Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL.
Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL.
Visceral leishmaniasis
(VL) is a poverty-linked disease,[1] and
occurs primarily in poor populations across
Asia, East Africa, and South America.[2] Current
estimates suggest 50 000–90 000 new cases of
VL occur each year.[3] VL is caused by infection
with the protozoan parasites Leishmania donovani and Leishmania infantum, with transmission
mediated by the bite of the female phlebotomine sand fly. Once the
human host is infected, parasites survive and multiply within macrophages,
leading to symptoms including prolonged fever, enlarged spleen and
liver, substantial weight loss, and progressive anemia.[4] If left untreated, the vast majority of clinically
symptomatic VL patients die within months.The current standard
treatment for patients with VL in East Africa
is sodium stibogluconate (SSG) combined with paromomycin (PM).[4] In South-East Asia, the standard treatment is
liposomal amphotericin B (LAB), with paromomycin and miltefosine as
a second-line treatment option.[5] While
these combination approaches are more effective than SSG monotherapy,
the mainstay of VL treatment for many years, they still have significant
limitations such as cost, route of administration (all given via the
parenteral route apart from the oral drug miltefosine), and toxicity.
Consequently, there is an ongoing need for effective new treatments
that are easy to administer via oral dosing, are effective against
VL in different regions of the world, have an improved safety profile,
and are affordable for the patients who require them. Research and
development for the treatment of VL has evolved rapidly in recent
years, with 5 drug candidates from 4 different classes now in phase-1
clinical studies (Table ).[6]
Table 1
Current Portfolio
of VL Drug Candidates
in Clinical Developmenta
compound ID
compound class
mechanism of action and/or molecular target
organization
DNDI-6148[6]
benzoxaborole
CPSF3
DNDi/Pfizer
DNDI-0690[6]
nitroimidazole
bioactivation by NTR2[7]
DNDi/TB Alliance
GSK3186899/DDD853651[6,7]
pyrazolopyrimidine
CRK12 (cyclin-dependent
kinase)[8]
GSK/DDU
GSK3494245/DDD1305143[6,8]
imidazopyridine
proteasome inhibitor[9]
GSK/DDU
LXE408[9]
triazolopyrimidine
proteasome inhibitor[10]
Novartis
DDU: Drug Discovery Unit, University
of Dundee and GSK: GlaxoSmithKline.
DDU: Drug Discovery Unit, University
of Dundee and GSK: GlaxoSmithKline.Here, we outline the optimization of a benzoxaborole
series toward
a possible new therapy for VL. The benzoxaborole class of compounds
has already successfully delivered a clinical candidate (acoziborole,
AN5568, 1, Figure ) for the treatment of stage 1 and stage 2 human African trypanosomiasis
(HAT, African sleeping sickness).[11] Stimulated
by the success of the acoziborole program, modifications to the benzoxaborole
template were undertaken to identify novel compounds that may have
utility for the treatment of VL, leading to the discovery of a preclinical
candidate 23 (DNDI-6148). This compound combines good in vitro potency against both L. infantum and L. donovani alongside very high
levels of in vivo efficacy (>98% reduction in
parasite
burden) in hamster models of infection. In addition, DNDI-6148 possesses
excellent pharmacokinetics and is predicted to have acceptable human
pharmacokinetics and an efficacious dose consistent with the requirements
of the published target candidate (TCP) and target product (TPP) profiles
for VL published by DNDi. Clinical trials with DNDI-6148
are now underway.[11]
Figure 1
Structures of acoziborole
(1), early hit 3, and preclinical
candidate DNDI-6148 (23).
Structures of acoziborole
(1), early hit 3, and preclinical
candidate DNDI-6148 (23).
Results
and Discussion
Chemistry
The synthesis of benzoxaborole
target compounds 3–31 consisted of
amide bond formation
between the key intermediate 2 and an appropriately functionalized
acid (Scheme ) using
one of three coupling methods, whose choice was driven by substrate
compatibility: Method A (HOBt/EDCI coupling), Method B (HATU coupling),
and Method C (acid chloride formation using SOCl2 followed
by coupling with intermediate 2). The synthesis of the
carboxylic acid intermediates R2CO2H is described
in detail in the Supplementary Information. Example 12 was synthesized using Method A. Examples 4, 6–11, 13–15, 17–18, 20–21, 23–27,
and 29–30 were synthesized using
Method B, while examples 5, 16, 19, 22, 28, and 31 were synthesized
using Method C.
Reagents and conditions:
Method A: HOBt, EDCI, N,N-diisopropylethylamine (DIPEA), CH2Cl2, rt,
2 h, 66%; Method B: HATU, DIPEA, N,N-dimethylformamide (DMF), rt, 16 h, 8–77%; and
Method C: (i) SOCl2, 50 °C, 4
h, (ii) DIPEA, tetrahydrofuran (THF), rt, 16 h, 4–68%.Preclinical candidate compound 23 was synthesized
using a 4-step synthesis (Scheme ).
Scheme 2
Synthesis of Compound 23
Reagents
and conditions: (i)
NaN3, CuI, NaOAc, DMEDA, EtOH, H2O, 1 h, reflux,
94%; (ii) ethyl 3-oxobutanoate, NaOEt, EtOH, 48 h, reflux; (iii) NaOH,
H2O, EtOH, 20 h, 45 °C, 48% for (ii) and (iii); and
(iv) 2, HATU, DIPEA, DMF, room temperature (RT), 3 h,
74%. Note, the material subsequently used in the mode of action studies
was prepared from commercially available 23-2 via a modification
of this route (see Scheme ).
Synthesis of Compound 23
Reagents
and conditions: (i)
NaN3, CuI, NaOAc, DMEDA, EtOH, H2O, 1 h, reflux,
94%; (ii) ethyl 3-oxobutanoate, NaOEt, EtOH, 48 h, reflux; (iii) NaOH,
H2O, EtOH, 20 h, 45 °C, 48% for (ii) and (iii); and
(iv) 2, HATU, DIPEA, DMF, room temperature (RT), 3 h,
74%. Note, the material subsequently used in the mode of action studies
was prepared from commercially available 23-2 via a modification
of this route (see Scheme ).
Scheme 5
Synthetic Route to DNDI-6148 (23) Employed
for the Material
Used in the MoA Studies
Reagents and conditions: (a)
NaOEt, EtOH, 150 °C, 2 h (flow), 38%; (b) LiOH, THF/water, room
temp., 16 h, 97%; and (c) 2, T3P, DIPEA, DMF/EtOAc, RT,
16 h, 29%.
Formation of the fused tetrazole 23-2 was achieved
by the reaction of 2-bromopyridine with sodium azide. Cyclo-addition
of 23-2 with ethyl 3-oxobutanoate gave the N1-substituted
triazole 23-3, which was hydrolyzed to the corresponding
acid 23-4. Coupling benzoxaborole scaffold 2 using Method B gave the final compound 23 in high overall
yield.Compound 1 was potent in in vitro assays against Trypanosoma brucei, the etiological agent of HAT (EC50 value of 795 nM),[12] but demonstrated only weak activity in intramacrophage
assays with both L. infantum and L. donovani (Table ). It should be noted that L. infantum-derived infections can be refractory to some current drug therapies.
Thus, new compounds in development are screened against both L. infantum and L. donovani at an early stage to ensure efficacy. However, the close analogue 3 (SCYX-6759), lacking the gem-dimethyl substitution on the
benzoxaborole ring, was considerably more potent in these assays.[13]
Table 2
Collated EC50 Values for
Compounds 1, 3 and the Current Front-Line
Antileishmanial Miltefosine
EC50 values, μMa
compound
intramacrophage L. donovani
intramacrophage L. infantum
PMM
1
59
>64
>64
3
1.2
2.7
>64
23
1.4
1.8
>64
miltefosine
10
10
33
Geometric mean
value of at least
three independent assays. PMM: primary mouse macrophages.
Geometric mean
value of at least
three independent assays. PMM: primary mouse macrophages.Previous studies demonstrated that
compound 3 has
low clearance in multiple animal models and is efficacious in a murine
model of HAT.[13] Consequently, compound 3 was progressed to an L. infantum hamster model of VL infection (Table ).[14] Compound 3 was highly efficacious at QD doses of 50 and 100 mg/kg, virtually
clearing all parasites from the liver and spleen. However, reductions
in parasite burden in the bone marrow were more modest and thus failed
to meet the DNDi target candidate profile (TCP) of
>95% reduction in parasite burden. Therefore, additional benzoxaboroles
were synthesized to identify compounds that were highly efficacious
in all tissues across a broad dose range.
Table 3
In Vivo Efficacy
of Compound 3 in an L. infantum Hamster Model of VL, after QD Dosing for 5 Daysa
reduction
in parasite burden, %
dose (mg/kg)
liver
spleen
bone marrow
25
87.8
81.8
81.6
50
97.4
95.0
91.2
100
98.6
96.0
88.3
n = 6 animals per
group, results were expressed as a percentage reduction in amastigote
burden compared to vehicle-treated, infected control animals.
n = 6 animals per
group, results were expressed as a percentage reduction in amastigote
burden compared to vehicle-treated, infected control animals.The simplified benzoxaborole ring
system, with the gem-dimethyl
substitution on the benzoxaborole ring removed, was retained in subsequent
analogues and the amide substituent became the focus for development.
Replacing the lipophilic aryl substituent with a more polar heteroaryl
group was investigated in detail to reduce the lipophilicity to values
more associated with orally bioavailable drugs.[15] Consistent with this design strategy, a range of substituted
pyrazoles was synthesized (Table ). Excellent levels of in vitro potency
were achieved with compounds 4–6,
which contain a trifluoromethyl R1 substituent and a fluorinated N-alkyl R2 substituent. Importantly, compounds 4–6 showed no evidence of cytotoxicity (CC50 > 38 μM) in the counter-screen with PMM. These analogues
also
had excellent in vitro metabolic stability in HLM,
but compounds 4 and 6 had poor stability
in hamster liver microsomes (HamLM), which precluded their evaluation
in the in vivo hamster model of VL. Compound 5 combined sufficient in vitro potency, selectivity
index (SI), and stability in both HLM and HamLM to be considered for
further progression. Compound 7 (R1 = Me)
was slightly less potent than its CF3 analogue 6, and this same trend was also observed for the N-cyclobutyl examples 8 and 9 as well as
for the 2-pyridyl examples 11 and 12. However,
the CF3-substituted analogues 6, 9, and 12 were all more cytotoxic in PMM than their CH3-substituted counterparts 7, 8,
and 11. Tetrahydropyranyl-substituted analogue 10 combined many of the necessary properties, but HamLM stability
was insufficient to deliver enough in vivo exposure
for efficacy studies. Typically, a HamLM clearance of <100 μL/min/mg
is required to observe meaningful exposure in vivo. Further lipophilic aryl R2 substituents such as 4-Cl-phenyl 13 and 2-MeO-phenyl 14 had potent in
vitroL. infantum activity
but were cytotoxic in PMM and/or MRC5 cells. The pyrazole 15 exhibited a significant drop in potency compared to its isomeric
analogue 6, whereas the pyridyl-substituted compound 16 was slightly more potent than its isomer 11. Further substituted pyridine examples 17 and 18 were also potent and demonstrated good SI. In addition,
compounds 16–18 were stable in both
HLM and HamLM. Additional pyrazole isomers 19–21 all failed to combine sufficient in vitro potency and weak cytotoxicity with good metabolic stability.
Table 4
Antileishmanial Activity of Pyrazoles 4–21
MRC5a
HLM
HamLM
compound
R1
R2
intramacrophage L. infantum EC50 (μM)a
PMMa CC50 (μM)
CC50 (μM)
SIb
Clint (μL/min/mg)
3
2.7
>64
>64
>24
0.8
n.d.
