apparatus was used to determine the free fraction
in plasma for each compound (HT Dialysis LLC, Gales Ferry, CT,
USA). Membranes (12–14 kDa cutoff) were conditioned in deion-
ised water for 60 min, followed by conditioning in 80:20 deionised
water/EtOH for 20 min, and then rinsed in isotonic buffer before
use. Female CD1 mouse plasma was removed from the freezer and
allowed to thaw on the day of experiment. Thawed plasma was
then centrifuged (Allegra X12-R, Beckman Coulter, USA, 10 min,
58C, 3270 g), spiked with test compound (10 mggÀ1), and 150 mL
aliquots (n=6 replicate determinations) loaded into the 96-well
Keywords: human African trypanosomiasis (HAT) · medicinal
chemistry
· N-myristoyltransferase · structure-based drug
design · Trypanosoma brucei
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Full Papers
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Published online on September 23, 2015
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1836 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim