Structural Insights into the Development of Cycloguanil Derivatives as Trypanosoma brucei Pteridine-Reductase-1 Inhibitors

Article abstract of DOI:10.1021/acsinfecdis.8b00358

Cycloguanil is a known dihydrofolate-reductase (DHFR) inhibitor, but there is no evidence of its activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of Trypanosoma brucei PTR1 (TbPTR1). A small library of cycloguanil derivatives was developed, resulting in 1 and 2a having IC₅₀ values of 692 and 186 nM, respectively, toward TbPTR1. Structural analysis revealed that the increased potency of 1 and 2a is due to the combined contributions of hydrophobic interactions, H-bonds, and halogen bonds. Moreover, in vitro cell-growth-inhibition tests indicated that 2a is also effective on T. brucei. The simultaneous inhibition of DHFR and PTR1 activity in T. brucei is a promising new strategy for the treatment of human African trypanosomiasis. For this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and DHFR inhibitors.

Full text of DOI:10.1021/acsinfecdis.8b00358

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Article
Structural Insights into the Development of Cycloguanil Derivatives
as Trypanosoma brucei Pteridine Reductase 1 Inhibitors
Giacomo Landi, Pasquale Linciano, Chiara Borsari, Claudia P. Bertolacini,
Carolina Borsoi Moraes, Anabela Cordeiro-da-Silva, Sheraz Gul, Gesa
Witt, Maria Kuzikov, Maria Paola Costi, Cecilia Pozzi, and Stefano Mangani
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00358 • Publication Date (Web): 23 Apr 2019
Downloaded from http://pubs.acs.org on April 24, 2019
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Structural Insights into the Development of Cycloguanil Derivatives as
Trypanosoma brucei Pteridine Reductase 1 Inhibitors
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Giacomo Landi , Pasquale Linciano , Chiara Borsari , Claudia P. Bertolacini , Carolina B. Moraes ,
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Anabela Cordeiro-da-Silva , Sheraz Gul , Gesa Witt , Maria Kuzikov , Maria Paola Costi , Cecilia
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Pozzi *, and Stefano Mangani *
1Department of Biotechnology, Chemistry and Pharmacy – Department of Excellence 2018-2020,
University of Siena, via Aldo Moro 2, 53100 Siena, Italy
2Department of Life Science, University of Modena and Reggio Emilia, via Campi 103, 41125
Modena, Italy
3National Laboratory of Biosciences, National Center for Research in Energy and Materials,
Campinas – SP, 13083-970, Brazil
4Instituto de Investigação e Inovação em Saúde, Universidade do Porto and Institute for Molecular
and Cell Biology, 4150-180 Porto, Portugal
5Fraunhofer Institute for Molecular Biology & Applied Ecology – ScreeningPort,
Schnackenburgallee 114, D-22525 Hamburg, Germany
†Present address: Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel,
Switzerland
*
Authors for correspondence: Cecilia Pozzi, pozzi4@unisi.it and Stefano Mangani,
stefano.mangani@unisi.it
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Cycloguanil is a known dihydrofolate reductase (DHFR) inhibitor, but there is no evidence of its
activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in
trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of
Trypanosoma brucei PTR1 (TbPTR1). A small library of cycloguanil derivatives was develop,
resulting in 1 and 2a having IC of 692 and 186 nM, respectively, towards TbPTR1. Structural
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analysis revealed that the increased potency of 1 and 2a is due to the combined contributions of
hydrophobic interactions, H-bonds and halogen bonds. Moreover, in vitro cell growth inhibition tests
indicated that 2a is also effective on T. brucei. The simultaneous inhibition of DHFR and PTR1
activity in T. brucei is a new promising strategy for the treatment of human African Trypanosomiasis.
On this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and
DHFR inhibitors.
Cycloguanil; triazine derivatives; Trypanosoma brucei; anti-parasite drugs; pteridine reductase;
folate pathway.
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Protozoan parasites belonging to the Trypanosoma and Leishmania species are the etiological agents
of various neglected tropical diseases, including human African Trypanosomiasis (HAT, also known
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as sleeping sickness), Chagas disease and different forms of leishmaniasis . HAT is caused by the
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bloodstream form of Trypanosoma brucei (T. brucei, or Tb) . Trypanosoma cruzi (T. cruzi), an
obligate intracellular parasite that invades various internal organs, is the etiological agent of Chagas
disease. Leishmania spp. infect macrophages and cause a wide spectrum of symptoms ranging from
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cutaneous lesions to potentially fatal visceral infections . Around 10 million people worldwide are
infected by these parasites. Since effective vaccines against these diseases have not been developed,
their treatment relies on chemotherapy and vector control. A very limited number of drugs is in use
and most of them are toxic, poorly effective and their clinical efficacy is compromised by the
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evolution of drug resistant parasites, therefore new therapies are required . In the field of HAT, the
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introduction of the nifurtimox-eflornithine combination therapy allowed a step forward towards the
eradication of the infection. It is hope that the introduction of fexinidazole, the first oral drug for the
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,7
treatment of this disease, will contribute further to the aim . However, drug resistance problems may
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always occur and the existence of animals that bear the infection justifies further the research in the
field.
The target-based drug-discovery approach represents a promising strategy and it has been
successfully applied to develop eflornithine, an ornithine decarboxylase inhibitor, which is currently
in use as a therapeutic for the late-stage HAT9,10. Although other targets such as cathepsin L11,
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trypanothione reductase , N-myristoyl transferase , and farnesyl transferase have been validated
for T. brucei, no drugs acting on them have been approved for the treatment of HAT. Enzymes of the
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folate pathway are established targets for bacterial infections and malaria and recently these have
also been exploited for the development of novel drugs against trypanosomatid parasites.
Nevertheless, classical anti-folate drugs targeting dihydrofolate reductase (DHFR), such as
methotrexate (MTX), are only poorly effective towards Leishmania and Trypanosoma because of
pteridine reductase (PTR, EC 1.5.1.33). PTR is a reduced nicotinamide adenine dinucleotide
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phosphate (NADPH)-dependent short-chain dehydrogenase/reductase (SDR) unique to
trypanosomatid parasites and mainly involved in the reduction of conjugated and unconjugated
pterins, such as biopterin (Figure SI-1)17,18. Under physiological conditions PTR accounts only for
the 10 % of tetrahydrofolate but, when the DHFR activity is blocked, the PTR gene is upregulated
and PTR provides reduced folates necessary for parasite survival, thereby creating a metabolic bypass
to DHFR inhibition19,20. PTR knockdown studies on the T. brucei bloodstream form, performed using
RNA interference methodologies, proved that the parasite growth is compromised both in vitro and
in the animal host20,21. Given the importance of this enzyme for parasite survival, PTR represents a
promising target for the treatment of HAT. During the NMTrypI European project, various inhibitors
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have been developed against Trypanosoma enzymes belonging to the folate pathway.
Cycloguanil (Figure 1), the active form of the pro-drug proguanil, is a known DHFR inhibitor22.
Kinetic studies performed on the isolated T. brucei DHFR (TbDHFR) yielded an inhibition constant
(K ) of 256 nM towards this target, whereas a K of 5.8 M was associated with L. major DHFR22,23.
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Cycloguanil has a modest anti-parasitic effect on T. brucei (EC up to 25 M) but it is almost inactive
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on L. major promastigotes (EC50 > 500 M)22,23. It is noteworthy that the two triazine-based
compounds melamine and cyromazine (Figure 1) which were previously investigated as TbPTR1
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inhibitors, yielded poor activity towards this target (K > 35 M) .
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In the present study, we provide experimental and structural evidence of cycloguanil being a TbPTR1
inhibitor. Structural information guided the design of cycloguanil derivatives based on a 1,6-
dihydrotriazine molecular core (Table 1). Two potent TbPTR1 inhibitors (1 and 2a), associated with
IC values in the nano molar range, were identified. The structure of TbPTR1 was solved in complex
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with these two inhibitors and compared with cycloguanil to explain their improved potency.
Following this, cell growth inhibition assays were performed on T. brucei and the early toxicity
profiles were determined for the compounds. Considering the efficacy of cycloguanil as DHFR
inhibitor against parasite diseases (e.g. malaria), the development of triazine derivatives acting as
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potent dual PTR1 and DHFR inhibitors would represent a step forward in the design of new effective
HAT treatments.
Cl
NH2
NH2
N
NH2
N
N
N
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N
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H
Cycloguanil
Cyromazine
Melamine
Figure 1. Chemical structures of cycloguanil, cyromazine and melamine.
RESULTS AND DISCUSSION
Synthesis of 2,4-diamino-1,6-dihydrotriazine derivatives and inhibition of TbPTR1
The chemical structures of the synthesized compounds are displayed in Table 1.
Table 1. Chemical structures of cycloguanil and the synthesized 2,4-diamino-1,6-dihydro-1,3,5-
triazine 1 and 2a-c.
Compound
Cycloguanil
Structure
Cl
NH2
N
N
N
N
N
NH2
Cl
Cl
NH2
N
1
N
NH2
Cl
Cl
NH2
N
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NH2
O N
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The dichloro-analogue of cycloguanil (1) was prepared by a one-step acid-catalysed cyclo-
condensation between equimolar 3,4-dichloroaniline and cyanoguanidine in acetone (3-component
synthesis) to give directly the desired triazine as hydrochloric salt (Scheme 1), as previously
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described .
Cl
Cl
NH2
NH2
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CN
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HCl
NH2
+
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H N
NH2
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Cl
N
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Scheme 1. a) 37 % w/w HCl aq., acetone, r.t., 2h.
In contrast, the 2-aryl-1,6-dihydrotriazines 2a-c were obtained through a two-component synthesis,
relying on a two-steps condensation of the preformed arylbiguanide 3a-b and an aromatic carbonyl
derivative, such as 3,4-dichlorobenzaldheyde (for 2a,c) or benzaldehyde (for 2b) (Scheme 2). The
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progress of the reaction was monitored by cuprammonium sulphate assay .
NH2
N
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H
HCl
CN
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N
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H N
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NH NH
N
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R1
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a R= 3,4-Cl
b R= -H
2a R= 3,4-Cl; R = 4-NO
1 2
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Scheme 2. a) 37 % w/w HCl aq. (1 eq.), neat, 170 °C, 10 min. b) aryl aldehyde (1.5 eq.), 37 % w/w
HCl aq. (1.1 eq.), MeOH, rifl. 2-12 h.
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Activity inhibition assays identified cycloguanil as a modest inhibitor of TbPTR1, yielding an IC50
of 31.6 μM (Table 2). The four synthesized derivatives (Table 1) were preliminary investigated
towards TbPTR1 through fixed-concentration inhibition assays. Compounds showing an inhibition
percentage >90 % at 50 M were selected for IC determination and toxicity evaluation. Among the
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synthesized 2,4-diamino-1,6-dihydrotriazines, 1 and 2a were the most potent derivatives of this
series, yielding IC of 692 nM and 186 nM, respectively (Table 2). However, 2b and 2c were less
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active (IC of 10.5 μM and 39.5 μM, respectively), suggesting that the introduction of halogen atoms
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on the phenyl moiety in position 1 improves the inhibitor potency.