4
CF3
CF3
2.1
>64
>64
>31
<11.9
1,035
5
CF3
CH2CF3
1.6
50
>64
>40
<11.9
41
6
CF3
(CH2)2CF3
1.3
38
49
38
<11.9
114
7
CH3
(CH2)2CF3
5.7
>64
>64
>11
n.d.
n.d.
8
CH3
c-butyl
16
>64
>64
>4
<11.9
57
9
CF3
c-butyl
0.59
32
26
53
<11.9
337
10
CF3
4-THP
3.0
>64
>64
>21
<11.9
109
11
CH3
2-pyridyl
5.7
>64
>64
>11
<11.9
n.d.
12
CF3
2-pyridyl
0.64
2.0
1.1
1.6
n.d.
n.d.
13
CF3
4-Cl-phenyl
1.3
8.0
2.6
2
n.d.
n.d.
14
CH3
2-MeO-phenyl
2.2
>64
15.7
7.3
n.d.
n.d.
15
CH3
(CH2)2CF3
14
>64
>64
>5
<11.9
48
16
CH3
2-pyridyl
3.1
59
61
15
<11.9
57
17
CH3
2-pyridyl-6-OMe
1.4
>64
40
31
<11.9
40
18
CH3
2-pyridyl-6-Me
1.6
>64
41
25
<11.9
44
19
H
2-pyridyl
0.5
32
4.0
8
<11.9
253
20
CH3
2-pyridyl
34
>64
>64
>2
18
121
21
CH3
2-pyridyl
1.8
>64
54
31
14
106
miltefosine
10
33
>64
>6.2
n.d.
n.d.
Geometric mean value of at least
two independent tests.
Selectivity
index representing the
ratio of CC50/EC50; data are presented as the
geometric mean value of ratios calculated using separate data from
at least two independent tests. n.d., not determined.
Geometric mean value of at least
two independent tests.Selectivity
index representing the
ratio of CC50/EC50; data are presented as the
geometric mean value of ratios calculated using separate data from
at least two independent tests. n.d., not determined.Of the compounds made up to this
point, 2-pyridyl-substituted analogue 16 was one of the
most interesting as it combined good in vitro potency,
low lipophilicity, promising SI, and was
stable in both HLM and HamLM. For the next set of compounds, the 2-pyridyl
substituent was retained and further changes to the 5-membered heterocycle
were investigated (Table ). Triazole 22, where R1 = H, showed
an encouraging improvement in both in vitro potency
and HamLM stability over pyrazole 16. The Me-substituted
example 23 had an even better profile with high levels
of L. infantum potency, no sign of
cytotoxicity in PMM and MRC5, and very high levels of metabolic stability
in HLM and HamLM. Further 1,2,3-triazoles demonstrated that the 2-pyridyl
substituent was important; for example, the phenyl example 24 and 3-pyridyl example 27 had significantly weaker in vitro potency. Substitution on the 2-pyridyl group could
be tolerated, with the 6-OMe analogue 25 showing an improved
potency profile but at the expense of metabolic stability. The 6-Me
compound 26 lost some potency and was therefore not progressed
to metabolic stability assessment. Furan example 28 was
very potent, and although it did exhibit some cytotoxicity in both
PMM and MRC5, the high level of potency meant that 28 still retained a high SI. Despite being more lipophilic, metabolic
stability was retained in both HLM and HamLM and, therefore, 28 was considered for further progression. The isomeric furan 29 had slightly reduced potency and SI compared to 28, and the thiophene 30 was highly cytotoxic in MRC5
cells. The more polar oxadiazole 31 delivered a good
overall profile and was also considered for further progression.
Table 5
Antileishmanial Activity of Compounds 22–31
MRC5a
HLM
HamLM
compound
R1
R2
intramacrophage L. infantum amastigote EC50 (μM)a
PMMa CC50 (μM)
CC50 (μM)
SIb
Clint (μL/min/mg)
22
H
2-pyridyl
1.7
>64
44.8
30
<11.9
18
23
CH3
2-pyridyl
1.8
>64
>64
>39
<11.9
<11.9
24
CH3
phenyl
37
>64
>64
>1.7
<11.9
n.d.
25
CH3
2-pyridyl-6-OMe
0.8
32
>64
>80
23
40
26
CH3
2-pyridyl-6-Me
6.4
>64
>64
>10
n.d.
n.d.
27
CH3
3-pyridyl
38
>64
>64
>1.8
n.d.
n.d.
28
CH3
2-pyridyl
0.5
49
18
37
<11.9
40
29
CH3
2-pyridyl
1.7
29
45
18
<11.9
82
30
CH3
2-pyridyl
0.6
32
2.3
4
<11.9
88
31
2-pyridyl
2.0
>64
>64
>32
19
12
Geometric mean value of at least
two independent tests.
Selectivity
index representing the
ratio of CC50/EC50; data are presented as the
geometric mean value of ratios calculated using separate data from
at least two independent tests. n.d., not determined.
Geometric mean value of at least
two independent tests.Selectivity
index representing the
ratio of CC50/EC50; data are presented as the
geometric mean value of ratios calculated using separate data from
at least two independent tests. n.d., not determined.The most promising compounds from
this SAR investigation, namely 5, 16, 23, 28, and 31, were also assessed
for their in vitro macrophage potency using intracellular L. donovani (Table ). Pleasingly,
four of these compounds retained excellent levels of L. donovani potency; however, the oxadiazole 31 was about 5-fold weaker against L. donovani compared with its L. infantum potency.
Table 6
Antileishmanial Activity of Selected
Compounds against L. donovani
MRC5a
compound
intramacrophage L. infantum EC50 (μM)a
intramacrophage L. donovani EC50 (μM)a
PMMa CC50 (μM)
CC50 (μM)
SIb
5
1.6
1.0
53.8
>64
>64
16
3.1
1.2
>64
61.1
52
23
1.8
1.4
>64
>64
>46
28
0.5
0.4
54
18
45
31
2.0
9.4
>64
>64
>7
Geometric mean value of at least
two independent tests.
Selectivity
index representing the
ratio of CC50/EC50; data are presented as the
geometric mean value of ratios calculated using separate data from
at least two independent tests.
Geometric mean value of at least
two independent tests.Selectivity
index representing the
ratio of CC50/EC50; data are presented as the
geometric mean value of ratios calculated using separate data from
at least two independent tests.Compounds 5, 16, 23, 28, and 31 were also progressed to hamster oral
pharmacokinetic (PK) studies and an L. infantum model of VL in hamsters (Table ). Triazole 23 exhibited noticeably higher
exposure after a single oral dose of 50 mg/kg compared with the other
lead compounds, which is consistent with its higher in vitro stability in HamLM and indicated that in vitro metabolic
clearance may be predictive of in vivo clearance
for this set of compounds.
Table 7
Oral Exposure and In Vivo Efficacy of Selected Compounds in an L. infantum Hamster Model of VL Following BID Dosing
for 5 and 10 Daysa
reduction
in parasite burden, %
liver
spleen
bone
marrow
compound
dose (mg/kg), BID
5 days
10 days
5 days
10 days
5 days
10 days
AUC0–24 after single p.o.
dose of 50 mg/kg (h.ng/mL)
5
25
89.7
n.d.
90.0
n.d.
77.0
n.d.
21 000
50
99.7
n.d.
99.2
n.d.
98.0
n.d.
16
25
87.8
n.d.
87.7
n.d.
72.2
n.d.
7900*
50
99.6
n.d.
98.5
n.d.
95.1
n.d.
23
25
98.3
100
98.2
99.9
93.6
99.4
120 000
50
99.8
n.d.
99.9
n.d.
99.0
n.d.
28
25
98.9
99.4
97.8
96.9
95.7
84
21 000
50
99.9
n.d.
99.9
n.d.
99.1
n.d.
31
25
67.3
n.d.
69.0
n.d.
55.0
n.d.
21 000
50
99.4
n.d.
99.4
n.d.
99.7
n.d.
n = 6 animals per
group, the results are expressed as a percentage reduction in amastigote
burden compared to vehicle-treated, infected control animals, *Study
run using a single 25 mg/kg p.o. dose.
n = 6 animals per
group, the results are expressed as a percentage reduction in amastigote
burden compared to vehicle-treated, infected control animals, *Study
run using a single 25 mg/kg p.o. dose.All five compounds demonstrated excellent efficacy,
with parasitemia
significantly reduced in all organs after a dose of 50 mg/kg BID for
5 days. Triazole 23 and furan 28 also maintained
excellent efficacy at a lower dosing of 25 mg/kg BID, consistent with
the combination of good potency and high exposure observed for 23 and the very high potency of 28. Extending
the duration of treatment from 5 to 10 days for compound 23 led to almost complete eradication of parasites in the liver, spleen,
and bone marrow. In contrast, the efficacy profile of compound 28 was not improved by extending the dosing period.Compounds 23 and 28 were then studied
in an L. donovani hamster model (Table ). Triazole 23 exhibited high levels of efficacy in the liver and spleen
when dosed orally at 50 mg/kg BID for 5 days, although the reduction
in parasite load in bone marrow was lower than the target of 95%.
However, by extending the dosing period to 10 days, very high efficacy
across all three tissues was achieved at a dose of 25 mg/kg BID. In
addition, furan 28 achieved excellent efficacy in all
tissues at both 25 mg/kg BID and 50 mg/kg BID for 5 days. These important
data demonstrated that for both L. infantum and L. donovani, potent in
vitro and in vivo activity could be achieved.
Table 8
In Vivo Efficacy
of Selected Compounds in an L. donovani Hamster Model of VL Following BID Dosing for 5 and 10 Daysa
reduction
in parasite burden, %
liver
spleen
bone
marrow
compound
dose (mg/kg),
BID
5 days
10 days
5 days
10 days
5 days
10 days
23
25
n.d.
99.9
n.d.
99.8
n.d.
99.6
50
99.3
n.d.
98.6
n.d.
83.4
n.d.
28
25
95.9
n.d.
95.9
n.d.
94.5
n.d.
50
99.9
n.d.
100
n.d.
100
n.d.
n = 6 animals per
group, results were expressed as a percentage reduction in amastigote
burden compared to vehicle-treated, infected control animals, n.d.,
not determined.
n = 6 animals per
group, results were expressed as a percentage reduction in amastigote
burden compared to vehicle-treated, infected control animals, n.d.,
not determined.Compounds 23 and 28 were also assessed
in mouse models of L. donovani and L. infantum; data confirmed that the high levels
of efficacy observed in the chronic hamster model of infection was
also achieved in the acute mouse model (Table ).
Table 9
In Vivo Efficacy
of Selected Compounds in L. donovani and L. infantum Mouse Models of VL
Following BID Dosing for 5 Days
reduction
in liver parasite burden following 5 days of p.o. dosing BIDa, %
compound
dose (mg/kg),
BID
L. infantum
L. donovani
23
25
96.1
97.1
50
98.6
99.7
28
25
n.d.
97.5
50
n.d.
99.5
n = 5 animals per
group, the results were expressed as a percentage reduction in parasite
burden compared to vehicle-treated, infected control animals, n.d.,
not determined.
n = 5 animals per
group, the results were expressed as a percentage reduction in parasite
burden compared to vehicle-treated, infected control animals, n.d.,
not determined.Based on
their in vitro potency, metabolic stability,
and in vivo pharmacokinetic and efficacy profiles,
compounds 23 and 28 were investigated in
more detail to determine if either could be progressed to further
preclinical development. Encouraged by the outstanding in
vivo efficacy of compound 28, a 14-day exploratory
toxicology study was conducted in male and female Sprague-Dawley rats.
