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. 2018 Sep 4;9(10):996–1001. doi: 10.1021/acsmedchemlett.8b00245

Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation

Jennifer L Woodring †, Ranjan Behera ‡, Amrita Sharma ‡, Justin Wiedeman ‡, Gautam Patel †, Baljinder Singh †, Paul Guyett ‡, Emanuele Amata †, Jessey Erath ∥,§, Norma Roncal ⊥, Erica Penn ⊥, Susan E Leed ⊥, Ana Rodriguez ∥,§, Richard J Sciotti ⊥, Kojo Mensa-Wilmot ‡, Michael P Pollastri †,*
PMCID: PMC6187419  PMID: 30344906

Abstract

graphic file with name ml-2018-002454_0007.jpg

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure–activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Keywords: Trypanosomes, human African trypanosomiasis (HAT), leishmaniasis, Chagas, malaria, protozoan parasite inhibitors, thienopyrimidines

Target class repurposing is a method for discovering new chemical matter against human African trypanosomiasis (HAT),1,2 a life-threatening disease caused by the protozoan parasite Trypanosoma brucei.3 By taking known human inhibitors of a well-characterized family of targets, inhibitors can be identified and reoptimized for antiparasitic activity. We have applied this approach against T. brucei.4−9 For example, during a screen of human tyrosine kinase inhibitors, lapatinib (Figure 1, compound 1), an FDA approved drug for breast cancer tumors,10−13 was found to be effective against T. brucei growth with an EC50 of 1.4 μM.14 Optimization of the lapatinib scaffold led to the synthesis of NEU-617 (2), which was 40-fold more potent than lapatinib (1) and had improved selectivity over human HepG2 cells.7 Further optimization of 2 to improve the physicochemical properties led to NEU-1961 (2a) with improved LogP and ligand-lipophilicity efficiency (LLE).15

Figure 1.

Figure 1

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Optimization of lapatinib.

Following our success in identifying lapatinib as a lead for anti-HAT drug discovery,15−17 we tested other lapatinib-related compounds synthesized by GlaxoSmithKline18 for activity against T. brucei. Compound GW837016X (NEU-391, Figure 2, compound 3) is a covalent inhibitor of human ErbB-2 kinase.18−21 This compound inhibits proliferation of bloodstream T. brucei in vitro (EC50 of 0.4 μM).22 Unlike compound 1, 3 features a thieno[3,2-d]pyrimidine scaffold and an alkyne warhead at the 6-position that covalently modifies a cysteine residue of ErB-2.21 We have previously explored bioisosteric replacement of the alkynyl functionality,22 and we herein report a wider range of substituted alkynes and other scaffolds (Figure 2). Besides the thieno[3,2-d]pyrimidine chemotype, we assessed matched isomeric thieno[2,3-d]pyrimidine and quinazoline scaffolds. Finally, we describe (i) the biological effects of 3 on T. brucei, (ii) characterization of the effects on trypanosome cell cycle, and (iii) efficacy of compound 3 in a mouse model of HAT.

Figure 2.

Figure 2

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Analog designs.

The thienopyrimidine (4, 5) and quinazoline (6) templates were synthesized by previously published methods.22−25 Each template was treated with a terminal alkyne via a Sonogashira coupling reaction, shown in Scheme 1, to achieve the desired product. Those alkynes that were not commercially available were prepared as outlined in Scheme S1 in the Supporting Information.

Scheme 1. Synthesis of Alkynyl Derivatives.

Scheme 1

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Reagents and conditions: (i) alkyne, PdCl2(PPh3)2, CuI, TEA, DMF, 55 °C, 12 h, 6–52%.