Table 2. TbPTR1 inhibition, anti-parasitic activity against T. brucei, and LogP(o/w) of cycloguanil
and 2,4-diamino-1,6-dihydrotriazine derivatives 1 and 2a-c.
T. brucei anti-parasitic activity
TbPTR1 inhibition
Compound
% cell growth inhibition at 50 µM
LogP(o/w)27
a
IC in μM
5
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a
and/or cEC in μM
5
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≤ 25.0 M23
23.0 % - > 100 M
99.8 % - 29.0 M
5.00 % - > 100 M
NI
Cycloguanil
1
31.6
1.61
2.02
2.26
2.03
2.92
0.692
0.186
10.5
2
a
2
b
2
c
39.5
a
Standard deviation is ±10 % of the value
NI. No inhibition
Overall structure and active site architecture of TbPTR1
Cycloguanil and the most active compounds 1 (IC : 692 nM) and 2a (IC : 186 nM) were used for
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structural investigations. The three complexes were characterized to resolutions ranging from 1.50 to
.92 Å (Table SI-1). In all complexes, the crystal asymmetric unit contains a whole TbPTR1 tetramer
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representing the enzyme functional unit, whose structure is highly conserved to those previously
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described18,28–30. NADP(H), acting as cofactor for PTR enzymes, binds in an extended conformation
stabilized by a tight network of conserved H-bonds18,31. Cofactor binding is essential to create both
the catalytic site and the substrate binding pocket. The pterin moiety of substrates and substrate-like
inhibitors bind in this pocket forming a peculiar π-sandwich between the nicotinamide ring of
NADP(H) and the aromatic side chain of Phe9718,31. Within the TbPTR1 active site, substrates and
inhibitors are further stabilized by the interactions with the flexible substrate binding loop (residues
207-215), which binds also the cofactor. A correlation between the occupancy of the cofactor binding
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pocket and the flexibility of the substrate binding loop was previously observed by us28,30
.
Cycloguanil and its derivatives were observed to populate the substrate binding pocket in all
complexes (inhibitor and cofactor estimated occupancies are summarized in Table SI-2).
TbPTR1–NADP(H)–cycloguanil
The 2,4-diamino-1,6-dihydrotriazine moiety of cycloguanil occupies the biopterin binding pocket
forming a tight network of H-bonds with the cofactor and several residues having a prominent role in
the catalytic process, such as Tyr174 and Ser95 (Figure 2A and SI-2A). The amino group in position
2
on the 2,4-diamino-1,6-dihydrotriazine core donates a H-bond to the Tyr174 hydroxyl that further
donates a H-bond to Asp161. The nitrogen in position 3 accepts a H-bond from the ribose hydroxyl
of NADP(H). Furthermore, the amine moiety in position 4, donates H-bonds to both the hydroxyl and
the backbone carbonyl of Ser95. One of the methyl groups in position 6 on the 1,6-dihydrotriazine
ring forms van der Waals interactions with both Phe97 and Pro210. The 4-chloro-phenyl moiety in
position 1 is rotated by  80° with respect to the 1,6-dihydrotriazine ring, placing the p-chlorine in an
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optimal position to form a halogen bond with the indole aromatic system of the Trp221 sidechain
(the halogen points exactly to the center of the indole five-membered ring, Figure 2A).
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Figure 2. Active site view of TbPTR1 (white cartoon and carbon atoms, interacting residues in sticks)
in complex with the cofactor NADP(H) (in sticks, grey carbons) and A) cycloguanil (in sticks, green
carbons), B) 1 (in sticks, cyan carbons), and C) 2a (in sticks, purple carbons). Hydrogen and halogen
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bond interactions within the active site are shown as tan dashed and light blue dotted lines,
respectively. In all panels, inhibitor molecules are surrounded by their omit map (displayed as green
wire, contoured at the 2.5 σ level). In all panels, oxygens atoms are coloured red, nitrogen blue,
phosphorous magenta, and chlorine black. Stereo-views are shown in Figure SI-2.
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TbPTR1–NADP(H)–1
The binding mode adopted by 1 within the TbPTR1 catalytic pocket closely resembles that displayed
by cycloguanil (showing also conserved interactions inside the cavity, Figure 2B and SI-2B). Two
binding modes were observed within the cavity, resulting from two alternative orientations of the 3,4-
dichloro-phenyl moiety of 1. In the main orientation, the m-chlorine is accommodated inside the
pocket formed by Val206, Val209, the backbone oxygen of Ser207, and the nicotinamide oxygen of
the cofactor (Figure 2B). The orientation of the m-chlorine within this pocket is compatible with a
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weak halogen bond involving the backbone carbonyl of Ser207 (omitted for clarity in Figure 2B).
The alternative orientation of the 3,4-dichloro-phenyl moiety is visible to a partial occupancy
(estimated to 30 %, Table SI-2) only in chain B. In this orientation, the 3,4-dichloro-phenyl moiety
is rotated by 180° with respect to the former, directing the m-chlorine substituent towards the solvent
exposed protein surface. Despite this rotation, the halogen bond between the p-chlorine and Trp221
is unaltered, supporting the prominent contribution of this interaction in inhibitor binding.