Unfortunately, compound 28 caused significant multiorgan
toxicity following daily dosing of 25 mg/kg and the NOAEL (No Observed
Adverse Effect Level) was not determined (<12.5 mg/kg). Consequently,
further development of compound 28 was halted.One potential concern associated with compound 28 was
the presence of the furan, a functional group that can undergo oxidative
metabolism to reactive metabolites.[16] Thus, 23 and 28 were investigated to determine potential
routes of metabolism and to understand if protein–ligand adducts
were formed when the compounds were incubated in microsomes in the
presence of glutathione. Metabolite identification studies with compound 28 showed evidence of ring-opening of the furan to an enone
M-12 metabolite in all species tested (rat, mouse, dog, human, hamster).
This metabolite has the potential to undergo conjugation to form protein–ligand
adducts (Scheme ).
Scheme 3
Metabolic Fate of Compound 28 in the Presence of Microsomes
from Multiple Species
In contrast, incubation of triazole 23 with microsomes
indicated very limited metabolism, with only small amounts of mono-oxidation
and hydrolysis/oxidative deboronation observed in dog and human microsomes,
respectively (Scheme ). No metabolism was detected in rat microsomes, and no evidence
of reactive metabolite formation was found in any species.
Scheme 4
Metabolic
Fate of Compound 23 in the Presence of Microsomes
In addition to the safety signals raised by
a moderate in vitro activity against the MRC5 cell
line (Table ) and
the lack of efficacy improvement
when treatment duration was extended in the hamster model (Table ), these in
vitro metabolic studies clearly identified a potential weakness
with furan 28 that may contribute to the observed in vivo toxicity. Therefore, 23 was prioritized
for further evaluation. CYP inhibition was weak (IC50 >
50 μM vs CYP1A2, 2C9, 2C19, 3A4, 2D6), hERG selectivity was
high (hERG inhibition < 20% at 30 μM), and no activity >
50% was observed when 23 was tested in a panel of 88
targets (Cerep panel, Figure S13) at a
concentration of 10 μM. Plasma-protein binding was moderate
and consistent across species (dog 87%, human 92%, mouse 93%, rat
92%, hamster 88%). Thermodynamic solubility in physiologically relevant
media was low to moderate (Fasted State Simulated Intestinal Fluid,
FaSSIF solubility = 5.4 μg/mL, Fed State Simulated Intestinal
Fluid, FeSSIF solubility = 6.3 μg/mL), and membrane permeability
was good with no evidence of P-gp-mediated efflux (MCDK efflux ratio
of 0.7, Table S3). Microsome and hepatocyte
stability was excellent across all species (Table ), although hepatocyte stability was slightly
worse in dogs compared with rats, monkeys, and humans. Compound 23 was also negative when tested in an Ames assay (TA98 and
TA100 bacterial strains) with and without metabolic activation over
a dose range from 1.5 to 1000 μg/well, suggesting no clastogenicity.
To complete the early safety profile of compound 23,
a 14-day exploratory toxicology study was conducted in male and female
Sprague-Dawley rats where the no adverse effect limit was set at 50
mg/kg/day.
Table 10
In Vitro Metabolic
Stability Across Species for Compound 23
species
mouse
rat
dog
human
microsome Clint (μL/min/mg protein)
<11.9
<11.9
5.8
<11.9
hepatocyte
Clint (μL/min/million cells)
n.d.
0.7
2.4
0.5
In vivo pharmacokinetics after i.v.
and p.o. dosing
was determined in rats and dogs (Table and Figure S12). Clearance in rats was low (4.5 mL/min/kg) with comparatively higher
clearance in dogs (13.7 mL/min/kg), in line with the higher turnover
in dog hepatocytes compared with rat hepatocytes. Oral bioavailability
in rats was very high, indicating complete oral absorption. Bioavailability
was somewhat lower in dogs and not fully understood at this point,
but still acceptable for future progression of the compound. Allometric
scaling from available rat and dog PK data suggests that compound 23 will have low clearance (Cl = 1–4 mL/min/kg) and
moderate–high bioavailability (40–90%) in humans. BID
dosing in humans at 3–20 mg/kg is predicted to achieve the
efficacious exposure observed in the L. infantum and L. donovani hamster and mouse
models of VL.
Table 11
In Vivo Pharmacokinetics
of 23 in Rat and Dog
species
rata
dogb
route of administration
i.v.
p.o.
i.v.
p.o.
clearance (mL/min/kg)
4.5
13.7
Vd (L/kg)
0.6
1.3
T1/2 (h)
2.8
1.5
F (%)
94
39
n = 3 male SD
rats, 2 mg/kg i.v., 10 mg/kg p.o.
n = 2 male Beagle
dogs, 1 mg/kg i.v., 5 mg/kg p.o.
n = 3 male SD
rats, 2 mg/kg i.v., 10 mg/kg p.o.n = 2 male Beagle
dogs, 1 mg/kg i.v., 5 mg/kg p.o.
Mode of Action Studies (MoA)
MoA studies as an integrated
part of a drug discovery program can provide vital information that
can be used to combat the high failure rates associated with the development
of phenotypically active compounds. The association of active compounds
with defined molecular targets during the development process can
be extremely powerful. Understanding compound MoA can allow toxic
liabilities associated with the target to be directly assessed, prevent
enrichment of drug candidates against the same molecular target, and
halt the development of inhibitors with an unattractive or invalidated
target. Furthermore, this knowledge can inform future drug combination
strategies.Our previous studies with acoziborole (AN5568, 1), in clinical development for HAT, revealed that this promising
benzoxaborole specifically targets the Cleavage and Polyadenylation
Specificity Factor 3 (CPSF3) in T. brucei.[17−19] Additional compounds from within this series were also shown to
target CPSF3, including 3.[13,19] CPSF3 is an
endonuclease that forms part of the CPSF complex, involved in the
control of polyadenylation and trans-splicing of pre-mRNA. Indeed,
CPSF3 orthologues have also been identified as the molecular targets
of benzoxaboroles active against Plasmodium falciparum,(20)Toxoplasma gondii,[21] and Cryptosporidium spp.[21] With this in mind, we hypothesized
that DNDI-6148 (23) might also target this important
endonuclease in L. donovani. To test
this hypothesis, we first assessed the potency of DNDI-6148 (23) against T. brucei bearing
a mutation in the active site of CPSF3 (Asn232His), previously
demonstrated to confer resistance to acoziborole (1).[19] Bloodstream trypanosomes bearing this specific
mutation were 1.8-fold less sensitive to DNDI-6148 (23) compared to wild-type parasites (Figure A). Similarly, trypanosomes overexpressing
the wild-type version of CPSF3 were 2.9-fold less sensitive to DNDI-6148
(23) (Figure B). Collectively, these data suggest that, like acoziborole
(1), this benzoxaborole specifically targets CPSF3 in T. brucei.
Figure 2
Potency of DNDI-6148 (23) against
wild-type and transgenic T. brucei and L. donovani. (A) EC50 values for DNDI-6148
(23) were
determined for wild-type T. brucei trypanosomes
(closed circles) and T. brucei trypanosomes
maintaining a mutated version of CPSF3 (Asn232His) (open
circles). EC50 values of 47 ± 0.9 and 90 ± 3.4
nM were determined for wild-type and transgenic trypanosomes, respectively.
(B) EC50 values of 67 ± 1 and 172 ± 3 nM were
determined for DNDI-6148-treated wild-type trypanosomes (closed circles)
and trypanosomes overexpressing the wild-type version of CPSF3, respectively.
(C) EC50 values of 2.5 ± 0.1, 2.8 ± 0.1, and
11.7 ± 0.4 μM were determined for acoziborole-treated wild-type
(open circles), CPSF3-overexpressing (blue circles), and CPSF3 (Asn219His)-overexpressing (red circles) L. donovani promastigotes, respectively. (D) EC50 values of 411 ±
12, 508 ± 13, and 1674 ± 51 nM were determined for DNDI-6148-treated
wild-type (open circles), CPSF3-overexpressing (blue circles), and
CPSF3 (Asn219His)-overexpressing (red circles) L. donovani promastigotes, respectively. All curves
are the nonlinear fits of data using a two-parameter EC50 equation provided by GraFit. EC50 values are the weighted
mean ± standard deviation of at three biological replicates (n = 3), with each biological replicate comprised of three
technical replicates.
Potency of DNDI-6148 (23) against
wild-type and transgenic T. brucei and L. donovani. (A) EC50 values for DNDI-6148
(23) were
determined for wild-type T. brucei trypanosomes
(closed circles) and T. brucei trypanosomes
maintaining a mutated version of CPSF3 (Asn232His) (open
circles). EC50 values of 47 ± 0.9 and 90 ± 3.4
nM were determined for wild-type and transgenic trypanosomes, respectively.
(B) EC50 values of 67 ± 1 and 172 ± 3 nM were
determined for DNDI-6148-treated wild-type trypanosomes (closed circles)
and trypanosomes overexpressing the wild-type version of CPSF3, respectively.
(C) EC50 values of 2.5 ± 0.1, 2.8 ± 0.1, and
11.7 ± 0.4 μM were determined for acoziborole-treated wild-type
(open circles), CPSF3-overexpressing (blue circles), and CPSF3 (Asn219His)-overexpressing (red circles) L. donovani promastigotes, respectively. (D) EC50 values of 411 ±
12, 508 ± 13, and 1674 ± 51 nM were determined for DNDI-6148-treated
wild-type (open circles), CPSF3-overexpressing (blue circles), and
CPSF3 (Asn219His)-overexpressing (red circles) L. donovani promastigotes, respectively. All curves
are the nonlinear fits of data using a two-parameter EC50 equation provided by GraFit. EC50 values are the weighted
mean ± standard deviation of at three biological replicates (n = 3), with each biological replicate comprised of three
technical replicates.To determine if these
boron-containing compounds specifically target
CPSF3 in L. donovani, transgenic promastigotes
were generated overexpressing either CPSF3WT or CPSF3 bearing
an Asn219His mutation, equivalent to the Asn232His mutation in the T. brucei enzyme.
Elevated levels of the wild-type and mutated CPSF3 in these cell lines
were confirmed by label-free quantification (Figure S2). Overexpression of CPSF3WT in L. donovani promastigotes did not substantively affect
susceptibility to either acoziborole (1) (Figure C) or DNDI-6148 (23) (Figure D). However,
promastigotes overexpressing the mutated version of this enzyme were
significantly less sensitive to both compounds, demonstrating a 5-fold
and 3.6-fold reduction in susceptibility to acoziborole (1) and DNDI-6148 (23), respectively (Figure C,D). The shift in potency
observed with parasites overexpressing mutated CPSF3 was found to
be specific for benzoxaboroles. Compound 3 elicited a
similar shift in potency, while the established N-myristoyltransferase inhibitor DDD100097[22] did not (Figure S3). The fact that this
single Asn219His mutation in the active site of CPSF3 has
a marked effect on the potencies of both acoziborole (1) and DNDI-6148 (23) provides strong evidence that this
endonuclease is the molecular target of this preclinical candidate.
Precision Base Editing of LdCPSF3
We next
utilized precision base editing with Cas9 to further probe
the interactions between DNDI-6148 (23) and L. donovani CPSF3. In the first instance, a template
encoding a specific Asn219His mutation was provided to
repair a Cas9-induced lesion within CPSF3. Transfected parasites were
then selected with DNDI-6148 (23) (6 μM). The resulting
DNDI-6148-resistant population was subcloned, genomic DNA was harvested,
and the CPSF3 was sequenced to ensure that the desired edit had been
successfully introduced. In all cases, these parasites maintained
the Asn219His encoded by the repair template and were consistently
3-fold less sensitive to DNDI-6148 (23) than wild-type
(Table S2). As in our previous studies
with T. brucei CPSF3, attempts to edit
Asn219 to Tyr to mimic the human CPSF3 enzyme at this position
were not tolerated by L. donovani.
