Improvement of Aqueous Solubility of Lapatinib-Derived Analogues: Identification of a Quinolinimine Lead for Human African Trypanosomiasis Drug Development

Article abstract of DOI:10.1021/acs.jmedchem.8b01365

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC₅₀)) was established, as part of the lead selection process. Increasing the sp³ carbon content of 1 resulted in 10e (0.19 μM EC₅₀ against T. brucei and 990 μM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC₅₀: 0.013 μM; aqueous solubility: 880 μM; and CEC₅₀: 0.18 μM). Compound 22a reduced parasitemia 10⁹ fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Full text of DOI:10.1021/acs.jmedchem.8b01365

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Article
Improvement of Aqueous Solubility of Lapatinib-derived
Analogs: Identification of a Quinolinimine as a Lead for
Human African Trypanosomiasis Drug Development
Kelly Ann Bachovchin, Amrita Sharma, SEEMA BAG, Dana M Klug, Katherine M Schneider,
Baljinder Singh, Hitesh B Jalani, Melissa Buskes, Naimee Mehta, Scott Tanghe, Jeremiah D
Momper, Richard J Sciotti, Ana Rodriguez, Kojo Mensa-Wilmot, Michael P. Pollastri, and Lori Ferrins
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01365 • Publication Date (Web): 19 Dec 2018
Downloaded from http://pubs.acs.org on December 19, 2018
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Improvement of Aqueous Solubility of Lapatinib-derived Analogs: Identification of a Quinolinimine
as a Lead for Human African Trypanosomiasis Drug Development
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Kelly A. Bachovchin,1,ɸ Amrita Sharma, Seema Bag, Dana M. Klug, Katherine M. Schneider, Baljinder
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Singh, Hitesh B. Jalani, Melissa J. Buskes, Naimee Mehta, Scott Tanghe, Jeremiah D. Momper,5
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3,4
6
3,4
2*
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1*
Richard J. Sciotti, Ana Rodriguez, Kojo Mensa-Wilmot, Michael P. Pollastri, Lori Ferrins.
1Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
2Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of
Georgia, Athens, GA 30602
3
th
New York University School of Medicine, Department of Microbiology, 430 E. 29 St. New York,
NY 10016
4Anti-Infectives Screening Core, New York University School of Medicine, New York, NY 10016
5Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla,
CA 92093
6Experimental Therapeutics, Walter Reed Army Institute for Research, 2460 Linden Lane, Silver
Spring, MD, 20910
ɸThese authors contributed equally
*
Corresponding author
Abstract
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Lapatinib, an approved EGFR inhibitor, was explored as a starting point for the synthesis of new
hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous
work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous
solubility. In this report, we present various medicinal chemistry strategies that were used to increase the
aqueous solubility and improve the physicochemical profile without sacrificing anti-trypanosome potency.
0
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8
9
0
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0
To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC )) was
50
3
established, as part of the lead selection process. Increasing the sp carbon content of 1 resulted in 10e (0.19
µM EC against T. brucei and 990 µM aqueous solubility). Further chemical exploration of 10e yielded
5
0
2
2a, a trypanocidal quinolinimine (EC : 0.013 µM, aqueous solubility: 880 µM, and CEC : 0.18 µM).
50 50
9
Compound 22a reduced parasitemia 10 -fold in trypanosome-infected mice; it is an advanced lead for HAT
drug development.
Introduction
Human African trypanosomiasis (HAT) is a neglected tropical disease that is caused by the
1
protozoan parasite Trypanosoma brucei. The two subspecies responsible for human infection, T.b.
gambiense (western and central Africa) and T.b. rhodesiense (eastern and southern Africa), cause chronic
2
and acute forms of the disease respectively, and infection is considered 100% fatal if left untreated. HAT
is a threat to over 70 million people in sub-Saharan Africa, where the tsetse fly vector can be found, and is
endemic in many rural areas.3, 4 In 2015, fewer than 3,000 cases were reported, largely due to disease
elimination efforts set by the World Health Organization (WHO), which include vector control, case
4
reporting, and availability of treatments. Vaccines are not available for HAT prevention, due to extensive
antigenic variation of the major surface protein, variant surface glycoprotein.
Chemotherapy for HAT is specific to the stage of disease as well as the subspecies of T. brucei.
The disease occurs in two stages; the first stage starts with trypanosomes in the blood and lymphatic
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systems, causing influenza-like symptoms. Stage two of the disease is coincident with parasites crossing
the blood-brain barrier, and is associated with disrupted sleep patterns, leading to coma and death.2, 4, 5
Current drugs for HAT have significant shortcomings in terms of toxicity, efficacy, and convenience of
dosing; treatment failure that could be a result of drug resistance has also emerged.4
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Two orally-administered leads, fexinidazole6-8 and acoziborole (SCYX-7158),9 are in the midst of
clinical trials. Because of well-documented high failure rates in drug development,10-12 and the possibility
1
3
of drug resistance, we believe that it is prudent to continue populating the pipeline of anti-trypanosome
leads with backup compounds that meet the target product profile (TPP) set by WHO and the Drugs for
Neglected Diseases initiative (DNDi).14, 15
To streamline our anti-trypanosome drug discovery effort, we used a target class repurposing
approach to improve the efficacy against T. brucei of the U.S. Food and Drug Administration approved
1
6
drug lapatinib. A series of medicinal chemistry optimization campaigns produced the highly potent NEU-
1
17
617; subsequent work led to 1 (NEU-1953 ), a compound that was moderately potent against T. brucei,
but had limited aqueous solubility and high microsomal clearance (Table 1).18, 19 Side-stepping the daunting
task of simultaneously improving anti-trypanosome potency, aqueous solubility and metabolic clearance of
these hits, we began efforts to improve physicochemical properties with a focus on increasing solubility of
the compounds.