TbPTR1–NADP(H)–2a
The 2,4-diamino-1,6-dihydrotriazine core of 2a adopts a pose similar to those observed for the
corresponding molecular scaffolds of cycloguanil and 1, showing also unaltered interactions within
the TbPTR1 active site (Figure 2C and SI-2C). In contrast to cycloguanil and 1, 2a possesses a 4-
nitro-phenyl substituent in position 6 on the 1,6-dihydrotriazine core (Table 1). The aromatic systems
in position 1 and 6 are rotated by  73° and  48°, respectively, with respect to the dihydrotriazine
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plane. The p-chlorine on the phenyl in position 1 is directed towards the sidechains of Trp221 and
Met213, without forming specific interactions with them (Figure 2C). On the other hand, the m-
chlorine is located in the pocket formed by Val206, Leu209, and the backbone carbonyls of Ser207
and Leu208. The orientation of the chlorine in this pocket supports the formation of two weak halogen
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bonds with the Ser207 and Leu208 backbone carbonyls (omitted for clarity in Figure 2C). In
contrast to cycloguanil and 1, the accommodation of the bulkier 4-nitro-phenyl substituent of 2a
induces a significant rearrangement of the substrate binding loop. This causes a shift of the 3,4-
dichloro-phenyl moiety toward the “floor” of the substrate binding cavity into the pocket formed by
residues 206-209, allowing optimal contact interactions but increasing the distance between the p-
chlorine and Trp221 by about 1.0 Å. The 4-nitro-phenyl substituent in position 6 points towards the
solvent exposed enzyme surface, allowing van der Waals interactions with the sidechains of Phe97
and Pro210. The orientation of the p-nitro group on this moiety is not clearly visible, indicating
rotational disorder of the group (modeled only in chain D, Figure 2C; in the other subunits the nitro-
oxygens were excluded from our model).
Comparison of the crystal structures and SAR
The comparison among the three structural complexes solved in this work showed a conserved
binding mode within the TbPTR1 active site (Figure 3A). Cycloguanil and its derivatives occupied
the biopterin binding pocket competing with the natural substrates (the comparison with the complex
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TbPTR1-folic acid is shown in Figure SI-3). All these compounds possess a phenyl substituent in
position 1 on the 2,4-diamino-1,6-dihydrotriazine core, rotated by 73-80° with respect to the
dihydrotriazine plane. The steric hindrance generated by this moiety, coupled with that of the
substituents in position 6, induce the rotation of the Phe97 aromatic system which is no longer parallel
to the nicotinamide of the cofactor (Figure 3B). The conserved -sandwich interaction involving the
aromatic sidechain of Phe97 and the cofactor nicotinamide is essential for the substrate placement
and has also a key role in inhibitor binding18,34
.
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Figure 3. A) Active site view of the superimposition among the complexes TbPTR1–NADP(H)–
cycloguanil (light green cartoon and carbon atoms; cycloguanil in sticks, green carbons), TbPTR1–
NADP(H)–1 (light cyan cartoon and carbon atoms, 1 in sticks, cyan carbons), and TbPTR1–
NADP(H)–2a (lilac cartoon and carbon atoms, 2a in sticks, purple carbons). The rotation of the
substrate binding loop in the complex with 2a is highlighted by a red dashed arrow. B) Active site
view of the superimposition between the complexes TbPTR1–NADP(H)–cycloguanil (light green
cartoon and carbon atoms; cycloguanil in sticks, green carbons) and TbPTR1–NADP(H)–cyromazine
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(
light yellow cartoon and carbon atoms; cyromazine in sticks, yellow carbons; PDB id: 2X9N ). In
the complex with cycloguanil the sidechain of Phe97 is rotated by  24° with respect to the orientation
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observed in the complex with cyromazine, generating, in the former, a distortion of the conserved -
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sandwich interaction.
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The characterization of TbPTR1 in complex with the triazines melamine (Figure 1, PDB id: 3JQF24)
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and cyromazine (Figure 1, PDB id: 2X9N ) were previously reported showing the inhibitors -
sandwiched inside the biopterin binding pocket (an active site view of the complex with cyromazine
is shown in Figure 3B). The comparison with cycloguanil clearly evidences that, despite the loss of
aromaticity, the 2,4-diamino-1,6-dihydrotriazine core retains the same binding mode of the triazine
moiety (Figure 3B). Furthermore, in the complex with cycloguanil the -sandwich is distorted by the
inhibitor binding that hinders Phe97, weakening this interaction but keeping close van der Waals
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contacts (Figure 3B). Melamine and cyromazine have reported K >35 M , indicating that they are
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modest TbPTR1 inhibitors. Thus, the aromaticity of the triazine core is not essential for the binding.
However, H-bond interactions provides a meaningful contribution for both the 2,4-diamino-1,6-
dihydrotriazine of cycloguanil and the triazine of melamine and cyromazine. Conserved H-bonds are
formed by their nitrogen atom in position 3 and amine moieties in positions 2 and 4 with the cofactor
and several residues having a prominent role in the catalytic process (e.g. Tyr174 and Ser95).
Cycloguanil and cyromazine also share hydrophobic substituents in position 6 of their triazine cores,
relying on two methyl groups and a N-cyclopropyl substituent, respectively (Figure 3B). In both
complexes, these hydrophobic moieties determine additional van der Waals interactions with Phe97
and residues belonging to the substrate binding loop. Nevertheless, the bulky N-cyclopropyl
substituent of cyromazine induces the displacement of the substrate binding loop (Figure3B). In
contrast to the triazine derivative, cycloguanil is further modified in position 1 by the introduction of
a 4-chloro-phenyl moiety, which forms a halogen bond with the indole aromatic system of Trp221
(Figure 2A and 3A). The same halogen bond is also visible in the complex with 1, having a chlorine
in the same position (Figure 2B and 3A). The additional m-chlorine on the phenyl moiety of 1 is
accommodated in a pocket adjacent to the active site, in which it entails further halogen bonds with
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the substrate binding loop. These additional interactions account for its increased potency (IC of
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92 nM) with respect to the parent compound (IC of 31.6 M).
