Hit-to-Lead Optimization of Benzoxazepinoindazoles As Human African Trypanosomiasis Therapeutics

Article abstract of DOI:10.1021/acs.jmedchem.9b01506

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by infection with either of two subspecies of the parasite Trypanosoma brucei. Due to a lack of economic incentive to develop new drugs, current treatments have severe limitations in terms of safety, efficacy, and ease of administration. In an effort to develop new HAT therapeutics, we report the structure-activity relationships around T. brucei for a series of benzoxazepinoindazoles previously identified through a high-throughput screen of human kinase inhibitors, and the subsequent in vivo experiments for HAT. We identified compound 18, which showed an improved kinase selectivity profile and acceptable pharmacokinetic parameters, as a promising lead. Although treatment with 18 cured 60% of mice in a systemic model of HAT, the compound was unable to clear parasitemia in a CNS model of the disease. We also report the results of cross-screening these compounds against T. cruzi, L. donovani, and S. mansoni.

Full text of DOI:10.1021/acs.jmedchem.9b01506

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Article
Hit-to-lead optimization of benzoxazepinoindazoles
as human African trypanosomiasis therapeutics
Dana M. Klug, Laura Tschiegg, Rosario Díaz González, Domingo Isaac Rojas-Barros,
Guiomar Perez-Moreno, Gloria Ceballos-Perez, Raquel García-Hernández, Maria Santos
Martínez-Martínez, Pilar Manzano, Luis M. Ruiz-Perez, Conor R. Caffrey, Francisco Gamarro,
Dolores Gonzalez-Pacanowska, Lori Ferrins, Miguel Navarro, and Michael P. Pollastri
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01506 • Publication Date (Web): 31 Oct 2019
Downloaded from pubs.acs.org on November 1, 2019
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Hit-to-lead optimization of benzoxazepinoindazoles as human African trypanosomiasis therapeutics
a
a
b
b
b
Dana M. Klug, Laura Tschiegg, Rosario Diaz, Domingo Rojas-Barros, Guiomar Perez-Moreno,
b
b
c
c
Gloria Ceballos, Raquel García-Hernández, Maria Santos Martinez-Martinez, Pilar Manzano, Luis
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Miguel Ruiz, Conor R. Caffrey, Francisco Gamarro, Dolores Gonzalez Pacanowska, Lori Ferrins,
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a*
Miguel Navarro, Michael P. Pollastri,
aNortheastern University Department of Chemistry & Chemical Biology, 360 Huntington Avenue,
Boston, MA 02115 USA.
bInstituto de Parasitología y Biomedicina “López-Neyra” Consejo Superior de Investigaciones Cientificas
(CSIC), Granada 18016 Spain.
cDiseases of the Developing World (DDW), Tres Cantos Medicines Development Campus,
GlaxoSmithKline, Tres Cantos 28760 Spain.
dCenter for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093
USA.
*
m.pollastri@northeastern.edu; 617-373-2703
Abstract
Human African trypanosomiasis (HAT) is a neglected tropical disease caused by infection with
either of two subspecies of the parasite Trypanosoma brucei. Due to a lack of economic incentive to
develop new drugs, current treatments have severe limitations in terms of safety, efficacy, and ease of
administration. In an effort to develop new HAT therapeutics, we report the structure-activity
relationships around T. brucei for a series of benzoxazepinoindazoles previously identified through a
high-throughput screen of human kinase inhibitors, and the subsequent in vivo experiments for HAT. We
identified compound 18, which showed an improved kinase selectivity profile and acceptable
pharmacokinetic parameters, as a promising lead. Although treatment with 18 cured 60% of mice in a
systemic model of HAT, the compound was unable to clear parasitemia in a CNS model of the disease.
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We also report the results of cross-screening these compounds against T. cruzi, L. donovani and S.
mansoni.
Introduction
Human African trypanosomiasis (HAT) is a parasitic infection caused by two subspecies of the
organism Trypanosoma brucei (T.b. gambiense and T.b. rhodesiense). Prevalent in sub-Saharan Africa,
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HAT is one of 20 neglected tropical diseases (NTDs) designated by the World Health Organization. The
disease proceeds in two stages. In the first stage, the parasite is present in the blood and lymph and causes
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mild, flu-like symptoms. A transition to stage 2 occurs when the parasite crosses the blood-brain barrier;
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this results in more severe symptoms such as disrupted sleeping patterns and mood fluctuation. There are
five compounds currently in use for the treatment of HAT: pentamidine, suramin, melarsoprol, and an
eflornithine/nifurtimox combination therapy, referred to as NECT. These treatments are reasonably
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efficacious but cause a variety of adverse effects and can be challenging to administer. Recent
developments include the approval of fexinidazole as an oral treatment for HAT in the Democratic
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Republic of Congo, and the progression of acoziborole to Phase II/III clinical trials. However, given the
attrition rate of compounds in clinical trials and the possibility that T. brucei could develop resistance to
current drugs, we believe it is prudent to continue to seed the drug discovery pipeline for HAT.
As part of our ongoing NTD drug discovery efforts, we employed a lead repurposing strategy to
find new chemical matter to use as a starting point for HAT therapeutics. Lead repurposing entails the
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screening of sets of targeted chemical matter based on homology between humans and parasites. As there
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are many examples of kinase essentiality in T. brucei, we undertook a high-throughput screen (HTS) of
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human kinase inhibitors against T.b. brucei cultures. A set of 42,444 known human kinase inhibitors
were screened against T.b. brucei at 4 μM; those that showed >50% inhibition of parasite growth were
progressed to dose-response assays against T.b. brucei and HepG2 cells. This screening process yielded
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97 compounds with a T.b. brucei pEC50 >6 that were >100x selective against HepG2 cells. These
compounds were grouped by structural similarity into 59 clusters and 53 singletons.
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One of the overall most promising clusters to emerge from this HTS comprised a series of
substituted benzoxazepinoindazoles (BOXIs). Structural features of note are highlighted in Table 1 and
include an aromatic “head” (red), the oxazepinoindazole “core” (blue) and an appended heterocyclic
“
tail” (yellow). The average potency of compounds in this cluster is a pEC of 7.6, and the three hits
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highlighted in Table 1 (NEU-1117, -1118, and -1119) all have pEC50 >8 while maintaining a good
margin of selectivity against mammalian cells. The physicochemical properties of these compounds are
also promising: topological polar surface area (TPSA) and molecular weight fall well within the targeted
range for lead compounds, and while the cLogP and LogD of these compounds are on average higher than
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desired, the lipophilic ligand efficiencies (LLEs) are high.
Additional parameters were also calculated or measured with the end goal of producing a HAT
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drug in mind. Central nervous system multiparameter optimization (CNS-MPO) scores were calculated
for the HTS hits in order to provide an indication of whether they were likely to be brain-penetrant,
essential for any treatment of stage 2 HAT. On average, the CNS-MPO scores of these compounds are
consistent with likely blood-brain barrier penetration. The rate of action and trypanosome cidality of hits
were also assessed; fast-acting, trypanocidal compounds are preferred to develop an effective treatment.
Most compounds in this cluster are fast-acting and cidal, including NEU-1117 and NEU-1118, though
which structural features gave rise to this property was not yet understood.