Table 1. Criteria used to select an advanced lead compound.
N
Head
HN
N
N
N
1
N
N
N
Tail
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Properties
Desired values
<0.03 µM, cidal
>100x EC50
1 (NEU-1953)
0.43 µM
>30 µM
180 µL/min/mg
87%
T.b. brucei EC50
HepG2 TC50
In vitro Human liver microsome clearance Cl <8.6 µL/min/mg
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int
Plasma protein binding (PPB)
<95%
Thermodynamic solubility (pH=7) >100 µM
44 µM
Pharmacokinetics
CNS exposure
>10x EC >6 h
nd
5
0
>10x EC >6 h
nd
5
0
In vivo
Mouse efficacy, bloodstream HAT <50 mg/kg p.o. x 10 days; >90% cure
Mouse efficacy, CNS HAT model
nd = not determined
Failed
<100 mg/kg p.o. x 10 days; >90% cure nd
Several medicinal chemistry strategies can be employed to increase the aqueous solubility of
compounds. These include increasing charge and polarity (through salt formation22, 23 or introduction of
3
24, 25
hydrophilic functional groups), reducing planarity by increasing sp carbon content,
or reducing the
aromatic ring content.26, 27 We present our application of different strategies to improve the aqueous
solubility and overall absorption, distribution, metabolism and excretion (ADME) profile of compound 1,
as part of an ongoing effort to produce an advanced lead that meets the criteria shown in Table 1. The
progression of our hit-to-lead optimization process followed the schematic highlighted in Figure 1, wherein
compounds were filtered through a series of selection parameters that were reflective of the TPP.14, 15 Given
that the TPP for HAT indicates a preference for compounds that act irreversibly against the trypanosome,
we developed a cidality assay that we used to prioritize compounds that exhibited such an effect.
Compounds were progressed into mouse model studies based upon these combined results (Figure 1).
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Figure 1. Hit-to-lead screening cascade employed in this project, along with the goal values used for the
various parameters, as determined when considering the TPP for HAT.
1
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Results and Discussion
Our goal was to improve the physicochemical properties, specifically focusing on the aqueous
solubility, of the previously reported compound 120 whose liabilities as an anti-trypanosome hit are
presented in Table 1. We also paid close attention to the lipophilic ligand efficiency (LLE) of each analog,
28
in comparison to 1. Calculated as the difference between the pEC and cLogP, LLE is now a frequently
5
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used metric in medicinal chemistry campaigns that normalizes the potency of an inhibitor to its lipophilicity;
2
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this allows easier comparison of compound quality as the optimization program proceeds. Furthermore,
given that compound lipophilicity can adversely impact pharmacokinetic properties, employment of LLE
helps to ensure that the best balance of lipophilicity and potency is struck to identify molecules with a good
ADME profile.
The approaches we pursued can be categorized as either increasing charge and polarity at biological
pH or decreasing planarity of the hits. Thermodynamic solubility (extent to which a compound dissolves)
is a more useful metric in guiding the optimization of compounds than kinetic solubility (the extent to which
3
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a compound precipitates from a solution). Furthermore, thermodynamic solubility of a compound is more
relevant in the biological assays used in the assessment of potency of our hits. As such, our hit optimization
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campaign tracked thermodynamic solubility throughout instead of the kinetic solubility of compounds,
although we have assessed kinetic solubility where necessary.
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Increasing charge and polarity
Salt screening. A common strategy used to increase the kinetic aqueous solubility of a series is through
the formation of salts at acidic or basic centers on compounds.22, 23 Initially, a range of salts of 1 were
studied (Table 2), taking advantage of the basic nitrogen atoms within the compound. The hydrochloride
(1a) and sulfate (1b) salt forms remained unchanged in both potency and kinetic solubility, while the citrate
salt (1c) showed a modest increase in kinetic solubility. The methanesulfonate salt (1d) demonstrated a 4-
fold increase in the kinetic solubility. However, the thermodynamic solubility decreased 2-fold for the
methanesulfonate (1d) salt form, remained unchanged for the hydrochloride (1a) and sulfate (1b) salt forms
and increased modestly for the citrate (1c) (25%).
Table 2. Salts of 1: Potency against T. brucei, thermodynamic and kinetic solubility, and metabolic
stability.
T. brucei
Kinetic
solubility
(µM)
25
Salt
Thermodynamic
solubility (µM)
HLM Clint
Salt form
EC (µM)
50
ratio
(µL/min/mg)
±
SEM
Free base
-
0.43 ± 0.015 44
0.33 ± 0.025 42
0.45 ± 0.024 39
0.51 ± 0.027 61
0.18 ± 0.023 16
180
160
170
190
170
Hydrochloride (1a)
Sulfate (1b)
Citrate (1c)
4:1
1:1
2:1
25
25
50
Methanesulfonate (1d) 2:1
100
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Modification of ionizable groups. A second strategy employed was introduction of functional groups that
are charged at physiological pH. For this purpose, we added ionizable groups to the piperazine. A general
synthetic scheme for these analogs is presented in Scheme 1. Briefly, 5-bromo-2-chloropyrimidine (2) was
substituted with the appropriate amines in good to excellent yields. The resulting bromide intermediates
1
1
1
1
1
1
1
1
1
1
2
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2
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a-3y were subjected to Miyaura borylation to give the corresponding boronic acid pinacol ester 4a-4p that,
2
0
in a Suzuki reaction with 5, led to the desired products 6a-6j and 10a-10j in acceptable to excellent yields.
Scheme 1. General synthetic scheme for addition of ionizable groups or increased sp3 carbon content as
new tail groups for analogs of 1.