All compounds in series 7, 8, and 9 had different alkyne groups, and they were first tested against T. brucei cultures at a single concentration (5 or 10 μM); those inhibiting proliferation >75% were advanced to EC50 determination. Several compounds demonstrated submicromolar potencies. Of the new analogs, 7t, 7j, and 7l were the most potent with EC50 values of 0.23 μM (selectivity index, SI = 43.6), 0.28 μM (SI = 17.5), and 0.76 μM (SI = 40.8), respectively. Compound 7t (EC50 = 0.22 μM) is the enantiomer of the reference compound 3 (EC50 = 0.4 μM) and is slightly better in terms of potency. Addition of the Boc-protecting group to the pyrrolidine ring in 7s resulted in a 5-fold loss of potency against T. brucei. This is likely due to an increase in steric encumbrance of the Boc group or reduction of cellular permeability and not due to the loss of the hydrogen bond donor, given the activity of both 7j and 7l. Furthermore, small aliphatic cyclic rings found in 7t, 7j, and 7l were more potent than both pyridine 7r (EC50 = 1.3 μM) and larger cyclic aliphatic systems such as morpholine 7a (16% inh/5 μM), piperazine 7b (EC50 = 2.5 μM), or thiomorpholine 7k (12% inh/5 μM). This may suggest either unfavorable steric interactions within the putative binding site of the target(s) or unfavorable effects on cellular permeability. Conversely, some steric bulk is needed in this region as evidenced by a loss in activity exhibited by smaller aliphatic substituted compounds such as 7i (EC50 = 3.4 μM) and 7g (52% inh/5 μM) when compared with 3 (EC50 = 0.26 μM). The methylated piperazine tail 8b (EC50 = 1.0 μM) was slightly more potent than the unsubstituted piperazine 8h (EC50 = 1.9 μM), though the analogous morpholine 8a showed little activity (48% inh/5 μM). The quinazoline series (9) generally displayed only modest potencies.

We have had previous success in “parasite hopping”,17 wherein compounds prepared as potential T. brucei inhibitors are tested against other protozoan parasites. Thus, this library was screened against intracellular amastigotes of T. cruzi (responsible for Chagas disease) and of Leishmania major (leishmaniasis). Based on our previous observation that thienopyrimidines have activity against Plasmodium falciparum,22 the library was also tested against different strains of Plasmodium falciparum (malaria). From these assays, compounds 8h (EC50 = 0.61 μM) and 9a (EC50 = 0.81 μM) (Table 2) showed submicromolar activity against T. cruzi. There does appear to be a preference for the quinazoline core over either thienopyrimidine regioisomer. This is apparent when comparing compounds 9a and 7a, where there is a ∼4-fold improvement in potency for the quinazoline core. A similar trend is observed for 7k (EC50 = 5.0 μM) versus 9k (EC50 = 2.5 μM) and 7l (EC50 = > 50 μM) versus 9l (EC50 = 2.0 μM). Interestingly, there appears to be a preference for the morpholine (9a; EC50 = 0.81 μM) and piperazine (8h; EC50 = 0.61 μM) over the thiomorpholine (9k; EC50 = 2.5 μM), indicating a sensitivity to electronegativity differences at this position.

Table 2. Inhibition Profile of Alkynes against T. cruzi amastigotes, L. major amastigotes, and P. falciparum D6.