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The same 3,4-dichloro-phenyl substituent also characterizes 2a, further having a 4-nitro-phenyl
moiety in position 6 that replaces the two methyl groups of 1 and cycloguanil (Table 1). In 2a, the
introduction of the bulkier 4-nitro-phenyl group induces a slightly different pose of the molecule with
respect to cycloguanil and 1 (Figure 3A). Nonetheless, the interactions of the 2,4-diamino-1,6-
dihydrotriazine core are unaltered in all complexes (Figure 2A-C and 3A). The p-chlorine of 2a is
shifted by 1.7 Å with respect to the same chlorine on 1 and cycloguanil, with subsequent loss of the
halogen bond with Trp221, but gain of van der Walls contacts with the sidechain of Leu209. In
addition, the m-chlorine substituent of 2a is involved in a large set of interactions that stabilizes its
binding and explains the increased potency of this compound (IC of 186 nM) compared to 1. The
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placement of 2a induces a shift of the substrate binding loop, reporting a maximal displacement of
3.7 Å on Ala211 (C) with respect to the corresponding residue in the complex with cycloguanil
and 1) (Figure 3A and 4). A similar movement of the substrate binding loop is also detected in the

(
complex with cyromazine, in which a maximal displacement of 3.4 Å on Val212 (C) is observed
by the comparison with the cycloguanil complex (Figure 3B and 4). The arrangement of this loop is
connected with the different set of interactions within the cavity (Figure 4A-D). In the complex with
cycloguanil, two H-bonds are formed by the nicotinamide of NADP(H) and the backbone nitrogen
and carbonyl of Ser207 and Leu208, respectively (Figure 4A,D). In the close proximity, Arg14
contributes to stabilize the orientation of the substrate binding loop by donating two H-bonds to the
backbone carbonyls of Leu208 and Leu209. Despite the movement of the substrate binding loop
observed in the complex with cyromazine the same set of interactions is formed (Figure 4B,D). On
the other hand, in the complex with 2a a slightly different arrangement is observed (Figure 4C,D).
The backbone carbonyl of Ser207 is rotated by 90° and accepts a H-bond from the nicotinamide
nitrogen of NADP(H) (Figure 4C,D). This carbonyl is located 3.11 Å away from the m-chlorine on
the 3,4-dichloro-phenyl moiety of 2a, suggesting the formation of a halogen bond with it. In the close
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proximity, Leu208 is shifted towards Arg14 that donates two H-bonds to the backbone carbonyl of
the former.
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Figure 4. Structural comparison among the complexes TbPTR1–NADP(H)–cycloguanil (light green
cartoon and carbon atoms; cycloguanil in sticks, green carbons), TbPTR1–NADP(H)–cyromazine
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(
light yellow cartoon and carbon atoms; cyromazine in sticks, yellow carbons; PDB id: 2X9N ), and
TbPTR1–NADP(H)–2a (lilac cartoon and carbon atoms; 2a in sticks, purple carbons) A-C) Set of H-
bonds (tan dashed lines) stabilizing the substrate binding loop in the complexes. The different set of
interactions involving Arg14, the cofactor nicotinamide and the substrate binding loop (especially
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residues 207-209) is displayed. D) Superimposition among the complexes showing the different
orientation adopted by the substrate binding loop. An aperture of the loop is observed in presence of
both cyromazine and 2a, having bulky substituents in position 6 of the triazine and the 2,4-diamino-
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,6-dihydrotriazine core, respectively. A stereo-view of the D panel is shown in Figure SI-4.
Biological profile of 1, 2a-c
The four synthesized 1,6-dihydrotriazines 1, 2a-c were evaluated for their in vitro anti-parasitic
activity against the T. brucei bloodstream form. The anti-parasitic potential was expressed as
percentage of parasitic cell growth inhibition at 50 μM (Table 2). Only 2a showed an anti-parasitic
activity >80 % at 50 μM with an estimated EC of 29 M, whereas the other 1,6-dihydrotriazines
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displayed a limited efficacy towards T. brucei (cell growth inhibition <23 % at 50 μM, corresponding
to calculated cEC >100 M). The trend of the anti-parasitic activity observed for the analyzed
50
compounds correlates with the PTR1 inhibition data, supporting a linkage between PTR and cell
growth inhibition in T. brucei. However, compound 1 shows an IC against PTR1 of 186 nM and an
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EC against cells of 29 µM, representing a 155-fold decrease in potency from target to parasite.
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Similar decreases in activity between enzyme and parasite are observed also for the other analogues.
The potency decrease is a general issue in medicinal chemistry studies and for our compounds, this
can be due to the difficulties in the trans-membrane passage or to off-target effects. The evaluation
of the LogP values (Table 2), showed the lowest value (1.61) and the highest hydrophilicity for
cycloguanil. Compounds 1, 2a-2c yielded very similar values, without an evident relationship with
their anti-parasitic potency.
To validate the 2,4-diamino-1,6-dihydrotriazine core as a safe scaffold for a drug discovery program,
the early in vitro toxicological properties of 1, 2a-2c were assayed. The inhibitory activity towards
the hERG and five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were
assessed together with the mitochondrial toxicity and the cytotoxicity against human A549 cells
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(adenocarcinoma human alveolar basal epithelial cells) and THP-1 macrophages (data are shown in
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Table SI-3). All the tested compounds inhibited hERG and CYP2D6 (IC in the range 0.51 – 77.3
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μM and 0.04 – 8.85 μM, respectively), resulting in a low Selectivity Index (SI, given by the ratio
between the off-target IC and the TbPTR1 IC , SIhERG/TbPTR1: 1.7 – 111; SICYP2D6/TbPTR1: 0.21 –
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.36). However, these were neither mitotoxic nor active against the other P450 CYP isoforms. Only
1 and 2a were cytotoxic against the A549 cell line with GI (compound concentration required for
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0 % of cell growth inhibition) of 0.31 μM and 11 μM, respectively. The cytotoxicity of 1 could be
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due to the inhibition of hDHFR (K of 0.11 μM ). Furthermore, the compounds were assessed for
i
cytotoxicity on THP-1 macrophage-like cells to determine the NOAEL (no observed adverse effect
level at 50 μM) and none of them showed meaningful cytotoxicity at the tested concentration.