However, using a degenerate repair template, parasites were recovered,
maintaining Asn219His and also Glu229Val homozygous
mutations. These doubly mutated parasites were >5-fold less sensitive
to DNDI-6148 (23) (Table S2). Collectively, these data provide compelling evidence that DNDI-6148
specifically targets the endonuclease CPSF3 in L. donovani. These data are also consistent with a recently published study
reporting CPSF3 as amongst the top “hits” following
the selection of the Cos-Seq genome-wide overexpression library with
DNDI-6148.[23]
Molecular Modeling
A homology model of the L. donovani CPSF3 was generated using the crystallographic
structure of the Thermus thermophilus TTHA0252 homologue as a template (PDB code 3IEM—Figure ). The Leishmania enzyme shares 30% sequence identity with this bacterial homologue.
The proposed binding mode for DNDI-6148 (23) in the model
is consistent with the binding of the benzoxaborole acoziborole to
the catalytic site of CPSF3 located at the interface of the metallo-β-lactamase
and β-CASP domains in T. brucei CPSF3.[19] The site comprises two zinc
atoms coordinated by a network of conserved histidine and aspartic
acid residues. Interaction with a zinc-activated water molecule leads
to the formation of a negatively charged tetrahedral boronate species
that coordinates the zinc atoms (Figure S4), mimicking the transition state of the phosphate of the RNA substrate.
The amide in position 6 of the benzoxaborole moiety directs the pyridyl-triazole
moiety of DNDI-6148 (23) toward the area occupied by
the terminal uracil base of the RNA substrate and establishes a π-stacking
interaction with Tyr370 (Figure A). Additionally, the amide NH forms a hydrogen
bond with the hydroxyl group in the Thr218 side-chain.
There seems to be no direct interaction between DNDI-6148 (23) and the Asn219 residue, where mutation to His is associated
with resistance (Figure B). However, we propose that mutation to a bulkier His residue in
this position has a negative impact on DNDI-6148 (23)
binding due to steric clashes with the methyl pyridyl-triazole moiety
of the compound (Figure C). This likely prompts the ligand to adopt a different binding mode
where a hydrogen bond with Thr218 is lost, the hydrophobic
methyl is directed toward the solvent, and the overall ligand conformation
is strained. Undoubtedly, there is a high degree of similarity surrounding
the proposed binding site of DNDI-6148 in the parasite enzyme and
the human homologue, with 21 identical residues out of the 26 within
5 Å from the bound ligand,[19] However,
Asn219 in the parasite enzyme is replaced by a tyrosine
residue in the human homologue. This bulkier tyrosine residue is likely
to cause severe steric hindrance that prevents DNDI-6148 from binding
to the human and is entirely consistent with the favorable selective
toxicity profile of this compound.
Figure 3
Molecular docking of DNDI-6148 into a
model of the L. donovani CPSF3 active
site. (A) Docking model
for DNDI-6148 (23) (yellow structure) bound to L. donovani CPSF3. Gray spheres represent zinc atoms,
blue dotted lines indicate a π stacking interaction, and the
purple dotted line indicates a hydrogen bond interaction. (B) Close-up
of key interactions involved in DNDI-6148 (23) (yellow
structure) binding in the active site of CPSF3. (C) Modified docking
model illustrating a steric clash between DNDI-6148 (23) (red patch) and the His residue (yellow) in Asn219His
substituted CPSF3. The wild-type CPSF3 structure is shown in green.
Molecular docking of DNDI-6148 into a
model of the L. donovani CPSF3 active
site. (A) Docking model
for DNDI-6148 (23) (yellow structure) bound to L. donovani CPSF3. Gray spheres represent zinc atoms,
blue dotted lines indicate a π stacking interaction, and the
purple dotted line indicates a hydrogen bond interaction. (B) Close-up
of key interactions involved in DNDI-6148 (23) (yellow
structure) binding in the active site of CPSF3. (C) Modified docking
model illustrating a steric clash between DNDI-6148 (23) (red patch) and the His residue (yellow) in Asn219His
substituted CPSF3. The wild-type CPSF3 structure is shown in green.The second mutation (Glu229Val) is at
the beginning
of helix 7 in the β-CASP domain of CPSF3. Helix 7 is some way
from the catalytic site but is connected to the loop that contains
Thr218, Asn219, and Ile221, defining
the pocket that recognizes the pyridine ring of DNDI-6148 (23). In our models, a mutation from Glu229 to Val would
result in a structural rearrangement of the loop, changing the morphology
of the binding site and ultimately impacting the ligand recognition
event.
Conclusions
We have optimized a
series of benzoxaboroles and identified a range
of compounds with very potent in vitro antileishmanial
activity against L. infantum and L. donovani, starting from benzamide-substituted
compound 3. Variation of the amide substituent was explored
extensively, and a range of heterocyclic amide groups was found to
deliver potent in vitro activity combined with good
metabolic stability. Several compounds were assessed in the L. infantum hamster model of VL, with compound 23 standing out by exhibiting very high levels of efficacy
in all organs at doses of 25 mg/kg BID for 5 or 10 days. Additional in vivo studies in the L. donovani hamster model also indicated high levels of efficacy, demonstrating
that 23 was equally efficacious against both Leishmania species responsible for causing VL. Efficacy
of 23 was also confirmed in L. donovani and L. infantum BALB/c mouse models.
Further study of compound 23 indicated that it had excellent
pharmacokinetics, a good in vitro safety profile,
and met all of the criteria in the DNDi TCP for VL.
Comprehensive MoA studies confirm that DNDI-6148 (23)
targets the endonuclease CPSF3, a previously unexploited drug target
in Leishmania spp. Consequently, 23 (DNDI-6148)
was nominated as a preclinical candidate and further development toward
clinical studies is ongoing. Furthermore, compound 23 demonstrates similar levels of in vitro and in vivo activity against species of Leishmania responsible for causing cutaneous leishmaniasis (CL), supporting
the development of this candidate for multiple forms of leishmaniasis.[24] It should be noted that we have also generated
data that supports the development of DNDI-6148 for Chagas’
disease. This information will be disclosed in due course. In conclusion,
the data package for DNDI-6148 (23) provides every reason
to believe that this benzoxaborole can become a much-needed safe oral
treatment for patients suffering from this devastating neglected tropical
disease.
Experimental Section
Purity
All compounds reported in this study are >95%
pure as determined by high-performance liquid chromatography (HPLC)
analysis. HPLC chromatograms of a representative compound (3) and all analogues used in in vivo experiments
(5, 16, 23, 28, 31) are shown in the Supplementary Information (Figures S6–S11).
General
Unless
otherwise indicated, all reactions were
magnetically stirred under an inert atmosphere. All reagents, including
solvents, were used as received. Anhydrous solvents were dried in-house
by passing through activated alumina. Thin-layer chromatography was
performed on glass-backed precoated silica gel 60 plates, and compounds
were visualized using UV light or iodine. Silica gel column chromatography
was performed using 200–300-mesh silica gel. Preparative HPLC
was performed using Gilson-281 liquid handlers equipped with one of
four columns, chosen from (1) Phenomenex Synergi C18 150 mm ×
30 mm, 4 μm, (2) YMC-pack ODS-AQ 150 mm × 30 mm, 5 μm,
(3) Agela Venusil ASB C18 150 mm × 21.2 mm, 5 μm, or (4)
Boston Symmetrix C18 ODS-R 150 mm × 30 mm, 5 μm; elution
was performed with 0.225% (by volume) of formic acid in water (solvent
A) and acetonitrile (solvent B); fractions containing products were
lyophilized. NMR spectra were recorded on a Bruker AVANCE 400 MHz
spectrometer in the solvents specified. Liquid chromatography–mass
spectrometry (LC-MS) spectra were recorded on an Agilent 1200 or Shimadzu
2020 spectrometer equipped with electrospray ionization, quadrupole
MS detector, and Chromolith Flash RP-18e 25 mm × 2.0 mm column,
eluting with 0.0375% (by volume) of trifluoroacetic acid (TFA) in
water (solvent A) and 0.01875% (by volume) of TFA in acetonitrile
(solvent B). Analytical HPLC was performed using a Shimadzu LC20AB
machine and one of two columns, chosen from (1) Xtimate C18 2.1 mm
× 30 mm, 3 μm or (2) CHROM-MATRIX Innovation C18 2.1 mm
× 30 mm, 2.6 μm; elution was performed with 0.0375% (by
volume) of TFA in water (solvent A) and 0.01875% (by volume) of TFA
in acetonitrile (solvent B). The purity of final compounds was ≥96%,
as determined by HPLC. Unless otherwise stated, final compounds were
isolated as amorphous solids without collection of melting point data.
General Procedures for the Preparation of Amides
General Procedure
A
To a solution of the corresponding
acid (14.4 mmol, 1.0 equiv), HOBt (17.3 mmol, 1.2 equiv), EDCI (17.3
mmol, 1.2 equiv), and DIPEA (36.1, 2.5 equiv) in dichloromethane (DCM)
(30 mL) was added 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol (14.4 mmol, 1.0 equiv). The reaction mixture was stirred
at room temperature for 2 h. Water (100 mL) was added, and the reaction
mixture was extracted with DCM (100 mL × 3). The combined organic
extracts were washed with brine (100 mL × 2), dried with anhydrous
Na2SO4, filtered, and concentrated in a vacuum
to give the crude product, which was purified by silica gel chromatography
or preparative HPLC.
General Procedure B
To a solution
of the corresponding
acid (1.5 mmol, 1.0 equiv), HATU (1.9 mmol, 1.3 equiv), and DIPEA
(2.8 mmol, 1.8 equiv) in DMF (3 mL) was added 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol (1.5 mmol, 1.0
equiv). The mixture was stirred at room temperature overnight. The
mixture was purified by preparative HPLC.
General Procedure C
A solution of the corresponding
acid (10.5 mmol) in SOCl2 (40 mL) was stirred at 50 °C
for 4 h. The mixture was concentrated in a vacuum to give acyl chloride,
which was used directly in the next step. To a solution of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol (1.2 g, 8.1 mmol)
and DIPEA (2.9 mL, 16.7 mmol) in THF (30 mL) was added the acid chloride
(8.1 mmol). The mixture was stirred at room temperature for 16 h.
Water (100 mL) was added, and the reaction mixture was extracted with
EtOAc (100 mL × 3). The combined organic extracts were washed
with brine (100 mL × 2), dried with anhydrous Na2SO4, filtered, and concentrated in a vacuum to give the crude
product, which was purified by silica gel chromatography or preparative
HPLC.
The title compound
was synthesized according to method B using a mixture of 1,3-bis(trifluoromethyl)-1H-pyrazole-4-carboxylic acid and 1,5-bis(trifluoromethyl)-1H-pyrazole-4-carboxylic acid (4 g, 16 mmol), HATU (7.3 g,
19.2 mmol), 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (2.9 g, 19.2 mmol), DIPEA (4.1 g, 32
mmol), and DMF (30 mL) to yield 6.5 g of crude product as a yellow
solid. 2 g of the crude product was purified by prep-HPLC to yield 4 as a white solid (200 mg). HPLC: 100% pure. MS (ESI) m/z = 380.1 [M + 1]+. 1H NMR (400 MHz, DMSO-d6) δ = 10.54
(s, 1H), 9.44 (s, 1H), 9.32 (brs, 1H), 8.18 (s, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H),
4.98 (s, 2H). HRMS (ES+): calcd for C13H9B1F6N3O3 [M+H]+ 380.0641, found 380.0644 (0.75 ppm).