N
N
Br
O
B
N
Br
HN
N
N
N
a or b
c
d
O
HN
N
N
N
R
N
+
Cl
N
R
N
N
Br
2
3a-3y
4a-4p
5
R
N
6a-6j, 10a-10j
Reagents and conditions: a) various substituted amines, Et N, EtOH, ambient temperature, 18 h (32-72%);
3
b) various substituted amines, DIPEA, tert-butanol, 150 °C, microwave, 30 mins (57-97%); c) B pin ,
2
2
KOAc, PdCl (dppf)·CH Cl , dioxane, 145 °C, 1 h (49%-assumed quant.); d) K CO , PdCl (dppf)·CH Cl ,
2
2
2
2
3
2
2
2
3
:1 dioxane/H O, 130 °C, microwave, 30 mins (21-99%).
2
Replacement of the piperazine ring with ethylene diamine (6a, Table 3) led to a loss of activity
against T. brucei and decreased aqueous solubility though the LLE remained high due to the reduced cLogP
0.81 compared with 2.1 for compound 1). The synthetic precursor tert-butyl carbamate (6b) was
(
predictably less soluble. The methylated, acyclic tail (6d) raised aqueous solubility but dropped anti-
trypanosomal potency ~4-fold compared to 1, however this was also accompanied by an improvement in
the human liver microsome intrinsic clearance (HLM Cl : 45 µL/min/mg) though this was still outside of
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our targeted range. The demethylated piperazine derivative 6f displayed a 6-fold loss in potency, the
solubility remained unchanged and the HLM Cl was significantly improved (30 µL/min/mg) suggesting
int
that demethylation of the piperazine is one of the major metabolites in this series. The tert-butyl carbamate
precursor (6e) was far less soluble than 1, with retention of moderate anti-trypanosome activity. Substitution
of the piperazine with methylene carboxylic acid (6h, presumably present as a zwitterion in vitro), and the
corresponding methyl ester (6g), made as a potentially more cell-permeable pro-drug of 6h, lost potency
and aqueous solubility compared to 1. Finally, the piperazine was replaced by proline (6i) and its methyl
ester derivative (6j); both were inactive against T. brucei, and no improvement in the ADME profile
compared to 1 was evident. Thus, analogs that are charged at biological pH do not consistently have better
aqueous solubility than 1.
0
1
2
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5
6
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8
9
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Table 3. Effect of incorporation of ionizable groups on 1 potency against T. brucei, metabolic stability, and
aqueous solubility.
N
HN
N
N
N
R
N
T. brucei
HepG2
TC50
Aq sol
Human
HLM Clint
ID
1
R
EC (µM)
LLE
5
0
(
µM)
PPB (%) (µL/min/mg)
±
SEMa
(µM)
N
0.43 ± 0.015 44
87
180
86
>30
ndd
4.3
3.9
N
H2N
6
a
N
21 ± 0.22
18
100
H
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H
N
6b
18 ± 1.6
>10
0.2
3.0
630
96
95
67
>300
>300
45
>30
8.0
2.5
Boc
N
H
N
6
c
-
Boc
N
N
H
1
1
1
1
1
1
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1
1
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6d
N
1.8 ± 0.076
>30
3.5
2.5
3.9
3.4
N
6
6
6
e
f
3.3 ± 0.10
2.6 ± 0.10
7.7 ± 0.88
0.1
39
16
99
nd
30
ndc
>30
>30
>30
N
Boc
HN
N
nd
O
N
N
g
ndb
N
O
O
6
6
h
i
>10
17
42
78
95
53
<3
>30
>30
-
N
HO
N
38 ± 1.6
3.6
O
HO
N
O
6
j
O
>50
0.3
93
220
>30
-
nd = no data
a
SCYX-7158 was used as a control, EC : 0.29 µM
5
0
bcompound lost, likely due to unspecific binding
ccompound detected in only the first sample
dnot tested initially and a combination of poor properties and potency meant that the compound was not
considered for progression
Reductions in planarity
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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
Pseudo-ring analogs. We looked to increase the conformational flexibility of the quinoline series by
breaking one of the rings of the central core; we hoped to maintain T. brucei activity by taking advantage
of a possible intramolecular hydrogen bond (Supplementary Information, Table S7). This scaffold also
has the advantage of incorporating additional hydrogen bond donors, increasing its potential interactions
with water. Applying this strategy to a compound related to 1, compound 7, led to compound 8 (Figure 2).
However, despite breaking the bicyclic aromatic system (8) this strategy did not show any significant
improvement to the aqueous solubility of the series or potency against T. brucei. An improvement in the
metabolic stability was observed (24 µL/min/mg) and was representative of this series of analogs, however
due to the lack of potency against T. brucei (and associated with a decreased LLE) this strategy was not
pursued further.
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 2. Pseudo-ring analogs of the previously reported compound 7.20
O
O
Cl
HN
Cl
HN
N
O
N
OH
N
N
N
N
N
N
7
N
8
T.b. brucei EC50: 0.91 ± 0.050 µM
Aq. sol. (µM): <1
T.b. brucei EC50: 4.8 ± 0.18 µM
Aq. sol. (µM): 6
PPB: 98%
PPB: 100%
HLM Clint (µL/min/mg): 170
HepG2 TC50: >3 µM*
compound dissolved at 0.5 mg/mL
HLM Clint (µL/min/mg): 24
HepG2 TC50: >6.5 µM*
*compound dissolved at 1 mg/mL
*
31
Methylation of the quinoline core. Methylation reduces planarity in biaryl motifs, and we hypothesized
that such a conformational change would increase solubility. Synthesis of compounds followed an
2
0
analogous route to a previously reported procedure, the details for which are presented in the Scheme S1,
Supplementary Information. However, introduction of the methyl groups led to a significant decrease in
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8
9
the potency against T. brucei causing a reduction in the LLE of the compounds was was accompanied by
increased toxicity toward HepG2 (human liver) cells, and a substantial decrease in aqueous solubility
(Table 4).