CONCLUSION
In this work, we have exploited cycloguanil, a known inhibitor of plasmodial and trypanosomal
DHFR-TS enzymes16,23,37, as a scaffold for the development of four derivatives with variable
substitution in positions 1 and 6 of the 2,4-diamino-1,6-dihydrotriazine ring (Table 1). The structural
characterization of TbPTR1 in complex with cycloguanil, elucidated the binding mode of this modest
inhibitor and supported the rational design of the four 2,4-diamino-1,6-dihydrotriazine derivatives.
Functional studies against TbPTR1 revealed that the two synthesized derivatives 1 and 2a were potent
TbPTR1 inhibitors (IC of 692 and 186 nM, respectively). The basis of the improved potency of
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these inhibitors was elucidated by structural analysis that evidenced the key binging contributions
provided by halogen and hydrogen bonds and by van der Waals interactions. Furthermore, the
complex with 2a highlights the displacement of the substrate binding loop induced by inhibitor
binging in the catalytic cavity. In addition, compound 2a was also active in blocking parasite cell
growth in vitro. While enlarging the compound library for SAR studies, we will pay attention to the
mechanism of parasite internalization and substituents/fragments will be introduced to modulate their
lipophilicity. Although the early in vitro toxicological studies indicated a few liabilities for 1 and 2a,
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all of which should be overcome by SAR studies, our data provides new important insights for the
development of 2,4-diamino-1,6-dihydrotriazine derivatives as potent TbPTR1 inhibitors. Giving the
effectiveness of cycloguanil on DHFR enzymes, 2,4-diamino-1,6-dihydrotriazine derivatives
represent a new resource exploitable for the development of dual PTR1 and DHFR inhibitors with
anti-parasitic activity. Additionally, the new compounds can be explored for their capacity to inhibit
PTR enzymes from other organisms, such as PTR1 from Leishmania spp and from Trypanosoma
cruzi. The similarity of the target structure among the different organisms suggest that the triazine
core can be developed further for a pan-PTR1 enzymes inhibition.
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EXPERIMENTAL SECTION
Synthetic procedures
All commercially available chemicals and solvents were reagent grade and were used without further
purification unless otherwise specified. Reactions were monitored by thin-layer chromatography on
silica gel plates (60F-254, E. Merck) and visualized with UV light, iodine vapours, cerium ammonium
sulphate or alkaline KMnO aqueous solution. The following solvents and reagents have been
4
abbreviated: ethyl ether (Et O), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane
2
(DCM), methanol (MeOH). All reactions were carried out with standard techniques or under
1
microwave irradiation. NMR spectra were recorded on a Bruker 400 spectrometer with H at 400.134
1
3
MHz and C at 100.62 MHz. Proton chemical shift was referenced to the residual solvent peak.
Chemical shifts are reported in parts per million (ppm, δ units). Coupling constants are reported in
units of Hertz (Hz). Splitting patterns are designed as s, singlet; d, doublet; t, triplet; q quartet; dd,
double doublet; m, multiplet; b, broad. Mass spectra were obtained on a 6520 Accurate-Mass Q-TOF
LC/MS and 6310A Ion TrapLC-MS(n). The melting point was recorded on a Stuart, SMP3
(Barloworld Scientific Limited Stone, Staffordshire, UK) and was uncorrected.
(a) 1-(3,4-dichlorophenyl)-6,6-dimethyl-1,6-dihydro-1,3,5-triazine-2,4-diamine hydrochloride (1)
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To a solution of 3,4-dichloroaniline (1 eq, 162 mg, 1 mmol) and N-cyano-guanidine (1 eq., 84 mg, 1
mmol) in acetone (15 ml) concentrated hydrochloric acid 37 % w/w (1 eq., 83 µL, 1 mmol) was added
dropwise. The resulting suspension was stirred at room temperature for 5 hours. The precipitate was
collected by filtration and washed with cold acetone to give the desired product as hydrochloric salt
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without further purification needed. White crystalline solid (71 % yield). HNMR (400 MHz, DMSO-
d ) δ: 1.37 (s, 6H, CH ); 6.37 (bs, 2H, NH ); 6.66 (bs, 1H, NH); 7.43 (dd, 1H, J = 4.0, 8.0 Hz, CH );
6
3
2
ar
1
3
7.78-7.80 (m, 3H, CH , NH ); 9.30 (s, 1H, CH ). CNMR (101 MHz, DMSO-d ) δ: 27.15; 27.24;
ar
2
ar
6
6
9.90; 130.61; 131.86; 132.35; 132.39; 132.80; 134.73; 156.82; 157.39. m.p. [> 200 °C]. HRMS m/z
+
35
35
37
[
M+H] Calcd for C H [ Cl] N : 286.0621, found: 286.0622; calcd for C H [ Cl][ Cl]N :
11 14 2 5 11 14 5
288.0591, found: 288.0590.