Entry T. cruzi amastigotes EC50 ± SEM (μM)a NIH3T3 TC50 (μM) SIb L. major amastigotes EC50 in μM (r2)c SId P. falciparum D6 EC50 in μM (r2)c SIe HepG2 TC50 (μM)
7a 3.4 ± 3.4 4.7 1.4 7.6 (0.92) 3.8 3.0 (0.98) >9.7 >29
8a nt nt -- 6.5 (0.94) 4.5 3.0 (0.90) >9.7 >29
9a 0.8 ± 0.06 33 41 5.5 (0.85) >5.5 5.9 (0.83) >5.1 >30
7b 2.1 ± 0.18 4.8 2.3 >15 -- 0.64 (0.99) 17 11
8b 3.5 ± 0.11 6.7 1.9 8.1 (0.94) 1.9 0.26 (0.94) 58 15
9b 2.5 ± 0.46 7.2 2.9 10 (0.81) 0.8 0.16 (0.98) 51 8.2
7c 9.6 ± 1.43 52 5.4 4.6 (0.85) 5.9 3.8 (0.91) 7.1 27
8c >50 >50 -- 9.0 (0.87) 3.1 >15 -- >28
9c 8.0 ± 0.30 >50 >6.3 >15 -- >15 -- >28
7d >50 >50 -- >15 -- >15 -- >27
8d 1.9 ± 0.08 2.5 1.3 11 (0.92) 2.0 4.8 (0.93) 4.6 22
7e >50 >50 -- >15 -- >15 -- 29
8e 30 ± 7.9 >50 >1.7 >15 -- 5.7 (0.95) >5.6 >32
7f >50 >50 -- 6.0 (0.85) 4.5 >15 -- 27
8f 1.7 ± 0.11 2.2 1.3 1.4 (0.92) 12 0.80 (0.99) 21 17
7g 8.2 ± 0.27 >50 >6.0 2.5 (0.91) 13 >15 -- >32
8g 2.6 ± 0.07 41 16 5.5 (0.89) >5.8 1.5 (0.98) >21 >32
7h >50 >50 -- 4.0 (0.86) 7.3 1.9 (0.97) >15 >29
8h 0.61 ± 0.16 3.9 6.4 9.1 (0.88) 0.85 0.38 (0.98) 20 7.7
7i 30.7 ± 8.7 >50 >1.6 9.4 (0.77) 3.5 5.9 (0.89) >5.6 >33
8i 1.5 ± 0.18 1.7 1.1 7.3 (0.93) 1.3 3.02 (0.97) 3.2 9.5
7j 1.8 ± 0.17 6.4 3.6 4.1 (0.77) 1.2 1.3 (0.96) 3.8 4.9
8j nt nt -- 4.3 (0.95) 7.0 0.14 (0.99) >214 >30
7k 5.0 ± 0.53 >50 >10 5.9 (0.81) 4.8 >15 -- >28
9k 2.5 ± 0.21 >50 >20 5.4 (0.93) >5.4 1.1 (0.96) >26 >29
7l >50 >100 -- >15 -- 3.6 (0.99) >8.6 >31
9l 2.0 ± 0.16 >50 >25 >2 -- >15 -- >3.0
7m 13 ± 3.5 47 3.6 6.9 (0.83) 3.5 1.5 (0.99) >19 >24
7n >50 >50 -- >15 -- >15 -- >32
7o >50 >50 -- >15 -- 5.7 (0.98) 4.9 >28
7p 19 ± 1.6 47 2.5 >15 -- 1.1 (0.97) 27 30
7q >50 >50 -- >3 -- >3.6 -- >5.0
7r 12 ± 0.45 27 2.3 0.38 (0.94) 63 2.0 (0.90) 12 24
7s >50 >100 -- 1.6 (0.97) 16 1.7 (0.99) >15 >26
7t 3.7 ± 0.23 10 2.7 4.2 (0.94) 2.3 0.94 (0.97) 10 9.6
Benznidazole 0.79 ± 0.01 -- --          
Amphotericin B       0.035 (0.90) -- -- -- --
Chloroquine       -- -- 0.0089 (0.97) -- --
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a

T. cruzi EC50 values are the result of triplicate experiments.

b

Selectivity index = NIH3T3TC50/Tc EC50. nt: not tested.

c

Compounds screened against L. major amastigotes or P. falciparum (D6 strain) either in duplicate or quadruplicate.

d

Selectivity index = HepG2 TC50/Lmj EC50.

e

Selectivity index = HepG2 TC50/Pfal EC50.

Against Leishmania major amastigotes, 15 compounds were active in the micromolar range (<10 μM), but only one compound had submicromolar activity (7r, EC50 value of 0.38 μM, SI = 62.1, Table 2). The P. falciparum screen yielded nine analogs with submicromolar activity. The most potent compounds were 8j (EC50 = 0.14 μM, Table 2) and 9b (EC50 = 0.16 μM, Table S2, Supporting Information). Several alkynes exhibited submicromolar activity irrespective of the central scaffold; they included 7b, 8b, 9b, 8f, 8h, 8j, 7t (Table 2), and 9b (Table S2, Supporting Information). These compounds were also screened against the drug-resistant W2 and C235 strains of P. falciparum (Table S2, Supporting Information); all compounds were within a 3-fold potency window compared to the drug-sensitive D6 strain, indicating no cross-resistance with chloroquine, mefloquine, or pyrimethamine.

HepG2 cells were employed to screen for overt toxicity and to calculate the selectivity index of the antiparasitic hits. Three compounds had selectivity index of 20 or more against T. brucei over HepG2 cells. The selectivity ranged from 43× (7t) to 4.3× (7b). In case of selectivity of compounds against P. falciparum D6 over HepG2 cells, there were eight compounds with selectivity of 20 or more; for P. falciparum, 8j had the best selectivity index of more than 200×. We also tested compounds against NIH 3T3 cells, which are the host cell line utilized for culturing the intracellular amastigotes of T. cruzi; this data is summarized in Table 2. Three compounds had selectivity index of 20 or more against T. cruzi over NIH 3T3 host cells; compound 9a had the best selectivity index of 41.