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
Table 4. Methylation of the quinoline reduces anti-trypanosome potency and increases toxicity of analogs
of 1.
N
HN
N
N
R1
N
R2
N
N
N
T. brucei
EC (µM)
Human
PPB
(%)
87
HepG2
TC50
Aq sol
µM)
HLM Clint
ID
R1
R2
LLE
5
0
(
(µL/min/mg)
±
SEMa
0.43 ± 0.015 44
2.5 ± 0.30 <0.02
-CH3 15 ± 1.4 5.0
(µM) (R2)
>30
1
-H
-H
180
130
120
4.3
3.0
2.2
9a
-CH3 -H
-H
97
5.9 (0.98)
9.6 (0.98)
9
b
97
nd = no data
a
SCYX-7158 was used as a control, EC : 0.29 µM
5
0
3
3
Increased sp atom content. We explored increasing the sp content of the tail group (Table 5), replacing
the piperazine ring with groups that have increased three-dimensionality. Bridged piperazines in 10a and
10b improved aqueous solubility 20-fold, as compared to 1, conferred a slight improvement in potency
against T. brucei, exhibited an improved LLE, and a 4-fold increase in metabolic stability. Ethyl and propyl
substituted piperazines (10c and 10d respectively) were ineffective at improving aqueous solubility, though
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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
potencies were comparable or better than 1. Swapping the piperazine moiety for alkyl-substituted
homopiperazines (10e, 10f and 10g) yielded highly soluble compounds coupled with a 2-to-4-fold
improvement in anti-trypanosome potency. In the case of 10f and 10g, there was a noticeable decrease in
metabolic stability that makes these hits less attractive for anti-trypanosome drug development, compared
to 10e. Similarly, the spirocyclic derivatives 10i and 10j showed 2-to-3-fold improvement in potency
compared with 1 and were both highly soluble. By far, the bridged piperazine (10a), the
methylhomopiperazine (10e) and the spirocyclic derivatives (10i and 10j) were the most improved
compounds in terms of anti-trypanosome potency, LLE, metabolic stability, and aqueous solubility. For
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
1
0e we also obtained calculated pK data and compared it with 1 (see Supplementary Information, Table
a
S4), the aliphatic nitrogen on the homopiperazine was found to be between 0.8-1.2 log units more basic
than 1. Closer examination of the LogD values (see Supplementary Information, Table S3) for this set
of analogs revealed that the addition of a one carbon bridge to the piperazine (10a and 10b) and replacement
with spirocyclic derivatives (10i and 10j) led to a decrease in the LogD compared with 1, which is consistent
3
2
with a recent report from AstraZeneca. We also observed a similar trend with two of the homopiperazine
analogs (10e and 10f), while 10g had the same LogD value as 1, despite the addition of 3 methylenes.
3
Table 5. Increased sp carbon content in the tail region can improve metabolic stability and aqueous
solubility of 1.
N
HN
N
N
N
R
N
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8
9
T. brucei
Aq
sol
Human
PPB
HepG2
TC50
HLM Clint
µL/min/mg)
ID
1
R
EC (µM)
LLE
5
0
(
±
SEMa
(µM) (%)
(µM) (R2)
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
N
N
N
0.43 ± 0.015 44
0.20 ± 0.020 910
0.15 ± 0.020 960
87
51
58
180
37
>30
>30
>30
4.3
4.6
4.7
N
N
N
1
1
0a
0b
41
0.070 ±
14
0.030
N
10c
94
95
>300
ndb
25 (0.93)
>30
4.7
3.4
N
N
1
0d
0.38 ± 0.030 44
N
0.19 ±
990
0.0080
N
10e
10f
75
80
77
>30
>30
4.6
4.1
N
N
N
0.25 ± 0.050 800
0.10 ± 0.014 78
200
N
10g
90
300
>10
4.0
N
N
1
1
1
0h
0i
25 ± 4.2
2.0
99
48
70
>300
43
3.4 (0.90) 1.1
N
N
0.25 ± 0.010 690
0.15 ± 0.010 700
>24
4.6
4.5
N
0j
77
16 (0.96)
N
nd = no data
a
SCYX-7158 was used as a control, EC : 0.29 µM
5
0
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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
bcompound detected in only the first sample
Saturated rings were also used to replace the pyrazine head group of 1 to explore the solubility
3
effect of increasing sp carbon content in this region. While the synthesis initially employed a Buchwald
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
amination to install the saturated primary amines (required for the preparation of compounds 15b and 15d)
(Scheme S2, Supplementary Information), subsequent optimization led to an alternative route where the
2
0
dihalogenated intermediate (11) was subjected to S Ar conditions with various primary amines to give
N
compounds 12a-12h. A subsequent Suzuki coupling utilizing both the N-methylpiperazine pyrimidine
boronic acid, pinacol ester (14, prepared from the bromo precursor 13)20 as well as the N-
methylhomopiperazine pyrimidine boronic acid, pinacol ester (4p, prepared from 3m) provided desired
compounds 15a, 15c, 15e-15g and 17a-17c.
Scheme 2. Synthetic route for various head group analogs.