To a
cold solution of 6-amino-3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol 2 (22.7 g, 152.3 mmol) and DIPEA (39.3
g, 305.2 mmol) in THF (500 mL) was added a solution of 1-(2,2,2-trifluoroethyl)-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl chloride (42.7 g, 152.5 mmol) in THF
(150 mL) dropwise. The mixture was stirred at room temperature for
4 h and concentrated in a vacuum to a residue that was recrystallized
from MeCN and water to yield 5 as a gray solid (41.1
g, 68%). HPLC: 98% pure. MS (ESI) m/z = 394.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.27 (s, 1H), 8.72
(s, 1H), 8.16 (s, 1H), 7.66 (dd, J = 2.0, 8.0 Hz,
1H), 7.39 (d, J = 8.4 Hz, 1H), 5.48 (q, J = 9.2 Hz, 2H), 4.97 (s, 2H).
The title
compound was synthesized according to method B using 3-(trifluoromethyl)-1-(3,3,3-trifluoropropyl)pyrazole-4-carboxylic
acid 6 (18.0 g, 65.2 mmol), HATU (34.7 g, 91.3 mmol),
1-hydroxy-3H-2,1-benzoxaborol-6-amine 2 (10.7 g, 71.8 mmol), DIPEA (16.9 g, 131.0 mmol), and DMF (150 mL)
to give a residue. The residue was purified by silica gel chromatography
(elution with ethyl acetate/petroleum ether/AcOH = 1: 3: 0.1) to give
the crude product, which was recrystallized from MeOH to yield 6 as a white solid (9.60 g, 36.2%). HPLC: 99% pure. MS (ESI) m/z = 408.1 [M + 1]+. 1H NMR (400 MHz, DMSO-d6): δ 10.22
(s, 1H), 9.25 (s, 1H), 8.66 (s, 1H), 8.17 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H),
4.96 (s, 2H), 4.56 (t, J = 6.8 Hz, 2H), 3.02 (m,
2H). HRMS (ES+): calcd for C15H13B1F6N3O3 [M+H]+ 408.0954, found 408.0957 (0.70 ppm).
N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-3-methyl-1-(3,3,3-trifluoropropyl)-1H-pyrazole-4-carboxamide (7) and N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-5-methyl-1-(3,3,3-trifluoropropyl)-1H-pyrazole-4-carboxamide (15)
The
title compounds were synthesized according to method B using a mixture
of 3-methyl-1-(3,3,3-trifluoropropyl)-1H-pyrazole-4-carboxylic
acid and 5-methyl-1-(3,3,3-trifluoropropyl)-1H-pyrazole-4-carboxylic
acid (469 mg, 2.1 mmol), HATU (890 mg, 2.3 mmol), 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (300 mg, 2.0 mmol), HATU (890 mg, 2.3 mmol), DIPEA (520 mg, 4.0
mmol), and DMF (5 mL) to yield the crude product, which was purified
by preparative HPLC to give 7 (207 mg, 29%) as a white
solid and 15 (205 mg, 29%) as a white solid.7: HPLC: 99% pure. MS (ESI) m/z = 354[M+H]+. 1H NMR (400 MHz, DMSO-d6): δ = 9.77 (s, 1H), 9.21 (s, 1H), 8.42
(s, 1H), 8.14 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H),
7.36 (d, J = 8.0 Hz, 1H), 4.97 (s, 2H), 4.38 (t, J = 6.8 Hz, 2H), 2.90 (m, 2H), 2.40 (s, 3H). HRMS (ES+): calcd
for C15H16B1F3N3O3 [M+H]+ 354.1237, found 354.1240
(0.90 ppm).15: HPLC: 99% pure. MS (ESI) m/z = 354[M+H]+. 1H NMR (400 MHz, DMSO-d6): δ = 9.79
(s, 1H), 9.22 (s, 1H), 8.16
(s, 1H), 8.12 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H),
7.36 (d, J = 8.4 Hz, 1H), 4.96 (s, 2H), 4.35 (t, J = 6.8 Hz, 2H), 2.87 (m, 2H), 2.57 (s, 3H). HRMS (ES+): calcd
for C15H16B1F3N3O3 [M+H]+ 354.1237, found 354.1240
(0.90 ppm).
A solution
of
5-methyl-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylic
acid (15.0 g, 73.9 mmol) in SOCl2 (100 mL) was stirred
at 50 °C for 4 h. The mixture was concentrated in a vacuum to
give acyl chloride, which was used directly in the next step. To a
solution of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (11.0 g, 73.8 mmol) and DIPEA (15 mL)
in DCM (30 mL) was added a solution of acyl chloride in DCM (20 mL)
dropwise at 0 °C. The mixture was stirred at room temperature
for 1 h. The precipitate was filtered and the cake was washed with
MTBE (100 mL) and water (50 mL), dried in a vacuum and recrystallized
from THF (50 mL) to yield 16 as a white solid (16.0 g,
65%). HPLC: 100% pure. MS (ESI) m/z = 335.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.24 (s, 1H), 8.57
(dd, J = 5.2, 1.2 Hz, 1H), 8.36 (s, 1H), 8.14 (d, J = 1.6 Hz, 1H), 8.07 (m, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.72 (m, 1H), 7.40 (m, 1H), 7.37 (d, J = 8.4 Hz, 1H), 4.98 (s, 2H), 2.85 (s, 3H).
To a solution of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (357.0 mg,
2.4 mmol) and DIPEA (619.0 mg, 4.8 mmol) in MeCN (10 mL) was added
a solution of 3-(pyridin-2-yl)-1H-pyrazole-5-carbonyl
chloride (500 mg, 2.4 mmol) in MeCN (10 mL) dropwise. The mixture
was stirred at 60 °C for 20 h. The mixture was concentrated in
a vacuum to give a crude product, which was purified by preparative
HPLC to yield 19 as a white solid (28.4 mg, 3.7%). HPLC:
96% pure. MS (ESI) m/z = 321.1 [M
+ 1]+. 1H NMR (400 MHz, DMSO-d6) δ = 8.65 (d, J = 4.4 Hz, 1H),
8.21 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.90 (t, J = 7.8 Hz, 1H), 7.82 (dd, J = 2.0, 8.0
Hz, 1H), 7.51 (s, 1H), 7.38 (m, 2H), 4.98 (s, 2H).
N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-1-methyl-3-(pyridin-2-yl)-1H-pyrazole-5-carboxamide (20) and N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-1-methyl-5-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (21)
The
title compounds were synthesized according to method B using a mixture
of 1-methyl-3-(pyridin-2-yl)-1H-pyrazole-5-carboxylic
acid and 1-methyl-5-(pyridin-2-yl)-1H-pyrazole-3-carboxylic
acid (300 mg, 1.5 mmol), HATU (722 mg, 1.9 mmol), 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (222.0 mg, 1.5 mmol), DIPEA (361.0 mg, 2.8 mmol), and DMF (3 mL)
to yield the crude product, which was purified by preparative HPLC
to give 20 (123 mg, 25%) as a white solid and 21 (40.7 mg, 8%) as a white solid.20: HPLC: 99%
pure. LC-MS (ESI) m/z = 335.1 [M
+ 1]+, t = 0.931 min. 1H NMR
(400 MHz, DMSO-d6) δ = 10.42 (s,
1H), 9.29 (s, 1H), 8.63 (d, J = 4.4 Hz, 1H), 8.23
(s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.87 (m, 1H),
7.78 (m, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.37 (m,
1H), 4.98 (s, 2H), 4.20 (s, 3H). HRMS (ES+): calcd for
C17H16B1 N4O3 [M+H]+ 335.1315, found 335.1319
(1.06 ppm).21: HPLC: 99% pure. LC-MS (ESI) m/z = 335.1 [M + 1]+, t = 1.026 min. 1H NMR (400 MHz, DMSO-d6) δ = 10.18 (s, 1H), 9.26 (s, 1H), 8.73
(d, J = 4.8 Hz, 1H), 8.23 (s, 1H), 7.95 (m, 2H),
7.80 (d, J = 6.8 Hz, 1H), 7.45 (m, 1H), 7.36 (m,
2H), 4.97 (s, 2H),
4.28 (s, 3H). HRMS (ES+): calcd for C17H16B1N4O3 [M+H] + 335.1315, found 335.1319 (1.06 ppm).
To a solution of 6-amino-3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol 2 (1.6 g, 10.5 mmol) and DIPEA (2.7 g, 21.0 mmol) in THF (20 mL) was
added a solution of 1-(pyridin-2-yl)-1H-1,2,3-triazole-4-carbonyl
chloride (2.2 g, 10.5 mmol) in THF (80 mL) at 0 °C. The mixture
was stirred at room temperature for 4 h. The solvent was removed in
a vacuum and the residue was recrystallized with MeCN and water to
yield 22 as a gray solid (1.7 g, 51%). HPLC: 100% pure.
MS (ESI) m/z = 321.9 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.61 (s, 1H), 9.39 (s, 1H), 9.27 (s, 1H), 8.67 (d, J = 4.4 Hz, 1H), 8.20 (m, 3H), 7.82 (dd, J = 8.8, 2.0 Hz, 1H), 7.63 (m, 1H), 7.41 (d, J =
8.4 Hz, 1H), 4.98 (s, 2H). HRMS (ES+): calcd for C15H12B1N5O3Na1 [M+Na] + 344.0931, found 344.0934
(0.90 ppm).
To a solution of 3-methyl-4-(pyridin-2-yl)furan-2-carboxylic
acid (1.7 g, 8.3 mmol) in DCM (20 mL) was added (COCl)2 (2.4 mL, 25.1 mmol) and DMF (50 μL). The mixture was stirred
at room temperature for 8 h. The mixture was concentrated in a vacuum
to give acyl chloride. To a solution of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (1.2 g, 8.1
mmol) and DIPEA (2.9 mL, 16.7 mmol) in THF (30 mL) was added acyl
chloride. The mixture was stirred at room temperature for 16 h. Water
(150 mL) was added and the mixture was extracted with EtOAc (150 mL
× 4). The combined organic extracts were washed with brine (100
mL × 2), dried with anhydrous Na2SO4, filtered,
and concentrated in a vacuum to give a residue. The residue was purified
by silica gel chromatography (elution with DCM/EtOH = 50: 1) to give
a crude product, which was recrystallized from MeCN (100 mL) to yield 28 as a white solid (1.6 g, 57%). HPLC: 98% pure. MS (ESI) m/z = 335.0 [M + 1]+. 1H NMR (400 MHz, DMSO-d6) δ 10.20
(s, 1H), 9.24 (s, 1H), 8.65 (d, J = 4.0 Hz, 1H),
8.37 (s, 1H), 8.18 (s, 1H), 7.87 (m, 1H), 7.75 (m, 1H), 7.30 (m, 3H),
4.96 (s, 2H), 2.62 (s, 3H).