The dose response curves of selected compounds against T. brucei, P. falciparum, and L. major are provided in Figures S2–S4, respectively, in the Supporting Information. Protocols for in vitro proliferation inhibition assays of T. brucei, T. cruzi, L. major, P. falciparum, and host cells are also provided in the Supporting Information. These methods have been previously described.8,26−28

In the end, compounds 3, 7j, and 7t were the most promising anti-T. brucei agents (Table 1). Noting that compound 3 was previously dosed in mice orally at 100 mg/kg,21 we evaluated its pharmacokinetics at 130 mg/kg for possible anti-HAT efficacy studies in mice: its in vitro ADME properties were also evaluated. These data, along with an optimized synthetic route for scaling up compound 3, are described in the Supporting Information.

Table 1. Inhibition Profile of Alkynes against T. brucei*.

graphic file with name ml-2018-002454_0006.jpg

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*

Compare compounds to 3, T. brucei EC50 = 0.26/0.4 μM. EC50 = 0.26 μM is obtained from our initial screening program with parasite concentration of 2 × 103 cells/mL, and for direct comparison with other compounds, 3 was retested in the same assays that its derivatives were tested in at 5 × 104 cells/mL (EC50 = 0.4 μM).

a

Ar is 3-chloro-4-((3-fluorobenzyl)oxy)phenyl.

b

Compounds showing >75% growth inhibition at 5 μM for T. brucei were tested for EC50 values. T. brucei EC50 values are the result of triplicate experiments.

c

SI is selectivity index = HepG2 TC50/Tbb EC50.

Compound 3 was dosed at 130 mg/kg orally (formulated in 10% NMP and 90% PEG) to female Swiss Webster mice, blood samples were collected at 0.5, 1, 2, 4, 8, and 24 h and analyzed by LC–MS/MS. The results are summarized in Figure S1 and presented in Tables S4 and S5 in the Supporting Information. The drug concentration observed in plasma was above EC50 value for up to 4 h. Given the good in vitro potency and acceptable plasma exposure in mice, we performed an efficacy study of 3 in a mouse model of acute phase human African trypanosomiasis.

Mice infected with T. brucei (Lister427) were dosed orally with 130 mg/kg once daily for 10 days. The control group was treated with vehicle (10% NMP and 90% PEG). Animals were monitored on alternate days for parasitemia. In untreated (control) mice, parasitemia reached 108/mL of plasma by day 5 (mean parasitemia = 9.30 × 108/mL) (Figure 3b), and the mice were euthanized. The mean survival of compound 3-treated mice was 7.5 days compared to 5 days for untreated mice (Figure 3a). The difference in mouse survival times (2.5 days) was not statistically significant (determined from chi-square analysis of Kaplan–Meier survival data (p = 0.127)). Mice treated with compound 3 did not show signs of toxicity. By day 5, the treated group had two animals with 3-fold decrease in parasitemia and two animals with 400-fold decrease in parasitemia. One of the latter mice had parasitemia of 4 × 104/mL on day 9, representing a projected 108-fold decrease of trypanosomes in blood compared to the untreated mice (Figure 3b). Drug dosing was stopped on day 10 after which trypanosome proliferation resumed; reaching 8 × 108/mL on day 13, all the mice were euthanized (Figure 3b).

Figure 3.

Figure 3

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Evaluation of compound 3 efficacy in mouse model of acute HAT. (A) Mouse (n = 4) survival curve postinfection for control and compound 3 treated mice. (B) Parasitemia during compound 3 treatment. Mice (four per group) were infected with 105T. brucei Lister427 (day 0). After 24 h, compound 3 (130 mg/kg) was administered by oral gavage once daily for 10 days. Control mice received vehicle (10% NMP and 90% PEG). Parasitemia was determined every 24 h. Different shapes in the graph represent individual mice; DPI = days postinfection. Statistical significance of the difference in mean parasitemia was determined with Student’s t test.