R
HN
R
Cl
HN
3m, 13
p, 14
i) c
4
a or b
N
N
Br
N
Br
N
ii) d or e
N
N
11
12a-12h
N
15a, 15c, 15e-15g, 17a-17c
n
n = 1 or 2
Reagents and conditions: a) saturated primary amine, DIPEA, n-butanol, 200 °C, microwave, 3 h (22-98%);
b) aryl primary amine, NaH, DMF, 60 °C (38-75%); c) B pin , KOAc, PdCl (dppf)·CH Cl , dioxane, 145
2
2
2
2
2
°
C, microwave, 1 h (assumed quantitative); d) Cs CO , Pd(PPh ) , 3:1 dioxane/H O, 80 °C, 48 h (11-44%);
2 3 3 4 2
e) K CO , PdCl (dppf)·CH Cl , 3:1 dioxane/H O, 130 °C, microwave, 30 mins (7-29%).
2
3
2
2
2
2
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The analog bearing the cyclohexylamine, 15a (Table 6), showed a modest improvement in
solubility and potency over 1, but was less metabolically stable and showed a reduction in LLE due to an
increase in the cLogP (3.8 compared with 2.1 for compound 1). Tertiary amines (15b, 15c and 15d) all
exhibited improved solubility with moderate-to-excellent anti-trypanosome potencies. Compound 15d is
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
2
0-fold more potent than 1 with a 10-fold improvement in aqueous solubility and significantly improved
LLE (5.3). However, its high plasma protein binding (PPB; 100%) and toxicity against HepG2 cells are
liabilities that make it less desirable for progression. Replacement of the headgroup with the
tetrahydropyrans (15e and 15f) maintained activity against T. brucei, when compared to 1, and improved
aqueous solubility 15-fold but again, increased toxicity against HepG2 cells.
In summary, modulation of the aqueous solubility of the headgroup of 1 could be achieved through
3
increasing the sp content. However, these analogs typically exhibited increased toxicity in HepG2 cells
and were less metabolically stable than those presented in Table 5.
3
Table 6. Increase in sp carbon content of the head group region of 1: Effect of anti-trypanosome
potency, toxicity to HepG2, aqueous solubility, and metabolic stability.
R
N
N
N
N
N
T. brucei
HepG2
TC50
EC50
Aq sol Human PPB
(µM) (%)
HLM Clint
ID
R
LLE
(µM) ±
(µL/min/mg) (µM)
(R2)
SEMa
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2
3
4
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
N
N
0
0
0
0
.43 ±
.015
.29 ±
.010
1
1
44
98
87
85
180
300
ndc
>30
ntf
4.3
2.8
5.3
HN
HN
HN
5a
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
N
0.053 ±
0.00
15b
>1000 ndb
ntf
N
0.17 ±
2.5
1
5c
660
ndd
ndd
3.7
HN
HN
0
.010
(0.95)
0.022 ±
0.001
3.0
N
nde
94
15d
590
760
760
980
100
50
5.3
4.5
4.0
3.0
(0.90)
15
O
0.38 ±
1
1
1
5e
5f
HN
HN
0
0
0
1
.040
.36 ±
.030
.9 ±
(0.96)
16
47
170
160
O
(0.98)
5g
63
>3.6
HN
OH
0.26
nt = not tested
nd = not determined
a
SCYX-7158 was used as a control, EC : 0.29 µM
5
0
bcompound lost, likely due to unspecific binding
cassay failed due to poor curve fitting
dpoor MS response
elow recovery
fnot tested initially and compounds with a superior profile overall had already been identified
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Insertion of -NH linker. Addition of an -NH linker between ring systems in the molecule could be explored
using a previously described synthetic route,20 and increased aqueous solubility in a position-dependent
manner (Table 7). Placing a nitrogen atom between the aromatic rings of the tail and core groups of the
molecule increased the aqueous solubility. For example, compare 1 to the matched analog 16c, which
showed a ~12-fold reduction in potency, although a 3-fold improvement in aqueous solubility occurred.
Moving the amine linker from the 5- to the 4-position of the pyrimidine (16d) restored some of the potency
when compared to 16c, but at a loss of aqueous solubility. Shifting the pyrimidine nitrogen (16e) restores
some of the aqueous solubility that was lost in 16d and retains potency compared to 1. Inclusion of the
nitrogen between the phenyl ring of the tail group and the quinoline core (16b), greatly improved the
solubility whilst maintaining potency compared to 16a. The addition of a nitrogen atom between the
piperazine and pyrimidine rings (16f) increased solubility over 20-fold, decreased plasma protein binding
to 51%, and increased metabolic stability 10-fold but dropped anti-trypanosomal potency 5-fold. Given the
lack of broad applicability of this strategy to the series, we sought other avenues by which to increase the
aqueous solubility.
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
Table 7. Influence of adding -NH linkers to analogs on metabolic stability, solubility, and potency against
T. brucei.
N
HN
N
N
R
T. brucei
HepG2
TC50
EC50
Aq sol Human
HLM Clint
ID
R
LLE
(
µM) ±
(µM) PPB (%) (µL/min/mg)
µM) (R2)
(
SEMa
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2
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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
N
0.43 ±
0.015
1
1
44
87
91
180
61
>30
>30
4.3
2.7
N
N
N
N
N
0
.91 ±
6a19
1.7
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
0.027
H
N
0
0
.91 ±
.092
1
6b
810
71
88
ndb
46
17 (0.98)
24 (0.96)
2.9
3.4
N
N
H
N
N
N
16c19
5.1 ± 0.20 130
N
N
N
N
N
0.81 ±
2
0.023
1
1
6d19
6e19
N
N
N
H
ntc
ntc
nd
21 (0.98)
22 (0.98)
3.6
3.9
N
N
0
.38 ±
26
ndb
N
N
N
H
N
N
0.029
H
N
2
0
.3 ±
1
6f
>1000 51
16
ndc
4.1
N
.070
nd = no data
a
SCYX-7158 was used as a control, EC : 0.29 µM
50
blow MS/MS signal
cnot tested initially and compounds with a superior profile overall had already been identified
At this point, 10e (Table 5) emerged as the preferred chemotype for optimization because of its
improved metabolic stability, aqueous solubility, plasma protein binding, and potent anti-trypanosomal
activity. To further explore the SAR, we performed a methyl scan on the pyrazine headgroup (Table 8).