Targeted Cluster
NEU-1117
NEU-1118 NEU-1119
Value
Average
R =
R = NH2
R = H
CH OCH
2
3
T.b.b. pEC50
≥7
7.6
4.8
8.1
8.9
5.5
8.4
4.0
HepG2 pTC50
≤pEC –
5.1
50
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cLogP
TPSA (Å2)
MW (Da)
LLE
≤3
40<x≤90
≤360
3.9
106
332
3.7
2.2
110
360
5.8
3.7
130
330
5.2
3.7
100
320
4.7
≥4
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CNS-MPO
Score
≥
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3.9
4.4
3.9
4.5
Fast
Cidal
Yes
Yes
>10
<95
85%
69%
--
Yes
Yes
11
Yes
Yes
4
Yes
No
8
Aq. Sol. (µM)
PPB (%)
--
98
97
nd
Table 1. Targeted, cluster average, and individual cluster member values for properties of interest. Values
highlighted in green meet or exceed targeted values; yellow highlighting indicates mid-range values, and
red highlighting indicates values that are well outside the targeted value. nd = no data.
Despite their promising characteristics, these compounds were not without room for optimization;
Table 1 highlights some challenges to be overcome in this cluster of compounds. In particular, in vitro
absorption, distribution, metabolism, and excretion (ADME) properties, such as aqueous solubility and
plasma protein binding (PPB), were outside of the desired range. As our end goal is to develop an orally
available HAT therapeutic, it was essential that these properties be improved. Additionally, a
representative compound was selected for assessment against a panel of human kinases. Given the
cluster’s origin as human kinase inhibitors, it is perhaps unsurprising that this compound, NEU-1328, is a
potent (<100 nM) inhibitor of at least five human kinases (Table S1). Our overall goal, therefore, was to
improve the ADME properties of BOXIs while maintaining their potency and favorable physicochemical
properties, and to assess how these changes impacted human kinase selectivity.
Results
Initial SAR studies focused on exploring the effect of various nitrogen-containing heterocyclic
tails coupled to the BOXI core. The synthesis of such compounds is shown in Scheme 1. Synthesis began
with the conversion of 4-bromo-2,6-difluorobenzoic acid 1 to the acid chloride 2; this intermediate was
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coupled with 2-aminophenol to produce amide 3. The benzoxazepine ring was then formed upon
treatment with potassium carbonate. Compound 4 was converted to the thioamide 5 using Lawesson’s
reagent, which then yielded benzoxazepinoindazole 6 upon treatment with hydrazine. Protection with
acetic anhydride and subsequent borylation gave boronic ester 8, which was then coupled via a Suzuki
reaction with the desired aryl halide to yield compounds 9b-s. Analog 9a was synthesized using 2,6-
difluorobenzoic acid and following steps a-e.
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O
R
OH
b
O
NH
c
e
F
F
O
NH
X
F
F
Br
Br
F
Br
1
2
R = OH
R = Cl
4 X = O
5 X = S
a
3
d
O
f
NH
N
h
O
O
NH
NH
N
N
N
R
N
N
Br
Ar
H
O
H
7
8
R = Br
R = B(pin)
6
g
9b-s
Scheme 1. Synthesis of compounds 9a-s. Reagents and reaction conditions: a) SOCl , 75 °C, 4 h (quant.).
2
b) 2-aminophenol, TEA, DCM, 0 °C to rt, 12 h (87%). c) K CO , DMF, rt, 36 h (72%). d) Lawesson’s
2
3
reagent, toluene, 100 °C, 12 h (68%). e) Hydrazine, dioxane, 85 °C, 3 h (89%). f) Acetic anhydride, 100
C, 3 h (93%). g) B (pin) , KOAc, PdCl (dppf)·CH Cl , dioxane, 145 °C, μw, 1 h (65%). h) Aryl halide,
°
2
2
2
2
2
K CO , Pd(PPh ) , 3:1 dioxane:water, 100 °C, 4 h (10-74%).
2
3
3 4
The biological activity data of the initial tail replacement analogs is shown in Table 2. Of these,
the 2-aminopyrimidine motif remained the best option in terms of potency. Excising the tail altogether
(9a) led to a drop in potency of ~2 log units; although notably, this compound still retained sub-
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micromolar activity. Removal of the -NH of the tail group resulted in a compound (9b) that was more
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than 10-fold less active than NEU-1119. Analogs where the 2-aminopyrimidine was replaced with other
aminopyrimidines (9c, 9d), aminopyridines (9e, 9f) or a phenyl ring (9g) all lost 1-2 log units of potency
compared to NEU-1119.
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Small substitutions at the 6-position of the pyrimidine, such as the ethyl of 9h, the cyclopropyl of
9
i, the methylamine of 9j, and the ethylamine of 9k were well-tolerated, with only a slight loss in potency
as compared to the original hits. Bulkier amines such as the isopropylamine, aniline, and methylaniline of
9l, 9m, and 9n, respectively, were also tolerated from a potency standpoint but showed signs of toxicity in
mammalian cells. Tertiary amines at the 6-position, including a dimethylamine (9o), morpholine (9p),
pyrrolidine (9q), piperidine (9r), and N-methylpiperazine (9s), were not tolerated and consistently showed
a drop in potency by 1-2 log units as compared to the primary amines or the unsubstituted 2-
aminopyrimidine. In general, compounds that were active at sub-micromolar concentrations maintained
LLEs between 3 and 4. Moving forward, analogs were primarily synthesized with an unsubstituted 2-
aminopyrimidine as the “tail” group with the idea that, once an improved core was identified, it could be
matched with other “tails” identified in this early SAR exploration if necessary.
Table 2. Biological activity and LLE of tail replacement analogs. *HTS hit, not resynthesized. Data
obtained against MRC5 cells are indicated with †; otherwise, HepG2 data is presented. All SD within ±
0
.20.
O
NH
N
N
R
H
T.b.b.
pEC50
T.b.b.
pEC50
ID
9a
R
pTC50
LLE
ID
R
pTC50
LLE
H
N
H
6.2
4.0
2.1
9k
7.8
4.6†
5.1
N
N
NH2
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H
N
9b
6.9
7.2
6.6
6.7
<4.3†
4.8
4.2
3.5
3.5
3.6
9l
9m*
9n*
9o
6.9
7.2
7.5
6.3
<4.3†
5.0
3.7
0.85
0.66
3.2
N
N
N
N
N
NH2
H
N
N
9
c
N
N
N
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0
NH2
NH2
H
N
N
9
d
N
<4.3†
<4.3†
5.3
N
NH2
NH2
N
9
e
N
4.8†
N
N
NH2
NH2
O
N
N
9f
6.9
5.7
7.9
nd
<4.3†
4.8
4.8
1.5
3.2
9p
9q
9r
6.3
6.1
4.8
<4.3†
3.4
2.7
N
N
NH2
NH2
N
9
g
4.7†
N
N
NH2
N
<4.3†
9
h
N
N
0.91
N
N
NH2
NH2
N
N
9
i*
N
N
N
7.7
7.7
4.6
5.3
3.1
3.2
9s
5.7
<4.3†
2.3
N
N
NH2
NH2
H
N
9
j
N
NH2
The importance of each potential hydrogen bond donor on the 2-aminopyrimidine-substituted
core was explored by systematically installing methyl groups at each position. These compounds were
synthesized in a manner similar to the other BOXIs in the series (Scheme S1-S2). Methylation of the
benzoxazepine nitrogen (15), as well as bis-methylation of the aminopyrimidine (19) resulted in complete
loss of activity; however, methyl groups on the indazole nitrogen (18), as well as mono-methylation of
the aminopyrimidine 20) were reasonably well-tolerated.
