Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors

Abstract

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure-activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC₅₀ < 50 μM), with 17z showing excellent cell permeability and no cytotoxicity.

Graphical Abstract

Topoisomerase II (TOP2) resolves DNA catenanes and knots and relax both positive and negative supercoils generated during normal physiological processes, such as transcription and replication.¹ TOP2 manages these topological transactions via producing transient TOP2 cleavage complexes (TOP2cc) which feature a unique covalent bond between its active site tyrosine and the 5´-phosphate terminus of the DNA backbone at the break sites.¹ In the presence of TOP2 poisons, such as anticancer drugs etoposide and doxorubicin, these transient TOP2cc can be stabilized and become abortive, resulting in DNA double-strand breaks (DSBs) and ultimately leading to cell death.^2–3 Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs these DSBs by specifically cleaving the 5’-phosphotyrosine bond that links TOP2 to DNA to create competent DSB termini ready for ligation through the non-homologous end joining (NHEJ).^4–7 By virtue of its cellular function TDP2 allows cancer cells to become resistant toward TOP2 poisons,⁶ a major class of anticancer drugs widely used in clinic. Evidence of TDP2 function in cancer cell damage repair also includes the observation that TDP2-deleted chicken lymphoma DT40 cells were hypersensitive to TOP2 poison etoposide, but not to the TOP1 poison camptothecin.⁸ Therefore, TDP2 represents an attractive anticancer drug target since its inhibition can sensitize cancer cells against TOP2 poisons. In addition, since treatment with TOP2 poisons is associated with non-specific toxicity due to limited tumor selectivity, co-administration with TDP2 inhibitors will potentially allow therapeutic efficacy with much lower doses of TOP2 poisons.^5,9

Interestingly, TDP2 is also implicated in the genome repair of certain viruses. TDP2 was identified as the host VPg-unlinkase enzyme necessary to cleave VPg-RNA bonds during picornavirus replicative cycle.^10–12 In addition, TDP2 could also play a role in hepatitis B virus (HBV) cccDNA formation by facilitating the release of P-protein from viral DNA.¹³

Phosphodiesterase activity of TDP2 was only discovered in 2009,⁸ and since then only a small number of scaffolds have been reported with TDP2 inhibitory activity (Fig. 1).^{9, 14–23} In addition, most of these scaffolds, such as compound 1 (NSC111041)²³ are poorly characterized, moderately active and lacking drug-like properties. Deazaflavin (e.g. 2, Fig. 1),^19–21 is the only inhibitor type with nanomolar biochemical potency¹⁹ and well characterized mechanism of inhibition,²¹ yet these inhibitors did not exhibit strong efficacy in cells²⁰ due to their poor cellular permeability.¹⁹ As part of our own efforts in developing drug like small molecule TDP2 inhibitors, we have previously identified an attractive inhibitor core isoquinoline-1,3-diones (e.g. 3).²²

Figure 1.

Representative reported TDP2 inhibitors

Recently we also developed the first fluorescence-based high throughput assay¹⁴ and identified new scaffolds with TDP2 inhibitory activity (e.g. compound 4, 5, and P10A10, Fig.1) during HTS of small library of 1600 compounds curated via virtual screening. In particular, [1,2,4]triazolo[1,5-a]pyrimidine compound P10A10 was found to inhibit 14M_zTDP2, a validated surrogate protein for hTDP2. Triazolopyrimidine is a highly privileged core in medicinal chemistry, with several biological activities reported just in the past three years: anti-Alzheimer’s disease,^24–25 antibacterial,^26–27 anticancer,^28–29 antileishmanial,³⁰ antimalarial,^31–32 antiviral,^33–34 CB2 cannabinoid receptor inverse agonists,³⁵ and phosphodiesterase 2 (PDE2a) inhibition.³⁶ Similar core is also featured in various herbicidal compounds.³⁷ In the present report, we describe our hit confirmation efforts including resynthesis and structural elucidation of hit P10A10, and the subsequent SAR of the triazolopyrimidine scaffold as TDP2 inhibitors.