These analogs are matched pairs with 10e and were synthesized following the procedure outlined in Scheme
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9
1
3
2
. Addition of a methyl group at R (17a) or R (17c) was tolerated, whilst maintaining excellent aqueous
2
solubility. Conversely, substitution with a methyl group at the R position (17b) reduced anti-trypanosome
potency 2-fold, compared to 10e, and reduced aqueous solubility, although it is still significantly higher
than our starting point (1) and the targeted threshold of 100 µM (Table 1). Finally, alkylation of the
endocyclic nitrogen (18) was detrimental to anti-trypanosome potency, whilst the physicochemical features
were relatively unchanged compared to 10e.
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
Table 8. Headgroup modifications of 10e: Effect on anti-trypanosome potency, metabolic stability,
aqueous solubility, and plasma protein binding.
R1
R2
N
N
N
N
HN
R3
N
N
N
N
N
N
N
N
N
N
N
18
T. brucei
HepG2
TC50
Aq
sol
Human
PPB
EC50
HLM Clint
ID
R1
R2
R3
LLE
(
µM) ±
(µL/min/mg) (µM)
(R2)
(µM) (%)
SEMa
0.19 ±
0.0080
10e
H
H
H
H
H
H
990
830
650
75
58
78
77
>30
>20
>30
4.6
4.2
4.1
0
0
0
0
.31 ±
.009
.46 ±
.049
1
1
7a
7b
-CH3
H
79
-CH3
100
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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
0
2
0
.13 ±
.027
.7 ±
18
1
1
7c
8
H
H
-CH3
>1000 77
72
4.6
3.2
(0.90)
810
67
170
>30
.070
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
a
SCYX-7158 was used as a control, EC : 0.29 µM
50
Alkylation of the -NH was attempted using the conditions outlined in Scheme 3 however,
alkylation occurred on the quinoline nitrogen (19a) rather than the endocyclic nitrogen as expected.
Alkylation on the quinoline nitrogen, and the E configuration of the imine was confirmed by x-ray
crystallography (Figure S2, Supplementary Information). A compound with a related structure has
3
3
previously been reported utilizing reaction conditions similar to those employed here and there are only a
few other examples of the quinolinimine scaffold available in the literature.34-36 Further, analysis of the
proton NMR reveals a characteristic chemical shift (~6.8 ppm) for the proton at the 3-position of the
quinolinimine. A Suzuki coupling with the relevant boronic acid was used to obtain the final product 22a,
whose hydrolytic stability was confirmed under both acidic and basic conditions. Further, the stability of
2
2a was assessed in the presence of a hard and soft nucleophile; it was found to be stable in the presence
of both glutathione and cyanide (Supplementary Information). To access the originally desired analog
18, Scheme 3b), methylation of the 2-aminopyrazine (23) was readily achieved to give 24 which could be
(
used to displace the chloro 11, before a Suzuki coupling to provide 18 in good yield.
Scheme 3. General synthetic route to quinolinimine core analogs, and 18.
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a)
N
N
N
N
N
N
HN
N
N
N
a
b, c
N
N
R
Br
Br
N
R
N
N
N
5
19a-c R=Me, Et or Pr
22a-22c
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
N
N
b)
N
N
N
N
N
N
d
e
b, c
N
N
N
N
H N
N
N
2
H
N
N
Br
N
2
3
24
25
18
Reagents and conditions: a) NaH, R-I, THF, 0 °C-ambient temperature (27-63%); b) 3m, B pin , KOAc,
2
2
PdCl (dppf)·CH Cl , dioxane, 145 °C, microwave, 1 h; c) K CO , PdCl (dppf)·CH Cl , 3:1 dioxane/H O,
2
2
2
2
3
2
2
2
2
1
30 °C, microwave, 30 mins (36-53% over two steps); d) 2-aminopyrazine, NaH, CH I, THF, 0 °C-ambient
3
temperature (49%); e) 11, NaH, DMF, ambient temperature (82%).
Alkylation of the quinoline nitrogen with the methyl, ethyl and propyl groups (22a, 22b and 22c
respectively) maintained excellent aqueous solubility, reduced PPB, and increased metabolic stability.
Compound 22a demonstrated a significant boost in potency (~15-fold) when compared to 10e, which
translated to an improved LLE (5.4 compared with 4.6 for 10e). The ethyl and propyl derivatives (22b and
2
2c) proved to be more metabolically labile, although they were improved over our original compound 1.
A comparison of the melting points of 1 (aqueous solubility: 44 µM), 10e (aqueous solubility: 990
µM) and 22a (aqueous solubility: 880 µM) (see Table S2, Supplementary Information) revealed a
decrease in the melting point range with the increased solubility of 10e and 22a. This suggests that one of
the factors influencing the increased solubility of 10e and 22a is the disruption of the crystal packing in
these analogs.
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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 9. Quinolinimine derivatives of 10e: potency against T. brucei, metabolic stability, aqueous
solubility and plasma protein binding.