b) 1-(3,4-dichlorophenyl)-6-(4-nitrophenyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine (2a)
(
Neat 3,4-dichlroaniline (1 eq., 162 mg, 1 mmol), N-cyano-guanidine (1 eq., 84 mg, 1 mmol) and
concentrated hydrochloric acid 37 % w/w (1 eq., 83 μL, 1 mmol) were gradually heated at 170 °C
and reacted for 10 minutes. The solid formed was chilled down, triturated over acetone and filtered
1
to give the N -(3,4-dichlorophenyl)-diguanide hydrochloride (3a) used in the next step without
further purification. 3a (1 eq., 167 mg, 0.60 mmol) was suspended in MeOH (15 mL) and 4-nitro-
benzaldehyde (1.5 eq., 136 mg, 0.90 mmol) and concentrated hydrochloric acid 37 % w/w (1.1 eq.,
5
8 µL, 0.7 mmol) were added. The mixture was refluxed for 12 hours. Thereafter, the mixture was
neutralized with Na CO and the solvent evaporated. The crude was directly purified on silica gel
2
3
1
(eluent DCM:MeOH 9:1) obtaining a brown solid (38 % yield). H NMR (400 MHz, Methanol-d ) δ
4
6
.29 (s, 1H), 7.18 (dd, J = 2.5, 8.6 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.70
1
3
(
d, J = 8.8 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H). C NMR (101 MHz, MeOD) δ 71.37, 125.37, 129.39,
130.02, 131.59, 133.34, 134.94, 134.98, 138.12, 145.56, 150.27, 159.05, 159.22. m.p. [dec. 185 °C].
+
35
35
HRMS m/z [M+H] Calcd for C H [ Cl] N O : 379.0472, found: 379.0469; calcd for C H [ Cl]
1
5
12
2
6
2
15 12
[37
c) 1,6-diphenyl-1,6-dihydro-1,3,5-triazine-2,4-diamine hydrochloride (2b)
Cl]N O : 381.0442, found: 381.0440.
6 2
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Neat aniline (1 eq., 452 µL, 5 mmol), N-cyano-guanidine (1.1 eq., 462 mg, 5.5 mmol) and
concentrated hydrochloric acid 37 % w/w (1 eq., 500 µL, 1 mmol) were gradually heated at 170 °C
and reacted for 10 minutes. The solid formed was chilled down, triturated over acetone and filtered
1
0
1
2
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0
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to give the N -phenyl-diguanide hydrochloride (3b) as a pink solid used in the next step without
further purification. 3b (1 eq., 500 mg, 2.20 mmol) was suspended in MeOH (25 mL) and
benzaldehyde (1.5 eq., 290 µL, 3.30 mmol) and concentrated hydrochloric acid 37 % w/w (1.1 eq.,
1.67 mL 2.10 mmol) were added. The mixture was refluxed for 4 hours and concentrated. The crude
was suspended in water and washed with AcOEt. The aqueous phase was concentrated and the residue
1
triturated over Et O to give a white crystalline solid (52 % yield). HNMR (400 MHz, DMSO-d ) δ:
2
6
5.28 (bs, 2H, NH ), 6.08 (s, 1H, CH
), 7.15 (d, 2H, CH ), 7.31-7.44 (m, 10H, CH ), 7.70 (s, 2H,
2
triazine
ar
ar
13
NH ), 9.37 (bs, 1H, NH). CNMR (101 MHz, DMSO-d ) δ: 69.88, 121.38, 123.31, 126.80, 127.37,
2
6
1
28.54, 128.76, 128.80, 129.32, 129.60, 129.95, 137.92, 138.72, 157.05, 157.26. m.p. [> 200 °C].
+
HRMS m/z [M+H] Calcd for C H N : 266.3275, found: 266.3272.
15 16
5
(d) 6-(3,4-dichlorophenyl)-1-phenyl-1,6-dihydro-1,3,5-triazine-2,4-diamine hydrochloride (2c)
3
b (1 eq., 28 mg, 0.15 mmol) was suspended in MeOH (5 mL) and 3,4-dichlorobenzaldehyde (1.5
eq., 40 mg, 0.23 mmol) and concentrated hydrochloric acid 37 % w/w (1.1 eq., 0.21 mL, 0.17 mmol)
were added. The mixture was refluxed for 24 hours and concentrated. The crude was triturated over
EtOH and the precipitate obtained crystalized from Et O to give 35 mg of a white crystalline solid
2
1
(
69 % yield). H NMR (400 MHz, DMSO-d ) δ 6.43 (s, 1H, CH
), 7.03 - 7.07 (m, 3H, CH , NH),
triazine ar
6
7.24 - 7.34 (m, 7H, CH , NH), 7.34 - 7.40 (m, 2H, CH ), 9.71 (s, 1H, NH). m.p. [> 200 °C]. HRMS
ar
ar
+
35
35
37
m/z [M+H] Calcd for C H [ Cl] N : 334.0621. Found: 334.0621; calcd for C H [ Cl] [ Cl]N :
1
5
14
2
5
15 14
5
3
36.0591, found: 336.0593.
TbPTR1 expression, purification, and enzymatic assays
Recombinant TbPTR1 was expressed and purified by established methods . TbPTR1 enzyme
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activity was measured using the in vitro coupled assay reported by Shanks et al. . The assay follows
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the reduction of H B to H B by PTR enzyme. H B is further oxidized to qH B by cytochrome c Fe ,
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whose reduction to cytochrome c Fe is spectrophotometrically monitored at 550 nm. The IC values
5
0
determined for the compounds are summarized in Table 2.
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Protein crystallization
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Crystallization was performed by the vapor diffusion hanging drop technique at room temperature .