The synthesis of triazolopyrimidines is well documented.^38–40 In our studies, [1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamides 6 and 7 (scheme 1) were synthesized in two steps starting with phenylpropene-2-one (8) or phenylpropan-2-one (9) to obtain 7-phenyl isomer 6 or 5-phenyl isomer 7, respectively. In both cases, compounds 8 or 9 were condensed with diaminotriazole 10 in the presence of acetic acid under MW conditions to yield [1,2,4]triazolo[1,5-a]pyrimidin-2-amine intermediates 11 and 12, respectively. Acylation with different acid chlorides, followed by treatment with ammonia solution 7N in methanol, provided the desired final compounds in good yields (73 – 83%). [1,2,4]Triazolo[1,5-a]pyridin-2-amine scaffold 13a was constructed by reacting 2-amino-5-bromopyridine (14) with ethoxycarbonyl isothiocyanate in dichloromethane at room temperature to form thiourea 15, which was cyclized by reacting with hydroxylamine under basic conditions at reflux in ethanol/methanol. Intermediate 13 then underwent microwave-irradiated Suzuki coupling with different boronic acids in the presence of Pd(PPh₃)₄ to afford [1,2,4]triazolo[1,5-a]pyridin-2-amines 16 in moderate yields (46 – 81%). Compounds 17 and 18 were obtain by acylation of 16 in the same manner as described for compounds 6 and 7 (45–96% yield). Compound 19 was synthesized via reductive amination involving compound 16a and 3-chlorobenzaldehyde. Treating compound 16a with 3-chlorobenzenesulphonyl chloride in methanol at room temperature for 2 h afforded compound 20 in 53% yield.

Scheme 1.

Reagents and conditions: (a) AcOH, reflux, 4h, 72%; (b) (i) acid chloride (2.5 equiv), pyridine, 0°C → r.t., 1–2 h, (ii) 7N NH₃ in MeOH, o.n., 45 – 96% (c) (i) AcOH, reflux, 6h; (ii) NBS, EtOH, reflux, 5h, 29% (d) ethoxycarbonylisothiocyanate (1 equiv), dioxane, under argon, 0°C to r.t., 12 h; (e) NH₂OH·HCl (5 equiv), DIPEA (3 equiv), EtOH/MeOH 1:1, 2 h r.t. then 60°C, 3 h, 65% (over 2 steps); (f) boronic acid, Pd(PPh₃)₄, K₂CO₃, EtOH, H₂O (1:1), M.W.,150°C, 30 min, 46 – 81% (g) 3 chlorobenzaldehyde (1.1 equiv), acetic acid (cat.), MeOH, r.t., 2 h; then sodium cyanoborohydride (2.0 equiv), r.t., 2 h, 76 %; (h) 3-chlorobenzenesulfonyl chloride (2.5 equiv), pyridine, 0°C → r.t., 2 h, 53%.

Our hit validation started with resynthesizing hit compound P10A10 (6a)⁴¹ and closely related analogues 6b-c to confirm the inhibitory capability of this scaffold (Scheme 1, Table 1). Surprisingly, all three analogues were found to be completely inactive at 100 μM, indicating that the structural assignment of compound P10A10 as 6a could be questionable. In further structural analysis, although mass spectrometry showed the same mass for P10A10 and 6a, from ¹H-NMR spectra the proton chemical shifts, and specially the coupling constant, for the pyrimidine hydrogens differed significantly (J= 7.1 versus J= 4.7 Hz, respectively, Figure 2) between P10A10 and 6a. In addition, literature search revealed that reacting aminoazoles with α,β-unsaturated compounds can generate different triazolopyrimidine regioisomers depending on the reaction conditions employed.^42–43 These observations suggest that either our synthesized compound 6a or the commercial P10A10 could be the 5-phenyl regioisomer 7a. To further elucidate the structure, we obtained the crystal structure for an advanced intermediate 11 (step a, Scheme 1), which confirmed that the phenyl ring is indeed on the 7 position (Figure 2) as reported in literature for the same reaction.⁴⁴ In the meantime, we synthesized compound 7a, the 5-phenyl isomer of compound 6a. Comparison of 7a to commercial compound P10A10 showed identical ¹H-NMR spectrum and same potency inhibiting TDP2. With all these, we corrected the structure for P10A10 (Chembridge ID 7236827) to be 7a, not 6a as assigned by the vendor.

Table 1.

TDP2 inhibitory activities of derivatives 6a-c, 7 a-f, 17a-o and 18.

