Synthesis and evaluation of ¹⁸F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression

Abstract

Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders. With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET) imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and MCF-7 cell lines. Based on these results, ¹⁸F-labeled analogs of these two compounds were synthesized and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts. [¹⁸F]-18 and [¹⁸F]-19b were synthesized from their corresponding protected tosylated derivatives by fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both compounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool very rapidly and getting metabolized over. The insights gained from the current study will surely aid in the design and construction of future generations of PET agents for the non-invasive delineation of Topo-II expression.

1. Introduction

Type II topoisomerase (Topo-II) is an ATPase that unravels DNA supercoiling in a very precise fashion through transient double-strand DNA cleavage followed by re-ligation. The enzyme is essential in the transcription, replication, and chromosome segregation processes, and its accurate functioning is a prerequisite for the development of normal mitosis [1–3]. In mammals, two isoforms of Topo-II exist: Topo-IIα (170 kDa) and Topo-IIβ (180 kDa) [4]. Despite a high degree of homology, recent studies have suggested that the two isoforms might play different roles in cellular processes and are regulated very differently. Topo-IIα is expressed in proliferating cells only. This isoform over-expression, a specific and sensitive marker for cell proliferation, has been observed in many types of cancer [5,6] and presents an attractive target for cancer therapy. Topo-IIβ, on the other hand, is expressed at lower levels at steady state levels during all stages of cell cycle.

Overall, inhibitors of Topo-II can be divided into two broad classes: poisons and catalytic inhibitors. Members of the first class interfere with Topo-II function by binding to and stabilizing the DNA–Topo-II complex, ultimately promoting the formation of extremely toxic double-strand breaks. The discovery of topoisomerase poisons has been tapped effectively in the development of several clinically successful chemotherapeutics [4,7–9]. Anthracyclines, for example, represent one of the most successful classes of topoisomerase poisons and are widely used in the treatment of breast cancer. Catalytic inhibitors of Topo-II, on the other hand, are a heterogeneous group of compounds with antineoplastic activity that act on various steps in the catalytic cycle [10]. These compounds might interfere with the binding of Topo-II to DNA, stabilize non-covalent DNA–topoisomerase II complexes, or prevent the catalytic turnover by inhibiting ATP binding [10,11]. The currently used Topo-II-targeted anticancer drugs are not specific to an isoform and inhibit both Topo-IIα and Topo-IIβ.

While inhibitors of Topo-II — both poisons and catalytic inhibitors — have proven to be effective anti-neoplastic drugs, several independent studies have established that the sensitivity of tumors to Topo-II inhibitors is highly dependent on both the expression and activity levels of the enzyme, and that lower Topo-II levels in tumors minimizes drug efficacy [5,12–17]. Currently, however, this information can be obtained only through biopsies. Therefore, it follows that there is an urgent need to develop noninvasive imaging methods that can provide this vital information without the risk, discomfort, and unreliability of invasive procedures. Indeed, in this regard, positron emission tomography (PET) represents an extremely attractive route. Over the past twenty years, PET and single photon emission computer tomography (SPECT) have revolutionized the field of diagnostic imaging by allowing clinicians to visualize biomarkers that provide functional information about tumor biology. In the case at hand, a PET radiotracer capable of the non-invasive delineation of the levels of Topo-II expression or activity in a tumor would be a powerful tool as a prognostic indicator of response to the Topo-II inhibitor therapy.

We have been interested in evaluating the efficacy of topoisomerase II targeted probes as non-invasive markers for imaging Topo-II expression levels. Previously, we investigated the potential of ⁶⁴Cu-radiolabeled thiosemicarbazone complexes as Topo-II expression probes and provided proof-of-concept that Topo-II-rich tumors could be visualized via PET using these radiotracers [18]. To date, this remains the only successful report of the use of PET agents for Topo-II delineation in tumor. However, the pharmacokinetic properties of the lipophilic copper complexes were suboptimal and led to high non-target tissue uptake. Additionally the exact mechanism of these complexes in targeting the Topo-II enzyme remains unclear and therefore complicates the interpretation of images.

More recently, as part of our continuing efforts toward the noninvasive delineation of Topo-II expression in vivo, we reasoned that a radiolabeled agent that is rationally designed to specifically bind to the ATP-binding pocket of Topo-II has potential to provide a direct measurement of Topo-II expression levels in the tumor. Following the identification of substituted purine derivatives as a novel structural class of catalytic topoisomerase II inhibitors, Furet et al. recently used a structure-based approach to rationally design a purine scaffold that acts as a ATP competitive inhibitor of Topo-II [11,19,20]. Quinoline aminopurine 1 (QAP1, Fig. 1) and its benzothiazole derivatives were described as first representatives of this new class of ATP competitive inhibitors. QAP1 was reported to have micromolar affinity for the enzyme with a fairly good selectivity over various protein kinases, and represents a promising starting point for lead optimization campaigns and further drug discovery efforts [20]. QAP1 inhibits topoisomerase ATPase activity as well as the decatenation reaction and targets both alpha and beta isoforms in cell-free assays. The ability of QAP1 to antagonize doxorubicin induced DNA damage and aberrant chromosome segregation further provides evidence that QAP1 is a catalytic inhibitor of Topo-II rather than a poison. [20,21]

Fig. 1.

The structure of QAP1.

The aim of this work is to identify fluorinated purine analogues that function as potent Topo-II inhibitors and have potential to be developed as radiotracers for the non-invasive assessment of Topo-IIα overexpression levels in the tumors. In addition, these probes have the potential to provide information on the pharmacokinetics and tissue biodistribution of these new Topo-II catalytic inhibitors. Herein, we describe the radiosynthesis and in vitro studies on fluorinated purine derivatives as Topo-II ATP competitive inhibitors and their in vivo evaluation as PET tracers for imaging Topo-II expression.

2. Results and discussion

2.1. Design of the compounds

The 2,6-diaminopurine moiety is critical for optimal binding of the molecule to the ATP binding site of Topo-II, whereas the aryl moiety sits outside the binding pocket and is amenable for modification [11]. Therefore this aryl group was chosen for derivatization and installation of fluorine substituent [20]. The aryl group is ideal for substitution because it does not disrupt the binding pocket interactions and is oriented away from important amino acid residues of the enzyme. As a consequence, all compounds (with the exception of compound 25b) were designed to follow these salient design features (Table 1). Another strategy for maximizing favorable interactions between the inhibitor and its target was used in the case of compound 25b, as we introduced a hydrophobic fluoroethoxy group in position 6 of the purine ring in order to make favorable hydrophobic contact with various residues and occupy a hydrophobic pocket. It was also important to anticipate the feasibility of ¹⁸F labeling and to consider synthetic routes amenable to incorporation of a suitable precursor for radiolabeling, which is developed in the radiochemistry section (vide infra).

Table 1.

Structures of fluorinated purine derivatives.

