Facile Synthesis and Preliminary Structure-Activity Analysis of New Sulfonamides Against Trypanosoma brucei

Article abstract of DOI:10.1021/ml400487t

The high throughput screening of a library of over 87,000 drug-like compounds against the African sleeping sickness parasite resulted in the discovery of hits with a wide range of molecular diversity. We report here the medicinal chemistry development of one such hit, a tetrahydroisoquinoline disulfonamide, with the synthesis and testing of 26 derivatives against the trypanosome subspecies. Activities in the 2-4 μM range were revealed with a selectivity index suitable for further development.

Full text of DOI:10.1021/ml400487t

Letter
pubs.acs.org/acsmedchemlett
Facile Synthesis and Preliminary Structure−Activity Analysis of New
Sulfonamides Against Trypanosoma brucei
Adel A. Rashad,† Amy J. Jones,‡ Vicky M. Avery,‡ Jonathan Baell,*,§ and Paul A. Keller*,†
†School of Chemistry, University of Wollongong, Wollongong 2522, Australia
‡Eskitis Institute for Drug Discovery, Griffith University, Brisbane Innovation Park, Don Young Road, Nathan 4111, Australia
§Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia
S
* Supporting Information
ABSTRACT: The high throughput screening of a library of over 87,000 drug-like compounds against the African sleeping
sickness parasite resulted in the discovery of hits with a wide range of molecular diversity. We report here the medicinal
chemistry development of one such hit, a tetrahydroisoquinoline disulfonamide, with the synthesis and testing of 26 derivatives
against the trypanosome subspecies. Activities in the 2−4 μM range were revealed with a selectivity index suitable for further
development.
KEYWORDS: T. brucei, SAR, tetrahydroquinoline, sulfonamides
untreated, the parasites penetrate the blood−brain barrier and
invade the central nervous system (CNS) (phase II) causing
neurological symptoms including progressive mental deterio-
ration, sleep disturbances, long lasting coma, and finally death if
not treated.6
Unfortunately, vaccines are not available and therefore, the
main line of defense against the parasite is chemotherapeutics.
The treatment options are limited with only four registered
drugs available. Suramin and pentamidine are effective against
early stage infections while melarsoprol (contains arsenic) and
eflornithine are used to treat late-stage disease (Figure 1).7,8
These drugs were developed approximately 30 years ago, and
suffer high toxicity, lack of efficacy, and emerging resistance are
concerns. Melarsoprol is the most toxic, causing a reactive
encephalopathy in 5−10% of treated patients, with a 1−5%
mortality rate.9
Trypanosoma brucei gambiense and T. brucei rhodesiense are the
causative agents of human African trypanosomiasis (HAT), also
known as sleeping sickness. T. b. rhodesiense is found in Eastern
and Southern Africa, whereas T. b. gambiense occurs in Western
and Central Africa and is responsible for over 90% of all
reported cases of infection.1 This disease threatens about 70
million people living in sub-Saharan Africa and causes an
estimated 25,000 deaths per year.2,3 It has a major impact on
the affected nations causing suffering and poverty, and if left
untreated, the disease is usually fatal.4 A lack of full-scale
screening programs and poor diagnostic tools leads to an
under-reporting of cases, which is likely to be at least 3-fold
higher than the measured value.5
Both subspecies are transmitted by the bite of the infected
tsetse fly. T. b. gambiense HAT is primarily a chronic disease,
and it can be many months to years before patients succumb to
the disease. In contrast, T. b. rhodesiense HAT is acute, with
death occurring within months of infection. After the bite, the
parasites start to multiply in the blood; that is, phase I. During
this phase, the parasite lives within the bloodstream and
subsequently migrates to other areas of the human body, such
as the lymph nodes and spleen, causing febrile illness with
symptoms similar to those caused by malaria (rash, fever,
shaking chills, body aches, and general fatigue). If phase I is left
Recently, there has been some progress in the treatment of
HAT10 with a nifurtimox−eflornithine (Figure 1) combination
(NECT) therapy developed, which is as effective as eflornithine
monotherapy but was easier and cheaper to administer.11,12
Received: November 26, 2013
Accepted: March 10, 2014
Published: March 10, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a mammalian cell line HEK293, to determine a selectivity index
(SI) for each compound. Although T. b. brucei is nonhuman
infective, it is frequently used in HAT drug discovery
campaigns and lead optimization programs as a model for the
human infective subspecies (T. b. gambiense and T. b.
rhodesiense), which are more difficult to maintain and culture
in vitro. Cluster analysis, considering chemical alerts such as
toxicophores, the likelihood of CNS penetration, and drug-like
structural features, yielded a subset of 12 compounds as
promising medicinal chemistry starting points for drug
development.