Compds	R1	IC50 (μM)a
6a	3-NO2	>100
6b	H	>100
6c	4-NO2	>100
7a	3-NO2	22.0 ± 2.5
7b	H	>100
7c	4-NO2	>100
7d	3-CN	>100
7e	3-CF3	>100
7f	3-Br	80.9 ± 4.0
17a	3-NO2Ph	16.6 ± 0.4
17b	3-OCH3Ph	>100
17c	3-CH3Ph	>100
17d	3-FPh	>100
17e	3-ClPh	43.8 ± 3.9
17f	pyridin-3-yl	>100
17g	pyridin-4-yl	>100
17h	4-OCH3Ph	>100
17i	4-N(CH3)2	>100
17j	3,4-FPh	>100
17k	furan-2-yl	>100
17l	4-ClPh	>100
17m	2-ClPh	>100
17n	3,5-ClPh	>100
17o	2-Cl, 4-FPh	>100
18	-	77.3 ± 7.5

^aIC₅₀ values are the mean of three independent experiments performed in triplicate.

Figure 2.

Comparison of pyrimidine hydrogens NMR coupling constant from both isomers 6a and 7a, and X-ray crystallography studies of intermediate 11 (CCDC 1869530) reveals that P10A10 is not isomer 6a, but 7a instead.

All 47 final compounds were evaluated in a fluorescence-based biochemical assay¹⁴ measuring the catalytic activity of TDP2. In addition, permeability of selected derivatives was assessed by parallel artificial membrane permeability assay (PAMPA) and cytotoxicity was determined for the two overall best derivatives.

With the confirmation of 7a as a TDP2 inhibitor hit, we started exploring the SAR to see if we could improve the hit. We were particularly interested in replacing the nitro group as nitroarenes are associated with potential toxicity in medicinal chemistry with negative impact to the pharmacokinetic and physicochemical profile.^45–46 To probe the importance of meta-nitro group of the benzamide ring to activity, we first synthesized analogues without nitro (7b), with nitro at para position (7c), or with nitro replaced with another electron withdrawing group, such as nitrile (7d), trifluoromethyl, (7e) and bromo (7f). As shown in Table 1, these substitutions had a dramatic impact on TDP2 inhibition as significant activity was not observed with compounds 7b-e whereas compound 7f exhibited only weak inhibitory activity (IC₅₀= 80.9 μM). One of the challenges we had technically with these compounds was their poor solubility in DMSO. In an attempt to improve solubility and to further probe the SAR, we decided to synthesize the 4-deaza bioisosteric core, the triazolopyridine (17). Interestingly, compound 17a which bears the same meta-nitrophenyl moiety as hit 7a was found to be slightly more active (IC₅₀= 16.6 μM vs. 22.0 μM) with a better solubility profile in both DMSO and buffer. Therefore, we decided to continue our study focusing on the triazolopyridine scaffold 17 instead. Successful nitro bioisosters⁴⁷ described in literature include nitrile,^48–50 pyridine,^51–52 and halogenated analogues.^53–54 In addition, nitroarenes can behave as halo- or alkylarenes when noncovalently interacting with proteins.⁴⁶ Unfortunately, all these bioisosteric replacements led to less active compounds (17b-o), with only 3-chloro derivative (17e) exhibiting an IC₅₀ lower than 50 μM. Moreover, changing chloro to para and ortho positions (17l-m) or adding a second chloro (17o) further reduced activity.

Another SAR trend was that the phenyl group at different positions in the main core favored position 7 (17a, IC₅₀= 16.6 μM vs. 18, IC₅₀= 77.3 μM vs. 6a, IC₅₀> 100 μM). Although the potency of compound 17e was found to be 2-fold lower than the hit compound (IC₅₀= 43.8 μM vs 22.0 μM), its permeability in PAMPA assay was 6-fold higher (Table 3), and therefore we decided to maintain a 3-chlorobenzamide moiety when probing substitutions at position 7 of the pyridine ring (17p-ac, Table 2). Only 17s with 4-methoxyphenyl group was able to retain almost the same activity (IC₅₀= 59.0 μM), with pyridine-3-yl substitution leading to a two-fold improve of potency (17z, IC₅₀= 21.0 μM vs. 17e, 43.8 μM) and 7-fold increase in permeability (Table 3). The increase in potency observed for derivative 17z led us to further test substitution at meta position with potential H bond acceptor properties (3-methylsulfonyl, 17ad; 3-acetyl, 17ae; 3-carboxylate 17af) and also additional derivatives with aromatic nitrogens (pyridine-4-yl, 17ag; quinoline-3-yl, 17ah; pyrimidin-5-yl, 17ai). However, all modifications led to a decrease in potency.