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MRC
5
pTC50
T.b.b.
pEC50
ID
R1
R2
R3
LLE
R1
O
N
1
1
5
8
CH3
H
CH3
H
NH2
5.1
7.6
5.3
7.0
<4.3
<4.3
<4.3
<4.3
2.3
4.9
1.9
3.8
N
N
R2
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0
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9
0
H
NH2
N
N
19
H
H
N(CH3)2
NHCH3
R3
2
0
H
Table 3. Biological activity of N-methylated analogs. All SD within ± 0.08.
6
All analogs with a pEC >6 were assessed in a speed-to-kill assay. Figure 1 shows the rate of
5
0
action profile of three slow-acting compounds (9a, 9b, and 9f) and three fast-acting compounds (9j, 9l,
and 18), where fast-acting compounds are defined as those that reach a pEC >6 in less than 18 h of
5
0
incubation time. Although all six analogs eventually reached a pEC50 >6.5, slow-acting compounds
required incubation times of up to 72 h. Interestingly, all fast-acting compounds were analogs that
retained the benzoxazepine core and 2-aminopyrimidine tail, while slow-acting compounds were analogs
where the 2-aminopyrimidine was either removed or changed to a different heterocycle altogether. These
data illustrate the importance of the 2-aminopyrimidine motif to producing not only potent, but fast-acting
analogs, and this tail group was prioritized moving forward.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Rate of action of selected analogs. Slow-acting compounds 9a, 9b, and 9f are represented in red
and fast-acting compounds 9j, 9l, and 18 are represented in blue. Dotted gray lines show the cutoff of
pEC of 6 at 18 h incubation time.
5
0
Additionally, we assessed the in vitro ADME parameters of these compounds. The properties of
tail replacement and methylated analogs for which ADME data was available are shown in Table 4. As
was typical for all analogs synthesized as this point in the campaign, these compounds show moderate to
high clogP and high logD, as well as low solubility and high PPB. In addition, we observed moderate-to-
high microsomal and hepatocyte clearance. With this in mind, despite the high LLE of the BOXI analogs,
we felt that further reduction of the lipophilicity would serve the overall goal of developing a potent
analog with a good ADME profile. Thus, considering metabolic stability and lipophilicity, we turned our
attention to the benzoxazepine core as the next site of modification.
Rat Hepatocyte
HLM Clint
T.b.b.
pEC50
Aq. sol.
(μM)
Clint
(μg/min/10
cells)
ID
cLogP LogD
PPB (%) (μg/min/m
6
g protein)
9a
9b
6.2
6.9
2.9
3.0
4.1
3.5
19
<1
>99
>96
7.6
130
47
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
c
7.2
6.6
6.7
7.7
7.8
6.9
6.3
4.8
5.7
7.6
5.3
7.0
2.8
3.4
3.5
2.9
3.3
3.7
3.6
4.4
3.4
2.7
3.8
3.2
3.8
4.1
3.7
>4.2
4.6
4.9
>4.4
4.6
3.4
>4.3
>3.9
3.8
0.6
4
9
2
2
>99
>99
>81
>95
>99
>99
>99
>99
97*
nd
52
34
99
9.5
9
6.7
63
17
41
110
51
55
73
56
220
45
26
32
44
25
16
12
29
45
9
d
9
9
e
j
9
k
9
l
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9o
9r
9s
0.4
0.3
26
3
0.7
1.9
1
1
2
8
9
0
<99
99
Table 4. ADME properties of selected analogs. *Predicted PPB. nd = no data.
Initial analogs targeted a truncated oxazepinoindazole core, the synthesis of which is shown in
Scheme 2. Reaction of 2 with PMB-ethanolamine facilitated cyclization to the desired amide 21; direct
deprotection of the oxazepine ring allowed the use of Lawesson’s reagent to convert amide 23 to
thioamide 24. Subsequent transformations proceeded in an analogous manner to those required to make
the benzoxazepine core, with thioamide 24 converted to oxazepinoindazole 25 using hydrazine. This
reaction was followed by acetylation and conversion of the bromide 26 to the boronic ester 27 through a
Miyaura borylation using bis(pinacolato)diboron. Finally, a Suzuki reaction of this intermediate with the
requisite chloropyrimidines yielded the desired products 28a-b.
OH
O
R
O
N
PMB
F
O
N
R
O
NH
S
F
F
b
c
e
f
F
O
Br
F
Br
F
Br
Br
21
1
2
R = OH
R = Cl
22 R = PMB
23 R = H
24
a
d
O
NH
O
O
NH
NH
N
g
i
N
N
N
R
N
N
Br
Ar
H
O
H
25
26 R = Br
7 R = B(pin)
28a-b
h
2
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Scheme 2. Synthesis of oxazepinoindazole core. Reagents and reaction conditions: a) SOCl , 75 °C, 4 h
2
(quant.). b) 2-((4-Methoxy-benzyl)amino)ethan-1-ol or ethanolamine, TEA, DCM, 0 °C to rt, 12 h (54%).
c) NaH, DMF, rt, 12 h (96%). d) CAN, 3:1 acetonitrile:water, rt, 3 h (36%). e) Lawesson’s reagent,
toluene, 100 °C, 12 h (56%). f) Hydrazine, dioxane, 85 °C, 3 h (95%). g) Acetic anhydride, 100 °C, 3 h
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
60%). h) B (pin) , KOAc, PdCl (dppf)·CH Cl , dioxane, 145 °C, μw, 1 h (79%). i) Aryl halide, K CO ,
2 2 2 2 2 2 3
Pd(PPh ) , 3:1 dioxane:water, 100 °C, 4 h (12-34%).
3 4
We also explored truncation to substituted indazoles and aminoindazoles (Scheme 3). The
aminoindazole core was constructed from (4-cyano-3-fluorophenyl)boronic acid 29, which was subjected
to a Suzuki reaction with the desired aryl chlorides to form biaryl compounds 30. These intermediates
were then reacted with hydrazine to form final compounds 31a-c. To synthesize indazole analogs, 4-
bromoindazole was subjected to the same Miyaura and Suzuki conditions as previously described to
provide final compounds 34a-c. The syntheses of other core or headgroup replacement analogs, such as
4
0 and 49, were completed in an analogous route to that described in Scheme 1, and are described in
detail in Scheme S3-S4.
NH2
CN
F
CN
F
a
b
N
(HO) B
Ar
N
2
Ar
H
29
30a-c
31a-c
N
c
d
O
N
N
H
N
B
N
N
Br
Ar
H
O
H
32
33
34a-c
Scheme 3. Synthesis of indazoles and aminoindazoles. Reagents and reaction conditions: a) Aryl halide,
NaHCO (sat. aq.), Pd(PPh ) , dioxane, 95 C, 3 h (43-97%). b) Hydrazine, EtOH, 95 °C, 12 h (71-87%).
3
3 4
c) B (pin) , KOAc, PdCl (dppf)·CH Cl , dioxane, 145 °C, μw, 30 min – 1.5 h. d) Aryl halide, K CO ,
2
2
2
2
2
2
3
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PdCl (dppf)·CH Cl , 3:1 dioxane:water, 150 °C, μw 30 min, (6-26%). Where no yield is reported, crude
2
2
2
material was progressed without further purification.