This communication discusses the synthesis and antitrypa-
nocidal activity of new analogues for the bis-sulfonamide hit,
WEHI-1203255 (Figure 3), which showed an IC50 value of 1.3
Figure 1. Older generation African sleeping sickness drugs.
However, NECT is not ideal due to the parental mode of
administration, the need to hospitalize patients during treat-
ment, and the possible development of resistance.13,14
Increased research and investment into HAT chemotherapy
has resulted in the identification of numerous trypanocidal
compounds, a number of which have entered or are in clinical
development. The diminazene, pafuramidine15 (Figure 2), was
Figure 3. Lead compound (WEHI 1203255), IC50 = 1.3 μM.
μM with a SI > 32. This compound has excellent
physicochemical properties, good calculated aqueous solubility
of 100 μM, an acceptable polar surface area of 84 Å2, and an
acceptable CLogP value20 of 2.5. The analogues were
synthesized and tested for their ability to inhibit the growth
of T. b. brucei limiting the changes to the two sulfonamide
moieties to study the preliminary structure−activity relation-
ships (SARs). The cytotoxicity profiles of the compounds were
evaluated using HEK293 cell line, and SI was estimated for each
analogue. The SI of the compounds was determined where
possible by directly comparing the IC50 values from the T. b.
brucei and HEK 293 assay. If this was not possible, an estimated
SI value was calculated by comparing the IC50 in the T. b. brucei
assay and the highest dose at which there was no activity
(<50%) in the HEK 293 assay.
Figure 2. New generation of potential African sleeping sickness
The strategy for the synthesis of sulfonamide analogues is
summarized in Scheme 1 and started from 7-nitrotetrahy-
droquinoline, which was sulfonated using the appropriate
sulfonyl chloride in pyridine at room temperature. The nitro
group was reduced, initially using acetic acid in ethanol in the
presence of tin with sonication;21 however, under these
conditions, yields were between 50 and 60%. The use of
Raney nickel in methanol in the presence of hydrazine hydrate
treatments.
the first oral drug to enter clinical development for early stage
HAT in 2005. However, the observation of severe hepatic
toxicity and renal insufficiency during a retrospective phase I
trial in 2008 lead to the compound being abandoned. In 2009, a
second orally available drug, fexinidazole16 (Figure 2), entered
phase I clinical trials for HAT. The drug is effective against both
stages of the disease and subsequently progressed to phase II/
III clinical development in 2012.
a
Scheme 1
More recently, the novel boron-containing molecule (SCYx-
7158) (Figure 2) emerged as an orally active drug candidate,
with promising activity against both acute and CNS stage
infections. The compound successfully completed preclinical
studies in 2011 and entered phase I clinical trials in 2012.17 The
mechanism by which the boronate acts as a trypanocidal agent
is still unknown. Overall, present treatment options for HAT
are limited, and the high attrition rates in drug discovery means
that new therapeutics with acceptable efficacies and safety
profiles are urgently needed.18
High throughput screening (HTS) is one approach that can
be used to identify new lead compounds for such neglected
diseases. Therefore, the HTS library of 87,926 compounds
(WEHI 2003)19 was tested against the nonhuman infective
trypanosome subspecies, Trypanosoma brucei brucei and against
a
Reagents and conditions: (a) dry pyridine, R1SO2Cl, rt, 24 h; (b)
methanol, NH2NH2·H2O, Raney nickel, reflux, 6−8 h; (c) dry
pyridine, R2SO2Cl, 0 °C to rt, 4−6 h.