Compound	R2	R6	R8
18	A	tBuNH–	H
19b	B	tBuNH–	H
20	B	tBuNH–	Et
21b	B	CYNH–	H
22b	C	tBuNH–	H
23b	C		Et
24b	D	tBuNH–	H
25b	E	F(CH2)2O–	H

2.2. Chemical synthesis

Our synthetic strategy involved utilization of C-6 functionalized 2-chloropurines as key intermediates and a synthetic route wherein these derivatives were obtained first and then coupled to the appropriate aryl- or heteroarylamine at a late stage was preferred. Two methods were used for the formation of the C–N bond linking the purine ring and the aryl/heteroaryl moiety (Scheme 1): reaction of an N-9 protected 2-chloropurine (2a, 3, 4 or 6b) with an aryl-/heteroarylamine in Buchwald–Hartwig Pd-catalyzed coupling conditions (method A) or from a non-protected purine (2b, 6a) and an aryl-/heteroarylamine via an acid-catalyzed S_NAr chlorine displacement (method B). Both methods were adapted from previously reported procedures [11,22]. A synthetic approach in which no protection and deprotection steps are required might seem more attractive, but 6-aminoquinolines and the 2-amino-6-fluoropyridine proved either unreactive or unstable in harsh acidic conditions at high temperature. In the end, both methods were tried for all compounds but compound 18, which was obtained in a satisfactory 73% yield using method B. For all other compounds, the highest-yielding procedure – method A in most cases – is reported. The Buchwald–Hartwig coupling step provided the products, but the yields were inconsistent. Removal of the THP protective group was achieved using trifluoroacetic acid in methanol.

Scheme 1.

Reaction scheme for purine compounds 18, 20, 19b, 21b–25b and QAP 1. Reagents and conditions: (i) Cs₂CO₃, binap, Pd(OAc)₂, 45 min, MW 160 °C, N-9 protected purine; (ii) TFA, MeOH, 1–3 h, RT (Method A) or (iii) HC1 cat., n-butanol, 24 h, 130 °C, non-protected purine (Method B).

The synthesis of 2-chloropurine derivatives was accomplished as follows: 2,6-dichloropurine 1a is commercially available and slightly modified reported methods for N-9 protection and selective C-6 functionalization led us to compounds 2a and 2b, 3 and 4 (Scheme 2) [22–24]. Notably, the order in which the two steps were performed had a limited impact on the two-step overall yield. In the case of compound 2b however, we found that a three-step procedure (protection, functionalization, and deprotection) was more favorable than the one-step introduction of the tert-butylamino group in position 6 from 2,6-dichloropurine. On the other hand, access to a C-6 functionalized 2-chloropurine with a small alkyl group in position 8 requires more complicated preparation procedures where the purine ring is built from barbituric acid derivative via the intermediate 2,4-dichloro-5,6-diaminopyrimidine 5 [20]. The ring closure step which led to compound 6a from 5 was accomplished in a moderate yield (44%, Scheme 3). It is important to note that while the introduction of a THP group on 2,6-dichloropurine proceeded in high yield (90%), the presence of an ethyl group in position 8 affected the product yield in the synthesis of compound 6b (67%).

Scheme 2.

Synthesis of 2-chloropurine derivatives 2a, 2b, 3 and 4. Reagents and conditions: (i) 3,4-DHP, PTSA cat, EtOAc, 3.5 h, 50 °C; (ii) t-BuNH₂ (compound 2a) or cyclohexylamine (compound 3), EtOH, 2–4 h, reflux; (iii) fluoroethanol, NaH, THF, 4 h, 60 °C; TFA/MeOH, 12 h, RT.

Scheme 3.

Synthesis of 2-chloropurine derivatives 6a and 6b. Reagents and conditions: (i) EtC(OEt)₃,1 h, MW 160 °C; (ii) t-BuNH₂, NMP, 2 h, MW 160 °C; (iii) 3,4-DHP, PTSA cat., EtOAc, 6 h, 60 °C

Apart from commercially available 2-amino-6-fluoropyridine and 6-aminobenzothiazole, all aryl- or heteroarylamines were obtained in multi-step syntheses. Aniline 9 was obtained in two steps starting from commercially available compound 7 (Scheme 4): following a DAST/CH₂Cl₂ fluorination step, the nitro group was reduced to the desired compound in near quantitative yield using Fe/AcOH. The synthesis of 6-aminobenzothiazole 12 was accomplished in two steps from compound 10 in accordance with Scheme 5. The intermediate 10 was obtained after regioselective nitration of 2-chlorobenzothiazole according to previous report [25]. Subsequent S_NAr displacement with in situ generated fluoroethanoate and reduction of the nitro group led to desired compound 12 in a good three-step overall yield. The synthesis of 6-aminoquinolines 16 and 17 (Scheme 6) began with chloroquinoline 13, an intermediate that was synthesized in two steps according to the literature [26]. Reaction of compound 13 with fluoroethanol and 2-morpholinoethanol afforded nitro compounds 14 and 15, respectively. The nitro group was subsequently reduced to the desired aminoquinolines 16 and 17 using Fe/AcOH.

Scheme 4.

Synthesis of 4-(2-fluoroethyl)aniline 9. Reagents and conditions: (i) DAST, DCM, 6 h, 0 °C to RT; (ii) EtOH/H₂O/AcOH, Fe powder, 1 h, reflux.

Scheme 5.

Synthesis of 2-(2-fluoroethoxy)benzo[d]thiazol-6-amine 12. Reagents and conditions: (i) fluoroethanol, NaH, THF, 2 h, RT; (iii) EtOH/H₂O/AcOH, Fe powder, 1 h, reflux..

Scheme 6.

Synthesis of 2-(2-fluoroethoxy)quinolin-6-amine 16 and 2-(2-morpholinoethoxy)quinolin-6-amine 17. Reagents and conditions: (i) fluoroethanol (compound 14) or 2-morpholinoethanol (compound 15), NaH, DMF, 12 h, RT; (iv) H₂, Pd/C, THF (compound 16) or DCM/EtOH (1:1) (compound 17), 20 h, RT.

Full synthetic details and characterization data are given in the experimental section (vide infra). All intermediates and final purine derivatives were purified via flash column chromatography and identified and characterized by ¹H NMR, ¹³C NMR, and ESI–MS.

2.3. Topoisomerase IIα inhibition assay

After successful synthesis and isolation of QAP1 and fluorinated purine derivatives 18, 19b, 20, 21b–25b, we investigated their Topo-Ilα inhibition activity with an agarose gel electrophoresis experiment. Their ability to inhibit the relaxation of a supercoiled DNA substrate (plasmid pHOT-1) was tested in a qualitative assay, which was performed using a commercially available kit (TopoGen, Port Orange, FL). The DNA substrate was incubated with Topo-IIα in the presence of the derivatives in question at a concentration of 100 (µM. QAP1 and the well-known Topo-II poison etoposide were used as positive controls, and pHOT-1 DNA and DMSO vehicle were used as negative controls.

This experiment indicated that compounds 21b and 24b are not active towards Topo-IIα inhibition (Fig. 2). In contrast, all other fluorinated compounds were found to be active Topo-IIα inhibitors at 100 (µM. Indeed, the absence of relaxed DNA in lanes 21b and 24b reveals that the enzyme is not inhibited as opposed to other lanes and positive controls in which supercoiled DNA, the initial substrate, is present. A wider range of fluorinated compounds would be necessary to discern strong structure–activity relationships (SARs) and possibly discover new important structural features, leading the way for optimizing the purine-based reported model. Now, in any case, given the relatively high tested concentration, it seems reasonable to conclude that neither an aminopyridyl group in position 2 of the purine scaffold nor an aminocyclohexyl group in position 6 allows for efficient Topo-IIα inhibition.

Fig. 2.

Agarose gel assay for Topo-IIα inhibition in the presence of 100 µM QAP1,18, 19b, 20, 21b-25b.

2.4. Cytotoxicity assay

QAP1 and fluorinated purine derivatives were tested in vitro to evaluate their anti-proliferative activities and how the anti-proliferative activities related to Topo-IIα expression level. This was accomplished using cytotoxicity assays (MTT) in two breast cancer cell lines. SK-BR-3 and MCF-7 cell lines, that express high and low levels (10-fold) of the enzyme, respectively, were chosen as positive and negative controls, respectively [27]. MTT assays were performed using 72 h drug incubations. These cell proliferation assays were aimed at the evaluation of the cytotoxic properties of our novel purine derivatives towards breast cancer cell lines, and provide a leading candidate for labeling with fluorine-18 and evaluate its efficacy in relevant in vivo models. Growth-inhibition values obtained for both cell lines are compiled in Table 2.