The biological activity and ADME properties of these core replacement analogs are presented in
Table 5 (additional ADME data in Table S2). Systematic truncation of the benzoxazepine core to an
oxazepinoindazole (28a-b), aminoindazole (31a-c), or indazole (34a-c) core uniformly resulted in a loss
in potency of ~3 log units. However, these analogs were much more soluble than the original hits.
Interestingly, despite their low potency, they also retained LLEs of around 4, suggesting that the
lipophilic benzoxazepine group is contributing to potency in proportion to its lipophilicity. Excision of the
indazole ring, as in compound 49, also led to a drop in potency, but without a corresponding lowering of
clogP or boost in solubility.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Given the importance of the full BOXI core to potency and its detrimental effect on solubility, we
sought to replace the benzene “headgroup” of this core with other features that would improve solubility
3
while filling the space occupied by the aromatic ring. We hypothesized that increased sp content or more
polarity might increase solubility without a loss in potency. However, while analogs with either a
saturated cyclopentane ring (49) or a heterocyclic pyridine (50) did indeed retain sub-100 nM activity, we
did not observe an increase in solubility. Interestingly, the methoxy-substituted aminoindazole 51 retained
sub-micromolar potency with solubility of >100 μM; its reduced clogP also translated into an LLE of 5.2.
Aq.
sol.
T.b.b. MRC5
pEC50 pTC50
ID
Core
R
LLE
cLogP
(μM)
H N
2
N
N
N
2
8a
O
NH
N
4.8
5.4
<4.3
<4.3
4.1
65
0.61
NH2
N
R
H
28b
N
4.7
nd
0.76
NH2
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H N
2
N
N
N
3
1a
4.8
<4.3
4.2
58
0.64
NH2
NH2
N
N
31b
R
N
N
4.8
5.5
<4.3
<4.3
4.0
4.6
5
0.79
0.87
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
31c
N
<13
NH2
H N
2
N
N
3
4a
5.3
<4.3
3.9
660
1.4
NH2
N
N
N
34b
R
N
N
4.3
5.2
<4.3
<4.3
2.8
3.6
332
215
1.5
1.6
H
NH2
3
4c
N
NH2
O
NH
O
4
0
9
N
N
5.9
7.1
<4.3
<4.3
3.4
5.3
0.3
13
2.6
1.8
NH2
R
R
O
NH
N
4
N
N
N
NH2
H
N
O
O
NH
5
5
0*
1*
N
N
8.2
6.2
5.9
4.0
5.1
5.2
10
1.4
N
NH2
N
R
R
H
NH2
N
N
N
>100
0.71
N
H
NH2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Biological activity and selected ADME data for headgroup truncation and replacement analogs.
†Kinetic aqueous solubility. *Compound and data provided by GSK; HepG2 toxicity and kinetic aqueous
solubility. All SD within ± 0.19.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Capitalizing on the reasonable potency and excellent solubility of 51, we pursued a series
substituted aminoindazoles, the synthesis of which is shown in Scheme 4. Starting with 4-bromo-2,6-
difluorobenzonitrile 52, desired alkoxybenzonitriles 53 were synthesized by substitution with the
corresponding alcohol. The indazole ring was closed using hydrazine to produce 54, then reacted with
acetic anhydride to yield the protected indazoles 55. Miyaura borylation of the core and subsequent
Suzuki coupling with 2-amino-4-chloropyrimidine gave the final ring-opened products 57a-j. The
syntheses of compounds 63 and 68 are presented in Scheme S5-S6.
R
O
R
NH2
N
R
O
R
NH2
F
O
O
NH2
N
CN
F
a
b
c
e
CN
F
N
N
N
H
X
Br
Br
N
N
N
Br
H
O
52
53
54
57a-j
NH2
55 X = Br
6 X = B(pin)
d
5
Scheme 4. Synthesis of ring-opened analogs. Reagents and reaction conditions: a) Alcohol, LHMDS (1
M in THF), THF, 0 °C to rt, 2 days (55-86%). b) Hydrazine, ethanol, 95 °C, 12 h (71-87%). c) Boc O,
2
DMAP, DCM, rt, 12 h (75%); or Ac O, pyridine, 100 C, 3.5 h (24-75%). d) B (pin) , KOAc,
2
2
2
PdCl (dppf)·CH Cl , dioxane, 145 °C, μw, 30 min – 1.5 h. e) Aryl halide, K CO , PdCl (dppf)·CH Cl ,
2
2
2
2
3
2
2
2
3
:1 dioxane:water, 150 °C, μw 30 min, (6-26%). Where no yield is reported, crude material was
progressed without further purification.
The biological activity of these compounds is shown in Table 6. Although we were able to
1
slightly improve upon the potency of 51 by introducing bulkier alkoxy substituents at R (57a-f), the LLE
either remained the same or dropped, suggesting that more lipophilic substituents such as the t-butyl or
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phenyl were, on balance, making the compound more lipophilic without a corresponding boost in
potency. Furthermore, the phenyl substituent 57e introduced some toxicity to mammalian cells.
We hypothesized that some potency could be gained by introducing substituents containing a
hydrogen bond acceptor which could interact with the aminoindazole. This potential intramolecular
hydrogen bond could position the substituent in such a way as to mimic the orientation of the benzene
ring of the full BOXI core. Compounds 57g-j were synthesized in order to test this hypothesis;
unfortunately, none of these analogs showed increased activity. However, they displayed some of the best
overall ADME data of the series (Table S2). Finally, compounds with a phenyl- (63) or benzyl- (68)
substituted amine were tested; these compounds displayed potency in the micromolar range and did not
show significant improvement in ADME profile.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
R2
N
N
H
N
N
NH2
T.b.b. MRC5
pEC50 pTC50
Aq. sol.
(μM)
<13
196
861
354
68
nd
117
85
ID
1c
R1
R2
LLE
cLogP
3
-H
-OMe
-OEt
-OiPr
-OtBu
-OcyBu
-OPh
-NH2
-NH2
-NH2
-NH2
-NH2
-NH2
-NH2
-NH2
5.5
6.2
6.4
6.4
6.2
6.6
5.7
5.9
<4.3
<4.3
5.0
5.0
4.6
5.0
5.7
5.0
4.4
5.2
5.3
4.9
4.4
5.0
3.3
3.4
0.87
0.71
1.1
1.5
1.8
1.6
2.4
2.4
5
1
57a
57b*
57c
5
7d
7e*
7f*
5
5
-OBn
O
5
7g
-NH2
-NH2
-NH2
5.5
5.8
6.1
<4.3
<4.3
<4.3
4.9
5.1
5.0
751
770
0.62
0.68
1.13
O
O
5
7h
O
5
7i
O
1000
O
O
5
7j
-NH2
5.7
5.4
<4.3
<4.3
5.1
2.1
383
3
0.56
3.1
O
6
3
-H
-NHPh
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
8
-H
-NHBn
6.1
<4.3
2.2
17
2.9
Table 6. Biological activity and selected ADME data for ring-opened analogs. *Compound and data
provided by GSK; HepG2 toxicity and kinetic aqueous solubility. All SD within ± 0.16.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
At this point, we assessed the overall profile of several promising compounds. Table 7 represents
a heat map of an original HTS hit, NEU-1117, as well as three analogs, 18, 49, and 57a. Values
highlighted in green meet or exceed targeted values; yellow highlighting indicates mid-range values, and
red highlighting indicates values that are well outside the targeted range. All three compounds show an
improved overall profile over NEU-1117. Compounds 18 and 49 both maintain high potency against T.b.
brucei and are less toxic against MRC5 cells than NEU-1117, although 49 is highly cleared in both rat
hepatocytes and human liver microsomes. These limitations were deemed sufficient to preclude 49 from
further assessment. Compound 57a, although approximately 1.5 log units less potent than NEU-1117,
nevertheless, exhibits excellent ADME properties including high aqueous solubility, low clearance, and
low PPB. Based on these data, we selected 18 and 57a for progression to pharmacokinetic (PK) studies.