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Table 1. R1, R2, Molecular Weight, ClogP, IC50 and Selectivity Index for Compounds 10−35
a
Calculated using ACDLabs v.12.0 (ACD/Laboratories, Toronto, Canada); S.I. = selectivity index.
as a source of hydrogen reliably gave the aniline derivatives in
gram quantities in 80−90% yields. The amino group was then
reacted with the different sulfonyl chlorides in pyridine at room
temperature (Scheme 1), giving the final analogues in 70−80%
yield. HPLC analysis of these bis-sulfonamides showed a purity
range >95% for all the synthesized derivatives. This facile three
step synthetic strategy enabled us to access 26 separate
derivatives in a short time frame, reliable yields as well and at
reasonable cost.
The results for the testing against T. b. brucei, the calculated
ClogP, and the SI are listed in Table 1. The initial activity was
determined by screening at 1 and 10 μM, and derivatives
showing >80% activity at 10 μM and >50% activity at 1 μM
were then tested to obtain the IC50 values. The first series of
derivatives examined the changes in the aromatic sulfonyl
moiety where the lead compound 10 has a fluorine atom in the
ortho position. This lead compound was also resynthesized,
confirming the activity results from the initial HTS. Changing
the ortho-fluoro substituent to the para (11) and meta (12)
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ACS Medicinal Chemistry Letters
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positions did not improve activity where the IC50/SI profile was
7.8 μM/>10 for both derivatives, indicating a slightly decreased
trypanocidal activity and increased toxicity compared to 10.
Increasing the number of the fluorine atoms had a negative
effect on the activity, where the addition of a second fluoro
substituent into the adjacent ortho position (13) resulted in a
decreased activity (82% activity at 10 μM) as did the presence
of five fluoro substituents (14, 94% activity at 10 μM). This
implied the importance of the monofluoro atom only in the
ortho position, with no advantages with the presence of the
extra fluorine atoms.
Figure 4. Structure−activity relationships for the bis-sulfonamides.
The fluorine atom is the smallest halogen size; therefore, it
was interesting to test the presence of other halogen atoms.
The addition of a bromo substituent at the para position
(compound 15) resulted in increased toxicity and did not
improve the activity (IC50/SI = 9.9 μM/>8).
inactive, whereas compound 25 showed similar activity and
selectivity (IC50/SI = 3.5 μM/>24) compared to the lead 10.
Increasing the bulkiness of compound 22 to 23 (replacing the
small methyl hydrophobe with the more bulky isopropyl
group) resulted in a similar activity profile but increased toxicity
(IC50/SI = 1.71 μM/>8). Further increases in the bulkiness by
replacing the isopropyl in 23 by a phenyl ring (24) decreased
the activity (IC50/SI = 5.7 μM/>16) by 3-fold compared to 23.
This difference could be attributed to a size effect. This also
confirms that the π system of the aromatic ring (directly
attached to the sulfonamide group) might be involved in the
activity.
When the aliphatic sulfonyl side chain (sulfonyl group
attached to the tetrahydroquinoline N) was replaced with
aromatic ring, the activity was either completely abolished as in
the case of compound 32 or toxicity was increased as in
compounds 33−35. Interestingly, compound 33 carrying the
para-methyl group was the best in this series (IC50/SI = 3.1
μM/>2), confirming the importance of the para position on
this aromatic moiety. The aliphatic sulfonyl groups directly
attached to the tetrahydroquinoline ring is important for
activity rather than an aromatic replacement.
An initial SAR model can be generated from this information
(Figure 4). Treating the tetrahydroisoquinoline unit as a
scaffold, toxicity is minimized if the sulfonamide (blue) is
aliphatic with the hydrophobicity tolerated up to 3 methylene
units. Larger moieties reduce the activity. In contrast, the
second sulfonamide unit (red) must be aromatic, indicating
that the π-electrons are likely to be significant. Aliphatic
substituents abolish activity.
The activity is maximized when this aromatic unit is
substituted in the para position. The substituent is best as a
small hydrophobic unit with a positive inductive effect.
This study revealed a new structural class of T. brucei
inhibitors with a good selectivity index, suitable for further
investigations. Close adherence to drug-like properties
throughout the study kept the molecular weight of synthesized
derivatives low, and ClogP values were used as a guide to
pharmacokinetic properties that needed to be maintained.