Table 2.

Topo-IIα inhibition activity and antiproliferative activity (GI50/µM) of the fluorinated purine derivatives and QAP1.

Compound	Topo-IIα inhibition/ 100 µM	SK-BR-3 cells		MCF-7 cells
		GI50/µM	R2	GI50/µM	R2
18	+	6.8 ± 0.8	0.975	14.5 ± 0.5	0.903
19b	+	8.0 ± 0.8	0.936	46.3 ± 0.9	0.958
20	+	13.6 + 0.9	0.954	4.9 ± 0.3	0.966
21b	−	>100	N/Aa	>100	N/A
22b	+	2.0 + 0.9	0.988	13.6 ± 0.7	0.954
23b	+	7.0 ± 0.9	0.981	3.2 ± 0.7	0.966
24b	−	>100	N/A	>100	N/A
25b	+	11.4 + 0.8	0.965	>100	N/A
QAP1	+	10.2 ± 0.9	0.977	32 ± 2	0.961

^aNot applicable.

The MTT data indicates that after 72 h incubation, QAP1 has GI₅₀ values of 10.2 ± 0.9 µM and 32 ± 2 µM with SK-BR-3 and MCF-7 cell lines, respectively. These rather high values are in accordance with and previously reported results that indicated that QAP1 is less potent in the cellular context as compared to other catalytic inhibitors [19]. Nevertheless, in addition to the fact that QAP1 targets topoisomerase II in cells, the slight differential antiproliferative effect that is observed on high versus low Topo-IIα expressing cell line suggests that the inhibition of the enzyme may play a role in the antiproliferative effects of this derivative.

Most importantly, the MTT assay reveals that compounds 21b and 24b — both inactive in the Topo-IIα inhibition assays — displayed no antiproliferative activity in the tested cell lines, even at concentrations where a general cytotoxic effect is usually observed. Many cell lineage dependent factors, like the cellular uptake, can modify the activity of a drug, but the antiproliferative activity observed in our MTT assay relates relatively well with the in vitro Topo-II inhibition assay results, which again suggests that Topo-II inhibition is a possible cause for the antiproliferative effect. The other fluorinated purines displayed antiproliferative activity at concentrations ranging from 2 to 46 µM, in general with higher potency than QAP1 in both cell lines. Compound 22b was the most active towards growth inhibition of SK-BR-3 cells, with a GI₅₀ value of 2.0 ± 0.9 µM. Compound 23b, which only differs from 22b with the ethyl group on position 8, was most active towards growth inhibition of MCF-7 cells, with a GI₅₀ value of 3.2 ± 0.7 µM. With regard to the in vitro potency, no general trend is observed, but GI₅₀ values are lower in the Topo-IIα overexpressing SK-BR-3 cells than in the MCF-7 cells for compounds 18, 19b, 22b and 25b, with a highly marked difference in some cases. More data would be necessary to support the hypothesis that Topo-IIα inhibition is the major mechanism involved in growth inhibition and to discern further SARs. However, at present, some of the fluorinated compounds reported herein as new purine based Topo-II catalytic inhibitors showed a higher potency than the parent QAP1.

2.5. Radiolabeling precursors synthesis and radiochemistry

After demonstrating that the fluorinated purines analogues were comparable to QAP1 in cell growth inhibition and potency towards Topo-IIα, our next objective was to use the corresponding ¹⁸F-labeled derivatives for in vivo studies. Our ultimate goal is the design and development of PET imaging agents to provide information on Topo-II expression level in vivo non-invasively. Further, to date, no information exists in the published scientific literature on the pharmacokinetic and tissue distribution of QAP1 or other members of this promising new class of purine based Topo-II inhibitors in murine models. PET imaging gives access to such knowledge, which will be critical in the further development and potential clinical applications as imaging agents of our fluorinated purines.

Based on the biological assay results discussed above, two compounds – 18 and 19b – were chosen for radiolabeling with ¹⁸F. The feasibility of both the synthesis of a suitable radiolabeling precursor and the radiolabeling reaction itself were also taken into account for the selection of these two compounds. Indeed, we initially hoped to ¹⁸F-radiolabel the active, quinoline-containing compound 22b to complete our studies, but the lack of access to a precursor that can be directly radiolabeled hindered our efforts, and this idea was not pursued further.

Typically, a one-step synthesis is the ideal scenario for radiochemical synthesis with short-lived isotopes, as it reduces the overall synthesis time and allows for higher real overall (non-decay corrected) yields. However, we anticipated that protecting the free amine in position 9 of the purine scaffold was necessary with our compounds. Our efforts to synthesize [¹⁸F]-18 from a precursor that was not protected at position-9 proved unsuccessful (data not shown), strongly supporting the importance of protecting group. Therefore, the need for a suitable fluorination precursor led us to design new synthetic routes to tosylated compounds 29 and 34 (Schemes 7 and 8). Tosylate is one of the most common precursors used for ¹⁸F labeling of organic molecules as it provides a good balance between the stability and its affinity to undergo nucleo-philic substitution, which is also the reason why we chose to introduce the tosyl group at a late stage of the syntheses.

Scheme 7.

Synthesis of precursor 29 and radiosynthesis of [¹⁸F]-18. Reagents and conditions: (i) 2a, Cs₂CO₃, binap, Pd(OAc)₂, 45 min, MW 160 °C; (ii) TsCl, Et₃N, DCM, 72 h, RT; (iii) K[¹⁸F]F/K222/K₂CO₃, MeCN, 12 min, 115 °C; (iv) 1 N HC1 in MeOH, MeCN, 10 min, 115 °C.

Scheme 8.

Synthesis of precursor 34 and radiosynthesis of [¹⁸F]-19b. Reagents and conditions: (i) 2-((triisopropylsilyl)oxy)ethanol, NaH, THF/DMF, 48 h, 90 °C; (ii) 2a, Cs₂CO₃, binap, Pd(OAc)₂, 45 min, MW 160 °C; (iii) TBAF, THF, 1 h, RT; (iv) TsCl, pyridine, 12 h, RT; (v) K[¹⁸F]F/K222/K₂CO₃, MeCN, 12 min, 115 °C; (vi) TFA, MeOH, MeCN, 10 min, RT.

The synthesis of compound 29, the immediate precursor to [¹⁸F]-18, was accomplished in two steps from commercially available aniline 27. First, conditions B (see Chemical Synthesis) were used for the Pd-catalyzed coupling step with purine 2a. Subsequent tosylation of intermediate 27 with TsCl/Et₃N led to the desired compound in a 63% two-step overall yield.

The synthesis of the radiolabeling precursor for [¹⁸F]-19b was less straightforward, as it was found that the hydroxyl group that was to be tosylated had to be introduced and appropriately protected early in the synthetic route. The synthesis of 6-aminobenzothiazole 31 was accomplished via S_NAr displacement with 2-((triisopropylsilyl)oxy)ethanoate generated in situ. Subsequent coupling with purine 2a and the removal of the TIPS protecting group led to intermediate 33 in a 35% three-step overall yield. Before TIPS was identified as a suitable protecting group, other synthetic strategies involving protection with Bz, or TBDPS groups were attempted with little success, the failures occurring either at the coupling or at the deprotection stage. The final tosylation of compound 33 with TsCl/pyridine gave precursor 34 in a 68% yield. Both precursors, like all other intermediates, were identified and characterized by ¹H NMR, ¹³C NMR, and ESI–MS, as detailed in the experimental section.