Targeted Value NEU-1117
18
7.6
4.3
330.34
2.7
>4.3
4.9
49
7.1
4.3
308.3
2.0
2.8
5.1
4.2
13
57a
6.4
4.3
270.3
1.1
1.7
5.3
4.1
861
T.b.b. pEC50
MRC5 pTC50
MW
cLogP
LogD (7.4)
LLE
>7.5
<5
≤360
≤3
≤2
≥4
8.1
5.1
360.4
2.6
>4
5.5
3.9
1
MPO Score
Aq. sol. (μM)
≥4
>10
3.9
3
HLM Cl (μg/min/mg
int
<
<
9
5
23.6
108
nd*
25
protein)
Rat Hepatocyte Cl
(μg/min/10 cells)
int
41.4
>99
12.4
52
88
20.3
89
6
PPB (%)
≤95
nd
Table 7. Heat maps of NEU-1117, 18, 49, and 57a. nd = no data. *Compound was cleared too rapidly for
detection.
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The results of the mouse PK studies of 18 and 57a are shown below. Figure 2 shows PK results
for two separate experiments: the green line represents blood concentration over time, while the red and
blue dots represent sparse sampling of blood and brain concentrations. Parameters calculated from these
curves, including Cmax, tmax, and t , are presented in Table 8. Both compounds are cleared from the blood
1/2
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within 4 h after injection, although 18 has a slightly longer half-life. Additionally, both compounds
achieve a Cmax >30× their T.b. brucei EC . Of the two compounds, 18 demonstrates higher brain
50
penetration and maintains its blood/brain ratio for 4 h.
A
B
Figure 2. Brain and blood concentrations of (A) 18 and (B) 57a over time after a 10 mg/kg
intraperitoneal dose.
T.b.b.
Cmax
ng/mL)
Brain/blood
ratio (t = 0.5 h)
Brain/blood
ratio (t = 4 h)
ID
EC50
ng/mL)
8.9
tmax (h)
t1/2 (h)
(
(
1
8
615 ± 85.8 0.25-0.5 1.06 ± 0.235
2.39 ± 1.12
2.12 ± 0.52
0.214
3
603 ±
36
0.893 ±
0.500
57a
96.2
0.25-0.5
0.155 ± 0.021
6
Table 8. PK parameters for 18 and 57a.
Because the compounds included in the HTS were designed as human kinase inhibitors, and a
compound in this cluster had known human kinase activity, we also assessed the activity of both 18 and
57a against a panel of 46 human kinases at a 1 μM concentration; Figure 3 shows the percent inhibition
of each kinase for both compounds. In general, 18 is a much more selective compound than 57a,
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inhibiting only three kinases >50% (Syk, MAP4K4, and JAK3), and two additional kinases (PKA and
IRAK4) when the cutoff is lowered to 30% inhibition. In contrast, at 1 μM 57a is a potent inhibitor of 19
kinases included in the panel and a moderate inhibitor of an additional nine (Table S3).
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Figure 3. Percent inhibition of a human kinase panel for 18 (blue) and 57a (green). Compounds were
tested at a 1 μM concentration.
Given its superior brain penetration and more selective kinase panel results as compared to 57a,
compound 18 was progressed to an in vivo efficacy model of blood-stage HAT. Infected mice with T.
brucei brucei STIB795 were treated with 18 once daily at 30 mg/kg intraperitoneally (IP) for five days;
the results are shown in Figure 4. Compared to vehicle control mice, which did not survive past day 5, all
of the mice treated with 18 survived up to day 23, and 60% of mice were cured of infection (undetectable
parasitemia on day 30). Furthermore, 18 is a fast-acting, trypanocidal compound: 80% of treated mice had
undetectable parasitemia 24 h after treatment began (Table S6).
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Treatment
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0
0
0
Control (Vehicle)
8 (30 mg/kg/day)
1
2
Infection
0
5
10
15
20
25
30
Days
Figure 4. Efficacy model results. Control mice are represented by closed circles and mice treated with 30
mg/kg/day 18 (IP dosing) are represented by open triangles.
Based on the promising results of this experiment, compound 18 was further progressed to a CNS
efficacy model to assess its potential as a stage 2 HAT therapeutic. Mice infected with T. brucei brucei
GVR35 were divided into three groups: control (Berenil 40 mg/kg pretreatment + vehicle), Treatment 1
(
Berenil 40 mg/kg pretreatment + 25 mg/kg BID 18), and Treatment 2 (25 mg/kg BID 18). Berenil®, an
anti-trypanosomal therapeutic used in livestock, was administered to control and Treatment 1 groups on
day 18 to clear blood stage parasitemia. Compound 18 was administered to Treatment 1 and 2 groups on
day 21-25, while vehicle was administered to the control group.
Figure 5 shows the relative survival and relative survival means of the three groups. Treatment
with 18 in combination with Berenil did not significantly extend life expectancy over the control group,
and the Treatment 2 group (18 alone) showed a lower relapse mean than either the control or Treatment 1
groups. In addition, toxicity was observed when dosing 18 at 50 mg/kg QD with 50% of animals
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experiencing inflammation after dosing, which resolved with time, and 50% of animals dying within 30
minutes of treatment. For these reasons, 18 was not progressed for further evaluation.
(a)
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Berenil+18 (50 mg/kg/day)
60
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2
Infection
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10
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30
40
50
Days
(b)
5
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0
0
Berenil+Control (Vehicle)
1
8 (50 mg/kg/day)
30
20
Berenil+18 (50 mg/kg/day)
1
0
0
Figure 5. CNS efficacy model results. (a) Relative survival of control mice (closed circles), Treatment 1
mice (closed triangles) and Treatment 2 mice (open circles). (b) Relative survival mean of the control
(black), Treatment 1 (white), and Treatment 2 (gray) groups.
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In addition to assessing T. brucei activity, we cross-screened selected compounds against other
parasites, including Trypanosoma cruzi (the causative agent of Chagas disease), Leishmania donovani
(one of the causative agents of leishmaniasis) and Schistosoma mansoni (a trematode flatworm and a
causative agent of schistosomiasis). The detailed activity data for T. cruzi and L. donovani are presented
in Table S4. Although only 9o showed a modest activity against L. donovani intracellular amastigotes
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with an acceptable selectivity over host cells, two compounds (NEU-1117 and 18) had pEC >7.0 against
50
T. cruzi, and three additional analogs (9j, 9k, and 20) had pEC50 ~ 6.0. Further work is ongoing to
elaborate the SAR of this series and assess its potential as anti-T. cruzi agents. Overall, the data highlight
the importance of the fully elaborated BOXI core and the 2-aminopyrimidine moiety for activity.