N
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
N
N
N
N
R
N
N
N
T.
brucei
HepG2
TC50
Aq
sol
Human
PPB
25×
6 h
EC50
HLM Clint
ID
R
LLE EC50 CEC50
(
µM)
(µL/min/mg) (µM)
(R2)
(µM) (%)
(µM) (µM)
±
SEM
0.19 ±
10e
990
880
75
15
77
22
>30
>30
4.6
5.4
nt
nt
0
0
.0080
.013
2
2
2a
2b
-CH3
±
0.33 180
0
0
.001
.069
-CH CH
±
1000 41
38
51
>30
>20
4.3
3.4
1.7
3.9
1700
2
3
0
.010
-
0.15 ±
22c
890
41
nda
(
CH ) CH 0.00
2 2 3
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9
SCYX-
158
0
.29
7.2
1100
7
nt = not tested
acompound did not meet the threshold of 50% cidality in either of 6 h or 12 h (data not shown) assays using
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
2
5×EC50
Cidal effective concentration (CEC ) can inform selection of hits for advancement
5
0
Given that the TPP for HAT indicates a preference for compounds that act irreversibly against the
trypanosome,15 an assessment of trypanocidality was undertaken for hits with EC50 ≤ 200nM. Here
5
trypanosomes (1×10 /mL) were incubated with hit (concentration 25-times EC , i.e. 25×EC ) for 6 h
5
0
50
(
Figure 3) or 12 h (data not shown). The number of trypanosomes used in this cidal assay was adjusted to
maintain the ratio of trypanosome density-to-hit concentration as was used in the 48 h proliferation assay
employed to determine EC . At the end of incubation, cell density was checked, compound was washed
5
0
4
off, and trypanosomes were incubated in fresh drug-free medium at density of 10 /mL. After 48 h culture
in absence of hits, trypanosomes were enumerated using a hemocytometer.
Data presented on the left side of Figure 3 shows that in the first 6 h of incubation with hits,
trypanosomes were viable. For cells treated with cidal hits, the expected density of trypanosomes is 50%
of the control (i.e., DMSO-treated) at the end of 48 h culture at the next stage of this cidal assay.
Trypanosomes treated with 22c (Table 9) had a density above 50% of the control value, meaning that the
compound is not cidal at 6 h, based on data expected from the EC values. Trypanosomes treated with 22b
5
0
(
Table 9) had densities that were approximately 50% of the controls, implying that the hit is cidal. The
most trypanocidal compounds, using the parameters presented above was 22a (Table 9); for this compound
trypanosome density was 25% that of the controls. In comparison to the clinical candidate, acoziborole
(
SCYX-7158), compound 22a appears to exhibit less of a cidal effect. However, because the anti-
trypanosomal potency of acoziborole (T. brucei EC : 0.29 µM) is higher than the other compounds, a
5
0
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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
significantly higher concentration of acoziborole (7.2 µM) was used in the cidal assay compared to 22a
(0.33 µM) making a direct comparison difficult.
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
5
Figure 3. Trypanocidal effect of test compounds. Log phase T.b. brucei (1×10 /mL) were incubated with
the hit (at 25×EC concentration) for 6 h and transferred into drug free HMI-9 medium for 48 h at a starting
5
0
4
cell density of 1×10 cells/mL. Trypanosomes were enumerated with a hemocytometer after 48 h
(*indicates student’s T-test p<0.01; error bars indicate standard deviation). The dashed line corresponds to
expected density of cells if 50% were killed in the earlier 6 h exposure to a hit.
To obtain a more robust measure of cidality, the study summarized in Figure 4 was performed.
Using the protocol described in Figure 3, we tested several concentrations of hits around the amount used
in the single-point assay (25×EC ), to arrive at CEC , the drug concentration at which 50% of
5
0
50
trypanosomes are killed after a 6 h exposure (Figure 4). When comparing CEC with 25×EC for multiple
5
0
50
hits a linear relationship is expected in a plot, because the assumption behind the 48 h proliferation assay,
used routinely in the field, is that trypanosome growth arrest occurs because a drug kills the parasite cells.
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For a drug development project, it is preferable that killing of the trypanosome takes place within one cell
division cycle (6 h) of drug treatment. Compounds that kill in >6 h are revealed by significant deviation of
a compound’s data point from an idealized linear correlation between 25×EC and CEC (see trend line
5
0
50
in Figure 4). For 22a and 22b the CEC values fall on the trend line (CEC /25×EC is between 0.95-1.1),
5
0
50
50
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
indicating that they are cidal after trypanosomes are exposed to them for 6 h.
Figure 4. Plot of 25×T. brucei EC against CEC of hits.
5
0
50
8103
22a
22b
SCYX-7158
6
4
2
103
103
103
0
0
2103
4103
6103
8103
25*GI50
Efficacy of 22a in a mouse model of HAT
Recognizing that it is difficult to attain a concentration in blood above 500 nM for most drugs
administered orally to vertebrates, we considered 22a as a candidate for testing in a murine model of HAT,
because of its low CEC (175 nM), potency (T. brucei EC = 0.013 µM), selectivity over mammalian cells
5
0
50
(selectivity index > 2000), low PPB (15%), excellent aqueous solubility (880 µM) and reasonable metabolic
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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
stability (22 μL/min/mg; Table 9). A preliminary toxicity study with 22a showed no adverse effects when
dosed orally to mice at 100 mg/kg. Pharmacokinetic (PK) parameters were measured in plasma following
oral administration of 100 mg/kg of 22a (see Table S6, Supplementary Information). To achieve efficacy
in a mouse model of HAT, we aimed to exceed 10×EC for 6-8 h (Table 1), and at this dose, we were in
5
0
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
excess of 0.013 µM (10×EC ) for 13 h in blood (see Figure S4, Supplementary Information).