Table 3.

TDP2 inhibition, permeability and cytotoxicity of the four best derivatives

Compds	TDP2 IC50 (μM)a	Pe (10−6 cm/s)b	HepG2 CC50 (μM)c	HeLa CC50 (μM)c
7a	22.0 ± 2.5	0.16	--	--
17a	16.6 ± 0.4	0.15	--	--
17e	43.8 ± 3.9	0.9	>100	>100
17z	21.0 ± 2.1	6.3	>100	>100
1	0.040	0.095	--	--

^aIC₅₀ values are the mean of three independent experiments performed in triplicate;

^bpermeability is considered high when Pe > 1.5;

^ccompounds were tested at 50 μM and 100 μM (two independent experiments were performed in triplicate).

Table 2.

TDP2 inhibitory activities of derivatives 17p-ai, 19–20.

Compds	R1	IC50 (μM)a
17p	4-ClPh	80.8 ± 7.5
17q	4-OHPh	>100
17r	4-CF3Ph	>100
17s	4-OCH3Ph	59.0 ± 3.7
17t	4-FPh	85.2 ± 4.6
17u	3-ClPh	>100
17v	3-OCH3Ph	>100
17w	3-CF3Ph	>100
17x	2-OCH3Ph	>100
17y	2-ClPh	>100
17z	pyridin-3-yl	21.0 ± 2.1
17aa	furan-2-yl	94.5 ± 6.8
17ab	thiophen-2-yl	>100
17ac	furan-3-yl	>100
17ad	3-SO2CH3Ph	>100
17ae	3-COCH3Ph	>100
17af	3-COOHPh	>100
17ag	pyridin-4-yl	>100
17ah	quinolin-3-yl	>100
17ai	pyrimidin-5-yl	>100
Compds	X	IC50 (μM)
19	CH2	>100
20	SO2	>100

^aIC₅₀ values are the mean of three independent experiments performed in triplicate.

Lastly, we synthesized compounds in which the carboxamide moiety of 17 is replaced by an amine (19) and or a sulfonamide (20). Both substitutions led to inactive derivatives, highlighting the importance of the amide group to activity.

Since the goal of developing TDP2 inhibitors is to sensitize cancer cells toward TOP2 poisons to ultimately overcome drug resistance and potentially also allow TOP2 therapeutic efficiency at lower and less toxic concentrations, it is imperative that these inhibitors are cell permeable and non-cytotoxic. Toward these ends, selected active compounds (7a, 17a, 17e, and 17z) were first tested in the PAMPA permeability assay along with the most potent deazaflavin inhibitor 1 (Table 3). From this assay, excellent permeability was observed only with our compound 17z. Another analogue 17e also exhibited borderline P_e value which is about 10 times higher than that of 1. These findings are consistent with the report that deazaflavin compounds lacked cellular permeability. We then assessed the two cell-permeable compounds 17e and 17z for cytotoxicity in two different cell lines (HepG2 and HeLa), from which no cytotoxicity was observed at concentrations up to 100 μM (Table 3). These results indicate that despite the relatively moderate TDP2 inhibitory activity, scaffold 17 has the potential to be developed into cell permeable and noncytotoxic chemical probes for studying cellular functions.

From a previous screening campaign against TDP2, compound P10A10, reported as 7-phenyl triazolopyrimidine 6a, was identified as a hit. The present study on hit validation via resynthesis and structure elucidation revealed that the correct structure for P10A10 (Chembridge ID 7236827) should be 5-phenyl isomer 7a. To probe TDP2 inhibition potentialities of this scaffold we synthesized a total of 47 compounds: 11 derivatives inhibited TDP2 (IC₅₀ < 100 μM), with four derivatives (7a, 17a, 17e, and 17z) reaching potencies below 50 μM. Although we were unable to increase significantly the potency of the original hit, compounds 17e and 17z, particularly the latter, demonstrated good cellular permeability and no cytotoxicity and can be further developed into useful chemical probes for studying cellular functions of TDP2 in host DNA repair as well as virus genome repair.