The data for four of the BOXIs vs. adult S. mansoni are presented in Table S5. When these
compounds were screened at 10 µM over 48 h against S. mansoni adults, a variety of phenotypic
responses and associated severity scores were elicited (Table S5). Thus, 9c, at the earliest observation
time point of 3 h, induced uncoordinated muscular activity and an inability of the oral and ventral suckers
to adhere to the substratum, which decreased over time; 9d produced a pronounced and sustained flaccid
paralysis at 3 h that was reversible upon removal of the compound; 63 elicited an uncoordinated response
at 3 h that was then replaced by decreased motility, darkening and an inability of the oral and ventral
suckers to adhere to the substratum; finally, 31c was inactive. Investigations into the anti-schistosomal
activity of other molecules in this series is continuing.
Discussion and Conclusion
Through a lead repurposing approach, we have evaluated a series of benzoxazepinoindazoles and
related compounds as potential HAT therapeutics. Detailed structure-activity and structure-property
relationships were established that enabled us to select two compounds, 18 and 57a, for further
evaluation. Given its high potency, favorable PK profile, and improved selectivity against human kinases,
18 was assessed for in vivo efficacy. Promising results were obtained with 18 in the systemic T. brucei
infection model. This, coupled with a high brain-blood ratio in pharmacokinetic experiments, led to the
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progression of 18 into the CNS model, though it was not successful in clearing parasitemia and toxicity
was observed. Further investigation to understand these findings is ongoing. In addition, several BOXIs
have been identified as potent inhibitors of T. cruzi, the causative agent of Chagas disease, and S.
mansoni, the causative agent of Schistosomiasis. Investigation of this class of compounds as anti-Chagas
agents and their potential as schistosomiasis therapeutics is likewise ongoing.
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Experimental Section
In vitro Biology. In order to determine the T. b. brucei EC50 values, 4 μL per well from
compound master plates were dispensed into a new plate and 96 μL of HMI-9 per well were added to
generate a 4% DMSO intermediate plate. Mid-log phase growth T. b. brucei was diluted to a working cell
density of 2,750 cells/mL and 90 μL/well dispensed into 96-well flat-bottom transparent assay plates
(Nunc®). Ten μL/well from intermediate plates were added so final cell concentration was 2,500 cell/mL,
and final top concentration of compounds was 40 μM in 0.4% DMSO per well. Assay plates were
incubated for 72 h at 37 ºC and 5% CO . Four hours prior to the end of the incubation, 20 μL of a 440 μM
2
resazurin solution in prewarmed HMI-9 was added to each well and incubated for another 4 h.
Fluorescence was then measured in an Infinite F200 plate reader (Tecan®) at 550 nm (excitation filter)
and 590 nm (emission filter). A four-parameter equation was employed to fit the dose-response curves
and determine EC50 using the SigmaPlot® 13.0 software. Assays were performed in duplicate at least
twice, to achieve a minimal n=2 per dose response.
Pharmacokinetics Protocols. Animals and ethical statement: All animal studies were ethically
reviewed and carried out in accordance with European Directive 2010/63/EEC and the GSK Policy on the
Care, Welfare and Treatment of Animals. Compound was administered intraperitoneally (IP) to two
groups of female NMRI mice (Group 1 n=3; Group 2 n=6) supplied by Charles River (Germany) Ltd. The
compound was prepared in 1% (v/v) DMSO:99% (v/v) 20% (w/v) sulfobutyl ether-beta-cyclodextrin
(SBE-β-CD/Captisol®, used as a solubilizing agent) in water and the dosing volume was 10 mL/kg for a
total dose of 10 mg/kg. Food and tap water were available ad libitum. Following IP dosing, Group 1
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blood samples were collected from the tail vein into capillary tubes containing K2EDTA at the following
time-points: 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h.
In order to obtain simultaneous blood and brain samples, Group 2 mice were placed under
terminal anaesthetic (isoflurane) and blood samples (0.3 mL) collected from the retro-orbital sinus into
K2EDTA tubes at 0.5 h (n= 3) and 4 h (n=3) after compound administration. Immediately following
blood sample collection, death was confirmed by cervical dislocation and the brain removed. Aliquots of
each blood sample were diluted in an equal volume of water. Mouse brain samples were weighed, water
was added at a ½ (w/v) ratio (brain/water), and then homogenized. Both blood and brain samples were
stored -80 ºC until analysis.
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Diluted blood and brain homogenates were processed under standard liquid-liquid extraction
procedures using CAN containing an internal standard (Nifedipine) and analysed by LC-MS/MS. Non-
compartmental analysis was performed using the Phoenix pharmacokinetic software version 1.4 (Certara)
and Cmax, tmax, AUClast, AUC, and t were estimated.
1/2
In vivo Efficacy Experiment Protocols. Animals and ethical statement: All procedures were
approved by ethical committee of Institute of Parasitology and Biomedicine Lopez-Neyra (Spanish
National Research Council, CSIC), code MNC.2/2015. This Institute has joined the Agreement on
Transparency in Animal Experimentation, promoted by the Confederation of Scientific Societies of Spain
(COSCE), with the collaboration of the European Association for Animal Research released on
September 20, 2016. The Animal Experimentation Unit is under the control of the competent authority,
registered in the national register as Center of Breeding and User of experimental animals code ES-
1
80210000022, according with the European and Spanish regulations. Female NMRI mice (Charles River
Laboratories) were provided with sterilized water and commercial pellets ad libitum and maintained
under the standard conditions in a conventional room at 20–24 °C with a 12/12-h light/dark cycle.
Compound 18 solution for treatment was resuspended in vehicle: 5 % DMSO in 20 %Captisol®
sulfobutyl ether β-cyclodextrin that improves solubility and stability for drug dosing.
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Blood-stage efficacy model. Infection was performed by i.p. injection of 10 bloodstream forms in
0
.2 mL TDB glucose of T. brucei brucei (STIB795) from a cryopreserved stock. Three days later, 9
infected animals with confirmed parasitemia were divided into two groups: control (infected mice treated
with vehicle, n=4) and treated (infected mice treated with 30 mg/kg/day of 18, n=5). For treatment, both
control and 18 were heated at 50 °C for 10 min to solubilize. Control and drug-treated mice received a 0.2
mL i.p. injection at the 3rd day from infection during 5 consecutive days. Parasitemia was individually
checked by direct microscopic counting of parasites in a Neubauer chamber using 2 µL of blood from
infected mice tail, diluted in 100 µL of TDB glucose. Parasitaemia of all mice were periodically checked
by tail blood examination up to day 31th. The day of death was recorded.
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CNS efficacy model. Infection was performed by i.p. injection of 2·10 bloodstream forms in 0.2
mL TDB glucose of T. brucei brucei (GVR25 strain) from a cryopreserved stock. Fourteen days later, 13
infected animals with confirmed parasitemia were divided into three equal groups: control (infected mice
pretreated with a single dose of 40 mg/kg diminazene aceturate -Berenil®- and treated with vehicle BID,
n=5); treatment 1 (infected mice pretreated with 40 mg/kg Berenil® + 25 mg/kg /day BID 11, n=4) and
treatment 2 (infected mice just treated with 25 mg/kg /day BID 18, n=4): the total concentration of 18
administered to mice was 50 /mg/kg/day. For treatment, vehicle and 18 were heated at 50 °C for 10 min
to solubilize. Mice received a 0.2 mL i.p. injection of vehicle or 18 at the 21th day from infection during 5
consecutive days. Parasitemia was individually checked by direct microscopic counting of parasites in a
Neubauer chamber using 2 µL of blood from infected mice tail, diluted in 100 µL of TDB glucose.