The ¹⁸F radiosyntheses proceeded from 29 or 34 in one-pot, two-step reactions to [¹⁸F]-18 and [¹⁸F]-19b, respectively. First, precursors were treated with [¹⁸F]-KF/Kryptofix 2.2.2 in CH3CN at 110 °C to produce the N9-THP protected fluorinated intermediates. Subsequently, the ¹⁸F-labeled purines were converted to the desired compounds by removal of the THP protecting groups. Notably, acidic conditions used for the synthesis of [¹⁸F]-18 (HC1 in MeOH at RT) were too harsh and could not be used for the successful synthesis of [¹⁸F]-19b. In the latter case, a mixture of MeOH and TFA (50% v/v) at RT were later found to be suitable conditions. Radiosyntheses were carried out in just over 2 h including purification and formulation in saline for injection. Both compounds were isolated in radiochemical purity greater than 98% (Fig. 3) with dcRCY of 26% ([¹⁸F]-18) and 6% ([¹⁸F]-19b). As both radiolabeled purines were synthesized for preliminary in vivo studies, RCYs were not optimized and specific activities were not determined. Notably, these two-step radiolabelings were remarkably clean, as very minor (if any) fluorinated side-products were formed in both steps.

Fig. 3.

Analytical radio-HPLC chromatograms.

2.6. Small animal PET imaging

Following successful radiosyntheses of [¹⁸F]-18 and [¹⁸F]-19b, small animal PET imaging experiments were performed in mouse bearing SK-BR-3 xenografts to evaluate the in vivo pharmacokinetic behavior of the two radiotracers. [¹⁸F]-18 or [¹⁸F]-19b in 150 µL saline was administered to athymic nude mice (n = 4) bearing SK-BR-3 xenografts on the right shoulder via tail vein injection. The animals were subsequently imaged at 30 min, 1, 2 and 4 h post administration. PET images (showed for [¹⁸F]-18 only, Fig. 4) clearly indicate a very fast clearance from blood circulation with excretion mainly into the liver and the gastrointestinal (GI) tract and also into the bladder. Subsequently, we observe as soon as 1 h p.i. that strong activity remains principally in the GI tract, revealing a rapid hepatic clearance. However, intense focal uptake in the GI tract regions suggests activity accumulation in the gallbladder that is visible at early time point and remain the most prominent feature at later time points. Notably, there was some uptake observed in bone at late time points, suggesting that our tracers did undergo a slow metabolic defluorination. A similar biodistribution profile was observed for the two tracers starting from 30 min p.i. Unfortunately, we observed that the tumor uptake was extremely low which demonstrates a non-favorable pharmacological profile. This PET study indicates that the pharmacokinetic properties of these potent QAP1 analogs render them unsuitable as imaging agents. Indeed, significant modifications to their design will be necessary to improve their in vivo behavior and lead to better uptake in Topo-II-expressing tumors.

Fig. 4.

Representative coronal PET images (Maximum Intensity Projection) of SCID mice bearing SK-BR-3 xenografts on the right shoulder. Images A, B and C are the results of 15-min static scans following iv injection of the mouse with [¹⁸F]-18. Images A, B and C were obtained at 30, 60 and 240 min after injection, respectively.

3. Conclusion

Herein, we have presented the synthesis of ATP competitive Topo-II inhibitor QAP1 and its fluorinated derivatives. Structural modifications were well tolerated, as most of the derivatives retained fairly high potency toward Topo-II inhibition in vitro and showed similar antiproliferative activity to QAP1 in two breast cancer cell lines. Some SARs were found, and the library of purine based Topo-II catalytic inhibitors was broadened. The successful radiosynthesis of two ¹⁸F-labeled purine derivatives is also reported. Both agents were isolated in high radiochemical purity; however, in vivo PET imaging experiments revealed suboptimal biodistribution profiles. It is clear from our in vivo studies that improvements of the pharmacokinetic profile of the QAP1 analogs is needed to make them suitable PET imaging agents. Additionally, the pharmacokinetic data also indicates that the compounds might fare poorly in the in vivo tumor regression studies, because of suboptimal bioavailability resulting from rapid clearance. Indeed, the question remains whether purine based derivatives could be efficient imaging agents for the delineation of high Topo-II expressing tumors, but further pharmacomodulation studies are currently underway. It is our hope that the work presented here will encourage further drug discovery efforts and provide useful information towards the identification of Topo-II imaging agents and the development of much-needed novel catalytic inhibitors.

4. Experimental section

4.1. General chemistry

All chemicals and solvents were obtained from Sigma—Aldrich Co., Fisher Scientific or TCI America and, unless stated otherwise, were used as received without further purification. All DMSO used for biological assays was of molecular biology grade (>99.9%; Sigma, D8418). Water (>18.2 MΩ cm⁻¹ at 25 °C) was obtained from an Alpha-Q. Ultrapure water system (Millipore). All MTT assay kit materials were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). All Topo-IIα inhibition assay materials were purchased from TopoGEN, Inc. (Port Orange, FL, USA). All instruments were calibrated and maintained in accordance with standard quality-control procedures. ¹H and ¹³C NMR spectra were recorded on Bruker instruments (500 MHz or 600 MHz). Chemical shifts (δ) are determined relative to CDCl3 (referenced to 7.26 ppm for ¹H NMR and 77.16 ppm for ¹³C NMR) or DMSO-d₆ (referenced to 2.50 ppm for ¹H NMR and 39.52 ppm for ¹³C NMR). Coupling constants (J) are given in hertz and spectral splitting patterns are designated as singlet (s), doublet (d), triplet (t), quadruplet (q), multiplet or overlapped (m), doublet of doublets (dd), and broad (br). Low resolution mass spectra (LRMS) were acquired on a Waters Acquity Ultra Performance LC with electrospray ionization and SQ detector (ESI). No MS data is given for compounds 1b, 4, 11, 14, 15 and 17, which did not respond in either negative or positive ionization mode. Thin-layer chromatography (TLC) was performed on E. Merck (Darmstadt, Germany) silica gel 60 F-254 aluminum-backed plates with visualization under UV (254 nm). Flash chromatography was performed using an Isco (Lincoln, NE) Combiflash Companion with RediSep Rf Gold Silica^™ columns and Fisher Optima^™ grade solvents. Microwave reactions were performed in a Biotage® Initiator microwave reaction system. All compounds employed were >98% pure, as determined by ¹H NMR and/or analytical RP-HPLC.

4.2. Chemistry

4.2.1. 2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine 1b

To a solution of 2,6-dichloropurine 1a (2 g, 10.58 mmol) and PTSA (200 mg, 0.11 mmol) in EtOAc (35 mL) at RT was added 3,4-DHP (2.4 mL, 26.45 mmol). The mixture was stirred at 50 °C for 3.5 h. After cooling to RT, saturated aqueous NaHCO₃ (35 mL) was added and layers were separated. The aqueous layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were dried over MgSO₄ and concentrated under vacuum. The oily residue was triturated with Et₂O (20 mL) and subsequent filtration gave compound 1b (2.60 g, 90%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.33 (s, 1H), 5.76 (dd, J = 2.4, 10.8 Hz, 1H), 4.19 (dt, J = 2.1, 11.9 Hz, 1H), 3.78 (td, J = 2.6, 11.7 Hz, 1H), 2.22–2.14 (m, 1H), 2.13–2.06 (m, 1H), 1.97 (qd, J = 3.9, 12.4 Hz, 1H), 1.87–1.71 (m, 2H), 1.71–1.65 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 153.08, 152.29, 151.87, 143.81, 130.91, 82.62, 69.07, 32.17, 24.84, 22.64.