To a solution of 5-methyl-4-(pyridin-2-yl)furan-2-carboxylic
acid (5.7 g, 28.1 mmol), DIPEA (7.5 g, 58.1 mmol), and HATU (16.0
g, 42.1 mmol) in DMF (60 mL) was added 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol 2 (4.2 g, 28.2
mmol). The mixture was stirred at room temperature overnight. Water
(200 mL) was added and a lot of solid formed. The suspension was filtered
and the cake was collected and purified by preparative HPLC to yield 29 as a white solid (6.5 g, 65.6%). HPLC: 100% pure. MS (ESI) m/z = 334.9 [M + 1]+. 1H NMR (400 MHz, DMSO-d6) δ 10.20
(s, 1H), 9.27 (s, 1H), 8.65 (d, J = 4.0 Hz, 1H),
8.17 (s, 1H), 7.88 (m, 2H), 7.75 (m, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.30 (m, 1H), 4.98 (s, 2H), 2.76 (s, 3H). HRMS (ES+): calcd
for C18H16B1N2O4 [M+H]+ 335.1203, found 335.1206
(0.86 ppm).
To a reaction mixture of 5-(pyridin-2-yl)-1,3,4-oxadiazole-2-carbonyl
chloride were added DIPEA (6.8 g, 52.7 mmol) and a solution of 1-hydroxy-3H-2,1-benzoxaborol-6-amine 2 (2.5 g, 17 mmol)
in MeCN (50 mL) at 0 °C. The mixture was stirred at 0 °C
for 1 h. The reaction mixture was quenched by adding water (100 mL)
at room temperature and extracted with EtOAc (100 mL × 2). The
combined organic extracts were washed with brine (200 mL × 2),
dried over Na2SO4, filtered, and concentrated
under reduced pressure to give a residue. The residue was dispersed
in DCM (20 mL), filtered, and the cake was dried under reduced pressure
to give a crude product, which was purified by preparative HPLC to
yield 31 as a white solid (1.9 g, 45%). HPLC: 99% pure.
MS (ESI) m/z = 323.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 11.36 (s, 1H), 9.32 (s, 1H), 8.84 (d, J = 2.4 Hz, 1H), 8.32 (m, 1H), 8.25 (s, 1H), 8.12 (m, 1H), 7.85 (m,
1H), 7.72 (d, J = 3.6 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 5.00 (s, 2H).
Biology
Compounds
and Reagents
For in vitro assays, compound
stock solutions were prepared in 100% dimethyl
sulfoxide (DMSO) at 20 mM. Compounds were serially prediluted (2-fold
or 4-fold) in DMSO, followed by a further (intermediate) dilution
in demineralized water to assure a final in-test DMSO concentration
of <1%. For in vivo studies, compounds were formulated
in 2% ethanol, 1 mol equiv NaOH, and dextrose (5% solution) when administrated
orally to mice, hamsters, and dogs or intravenously (pH adjusted to
7) to rats and dogs. For oral administration in rats, 0.5% (w/v) methylcellulose
and 0.1% (w/v) sodium dodecyl sulfate in Milli-Q water were used.
Miltefosine was formulated in water at 20 mg/mL.
Cell Cultures
Primary peritoneal mouse macrophages
(PMM) were collected two days after peritoneal stimulation with a
2% potato starch suspension. MRC5SV2 cells (diploid human
embryonic lung fibroblasts) were cultured in minimal essential medium
(MEM) containing Earle’s salts, supplemented with l-glutamine, NaHCO3, and 5% inactivated fetal calf serum.
All cultures and assays were conducted at 37 °C under an atmosphere
of 5% CO2.
Parasites
L. infantum (MHOM/MA/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) were maintained in the golden (Syrian) hamster
(Mesocricetus auratus). Ex
vivo amastigotes were collected from the spleen of an infected
donor hamster using two centrifugation purification steps: 230g for 10 min, keeping the supernatant layer, and 4100g for 30 min, keeping the pellet. The spleen parasite burden
was assessed using the Stauber technique.[25] For in vitro assays, the inoculum was prepared
in RPMI-1640 medium, supplemented with 200 mM l-glutamine,
16.5 mM NaHCO3, and 5% inactivated fetal calf serum. For
the in vivo model, an infection inoculum containing
2 × 107 amastigotes/100 μL was prepared in phosphate-buffered
saline (PBS).
Animals
Female golden hamsters for
the in vivo model of visceral leishmaniasis were
purchased from Janvier, France
(body weight 80–100 g). This study using laboratory rodents
was carried out in strict accordance with all mandatory guidelines
(EU directives, including the Revised Directive 2010/63/EU on the
protection of Animals used for Scientific Purposes that came into
force on 01/01/2013, and the declaration of Helsinki in its latest
version) and was approved by the ethical committee of the University
of Antwerp, Belgium (UA-ECD 2011-74). Female golden hamsters for the
pharmacokinetic study were purchased from Vital River, Beijing, China.
This study was conducted following institutional review and in accordance
with institutional and national guidelines at WuXi AppTec (the Institutional
Animal Care and Use Committee (IACUC)). Balb/c mice for the in vivo model of visceral leishmaniasis (LSHTM) were purchased
from Charles River, U.K., and related studies were conducted under
license PPL 70/8427 from the U.K. Home Office.
Intramacrophage L. infantum and L. donovani Assays
The assay was performed
in sterile 96-well microtiter plates, each well containing 10 μL
of the compound dilution and 190 μL of the PMM/amastigote inoculum
(3 × 104 cells/4.5 × 105 parasites
per well). Parasite multiplication was compared to untreated infected
controls (100% growth) and uninfected controls (0% growth). After
five-day incubation, total parasite burdens were microscopically assessed
after staining the cells with a 10% Giemsa solution. The results were
expressed as a percentage reduction in parasite burden compared to
untreated control wells. EC50 values were determined using
an extended dose range (2-fold compound dilutions, 8-point concentration
curve). Miltefosine was used as the reference drug.[26] For selected compounds, this assay format was also run
using the L. donovani inoculum.
In Vitro MRC5 and PMM Cytotoxicity Assays
Assays
were performed in sterile 96-well microtiter plates, each
well containing 10 μL of the compound dilution and 190 μL
of MRC5SV2 or PMM inoculum (3 × 104 cells/mL).
Cell growth was compared to untreated controls (100% growth) and assay-media
controls (0% growth). After three-day incubation, cell viability was
assessed fluorometrically by adding resazurin (50 μL/well of
a stock solution in phosphate buffer (50 μg/mL)), incubating
for 4 h and measuring fluorescence (λex 550 nm, λem 590 nm). The results were expressed as a percentage reduction
in cell growth compared to untreated control wells. EC50 values were determined using an extended dose range (2-fold compound
dilutions, 8-point concentration curve) to a highest concentration
of 64 μM. Tamoxifen was included as the reference drug.
In Vivo Hamster Model of Visceral Leishmaniasis
Female golden hamsters were randomly allocated to experimental
groups of six animals each, based on body weight. At the start of
the experiment (Day 0), each animal was infected with L. infantum or L. donovani inoculum, delivered by intracardial injection. Six hamsters were
assigned to the vehicle-treated, infected control group. Six hamsters
were assigned per group (1 group/compound) for the evaluation of compound
and miltefosine. At Day 21 postinfection (21 dpi), all animals in
each group were dosed orally for five (or ten) consecutive days (21–25
dpi): compound was dosed at 25–50 mg/kg b.i.d.; miltefosine
was dosed at 40 mg/kg q.d. At Day 35 (10 days after the final oral
dose), all animals were euthanized and autopsies were conducted. The
study evaluated the following parameters.Adverse effects: all animals were observed
daily for the occurrence/presence of adverse effects.Body weight: all animals were weighed
twice per week to monitor general health.Parasite burden: amastigote burdens
in each target organ (liver, spleen, and bone marrow) were determined
at Day 35. The organs of individual animals were weighed (except for
bone marrow). Impression smears were stained with Giemsa for microscopic
examination of the total amastigote burden, defined as the mean number
of amastigotes per cell multiplied by the number of cells counted
(minimum 500 nuclei); the results were expressed as a percentage reduction
in amastigote burden compared to vehicle-treated, infected control
animals.
In Vivo Mouse Model of Visceral Leishmaniasis
Female BALB/c mice
were infected with 2 × 107L. donovani amastigotes/0.2 mL i.v. harvested from
the spleen of an infected donor RAG1B6 mouse (LSHTM). After 7 days,
the mice were treated with miltefosine or DNDI-6148 for 5 consecutive
days. Five days after the final dose, mice were humanely killed, liver
and spleens were dissected and weighed, and impression smears were
made for the calculation of parasite burden (Stauber equation).[25]
Liver Microsome Stability
Tests
were performed by WuXi
AppTec, China. Test compounds (at 1 μM) or positive controls
(testosterone, propafenone, and diclofenac) were incubated at 37 °C
with liver microsomes from Balb/c mouse, golden (Syrian) hamster,
Sprague-Dawley rat, Beagle dog, or human in the presence of a NADPH
regenerating system and phosphate buffer (100 mM, pH 7.4) at 0.4 or
0.8 mg/mL microsomal protein. The samples were removed at time intervals
from 0 to 60, 90, or 120 min and immediately mixed with cold methanol
supplemented or not with acid (3% formic acid) and centrifuged prior
to analysis by LC-MS/MS using tolbutamide or propranolol as internal
standards.
Hepatocyte Stability
Tests were
performed by WuXi AppTec,
China. Following a viability check of cryopreserved hepatocytes from
Sprague-Dawley rat, Beagle dog, or human, test compounds (at 1 μM)
or positive controls 7-ethoxycoumarin and 7-hydroxycoumarin (at 3
μM) were incubated at 37 °C, 5% CO2 with hepatocytes
in Williams’ Medium E. Following incubation of 15, 30, 60,
90, 120, 180, and 240 min, reactions were stopped by the addition
of acetonitrile, the samples were centrifuged, and LC-MS/MS analysis
was conducted using tolbutamide as an internal standard.
Metabolite
ID Studies
Metabolite identification studies
were conducted at WuXi AppTec, China. DNDI-6148 was incubated at 10
μM in the presence of Beagle dog and Sprague-Dawley rat liver
microsomes (1 mg/mL) for 60 min. Following precipitation with a solution
of formic acid and acetonitrile, metabolites were identified by LC-UV-MSn
(n = 1–2).
CYP Inhibition Studies
The study was conducted by WuXi
AppTec, Co., China, using the time-dependent (TDI) method. For detecting
any IC50 shift, the test compound (at concentrations of
0.05–50 μM) was first preincubated for 30 min at 37 °C
with pooled human liver microsomes in the presence or not of NADPH.
Following incubation with substrates (phenacetin for 1A2, diclofenac
for 2C9, S-(+) mephenytoin for 2C19, dextromethorphan
for 2D6, and midazolam or testosterone for 3A4), reactions were stopped
by adding ice-cold acetonitrile, the samples were analyzed for the
formation of metabolites by LC-MS/MS, and the percentage of inhibition
was determined.
hERG Inhibition
An automated patch-clamp
method (QPatch
HTX) was used by WuXi AppTec, China. Chinese hamster ovary cells expressing
hERG potassium channels were incubated at room temperature with test
compounds (0.12, 0.37, 1.11, 3.33, 10, and 30 μM, in triplicate)
or amitriptyline as a positive control.
Plasma-Protein Binding
Tests were conducted by WuXi
AppTec, Co., China, using the equilibrium dialysis method and a 96-well
plate format. The test compound (at 2 μM, in triplicate) in
solution in plasma (Balb/c mouse, golden (Syrian) hamster, Sprague-Dawley
rat, Beagle dog or human) was dialyzed against 100 mM phosphate-buffered
saline (pH 7.4) on a rotating plate incubated for 6 h at 37 °C.
Following the precipitation of protein with acetonitrile or acetonitrile/methanol
containing both 1% phosphoric acid, the amount of compound present
in each compartment was quantified by LC-MS/MS.