Initial SAR studies confirm the initial hit compound and
enabled basic design principles to be observed. Further
optimization of this sulfonamide series is possible, in particular
the para position of the terminal aryl sulfonamide group, and
further studies in this direction will be forthcoming. Therefore,
the discovery of the bis-sulfonamides as a novel class of
antiparasitic agents offers a new medicinal chemistry oppor-
tunity for targeting T. brucei spp.
Replacing the benzene moiety with its bioisostere
thiophene22,23 (16) resulted in a similar activity (IC50/SI =
4.0 μM/>20) compared to the lead 10. The same activity
profile of 10 and the simpler 16 might be attributed to the
lipophilic nature of the thiophene ring in 16 that might have
the same effect as the fluorine atom in 10. The addition of
another halogen to this thiophene (Br, 17) showed a decreased
trypanocidal activity (IC50/SI = 11.2 μM/>7) and a concurrent
reduction in the SI value compared to the unsubstituted
thiophene analogue 16. Replacement of the bulky bromo
substituent in 17 by a chloro substituent (18) was also not
tolerated, with a similar decrease in activity (IC50/SI = 13.1
μM/>6). The activity of 18 was 3-fold less than that of 16 but
similar to that of 17.
Replacing the aromatic moiety with aliphatic chains as in
compounds 20 and 21 completely abolished the activity, which
indicates the importance of the aromatic ring in that position
for the trypanocidal activity. The π system of the aromatic ring
may be involved in the interaction site, whereas with aliphatic
side chains such interactions do not exist. Interestingly, the
introduction of a small hydrophobe such as a methyl group on
the para position on the phenyl ring of the aromatic sulfonyl
moiety (compound 19) made little difference to the activity
(IC50/SI = 3.4 μM/>24) compared to 10. As the mode of
action and the target of the compounds are not yet known, the
role of this methyl group cannot be assured; however, it might
be involved in a hydrophobic interaction within the target site.
The extension of the ethyl side chain of the other sulfonyl
group (second changeable moiety) to a propyl group, as in
compound 22, was also tolerated (IC50/SI = 1.84 μM/>29)
with no significant difference in the activity compared to 19.
However, in the case of compound 26, the extra length of this
substituent was not tolerated. When the p-tolyl group of 22 was
replaced by a thiophene ring (compound 26), the activity was
completely abolished due to the propyl group compared to 16
(with an ethyl side chain).
The para position of the methyl hydrophobe in 22 was
important for activity as can be indicated by the inactivity of
compounds 27 (ortho position) and 28 (meta position). The
relatively good activity of 29 and 31 is moderated by a poor SI
when compared to 10 or 22. The inactivity of 30 (no p-methyl
group) also gives indications about the importance of the para
position. Figure 4 shows the SAR for this series of bis-
sulfonamides.
The addition of the hydrophobic methyl groups on either the
ethyl side chain or on the aromatic sulfonyl moieties seemed to
act as a tuner for the activity. Compound 26 was completely
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ASSOCIATED CONTENT
* Supporting Information
Synthetic and biological experimental procedures, selected
dose−response curves (active compounds), full characterization
of the synthesized compounds, and biological assay protocol
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
*(J.B.) E-mail: Jonathan.Baell@monash.edu.
*(P.A.K.) E-mail: keller@uow.edu.au.
Funding
This research was supported by the National Health and
Medical Research Fund (AUSTRALIA) Project Grant 1025581
and a University Postgraduate Award to AR from the University
of Wollongong.
■
Notes
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Noe, C. R. Synthesis of thieno[2,3-b]pyridinones acting as
cytoprotectants and as inhibitors of [H-3]Glycine binding to the N-
methyl-D-aspartate (NMDA) receptor. J. Med. Chem. 2006, 49, 864−
871.
(23) Hwang, K. J.; Lee, T. S.; Kim, K. W.; Kim, B. T.; Lee, C. M.;
Park, E. Y.; Woo, R. S. 4-Hydroxy-6-oxo-6,7-dihydro-thieno[2,3-b]
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The authors declare no competing financial interest.
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