4.2.2. N-(tert-butyl)-2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine 2a

A solution of compound 1b (500 mg, 1.83 mmol) and t-BuNH₂ (1.93 mL, 18.32 mmol) in EtOH (10 mL) was refluxed for 4 h. After cooling to RT, the solvent was stripped under vacuum and saturated aqueous NaHCO₃ (60 mL) was added. The aqueous mixture was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over MgSO₄ and concentrated under vacuum. Purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) yielded compound 2a (483 mg, 85%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.90 (s, 1H), 5.82 (s, 1H), 5.67 (dd, J = 2.2, 10. 8 Hz, 1H), 4.19–4.12 (m, 1H), 3.76 (td, J = 2.3,11.6 Hz, 1H), 2.14–2.06 (m, 1H), 2.03–2.00 (m, 1H), 1.92 (qd, J = 3.7, 12.3 Hz, 1H), 1.82–1.67 (m, 2H), 1.66–1.59 (m, 1H), 1.55 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 154.93, 154.07, 148.98, 137.44, 118.94, 81.57, 68.87, 52.86, 32.35, 29.09, 24.99, 22.85. ESI–MS m/z 310.23, 312.22 [M+H]⁺, 332.07, 334.08 [M+Na]⁺.

4.2.3. N-(tert-butyl)-2-chloro-9H-purin-6-amine 2b

Compound 2a (122 mg, 0.39 mmol) was stirred at RT for 12 h in a MeOH/TFA (3/1) mixture (20 mL). When no more starting material was detected on TLC, the mixture was concentrated under vacuum. The residue was partitioned between a saturated NaHCO₃ aqueous solution and EtOAc. Layers were separated and the aqueous layer was extracted with EtOAc (2–3×). The combined organic layers were washed with brine (20 mL), dried over MgSO₄ and concentrated under vacuum. Compound 2b (77 mg, 87%) was isolated after purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.90 (s, 1H), 6.04 (s, 1H), 1.58 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ 155.07, 153.10, 150.05, 137.99, 118.84, 53.14, 29.11. ESI–MS m/z 226.17, 228.18 [M+H]⁺, 248.14, 250.16 [M+Na]⁺.

4.2.4. 2-Chloro-N-cyclohexyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine 3

Compound 3 was synthesized according to the procedure described for compound 2a using cyclohexylamine (reflux: 2 h). Compound 3 (231 mg, 94%) was isolated as a colorless oil. ¹H NMR (600 MHz, CDCl₃) δ 7.92 (s, 1H), 5.77 (s, 1H), 5.67 (dd, J = 6.0, 9.4 Hz, 1H), 4.17–4.09 (m, 2H), 3.80–3.70 (m, 1H), 2.11–2.00 (m, 4H), 1.97–1.86 (m, 1H), 1.80–1.69 (m, 4H), 1.68–1.58 (m, 2H), 1.53–1.40 (m, 2H), 1.32–1.16 (m, 3H). ¹³C NMR (151 MHz, CDC13) δ 154.87, 154.66, 149.22, 137.73, 118.47, 81.58, 68.94, 49.31, 33.26, 32.38, 25.64, 25.02, 24.88, 22.90. ESI–MS m/z 336.06, 338.20 [M+H]⁺.

4.2.5. 2-Chloro-6-(2-fluoroethoxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine 4

To a solution of fluoroethanol (117 µL, 2.02 mmol) in dry THF (20 mL) at 0 °C under argon was added a 60% dispersion of NaH in mineral oil (88 mg, 2.21 mmol). The mixture was allowed to react for 30 min at 0 °C before a solution of compound lb (500 mg, 1.84 mmol) in dry THF (4 mL) was added. The reaction mixture was stirred at 60 °C for 4 h. After cooling to RT, water (25 mL) and EtOAc (25 mL) were added and layers were separated. The aqueous layer was extracted with EtOAc (2 × 25 mL). The combined organic layers were washed with brine (20 mL), dried over MgSO₄ and concentrated under vacuum to an oily residue. Purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) yielded compound 4 (500 mg, 90%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.15 (s, 1H), 5.74 (dd, J = 2.1, 10.7 Hz, 1H), 4.96–4.75 (m, 4H), 4.20–4.14 (m, 1H), 3.78 (dd, J = 2.4, 11.7 Hz, 1H), 2.17–2.05 (m, 2H), 2.02–1.93 (m, 1H), 1.84–1.71 (m, 2H), 1.69–1.62 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 160.62, 152.94, 152.77, 140.82, 120.35, 82.12, 81.10 (d, ¹J_C–F= 171.5 Hz), 68.97, 66.81 (d, ²J_C–F= 20.6 Hz), 32.22, 24.93, 22.78.

4.2.6. N-(tert-butyl)-2-chloro-8-ethyl-9H-purin-6-amine 6a

This compound was synthesized in two steps starting from 2,6-dichloropyrimidine-4,5-diamine 5 (Axon MedChem BV, Groningen, Netherlands), according to the procedure described by Baenteli R et al. [22]. Briefly, a sealed microwave vial was charged with a solution of 5 (400 mg, 2.25 mmol) in EtC(OEt)₃ (8 mL) and heated at 160 °C for 1 h. After cooling to RT, Et₂O (10 mL) was added and the mixture was placed in an ice bath for 1 h. The intermediate product was filtered off and rinsed with ice-cold Et₂O (10 mL). The solid was added to a sealed microwave vial charged with a mixture of NMP and t-BuNH₂ (10 mL; 1/4 v:v), and the mixture was heated at 160 °C for 2 h. After cooling to RT, the reaction mixture was partitioned between a saturated NaHCO₃ aqueous solution (10 mL) and EtOAc (10 mL). Layers were separated and the aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO₄ and concentrated under vacuum to an oily residue. Purification by flash chromatography (SiO₂, DCM/MeOH, 98/2) yielded compound 6a (250 mg, 44%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 13.13 (bs, 1H), 5.83 (s, 1H), 3.01 (q, J = 7.6 Hz, 2H), 1.57 (s, 9H), 1.44 (t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 154.28, 151.94, 136.58, 130.09, 118.91, 52.95, 29.23, 23.09, 13.06. ESI–MS m/z 254.24, 256.06 [M+H]⁺, 276.05, 278.06 [M+Na]⁺.

4.2.7. N-(tert-butyl)-2-chloro-8-ethyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine 6b

To a solution of compound 6a (240 mg, 0.95 mmol) and PTSA (10 mg, 0.05 mmol) in EtOAc (50 mL) at RT was added 3,4-DHP (170 µL, 1.90 mmol). The mixture was stirred at 60 °C for 6 h. After cooling to RT, saturated aqueous NaHCO₃ (35 mL) was added and layers were separated. The aqueous layer was extracted with EtOAc (2 × 40 mL). The combined organic layers were dried over MgSO₄ and concentrated under vacuum. Purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) yielded compound 6b (215 mg, 67%) as a colorless oil. ¹H NMR (600 MHz, CDCl₃) δ 5.76 (s, 1H), 5.68 (dd, J = 2.5, 11.4 Hz, 1H), 4.18–4.12 (m, 1H), 3.71 (t, J = 10.9 Hz, 1H), 3.06 (dq, J = 7.5, 15.2 Hz, 1H), 2.94 (dq, J = 7.4, 15.1 Hz, 1H), 2.33 (qd, J = 4.3, 12.1 Hz, 1H), 2.04 (dd, J = 2.7, 8.4 Hz, 1H), 1.89–1.82 (m, 1H), 1.77–1.69 (m, 2H), 1.63–1.58 (m, 1H), 1.54 (s, 9H), 1.40 (t, J = 7.6 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 154.18, 153.78, 152.88, 150.25, 117.67, 82.52, 69.12, 52.70, 30.63, 29.15, 25.06, 23.24, 22.77, 12.24. ESI–MS m/z 338.20, 340.22 [M+H]⁺.