Thermodynamic
Solubility
The thermodynamic solubility
was measured by WuXi AppTec, China. Aliquots of the compound DMSO
stock (10 mM) were transferred to fasted state simulated intestinal
fluid (FaSSIF) buffer (pH 6.5) or fed state simulated intestinal fluid
(FeSSIF) buffer (pH 5.0), and the mixtures were shaken for 24 h at
room temperature. Following sampling by a Whatman filter device, the
compound concentrations were determined by UV spectroscopy.
Membrane
Permeability
Bidirectional permeability tests
with MDCK-MDR1 cells were performed by WuXi AppTec, Co., China. The
test compound, at a concentration of 2 μM in 0.4% DMSO/HBSS
buffer with 10 mM HEPES pH 7.4, was applied to the apical or basolateral
side of the cell monolayer. Permeation of the compound from A to B
direction or B to A direction was determined in triplicate over a
150 min incubation at 37 °C and 5% CO2 (95% humidity).
Test and reference compounds were quantified by LC-MS/MS analysis
based on the peak area ratio of analyte/internal standard.
Ames
Assay
Mini-Ames reverse mutation screens were
conducted by WuXi AppTec, Co., China. Two Salmonella
typhimurium strains (TA98 and TA100) were employed,
both in the presence and absence of metabolic activation (induced
rat liver S9). Test compounds were assessed at doses of 1.5, 4, 10,
25, 64, 160, 400, and 1000 μg per well, 2-aminoanthracene, 2-nitrofluorene,
and sodium azide were used as positive controls, and the negative
control was DMSO.
In Vivo Pharmacokinetic
Studies
In vivo pharmacokinetic studies
were conducted at WuXi AppTec,
Co., China.
Hamster
Compounds were formulated
as a homogenous suspension
in 2% ethanol, 1 mol equiv NaOH, dextrose (5% solution), and dosed
twice p.o. on a single day (8 h apart, 25 or 50 mg/kg, BID) in female
golden Syrian hamsters. The K2-EDTA anticoagulant was added
to blood samples collected from 0.25 to 36 h, which were processed
to plasma and stabilized with phosphoric acid (1% final) before analysis
by LC-MS/MS. PK parameters were calculated using WinNonlin software
(version 6.3).
Rat
DNDI-6148 was formulated in
2% ethanol, 1 mol equiv
NaOH, dextrose (5% solution), and administrated to male Sprague-Dawley
rats, either i.v. after filtration and pH adjustment (at 2 mg/kg),
or p.o. as a homogenous suspension (at 10 mg/kg). The K2-EDTA anticoagulant was added to blood samples collected at predose,
0.083 (i.v. only), 0.25, 0.5, 1, 2 (p.o. only), 4, 8, and 24 h, and
were processed to plasma and stabilized with phosphoric acid (1% final)
before analysis by LC-MS/MS. PK parameters were calculated using WinNonlin
software (version 6.21 and 6.3).
Dog
DNDI-6148
was administrated intravenously at 1
mg/kg and orally at 5 mg/kg to male Beagle dogs. The formulation for
the i.v. leg was 2% ethanol, 1.0 mol equiv NaOH, dextrose (5% solution)
filtered, and adjusted to pH 7, and for the p.o. leg, the formulation
was 2% ethanol, 1.0 mol equiv NaOH, and dextrose (5% solution). The
K2-EDTA anticoagulant was added to blood samples collected
at predose, 0.033 (i.v. only), 0.083 (p.o. only), 0.25, 0.5, 1, 3,
6, 9, and 24 h, and were stabilized with phosphoric acid (1% final)
before analysis by LC-MS/MS. PK parameters were calculated using WinNonlin
software (version 6.3).
MoA Studies
Synthesis
of DNDI-6148, N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-5-methyl-1-(pyridin-2-yl)-1H-1,2,3-triazole-4-carboxamide (23) Used in
MoA studies
All NMR spectra for the compounds and intermediates
made in the course of these studies are available upon request.The samples of DNDI-6148 used in the MoA studies were prepared using
a three-step sequence (Scheme ) modified from a synthetic
route previously reported by Jacobs and co-workers.[27] In brief, triazole 23-3 was prepared using
an optimized flow chemistry procedure to alleviate the potential risk
of explosion when heating low molecular weight organic azides. Note,
tetrazole 23-2 is converted to the azide reactant (IV) under the thermal reaction conditions.[28] Treatment with LiOH hydrolyzed the ester function of 23-3 to give lithium salt II, which was converted
to DNDI-6148 (23) by reaction with aniline 2, T3P, and base.
Synthetic Route to DNDI-6148 (23) Employed
for the Material
Used in the MoA Studies
Reagents and conditions: (a)
NaOEt, EtOH, 150 °C, 2 h (flow), 38%; (b) LiOH, THF/water, room
temp., 16 h, 97%; and (c) 2, T3P, DIPEA, DMF/EtOAc, RT,
16 h, 29%.
A solution of tetrazolo[1,5-a]pyridine (23-2) (144 mg, 1.2 mmol) and ethyl 3-oxobutanoate
(I) (1.56 g, 5.99 mmol) in EtOH (10 mL) and a solution
of NaOEt (245 mg, 3.6 mmol) in EtOH (10 mL) were mixed in a 1:1 ratio
and applied to the flow coil (Vapourtec RS-400 flow chemistry system).
The reaction mixture was then heated at 150 °C with a residence
time of 2 h. The crude product mixture was collected into a vial and
acidified to pH 5 by the addition of HCl (2N aq.). The reaction mixture
was then concentrated under reduced pressure and directly purified
by silica flash column chromatography (10:90 → 50:50 EtOAc:heptane),
affording ester 23-3 as a colorless solid (110 mg, 38%
yield). 1H NMR (500 MHz, CDCl3) δ 8.59
(1H, d, J = 4.75 Hz), 8.00–7.93 (2H, m), 7.42
(1H, t, J = 6.03 Hz), 4.47 (2H, q, J = 7.12 Hz), 2.92 (3H, s), 1.45 (3H, t, J = 7.10
Hz). 13C NMR (126 MHz, CDCl3) δ 161.7,
150.2, 148.5, 139.7, 139.1, 137.4, 124.1, 118.3, 61.0, 14.3, 11.0.
HRMS (ES+): m/z [M+H]+ calcd for C11H13N4O2, 233.1033; found, 233.1061.
Lithium 5-Methyl-1-(pyridin-2-yl)-1H-1,2,3-triazole-4-carboxylate
(II)
A solution of LiOH (1 M, aq, 0.31 mL, 0.31
mmol) was added to a solution of ethyl 5-methyl-1-(pyridin-2-yl)-1H-1,2,3-triazole-4-carboxylate (23-3) (72 mg,
0.31 mmol) in THF/water (4:1, 10 mL), and the resulting mixture was
stirred at room temperature for 16 h. The reaction mixture was then
concentrated under reduced pressure, and the resultant aqueous residue
was lyophilized to furnish the title compound as a colorless solid
(64 mg, 97%). 1H NMR (500 MHz, D2O) δ
8.74 (1H, d, J = 4.05 Hz), 8.28 (1H, t, J = 7.80 Hz), 7.84 (1H, d, J = 8.00 Hz), 7.79 (1H,
dd, J = 7.15, 5.35 Hz), 2.67 (3H, s). 13C NMR (126 MHz, D2O) δ 168.4, 149.0, 147.9, 141.9,
140.7, 137.6, 125.9, 120.6, 9.3. HRMS (ES+): m/z [M+H]+ calcd for C9H9N4O2, 205.0720; found, 205.0686.
Neat DIPEA (130 mg, 1.28 mmol) was added to a stirred suspension
of carboxylate II (45 mg, 0.214 mmol) and 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol, hydrochloride
(5) (59.6 mg, 0.321 mmol) in dry DMF (3.0 mL) at room
temperature. A solution of propylphosphonic anhydride (50% w/v in
EtOAc, 0.757 mL, 0.642 mmol) was then added dropwise, and the resulting
mixture was stirred for a further 16 h at room temperature. The reaction
mixture was then concentrated under reduced pressure and directly
purified by silica flash column chromatography (2:98 MeOH:CH2Cl2), affording the title compound as a colorless solid
(21.7 mg, 95% purity, 29% yield). The product was further purified
by reverse phase preparative HPLC (C18 silica, 5:95 → 95:5
MeCN:water + 0.1% NH3) to give an assay/analytical sample
at >95% purity. 1H NMR (400 MHz, DMSO) δ 10.53
(1H,
s), 9.20 (1H, s), 8.71 (1H, ddd, J = 4.86, 1.78,
0.72 Hz), 8.27 (1H, d, J = 1.76 Hz), 8.19 (1H, dt, J = 11.79, 1.87 Hz), 7.98 (1H, d, J = 8.08
Hz), 7.84 (1H, dd, J = 8.22, 2.02 Hz), 7.68 (1H,
ddd, J = 7.51, 4.87, 0.95 Hz), 7.39 (1H, d, J = 8.32 Hz), 4.98 (2H, s), 2.82 (3H, s). 13C
NMR (126 MHz, DMSO) δ 159.8, 149.8, 149.7, 149.5, 140.5, 139.4,
138.1, 137.7, 125.5, 124.3, 123.0, 121.9, 119.5, 70.2, 10.7 (note,
the signal for the benzene quaternary carbon atom directly linked
to the boron atom was not observed in the 13C NMR due to
severe broadening from scalar relaxation caused by the spin dynamics
of the quadrupolar 11B nucleus). HRMS (ES+): m/z [M+H]+ calcd for C16H15BN5O3, 336.1262; found, 336.1271.
Cell Lines and Culture Conditions
The clonal L. donovani cell line LdBOB (derived from MHOM/SD/62/1S-CL2D)
was grown as promastigotes at 28 °C, as described previously.
Bloodstream form T. brucei MiTat 1.2
clone 221a and its derived subline, 2T1, were cultured at 37 °C
in the presence of 5% CO2 in HMI9-T medium, as described
previously. T. brucei cell lines overexpressing
CPSF3 and overexpressing a mutated version of CPSF3 (Asn232His) were
generated as part of a previous study and cultured as described.[19]
Generation of L. donovani Transgenic
Cell Lines
L. donovaniCPSF3 gene (LdBPK_343210.1) was custom synthesized by GeneArt
and ligated into the pIR1SAT vector via BglII restriction sites. Using
the Q5 mutagenesis kit (NEB) and the pIR1SAT-LdCPSF3WT construct as a template, mutations encoding A655C
and Asn219His change to CPSF3WT were introduced. Mutations
were introduced using the specific primers 5′-GAGTACACGGCATCCGC-3′
and 5′-TCCGCAATCAGGATGTCTGGT-3′ and as per manufacturers’
instructions. Wild-type and mutated pIR1SAT constructs were digested
with SwaI and transfected into L. donovani promastigotes, as previously described. Resistant clones were selected
with nourseothricin (100 μg/mL), and two clones per transfection
were selected for further study.
CRISPR-Cas9 Generated Mutations
in LdCPSF3
A cell line constitutively expressing
Cas9 and T7 RNA polymerase
was generated by transfecting 4 × 107 promastigotes
with the pTB007 vector (10 μg), as previously described.[29] Expression of Cas9 in selected clones was confirmed
by western blotting (Figure S1). In addition,
the CRISPR-Cas9 system in these parasites was confirmed as functional
following the successful tagging of the flagellar protein PF16 with
mNeonGreen, as previously described.[29]To introduce specific mutations into CPSF3, 3 ×
106 promastigotes expressing Cas9 and T7 RNA were resuspended
in a 110 μL of Human T-Cell Nucleofector reagent and transfected
with 18 μL of sgRNA and 4 μg of template using the Amaxa
Nucleofector electroporator (program V-033). sgRNA was produced according
to the previously established protocol from the Gluenz lab,[30] in which the scaffold primer G00 and a gene-specific
forward primer (FwsgRNA) were used (Table S1). The sgRNA template and templates introducing base edits are summarized
in Table S1. Twenty-four hours following
transfection, DNDI-6148 was added to cultures at 6 μM. Drug-selected
populations were cloned by limiting dilution, EC50s were
determined, and genomic DNA was extracted. The CPSF3 gene from CRISPR-edited parasites was amplified by PCR, and the
resulting products were sequenced.