Parasitaemia of all mice were checked twice a week by tail blood examination and thereafter mice with
parasitaemia relapses were euthanized and the day of parasitaemia relapse was recorded. Results are
expressed as percentage of accumulative mortality and in Mean Survival Days (MSD).
General Chemistry. Reagents purchased were used as received, unless otherwise noted.
Purification of intermediates and final compounds was performed using silica gel chromatography using
the Biotage® Isolera™One flash purification system. When required, preparative HPLC was conducted
for final compounds on Waters FractionLynx system using acetonitrile/water and 0.1% formic acid
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gradient and collected based on UV monitoring at 254 nm. LCMS analysis was performed using a Waters
Alliance reverse phase HPLC (columns Waters SunFire C18 4.6 × 50 mm, 3.5 μm, or Waters SunFire C8
4
.6 × 50 mm, 3.5 μm), using a multi-wavelength photodiode array detector from 210 nm to 600 nm and
Waters Micromass ZQ detector (electrospray ionization). All compounds tested had a purity of > 95% as
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measured by LCMS. H NMR spectra were obtained with Varian NMR systems, operating at either 400
or 500 MHz at room temperature, using solvents from Cambridge Isotope Laboratories. Chemical shifts
1
1
(δ, ppm) are reported relative to the solvent peak (CDCl : 7.26 [ H]; DMSO-d : 2.50 [ H]; Acetone-d :
3
6
6
1
1
2.05; or CD OD: 3.31 [ H]). Data for H NMR spectra are reported as follows: chemical shift (ppm),
3
multiplicity (s for singlet, d for doublet, t for triplet, dd for doublet of doublet, m for multiplet), coupling
constant (Hz), and integration. Compounds obtained from GSK in-house library were not resynthesized
unless otherwise noted.
General Procedure A. Desired boronate, desired aryl halide (1.5 equiv.), and potassium
carbonate (3.5 equiv.) were suspended in 3:1 dioxane:water (0.08 M), and the reaction vial was degassed
for ~10 minutes. Pd(PPh ) (5 mol%) was added and the reaction was run under nitrogen at 100 °C until
3
4
completion as indicated by LCMS analysis (~4 h). The reaction mixture was diluted with EtOAc or
MeOH, filtered through celite, and concentrated under reduced pressure. The resulting crude residue was
purified by the stated method to afford the desired product.
General Procedure B. The desired alcohol (1.2 equiv.) was dissolved in THF (0.64 M) and 1 M
LHMDS in THF (1.2 equiv.) was added dropwise. The reaction was stirred at room temperature for ~1 h,
then cooled to 0 °C. 4-Bromo-2,6-difluorobenzonitrile 52 was added and the reaction was allowed to
warm to room temperature and stirred until completion by TLC. The reaction mixture was diluted with
EtOAc, poured over water, and extracted three times. The combined organic layers were washed once
with brine, dried with sodium sulfate and rotovapped. The crude material was purified by the stated
method to afford the title compound.
General Procedure C. The desired 4-bromo-2-alkoxy-6-fluorobenzonitrile 53 was dissolved in
ethanol (0.10 M) and hydrazine monohydrate (10 equiv.) was added. The reaction was refluxed overnight
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5
5
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5
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6
at 95 °C. The reaction was cooled to room temperature and the solvent was removed under reduced
pressure. The crude material was purified by the stated method to afford the title compound.
General Procedure D. The desired 6-bromo-4-alkoxy-1H-indazol-3-amine 54 was dissolved in
pyridine (0.09 M) and acetic anhydride (3.5 equiv.) was added. The reaction was refluxed at 100 °C for
0
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~4 h. The reaction mixture was diluted with DCM and extracted three times with 1M HCl. The combined
aqueous layers were extracted once with DCM, and the combined organic layers were washed once with
water, once with brine, dried with sodium sulfate, and concentrated under reduced pressure. The crude
material was purified by the stated method to afford the title compound.
General Procedure E. The desired protected 6-bromo-4-alkoxy-1H-indazol-3-amines 55 (1.0
equiv.), potassium acetate (3.5 equiv.), bis(pinacolato)diboron (1.5 equiv.), and PdCl (dppf)·CH Cl (0.5
2
2
2
equiv.) were combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry,
degassed dioxane (0.10 M) was added and the reaction was run in the microwave (145 °C) for 0.5 h. The
reaction mixture was diluted with EtOAc, filtered through celite, and concentrated under reduced
pressure. The crude material was either taken forward without further purification or purified by the stated
method and used as a mixture of mono- and di-protected boronic acid and ester 56.
Potassium carbonate (3.0 equiv.), 2-amino-4-chloropyrimidine (1.2 equiv.), and
PdCl (dppf)·CH Cl (0.5 equiv.) were combined in a microwave vial that was filled with nitrogen and
2
2
2
evacuated three times. The requisite boronic ester 56 or mixture of mono- and di-protected boronic acid
and ester was dissolved in 3:1 dioxane:water (0.10 M) and added to the reaction mixture. The reaction
was degassed for ~10 minutes and run in the microwave (150 °C) for 0.5-1.5 h. The reaction mixture was
diluted with MeOH, filtered through celite, and concentrated under reduced pressure. The resulting crude
residue was purified by the stated method to afford the desired product.
4
-Bromo-2,6-difluorobenzoyl chloride (2). 4-Bromo-2,6-difluorobenzoic acid 1 (3.00, 12.66 mmol) was
suspended in thionyl chloride (15 ml, 207 mmol) and the reaction was refluxed at 75 °C for
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approximately 4 h. Excess thionyl chloride was removed by distillation as the product was azeotroped
with toluene three times, affording the title compound as a yellow oil which was used in the next reaction
without further purification.
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4
-Bromo-2,6-difluoro-N-(2-hydroxyphenyl)benzamide (3). 2-Aminophenol (1.38 g, 12.65 mmol) was
dissolved in DCM (30 ml, 0.42 M), and TEA (3.5 ml, 25.11 mmol) was added. Compound 2 (3.23 g,
12.64 mmol) was dissolved in DCM (15 ml, 0.22 M), and this solution was added to the reaction mixture
dropwise at 0 °C. The reaction was allowed to warm to room temperature and stirred overnight. After
pouring the reaction mixture over 1M HCl, the title compound was isolated by vacuum filtration as an
+
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off-white solid (3.62 g, 87%). LCMS [M+H] 327.95 m/z ( Br), 329.96 m/z ( Br); H NMR (500 MHz,
DMSO-d ) δ ppm 10.03 (s, 1 H) 9.88 (br. s., 1 H) 7.88 (dd, J=8.1, 1.2 Hz, 1 H) 7.60 (d, J=7.3 Hz, 2 H)
6
6
.97 - 7.03 (m, 1 H) 6.91 (dd, J=8.1, 1.2 Hz, 1 H) 6.78 - 6.84 (m, 1 H).