4.2.8. 1-(2-Fluoroethyl)-4-nitrobenzene 8

To a solution of 2-(4-nitrophenyl)ethanol 7 (800 mg, 4.79 mmol) in DCM (20 mL) under argon at 0 °C was added DAST (940 µL, 7.18 mmol). The mixture was allowed to warm to RT and stirred under argon for 7 h. The reaction mixture was partitioned between a saturated Na₂CO₃ aqueous solution (60 mL) and EtOAc (120 mL). The organic layer was washed with water (60 mL) and brine (60 mL), dried over MgSO₄, and concentrated under vacuum. Purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) yielded compound 8 (466 mg, 58%) as a solid. ¹H NMR (500 MHz, CDCl₃) δ 8.19 (d, J = 8.6 Hz, 1H), 7.42 (d, J = 8. 6 Hz, 1H), 4.69 (dt, J = 6.1, 46.9 Hz, 1H), 3.12 (dt, J = 6.0, 25.7 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 147.11, 145.26 (d, ³J_C–F = 3.9 Hz), 129.95, 123.88, 83.15 (d, ¹J_C–F= 170.3 Hz), 36.84 (d, ²J_C–F = 20.3 Hz). ESI–MS m/z 192.07 [M+Na]⁺.

4.2.9. 4-(2-Fluoroethyl)aniline 9

Compound 8 (90 mg, 0.53 mmol) was added to a suspension of 10% Pd/C (18 mg) in EtOH (15 mL) and stirred 5 h under hydrogen atmosphere (1 atm) at RT. The mixture was filtered through Celite® and evaporated under reduced pressure to yield compound 9 (72 mg, 97%) as a brown oil which was used without further purification. ¹H NMR (500 MHz, CDCl₃) δ 6.99 (d, J = 8.2 Hz, 1H), 6.60 (d, J = 8.2 Hz, 1H), 4.53 (dt, J = 6.7, 47.2 Hz, 1H), 3.50 (s, 1H), 2.88 (dt, J = 6.7, 22.3 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 145.11, 129.83, 126.78 (d, ³J_C–F = 7.2 Hz), 115.34, 84.56 (d, ¹J_C–F= 167.2 Hz), 36.09 (d, ²J_C–F= 20.3 Hz). ESI–MS m/z 139.90 [M+H]⁺.

4.2.10. 2-(2-Fluoroethoxy)-6-nitrobenzo[d]thiazole 11

To a solution of fluoroethanol (354 µL, 6.11 mmol) in dry THF (20 mL) at 0 °C under argon was added a 60% dispersion of NaH in mineral oil (270 mg, 6.72 mmol). The mixture was allowed to react for 15 min at 0 °C before a solution of compound 10 (1.31 g, 6.11 mmol) in dry THF (5 mL) was added. The reaction mixture was stirred at RT for 2 h before dilution with EtOAc (40 mL). The mixture was washed with water (30 mL), brine (30 mL), dried over MgSO₄, and concentrated under vacuum to a beige solid. Stirring in MeOH for 30 min at RT and filtration afforded compound 11 (1.33 mg, 90%) as a white solid. ¹H NMR (500 MHz, DMS0-d₆) δ 8.97 (d, J = 2.3 Hz, 1H), 8.25 (dd, J = 2.4, 8.9 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 4.99–4.71 (m, 5H). ¹³C NMR (126 MHz, DMSO-d₆) δ 176.72, 153.59, 143.28, 132.19, 121.95, 120.72, 119.19, 81.36 (d, ¹J_C–F= 167.0 Hz), 71.84 (d, ²J_C–F = 18.7 Hz).

4.2.11. 2-(2-Fluoroethoxy)benzo[d]thiazol-6-amine 12

To a solution of compound 11 (1.33 g, 5.49 mmol) in a mixture of EtOH (25 mL), H₂O (4 mL) and AcOH (2.5 mL) at RT was added Fe powder (1.50 g). The reaction mixture was refluxed for 1 h. After cooling to RT the mixture was filtered through Celite® and concentrated under vacuum. The residue was partitioned between a saturated NaHCO₃ aqueous solution (50 mL) and EtOAc (70 mL). Layers were separated and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over MgSO₄, and concentrated under vacuum to a brown solid. Purification by column chromatography (SiO₂, Hexane/EtOAc, 3/1) yielded compound 12 (930 mg, 80%) as an off-white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.45 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 2. 3 Hz, 1H), 6.73 (dd, J = 2.4, 8.5 Hz, 1H), 4.91–4.68 (m, 4H), 3.68 (s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 169.72, 143.25, 141.95, 133.50, 121.45, 114.88, 106.84, 81.39 (d, ¹J_C–F= 171.0 Hz), 70.04 (d, ²J_C–F = 20.2 Hz). ESI–MS m/z 213.08 [M+H]⁺, 235.08 [M+Na]⁺.

4.2.12. 2-(2-Fluoroethoxy)-6-nitroquinoline 14

To a solution of fluoroethanol (278 µL, 4.80 mmol) in dry DMF (30 mL) at 0 °C under argon was added a 60% dispersion of NaH in mineral oil (212 mg, 5.28 mmol). The mixture was allowed to react for 30 min at 0 °C before a solution of compound 13 (500 mg, 2.40 mmol) in dry DMF (10 mL) was added. The reaction mixture was stirred at RT for 12 h before water (100 mL) was added. The aqueous mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (60 mL), dried over MgSO₄, and concentrated under vacuum to a brown solid. Stirring in EtOH at 50 °C and filtration of the warm suspension afforded compound 14 (391 mg, 69%) as a pure beige solid. ¹H NMR (500 MHz, CDCl₃) δ 8.69 (d, J = 2.5 Hz, 1H), 8.41 (dd, J = 2.5, 9.2 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 9.2 Hz, 1H), 7.12 (d, J = 8.8 Hz, 1H), 4.95–4.71 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 163.97, 149.71, 144.11, 140.29, 128.71, 124.26, 124.10, 123.59, 115.57, 81.78 (d, ¹J_C–F= 169.8 Hz), 65.67 (d, ²J_C–F = 20.0 Hz).

4.2.13. 4-(2-((6-Nitroquinolin-2-yl)oxy)ethyl)morpholine 15

Compound 15 was synthesized according to the procedure described for compound 14 using 2-morpholinoethanol. Purification of the crude compound by stirring in EtOAc at 50 °C and filtration of the warm suspension afforded compound 15 (136 mg, 31%) as a pure orange solid. ¹H NMR (500 MHz, CDCl₃) δ 8.67 (d, J = 2.6 Hz, 1H), 8.40 (dd, J = 2.6, 9.0 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 9.3 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H), 4.67 (t, J = 5.8 Hz, 2H), 3.74 (t, J = 4.5 Hz, 4H), 2.87 (t, J = 5.8 Hz, 2H), 2.61 (t, J = 4.5 Hz, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 164.39, 149.94, 143.93, 139.95, 128.62, 124.23, 123.93, 123.49, 115.76, 67.07, 63.98, 57.53, 54.16.