In Vitro Drug Sensitivity Assays (MoA Studies)
Drug sensitivity
assays with wild-type and transgenic T. brucei and L. donovani cell lines were carried
out as previously described.[31,32]
Confirmation of CPSF3 Overexpression
Using MS
WT and L. donovani promastigotes (3 × 108) overexpressing CPSF3 were
washed twice with PBS (1000 g, 4 °C,
5 min). The pellet was resuspended in 1.5 mL of lysis buffer (50 mM
potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM DTT, and 1 ×
EDTA-free complete protease inhibitors (Roche)). Parasites were biologically
inactivated by 3 × freeze/thaw cycles prior to lysis using a
One-Shot Cell Disruptor (Constant Systems) at 30 kpsi. Lysed cells
were then centrifuged (10 min, 4 °C, 15 000g), and the soluble material was harvested. Soluble cell lysates (100
μg samples) were reduced by the addition of tris(2-carboxyethyl)phosphine
hydrochloride (TCEP, 25 mM final concentration) and incubated at 37
°C for 10 min. The samples were then alkylated by the addition
of iodoacetamide (25 mM final concentration) and incubated at RT for
1 h in the dark. Alkylated samples were digested by the addition of
1:50 endoproteinase LysC (Wako, Japan) in 100 mM triethylammonium
bicarbonate (TEAB), followed by incubation at 37 °C for 6 h,
then the addition of 1:50 trypsin (1.50 dilution) and incubation at
37 °C overnight. The resulting digested peptides were then vacuum-dried.
LC-MS/MS Analysis
Analysis of peptides was performed
on a Q-Exactive Plus mass spectrometer (Thermo Scientific) coupled
with a Dionex Ultimate 3000 RS (Thermo Scientific). LC buffers used
were as follows: Buffer A (0.1% formic acid in Milli-Q water (v/v))
and Buffer B (80% acetonitrile and 0.1% formic acid in Milli-Q water
(v/v)). Aliquots (15 μL per sample) were loaded at 10 μL/min
onto a PepMap nanoViper C18 column (100 μm × 2 cm, 5 μm,
100 Å, Thermo Scientific) equilibrated with 98% Buffer A. The
column was washed for 5 min at the same flow rate and then was switched
in line with a Thermo Scientific, resolving PepMap RSLC C18 column
(75 μm × 50 cm, 2 μm, 100 Å). Peptides were
eluted at a constant flow rate of 300 nL/min with a linear gradient
of 2–35% Buffer B in 125 min, then to 98% in 127 min. The column
was then washed with 98% Buffer B for 20 min and re-equilibrated in
2% Buffer B for 17 min prior to loading the next sample. The Q-Exactive
Plus was used in the data-dependent mode. The scan cycle comprised
MS1 scan (m/z range from 335–1600,
with a maximum ion injection time of 20 ms, a resolution of 70 000,
and an automatic gain control (AGC) value of 1 × 106), followed
by 15 sequential dependent MS2 scans (with an isolation window set
to 1.4 Da, resolution at 17 500, maximum ion injection time
at 100 ms, and AGC 2 × 105). The stepped collision energy was
set to 27 and fixed first mass to 100 m/z. Spectra were acquired in a centroid mode and unassigned charge
states. Charge states above 6, as well as singly charged species,
were rejected. To ensure mass accuracy, the mass spectrometer was
calibrated on day 1 of the analyses. LC-MS analysis was performed
by the FingerPrints Proteomics Facility (University of Dundee).
Data Analysis
MS data analysis was performed using
MaxQuant software (http://maxquant.org/, version 1.6.2.6a). Carbamidomethyl (C), oxidation (M), acetyl (Protein
N-term), deamidation (NQ), and Gln -> pyro-Glu were set as variable
modifications. Proteins were identified by searching a protein sequence
database containing T. brucei brucei TREU927 annotated proteins (downloaded from TriTrypDB 46, http://www.tritrypdb.org). LFQ
and “match between runs” features were enabled. Trypsin/P
and LysC/P were selected as the specific proteases with two potential
missed cleavages. The FDR threshold for peptides and proteins was
0.01. FTMS MS/MS mass tolerance was set to 10 ppm, and ITMS MS/MS
mass tolerance was 0.6 Da. Protein abundance was obtained using LFQ
intensity values. LFQ intensities were calculated using at least 2
unique peptides. Data was visualized using Perseus 1.6.2.1 (https://maxquant.org/perseus/).
Homology Modeling and In Silico Docking Studies
To identify suitable template structures for the generation of
the L. donovani CPSF3 homology model,
the sequence UniProtKB—E9BRB9 was used to query the PDB using
“BLAST” as implemented in the NCBI blastp suite (https://blast.ncbi.nlm.nih.gov/). Endonucleases from five different species, human CPSF73 (PDB code 2I7T and 6M8Q—sequence
identity 54%), Saccharomyces cerevisiae CPSF (PDB code 6I1D—sequence identity 47%), Pyrococcus horikoshii CPSF3 (PDB code 3AF5—sequence identity 29%), Cryptosporidium hominis (PDB code 6Q55—sequence identity 48%), and T. thermophilus TTHA0252 (several structures available exemplified by PDB code 3IEM—sequence
identity 30%), were identified as close analogues that could be used
as template structures. The T. thermophilus structure is complexed with a stable RNA analogue bound to the catalytic
site located at the interface between the metallo-β-lactamase
and β-CASP domains, whereas one human, S. cerevisiae and P. horikoshii structures are
apo forms where the Zn2+-containing binding site is inaccessible.
The C. hominis structure is in complex
with a small boron-containing inhibitor (AN3661) and where the binding
site is still inaccessible to a larger ligand. One of the human structures
(6M8Q) is in
complex with a small ligand that does not interact with the two Zn2+ ions and adopts the binding mode that causes structural
rearrangements in loop regions adjacent to the binding cavity. Thus,
the T. thermophilus structure was selected
as a suitable template. The alignment between the amino acid sequence
of LdCPSF3 that of the T. thermophilus CPSF3 was manually curated (Figure S5) and showed a sequence identity of 68% in the binding site area.
A homology model for LdCPSF3 was built using the
knowledge-based method in Prime in Schrödinger (Schrödinger
Release 2019-3: Schrödinger, LLC, New York, NY, 2020). Zinc
atoms were modeled into the structure but not the RNA substrate. Due
to its empty p-orbital, the boron atom in DNDI-6148 is a strong electrophile
(Lewis acid) that can react with solvent water molecules. The nucleophilic
attack of an activated water molecule on the trigonal boron atom leads
to the formation of a tetrahedral negatively charged boron species.
The three-dimensional structure of the hydroxylated form of DNDI-6148
was built in Maestro, minimized with the OPLS3 force field, and docked
in the catalytic site of the LdCPSF3 model using
GLIDE in Schrödinger. Docking results were subjected to a restrained
minimization procedure with the OPLS3e force field, as implemented,
where each heavy atom was allowed to deviate by up to 0.5 Å from
its original position in the model.
Authors: Bakela Nare; Stephen Wring; Cyrus Bacchi; Beth Beaudet; Tana Bowling; Reto Brun; Daitao Chen; Charles Ding; Yvonne Freund; Eric Gaukel; Ali Hussain; Kurt Jarnagin; Matthew Jenks; Marcel Kaiser; Luke Mercer; Elena Mejia; Andy Noe; Matt Orr; Robin Parham; Jacob Plattner; Ryan Randolph; Donna Rattendi; Cindy Rewerts; Jessica Sligar; Nigel Yarlett; Robert Don; Robert Jacobs Journal: Antimicrob Agents Chemother Date: 2010-07-26 Impact factor: 5.191
Authors: Robert T Jacobs; Bakela Nare; Stephen A Wring; Matthew D Orr; Daitao Chen; Jessica M Sligar; Matthew X Jenks; Robert A Noe; Tana S Bowling; Luke T Mercer; Cindy Rewerts; Eric Gaukel; Jennifer Owens; Robin Parham; Ryan Randolph; Beth Beaudet; Cyrus J Bacchi; Nigel Yarlett; Jacob J Plattner; Yvonne Freund; Charles Ding; Tsutomu Akama; Y-K Zhang; Reto Brun; Marcel Kaiser; Ivan Scandale; Robert Don Journal: PLoS Negl Trop Dis Date: 2011-06-28
Authors: Richard J Wall; Sonia Moniz; Michael G Thomas; Suzanne Norval; Eun-Jung Ko; Maria Marco; Timothy J Miles; Ian H Gilbert; David Horn; Alan H Fairlamb; Susan Wyllie Journal: Antimicrob Agents Chemother Date: 2018-07-27 Impact factor: 5.191
Authors: M Van den Kerkhof; D Mabille; E Chatelain; C E Mowbray; S Braillard; S Hendrickx; L Maes; G Caljon Journal: Int J Parasitol Drugs Drug Resist Date: 2018-01-31 Impact factor: 4.077
Authors: Katrien Van Bocxlaer; Diana Caridha; Chad Black; Brian Vesely; Susan Leed; Richard J Sciotti; Gert-Jan Wijnant; Vanessa Yardley; Stéphanie Braillard; Charles E Mowbray; Jean-Robert Ioset; Simon L Croft Journal: Int J Parasitol Drugs Drug Resist Date: 2019-02-26 Impact factor: 4.077
Authors: Magali Van den Kerkhof; Philippe Leprohon; Dorien Mabille; Sarah Hendrickx; Lindsay B Tulloch; Richard J Wall; Susan Wyllie; Eric Chatelain; Charles E Mowbray; Stéphanie Braillard; Marc Ouellette; Louis Maes; Guy Caljon Journal: Microorganisms Date: 2021-06-29
Authors: Susan Wyllie; Michael Thomas; Stephen Patterson; Sabrinia Crouch; Manu De Rycker; Rhiannon Lowe; Stephanie Gresham; Michael D Urbaniak; Thomas D Otto; Laste Stojanovski; Frederick R C Simeons; Sujatha Manthri; Lorna M MacLean; Fabio Zuccotto; Nadine Homeyer; Hannah Pflaumer; Markus Boesche; Lalitha Sastry; Paul Connolly; Sebastian Albrecht; Matt Berriman; Gerard Drewes; David W Gray; Sonja Ghidelli-Disse; Susan Dixon; Jose M Fiandor; Paul G Wyatt; Michael A J Ferguson; Alan H Fairlamb; Timothy J Miles; Kevin D Read; Ian H Gilbert Journal: Nature Date: 2018-07-25 Impact factor: 49.962
Authors: Angel M Padilla; Wei Wang; Robert T Jacobs; Rick L Tarleton; Tsutomu Akama; David S Carter; Eric Easom; Yvonne Freund; Jason S Halladay; Yang Liu; Sarah A Hamer; Carolyn L Hodo; Gregory K Wilkerson; Dylan Orr; Brooke White; Arlene George; Huifeng Shen; Yiru Jin; Michael Zhuo Wang; Susanna Tse Journal: Nat Microbiol Date: 2022-09-05 Impact factor: 30.964
Figure 1
Scheme 1
Scheme 2
Scheme 5
Scheme 3
Scheme 4
Figure 2
Figure 3