Well-ordered monoclinic crystals of histidine tagged TbPTR1 were grown within few days using a
precipitant solution composed of 2-2.5 M sodium acetate and 0.1 M sodium citrate, pH 5, as described
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elsewhere . The ternary complexes TbPTR1–cofactor–inhibitor were obtained by the soaking
technique. Compounds, solubilized in DMSO (50 mM), were added to the cryo-protectant solution
(
prepared by adding 30 % v/v glycerol to the precipitant) to reach final concentrations of 4-5 mM
without exceeding a 10 % v/v DMSO concentration). Preformed protein crystals were then
(
transferred to the cryo-protectant/soaking solution, and flash-frozen in liquid nitrogen after 24-48
hours.
Data collection, structure solution and refinement
Diffraction data were collected using the synchrotron radiation at the Diamond Light Source (DLS,
Didcot, United Kingdom) beamline I02 and at the European Synchrotron Radiation Facility (ESRF,
Grenoble, France) beamline ID30-B, both equipped with a Dectris Pilatus 6M-F detector. Diffraction
4
0
41
42
data were integrated using Mosflm and scaled with Scala from the CCP4 suite . Data collection
statistics are reported in Table SI-1. Crystals of TbPTR1 belonged to the primitive monoclinic space
group P2 , with a functional tetramer in the asymmetric unit. Structures were solved by the molecular
1
4
3
replacement technique, as implemented in the software MOLREP . A functional tetramer of TbPTR1
28
(
PDB id 5JDC ) was used as searching model (solvent and non-protein molecules were preventively
4
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excluded). All models were refined using REFMAC5 from the CCP4 suite. The molecular graphic
software Coot45,46 was used for visual inspection and manual rebuilding of missing atoms in the
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protein models. The occupancy of the exogenous ligands was adjusted and refined to a value resulting
in atomic displacement parameters close to those of neighboring protein atoms in fully occupied sites.
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Water molecules were added using the ARP/wARP suite and checked with Coot. The final models
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were inspected manually and checked with the programs Coot and Procheck . Data refinement
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statistics are reported in Table SI-1. The structural models were rendered using CCP4mg . Final
coordinates and structure factors were deposited in the Protein Data Bank under the codes 6HNC
(TbPTR1–NADP(H)–cycloguanil), 6HNR (TbPTR1–NADP(H)–1), and 6HOW (TbPTR1–
NADP(H)–2a).
Evaluation of activity against T. brucei
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Anti-trypanosomal activity tests were performed as described elsewhere . Briefly, T. brucei Lister
427 bloodstream forms were incubated in the presence of compounds for 72 h, followed by cell lysis
and addition of the Sybr Green I dye (Life Technologies). The plates were then read for fluorescence
at the microplate reader Envision (PerkinElmer). Compounds that showed percentage of inhibition
of parasite growth higher than 80 % at 50 M were selected for EC determination.
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Determination of in vitro toxicological properties
The determination of the early toxicity profile of 1, 2a-c, comprising the hERG, cytochrome P450
1A2, 2C9, 2C19, 2D6 and 3A4), mitochondrial toxicity, cytotoxicity were preformed as previously
(
5
1
described . The cytotoxicity of THP-1-derived macrophages was assessed by the colorimetric MTT
2
8
assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) .
ASSOCIATED CONTENT
Supporting Information contains additional figures, showing the PTR catalytic mechanism and
additional stereo-views of the complexes, and tables, including data collection, structure solution and
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refinement statistics, inhibitor and cofactor occupancies within the catalytic cavity, and the liability
profile for the synthesized compounds.
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AUTHOR INFORMATION
Corresponding authors
*
(C.P.) Phone: +39 0577232132. E-mail: pozzi4@unisi.it
*(S.M.) Phone: +39 0577234255. E-mail: stefano.mangani@unisi.it
Author Contributions
P.L. designed/synthesized the molecules and performed the medicinal chemistry.
G.W. carried out the in vitro enzyme assays.
M.K. carried out the in vitro toxicological assays.
C.P.B., C.B.M. carried out the in vitro antiparasitic tests and the in vitro toxicological profiles.
G.L. and C.P. prepared the TbPTR1 protein and carried out the X-ray crystallographic studies.
All authors analyzed the data and C.P., P.L., C.B., A.C.D.S., S.G. M.P.C. and S.M. wrote the
manuscript.
Notes
The authors declare no competing financial interest.
Authors will release the atomic coordinates and experimental data upon article publication.
ACKNOWLEDGEMENT
This project has received funding from the European Union’s Seventh Framework Programme for
research, technological development and demonstration under grant agreement n° 603240 (NMTrypI
-
New Medicine for Trypanosomatidic Infections). The authors acknowledge the COST Action
CM1307, http://www.cost.eu/COST_Actions/cmst/CM1307 for the contribution to the discussion of
the research results.
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The authors would also like to acknowledge Diamond Light Source (proposal MX11690) and the
European Synchrotron Radian Facility for provisioning of beamtime, and in particular the staff of the
DLS beamline I02 and the ESRF beamline ID30-B. We would also like to thank all the staff of the
synchrotron facilities for assistance in using the beamlines. The research leading to these results
received founding from the European Community Seventh Framework Programme (FP7/2007-2013)
under Biostruct-X (Grant Agreement 283570). We would also like to thank the financial support
provided by the MIUR Grant Dipartimento di Eccellenza 2018-2022.
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ABBREVIATIONS
PDB, Protein Data Bank; H B, dihydrobiopterin; H B, tetrahydrobiopterin; qH B, quinonoid
2
4
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dihydrobiopterin; Et O, ethyl ether; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; DCM,
2
dichloromethane; MeOH, methanol; hERG, human ether-ago-go-related gene; THP1, human
monocytic cell line; A549, human lung adenocarcinoma epithelial cell line; ATP, adenosine
triphosphate; DMEM, Dulbecco's Modified Eagle Medium; FCS, fetal calf serum
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