4.2.14. 2-(2-Fluoroethoxy)quinolin-6-amine 16

Compound 14 (334 mg, 1.41 mmol) was added to a suspension of 10% Pd/C (72 mg) in THF (25 mL) and stirred 20 h under hydrogen atmosphere (1 atm) at RT. The mixture was filtered through Celite® and concentrated under vacuum. Purification of the residue by gradient flash chromatography (SiO₂, Hexane/EtOAc) yielded compound 16 (246 mg, 86%) as an orange solid. ¹H NMR (500 MHz, CDCl₃) δ 7.79 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.07 (dd, J = 2.2, 8.8 Hz, 1H), 6.94–6.82 (m, 2H), 4.80 (dt, J = 3.8,47.6 Hz, 2H), 4.69 (dt, J = 4.2, 29.2 Hz, 2H), 3.78 (s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 159.68, 142.94, 140.73, 137.43, 128.32, 126.42, 121.06, 113.33, 109.16, 82.35 (d, ¹J_C–F= 168.8 Hz), 64.69 (d, ²J_C–F= 20.0 Hz). ESI–MS m/z 205.01 [M–H]⁻, 207.15 [M+H]⁺, 229.16 [M+Na]⁺.

4.2.15. 2-(2-Morpholinoethoxy)quinolin-6-amine 17

Compound 17 was synthesized according to the procedure described for compound 16 using a DCM/EtOH (1:1) mixture as solvent. Compound 17 (81 mg, 67%) was isolated as a brown solid. ¹H NMR (500 MHz, CDCl₃) δ 7.75 (d, J = 8.8 Hz, 1H), 7.63 (d, J= 8.8 Hz, 1H), 7.06 (dd, J = 2.7, 8.9 Hz, 1H), 6.87 (d, J = 2.6 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 4.57 (t, J = 5.8 Hz, 2H), 3.79 (bs, 1H), 3.73 (t, J = 4.6 Hz, 4H), 2.83 (t, J = 5.8 Hz, 2H), 2.59 (t, J = 4.6 Hz, 4H). ¹³C NMR (126 MHz, CDCL₃) δ 160.07, 142.79, 140.87, 137.12, 128.25, 126.23, 120.95, 113.45, 109.12, 67.06, 62.86, 57.78, 54.13.

4.3. General synthetic procedures for the synthesis of final derivatives

Two methods were employed for the synthesis of final derivatives. In cases where a compound was obtained via both methods, only the higher yielding one was reported. Compounds 19b, 21b–25b and QAP1 were obtained according to method A via THP protected intermediates 19a, 21a–25a and 26, respectively. Compounds 18 and 20 were obtained according to method B.

4.3.1. Method A (two-step procedure)

A microwave vial was charged under argon with a solution of the appropriate 2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H–purine derivative (2a, 3, 4 or 6b, 1 eq.) in dry degassed dioxane. To this solution were added Cs₂CO₃ (5 eq.), BINAP (0.2 eq.), Pd(OAc)₂ (0.1 eq.) and the appropriate arylamine (1.2 eq.). The mixture was heated under microwave irradiation for 45 min at 160 °C. After cooling to RT, water and EtOAc were added and layers were separated. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over MgSO₄ and concentrated under vacuum. Pure ⁹N-THP protected derivatives were isolated after purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) in moderate to good yields.

For the removal of the THP group, ⁹N-THP protected compounds were stirred at RT for 1–3 h in a MeOH/TFA(3/l) mixture. When no more starting material was detected on TLC, the mixture was concentrated under vacuum. The residue was partitioned between a saturated NaHCO₃ aqueous solution and EtOAc. Layers were separated and the aqueous layer was extracted with EtOAc (2–3 ×). The combined organic layers were washed with brine (20 mL), dried over MgSO₄ and concentrated under vacuum. Pure compounds were isolated after purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) in moderate to good yields.

4.3.2. Method B (one-step procedure)

To a solution of purine derivative 2b or 6a (1 eq.) in n-butanol was added the appropriate arylamine (1.1 eq.) and a catalytic amount of concentrated 37% HC1 (0.1 eq.). The mixture was heated at 117 °C until no more starting material was detected on TLC (typically 12–15 h). After cooling to RT, water and EtOAc were added and layers were separated. The aqueous layer was extracted with EtOAc (3×). The combined organic layers were washed with brine, dried over MgSO₄ and concentrated under vacuum. Pure compounds were isolated after purification by gradient flash chromatography (SiO₂, Hexane/EtOAc) in moderate yields.

4.3.3. N⁶-(tert-butyl)-N²-(4-(2-fluoroethyl)phenyl)-9H-purine-2,6-diamine 18

Starting from 9 and 2b. Yield 73%. ¹H NMR (600 MHz, CDCl₃) δ 13.10 (s, 1H), 7.48 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 6.87 (s, 1H), 6.56 (s, 1H), 5.65 (s, 1H), 4.62 (dt, J = 6.5, 47.0 Hz, 2H), 2.97 (dt, J = 6.5,23.3 Hz, 2H), 1.52 (s, 9H). ¹³C NMR(151 MHz, CDCl₃) δ 156.17, 155.03, 149.78, 138.53, 135.68, 132.00 (d, ³J_C–F= 6.2 Hz), 129.87, 121.88, 115.50, 84.29 (d, ¹J_C–F= 169.0 Hz), 52.15, 36.38 (d, ²J_C–F= 20.3 Hz), 29.28. ESI–MS m/z 329.25 [M+H]⁺, 351.23 [M+Na]⁺, 327.31 [M−H]⁻.

4.3.4. N⁶-(tert-butyl)-N²-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 19a

Starting from 12 and 2a. Yield 84%. ¹H NMR (500 MHz, CDC1₃) δ 8.38 (s, 1H), 7.70 (s, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 6.9 Hz, 1H), 6.93 (s, 1H), 5.62–5.50 (m, 2H), 4.96–4.70 (m, 4H), 4.17 (d, J = 10.4 Hz, 1H), 3.76 (t, J = 10.3 Hz, 1H), 2.15–2.04 (m, 3H), 1.88–1.60 (m, 3H), 1.53 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.92, 156.29, 154.81, 149.48, 143.76, 137.18, 135.23, 132.74, 120.65, 118.55, 115.88, 111.75, 81.93, 81.36 (d, ¹J_C–F= 171.5 Hz), 70.17 (d, ²J_C–F= 20.2 Hz), 68.76, 52.09, 31.48, 29.33, 25.18, 23.13. ESI–MS m/z 486.13 [M+H]⁺, 484.11 [M−H]⁻.

4.3.5. N⁶-(tert-butyl)-N²-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-9H-purine-2,6-diamine 19b

Yield 83%. ¹H NMR(500 MHz, CDC13) δ 12.02 (s, 1H), 8.09 (s, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 6.89 (s, 1H), 6.80 (s, 1H), 5.58 (s, 1H), 4.92–4.68 (m, 4H), 1.53 (s, 9H). ¹³C NMR(126 MHz, CDCl₃) δ 171.48, 156.37, 155.01, 149.88, 144.79, 136.34, 135.18, 133.02, 121.16, 120.17, 115.53, 113.57, 81.32 (d, ¹J_C–F= 171.4 Hz), 70.30 (d, ²J_C–F= 20.4 Hz), 52.21, 29.32. ESI–MS m/z 402.02 [M+H]⁺.