3
-Bromo-1-fluorodibenzo[b,f][1,4]oxazepin-11(10H)-one (4). Compound 3 (3.62 g, 11.04 mmol) was
dissolved in DMF (30 ml, 0.37 M) and potassium carbonate (3.05 g, 22.06 mmol) was added. The
reaction was stirred at room temperature for two days and was then poured over water. The title
+
compound was isolated by vacuum filtration as an off-white solid (2.44 g, 72%). LCMS [M+H] 307.99
7
9
81
1
m/z ( Br), 309.96 m/z ( Br); H NMR (500 MHz, DMSO-d ) δ ppm 10.74 (br. s., 1 H) 7.61 (s, 1 H) 7.57
6
(dd, J=9.8, 1.5 Hz, 1 H) 7.37 - 7.41 (m, 1 H) 7.22 (dd, J=8.3, 1.5 Hz, 1 H) 7.14 - 7.19 (m, 2 H).
3
-Bromo-1-fluorodibenzo[b,f][1,4]oxazepine-11(10H)-thione (5). Compound 4 (2.44 g, 7.91 mmol)
was suspended in toluene (160 ml, 0.05M) and Lawesson's reagent (3.49 g, 8.63 mmol) was added. The
reaction was refluxed at 100 °C overnight. Upon cooling to room temperature, a yellow precipitate was
observed and collected by vacuum filtration to afford the title compound as a yellow solid (1.75 g) which
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was carried forward without further purification. LCMS [M+H] 323.93 m/z ( Br), 325.95 m/z ( Br).
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4
-Bromo-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (6). Compound 5 (1.00 g, 3.09
mmol) was suspended in dioxane (10 ml, 0.3 M) and hydrazine monohydrate (0.30 ml, 6.11 mmol) was
added. The reaction was refluxed for 3 h at 85 °C. After cooling to room temperature, the reaction
mixture was diluted with DCM and concentrated under reduced pressure. The residue was purified by
flash chromatography (0-5% MeOH:DCM) to afford the title compound as an off-white solid (833 mg,
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9%). LCMS [M+H] 301.97 m/z ( Br), 303.99 m/z ( Br); H NMR (400 MHz, DMSO-d ) δ ppm 12.29
6
(s, 1 H) 9.52 (s, 1 H) 7.31 (d, J=1.5 Hz, 1 H) 7.26 (dd, J=8.1, 1.5 Hz, 1 H) 7.23 (dd, J=8.1, 1.5 Hz, 1 H)
7.04 - 7.09 (m, 1 H) 6.85 - 6.91 (m, 2 H).
1
-(4-Bromobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-11(2H)-yl)ethan-1-one
and
1-(4-
bromobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-2(11H)-yl)ethan-1-one (7). Acetic anhydride (12.0
ml, 127.18 mmol) was added to 6 (833 mg, 2.76 mmol) and the reaction was refluxed at 100 °C for ~3 h.
Upon cooling to room temperature, a bright yellow precipitate was observed and collected by vacuum
filtration to afford the title compounds as a mixture of isomers. (Bright yellow solid, 885 mg, 93%).
+
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LCMS [M+H] 343.99 m/z ( Br), 345.96 m/z ( Br).
1-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-
1
1(2H)-yl)ethan-1-one (8). Compound 7 (401 mg, 1.17 mmol), potassium acetate (405 mg, 4.13 mmol),
bis(pinacolato)diboron (443 mg, 1.74 mmol), and PdCl (dppf)·CH Cl (46 mg, 0.06 mmol) were
2
2
2
combined in a reaction vial that was filled with nitrogen and evacuated three times. Dry, degassed
dioxane (8.0 ml, 0.15 M) was added and the reaction was run in the microwave (145 °C) for 1 h. The
reaction mixture was then diluted with EtOAc, filtered through celite, and concentrated under reduced
pressure. The residue was purified by flash chromatography (0-50% EtOAc:hexanes) to afford the title
+
1
compound as an off-white solid (297 mg, 65%). LCMS [M+H] 392.13 m/z; H NMR (500 MHz,
DMSO-d ) δ ppm 10.03 (s, 1 H) 8.25 (s, 1 H) 7.27 (dd, J=7.6, 3.2 Hz, 2 H) 7.26 (s, 1 H) 7.11 (t, J=7.6
6
Hz, 1 H) 6.98 (t, J=7.3 Hz, 1 H) 2.62 (s, 3 H) 1.34 (s, 12 H).
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4
-(Pyrimidin-4-yl)-2,11-dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazole (9b). The title compound
was prepared according to General Procedure A on a 40-mg scale using 8 and 4-chloropyrimidine·HCl.
Upon cooling to room temperature, a precipitate was observed and collected by vacuum filtration. The
precipitate was suspended in DME (0.75 ml, 0.06 M) and concentrated HCl (0.25 ml, 3.00 mmol) was
added. This suspension was stirred at 85 °C for ~1 h, then cooled to room temperature. The resulting
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precipitate was collected to afford the title compound as an orange solid (12 mg, 40%). LCMS [M+H]+
1
302.09 m/z; H NMR (400 MHz, DMSO-d ) δ ppm 12.55 (br. s., 1 H) 9.59 (s, 1 H) 9.28 (s, 1 H) 8.89 (d,
6
J=5.9 Hz, 1 H) 8.24 (dd, J=5.1, 1.5 Hz, 1 H) 8.00 (s, 1 H) 7.58 (s, 1 H) 7.29 (dd, J=8.1, 1.5 Hz, 2 H) 7.08
(t, J=7.7 Hz, 1 H) 6.90 (t, J=6.6 Hz, 1 H).
6
-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-4-amine (9c). The title
compound was prepared according to General Procedure A on a 41-mg scale using 8 and 4-amino-6-
chloropyrimidine. The crude material was purified preparative HPLC (5-50% water:acetonitrile) to afford
+
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the title compound as a yellow solid (15 mg, 45%). LCMS [M+H] 317.11 m/z; H NMR (400 MHz,
DMSO-d ) δ ppm 12.36 (s, 1 H) 9.52 (s, 1 H) 8.46 (s, 1 H) 7.74 (s, 1 H) 7.23 - 7.32 (m, 3 H) 7.06 (t,
6
J=7.3 Hz, 1 H) 6.99 (s, 1 H) 6.92 (s, 2 H) 6.89 (t, J=8.1 Hz, 1 H).
2
-(2,11-Dihydrobenzo[2,3][1,4]oxazepino[5,6,7-cd]indazol-4-yl)pyrimidin-4-amine (9d). The title
compound was prepared according to General Procedure A on a 41-mg scale using 8 and 4-amino-2-
chloropyrimidine. The crude material was purified by preparative HPLC (5-50% acetonitrile:water) to
+
1
afford the title compound as a yellow solid (17 mg, 51%). LCMS [M+H] 317.11 m/z; H NMR (400
MHz, DMSO-d ) δ ppm 12.32 (s, 1 H) 9.49 (s, 1 H) 8.20 (d, J=5.9 Hz, 1 H) 8.07 (s, 1 H) 7.71 (s, 1 H)
6
7
1
.28 (d, J=8.1 Hz, 2 H) 7.06 (t, J=6.6 Hz, 1 H) 6.96 (br. s., 2 H) 6.88 (t, J=7.3 Hz, 1 H) 6.39 (d, J=5.9 Hz,
H).
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