Recently, we have reported that lapatinib (1) inhibited uptake of transferrin, a growth factor29 in bloodstream trypanosomes. Based upon the structure similarity of 3 to 1, we hypothesized that these two compounds might inhibit proliferation via the same mechanism. Thus, the possible effect of 3 on transferrin (Tf-647) uptake was tested (Biological Assay Protocols in Supporting Information). We found that compound 3 had no effect on transferrin uptake (data not shown), indicating that 3 inhibits T. brucei proliferation through a different mechanism than lapatinib.

Stages in trypanosome division can be classified by enumeration of several single organelles (e.g., kinetoplast (a nucleoid containing mitochondrial DNA termed kinetoplast DNA (kDNA), nucleus, flagellum).30,31 Synthesis of mitochondrial and nuclear DNA occurs in S-phase in cells containing one nucleus and one kinetoplast (1K1N) (Figure 4).32 Division of kinetoplasts is completed in G232 yielding 2K1N trypanosomes (Figure 4) in which nucleus division occurs, producing 2K2N cells (Figure 4). Cytokinesis takes place in 2K2N trypanosomes yielding new progeny with 1K1N genotype that can re-enter S-phase.33

Figure 4.

Figure 4

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Compound 3 inhibits mitosis and cytokinesis. Trypanosomes (5 × 105/mL) were treated with compound 3 (9.5 μM) or DMSO (0.1%) in HMI-9 medium for 3 h. Cells were incubated with mCLING, and the kinetoplast and nuclear DNA was counterstained with DAPI. The number of kinetoplasts and nuclei per trypanosome (n = >120) was quantitated. The mean percentage of trypanosomes with indicated number of nuclei (N) or kinetoplasts (K) is shown. Trypanosomes with two kinetoplasts and one or two nuclei are binned together for clarity of the 3 effect. Error bars represent standard deviation among three biological replicates. The difference in distribution of kinetoplasts and nuclei between control and compound 3-treated trypanosomes was evaluated with Pearson χ2 analysis (p = 5.85 × 10–7). Inset: Representative images of mCLING and DAPI-stained trypanosomes after treatment with DMSO. Scale bar, 2 μm.

In “mode of action” studies to learn how compound 3 affected the biology of bloodstream trypanosomes in vitro, mid log phase T. brucei (5 × 105 cells/mL) were treated with either 3 or DMSO for 3 h, fixed, stained with mCLING34,35 and DAPI, and examined with a fluorescence microscope. The number of kinetoplasts (K) and nuclei (N) per cell was enumerated (Figure 4). Compound 3 reduced the fraction of trypanosomes containing one nucleus and one kinetoplast (1K1N) (i.e., trypanosomes in G1 or S-phase of the cell cycle) but increased the proportion of cells with 2K1N or 2K2N. We conclude that 3 arrests proliferation of trypanosomes after division of the kinetoplast, that is, after G2 (i.e., leading to a buildup of 2K1N) and before cytokinesis (i.e., causing accumulation of 2K2N trypanosomes).

In conclusion, we have synthesized and assessed the biological activity of alkynyl-substituted thienopyrimidine and quinazoline compounds derived from 3, leading to the discovery of potent antitrypanosome agents, such as 7t (EC50 = 0.22 μM). The mode of action studies reveals compound 3 arrest proliferation of trypanosomes after G2 phase and before cytokinesis. Assessment of these compounds against other protozoan parasites produced potent compounds viz. 9a (T. cruzi EC50 = 0.81 μM, SI = 40.9), 7r (L. major EC50 = 0.38 μM, SI = 62.1), 8b (P. falciparum EC50 = 0.26 μM, SI = 57.7), 8j (P. falciparum EC50 = 0.14 μM, SI = 214.3), 9b (P. falciparum EC50 = 0.16 μM, SI = 51), and 8h (P. falciparum EC50 = 0.16 μM, SI = 20.3). Work is ongoing to improve compound properties toward discovery of novel antiparasitic agents.

Acknowledgments

We are grateful to AstraZeneca for performing the in vitro ADME experiments tabulated in Table S3 in the Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.8b00245.

  • Details of synthetic chemistry, biological assay protocols, and other data (annotated with NEU registry numbers), and ADME and PK properties of selected analogs (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding from the National Institutes of Health (R01AI124046, R56AI099476 to M.P. and K.M.-W.) is gratefully acknowledged.

The authors declare no competing financial interest.

Supplementary Material

ml8b00245_si_001.pdf (645.5KB, pdf)

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