5
0
In a mouse efficacy study of 22a (dose: 100 mg/kg, p.o., Figure 5) the compound was administered
one day post-infection, once daily for five days, and then on day six they received an additional dose of 50
8
8
mg/kg of 22a. In control (untreated) mice, parasitemia reached 10 /mL (mean parasitemia = 4.1×10 /mL)
and they were sacrificed. For mice treated with 22a, parasitemia decreased (compared to that recorded in
control mice) from day two post-infection. On day four, the difference in parasitemia between untreated
(control) and 22a-treated animals was 100-fold, which met the benchmark for classification of 22a as a lead
compound for HAT.37 In mice treated with 22a, parasitemia peaked at day five (3.7×10 /mL), and then
6
4
declined to a mean of 2×10 /mL, before becoming undetectable in three mice by day nine (cell density <
4
4
×10 /mL), although treatment was terminated on day seven. Had the control (untreated) mice lived until
1
3
day nine, the projected mean parasitemia would be 10 /mL, as density of trypanosomes in blood rises 10-
fold every 24 h. Thus, the estimated difference in parasitemia between 22a-treated and control mice is 109-
fold on day nine. However, as trypanosomes were detected in the blood of the treated mice on day 11, the
mice were not deemed to have been cured in this study (which is defined as no parasitemia on day 30).
Through this study, compound 22a has been identified as a promising lead for further optimization. Future
studies will attempt to find a dosing protocol that may lead to cure of the trypanosome infection in mice by
22a.
Figure 5. Effect of 22a on trypanosomes in a mouse model of HAT. Mice (four per group) were each infected
5
with 10 T. brucei brucei (day 0). Compound 22a (100 mg/kg) was administered p.o. for six days, (an
additional 50 mg/kg was dosed on day six). Control mice received vehicle. Different shapes represent
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individual mice, and horizontal lines in each group indicate mean parasitemia. Parasitemia was determined
with a hemocytometer. UND = undetectable.
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
Safety profiling of 22a
Given the promising outcome of the efficacy study into 22a, we sought to gain a better
understanding of the human kinase inhibition profile of this compound as compared to 1 (Figure S5,
Supplementary Information). While 1 showed moderate to potent inhibition of 28 out of the 45 human
kinases tested in the Eurofins ExpreS Diversity kinase panel, 22a demonstrated potent inhibition of only
three out of the 45 kinases tested. This indicates a significant improvement in the overall selectivity of this
compound as compared to the starting point for this work (full data for 22a presented in Table S9,
Supplementary Information).
To assess the genotoxicity of 22a, we used an Ames fluctuation assay offered by Eurofins to detect
frameshift mutations and base-pair substitutions in Salmonella typhimurium strains treated with 22a (full
data presented in Table S10, Supplementary Information). We also tested major metabolites identified
through metabolic activation using rat liver S9 fraction. Neither 22a or its metabolites were found to be
genotoxic under these conditions.
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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
Finally, given the improved selectivity profile of 22a, it was further assessed in a
HitProfilingScreen (Eurofins) consisting of 30 GPCRs and ion channels. Significant responses (>50%
inhibition at 10 µM) were recorded in five of these assays, against: muscarinic M2, muscarinic M3, nicotinic
acetylcholine α1, hERG and the norepinephrine transporter (full data presented in Table S11,
Supplementary Information). The activity of 22a against these receptors is the major liability of this
compound and is the subject of ongoing optimization efforts which will be reported in due course.
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
Conclusions
Previous studies focused on the optimization of lapatinib against T. brucei led to the identification
of 1, a potent and selective anti-trypanosomal compound. The work presented herein describes a number
of strategies that were employed to improve the ADME profile of 1, and it was found that increasing the
3
3
sp carbon content of the molecule was a highly successful approach. Increasing the sp content in the tail
portion of the molecule led to 10e which reduced parasitemia in vivo. Further exploration of the 10e
chemotype led to 22a, a quinolinimine that renders parasitemia undetectable in vivo in 75% of mice
following seven days of treatment. Further profiling of 22a reveals that it has an improved kinase selectivity
profile versus the starting compound and neither it, nor its metabolites, are genotoxic. We have identified
a hERG liability which we are conscious of correcting as our efforts to further optimize this chemotype for
efficacy in the murine model of HAT continue. We conclude that 22a is a promising lead for HAT drug
development, with an improved ADME profile and potent anti-trypanosomal activity over starting
compound 1.
Table 10. Summary table showing progress 1 and 22a.
Targeted lead
Properties
1
22a
values
in vitro
T. brucei EC50
<0.03 µM, cidal
0.43 µM
0.013 µM, cidal
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9
HepG2 TC50
>100x EC50
>30 µM
180
>30 µM
22
Cl <8.6
int
HLM clearance
µL/min/mg
Plasma protein
binding
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
<
95%
87%
15%
Solubility (pH=7) >100 µM
44 µM
880 µM
in vivo
Pass at 100
mg/kg, p.o.
Pharmacokinetics >10x EC for >6 h nd
5
0
1
00 mg/kg, p.o.,
Mouse efficacy,
<50 mg/kg p.o. x 5
7 days, 75%
UND
Failed
bloodstream stage days; >90% cure
parasitemia
nd = no data
Experimental
Biological assay protocols
Trypanosoma brucei proliferation.
New York University (NYU). In a 96-well plate, compounds were added in triplicates at 50 μM and in
3
serial dilutions 1:2 in HMI-9 medium. To each well, 2.5×10 T. b. brucei (strain 427) were added and
incubated at 37 °C, 5% CO for 48 h. Following incubation, 20 μL of PrestoBlue® were added to each well
2
and incubated for additional 4 h. Fluorescence was read at 530 nm excitation and 590 nm emission. Suramin
at 100 μM was used as positive control and reference for calculation of IC .
5
0
ACS Paragon Plus Environment