4.3.6. N⁶-(tert-butyl)-8-ethyl-N²-(2-(2-fluoroethoxy)benzo[d] thiazol-6-yl)-9H-purine-2,6-diamine 20

Starting from 12 and 6a. Yield 50%. ¹H NMR (500 MHz, CDCl₃) δ 10.46 (s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.32 (dd, J = 2.2, 8.7 Hz, 1H), 6.9 (s, 1H), 5.52 (s, 1H), 4.98–4.61 (m, 4H), 2.59 (q, J = 7.6 Hz, 2H), 1.54 (s, 9H), 1.25 (t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 171.10, 155.66, 154.22, 150.83, 150.29, 143.98, 136.96, 132.91, 120.87, 118.65, 115.12, 111.83, 81.35 (d, ¹J_C–F = 171.5 Hz), 70.19 (d, ²J_C–F= 20.4 Hz), 52.13, 29.40, 22.64, 12.49. ESI–MS m/z 428.12 [M+H]⁺, 430.15 [M−H]⁻.

4.3.7. N⁶-cyclohexyl-N²-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 21a

Starting from 12 and 3. Yield 100%. ¹H NMR (500 MHz, CDCl₃) δ 8.48 (s, 1H), 7.73 (s, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.28 (dd, J = 2.0, 8.7 Hz, 1H), 7.06 (s, 1H), 5.61–5.49 (m, 2H), 4.94–4.66 (m, 4H), 4.16 (d, J = 14.92 Hz, 1H), 3.75 (td, J = 2.23, 11.51 Hz, 1H), 2.21–2.03 (m, 5H), 1.90–1.59 (m, 7H), 1.45 (q, J = 12.50 Hz, 2H), 1.35–1.21 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.66, 156.45, 154.20, 143.41, 137.24, 135.39, 132.73, 120.56, 117.82, 110.75, 81.70, 81.39 (d, ¹J_C–F = 171.4 Hz), 70.02 (d, ²J_C–F= 20.3 Hz), 68.65, 53.43, 33.36, 31.39, 25.71, 25.23, 25.03, 23.01. ESI–MS m/z 510.4 [M−H]⁻.

4.3.8. N⁶-cyclohexyl-N²-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-9H-purine-2,6-diamine 21b

Yield 50%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.40 (s, 1H), 9.05 (s, 1H), 8.63 (s, 1H), 7.83 (s, 1H), 7.60 (dd, J = 2.2, 8.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.25 (s, 1H), 4.88–4.72 (m, 4H), 4.08 (s, 1H), 2.00–1.92 (m, 2H), 1.85–1.75 (m, 2H), 1.67 (d, J = 13.0 Hz, 1H), 1.44–1.30 (m, 4H), 1.24–1.13 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.21, 156.05, 153.54, 150.90, 141.91, 138.65, 136.15, 131.53, 120.19, 117.68, 113.93, 109.49, 81.54 (d, ¹J_C–F= 165.4 Hz), 70.55 (d, ²J_C–F = 18.8 Hz), 64.90, 32.51, 25.30, 15.14. ESI–MS m/z 428.06 [M+H]⁺, 426.16 [M−H]⁻.

4.3.9. N⁶-(tert-butyl)-N²-(2-(2-fluoroethoxy)quinolin-6-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6 diamine 22a

Starting from 16 and 2a. Yield 76%. ¹H NMR (500 MHz, CDCl₃) δ 8.34 (d, J = 2.4 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.74 (m, 2H), 7.67 (dd, J = 2.4, 9.0 Hz, 1H), 7.04 (s, 1H), 6.94 (d, J = 8.8 Hz, 1H), 5.64–5.56 (m, 2H), 4.92–4.66 (m, 4H), 4.18 (d, J = 9.9 Hz, 1H), 3.78 (t, J = 11.5 Hz, 1H), 2.12–2.08 (m, 3H), 1.85–1.61 (m, 3H), 1.57 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 160.43, 156.27, 154.83, 149.48, 142.16, 138.38, 137.03, 135.23, 127.41, 125.85, 123.75, 115.89, 114.53, 113.26, 82.17 (d, ¹J_C–F= 169.3 Hz), 81.90, 68.79, 64.80 (d, ²J_C–F= 20.2 Hz), 52.09, 31.56, 29.38, 25.17, 23.15. ESI–MS m/z 480.4 [M+H]⁺.

4.3.10. N⁶-(tert-butyl)-N²-(2-(2-fluoroethoxy)quinolin-6-yl)-9H-purine-2,6-diamine 22b

Yield 73%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.47 (bs, 1H), 9.08 (s, 1H), 8.46 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.93 (dd, J = 1.9, 9.0 Hz, 1H), 7.82 (s, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 6.35 (s, 1H), 4.81 (dt, J = 3.8 Hz, ¹J_H–F = 47.9 Hz, 2H), 4.62 (dt, J = 4.0 Hz, ³J_H–F = 30.5 Hz, 2H), 1.56 (s, 9H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.54, 155.68, 154.15, 150.33, 140.72, 138.41, 138.05, 136.17, 126.67, 126.47, 125.23, 123.81, 114.91, 114.70, 113.21, 112.68, 82.07 (d, ¹J_C–F= 165.7 Hz), 64.60 (d, ²J_C–F= 18.8 Hz), 51.12, 29.00. ESI–MS m/z 396.15 [M+H]⁺, 394.13 [M−H]⁻.

4.3.11. N⁶-(tert-butyl)-8-ethyl-N²-(2-(2-fluoroethoxy)quinolin-6-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 23a

Starting from 16 and 6b. Yield 26%. ¹H NMR (500 MHz, CDC1₃) δ 8.34 (d, J = 2.2 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.68 (dd, J = 2.3, 8.9 Hz, 1H), 6.99–6.88 (m, 2H), 5.58 (s, 1H), 4.93–4.65 (m, 4H), 4.21 (d, J = 11.8 Hz, 1H), 3.71 (t, J = 11.7 Hz, 1H), 3.01–2.86 (m, 2H), 2.81 (qd, J = 3.9, 12.8 Hz, 1H), 2.12 (d, J = 12.5 Hz, 1H), 1.89 (d, J = 13.1 Hz, 1H), 1.85–1.62 (m, 4H), 1.57 (s, 9H), 1.39 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 160.33, 155.36, 154.18, 151.01, 150.75, 141.98, 138.30, 137.35, 127.34, 125.91, 123.63, 114.66, 114.06, 113.22, 83.19, 82.34 (d, ¹J_C–F= 168.8 Hz), 69.20, 64.79 (d, ²J_C–F= 20.0 Hz), 52.00, 30.07, 29.46, 25.33, 23.78, 22.38, 12.58. ESI–MS m/z 507.8 [M+H]⁺.

4.3.12. N⁶-(tert-butyl)-8-ethyl-N²-(2-(2-fluoroethoxy)quinolin-6-yl)-9H-purine-2,6-diamine 23b

Yield 80%. ¹H NMR (600 MHz, CDCl₃) δ 10.34 (bs, 1H), 8.25 (d, J = 2.4 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.66 (dd, J = 2.4, 9.0 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 8.8 Hz, 1H), 5.58 (s, 1H), 4.82 (dt, J = 4.2 Hz, ²J_C–F = 47.6 Hz, 2H), 4.72 (dt, J = 4.12 Hz, ³J_C–F = 29.26 Hz, 2H), 2.61 (q, J = 7.6 Hz, 2H), 1.58 (s, 9H), 1.26 (t, J = 7.6 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 160.50, 155.63, 154.16, 150.78, 150.27, 142.21, 139.70, 138.33, 136.80, 127.63, 125.86, 123.77, 114.67, 113.39, 82.31 (d, ¹J_C–F= 169.2 Hz), 64.82 (d, ²J_C–F= 20.0 Hz), 52.12, 29.42, 22.69, 14.35. ESI–MS m/z 424.27 [M+H]⁺, 422.18 [M–H]⁻.

4.3.13. N⁶-(tert-butyiyl)-N²-(6-fluoropyridin-2-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 24a

Yield 40%. Starting from 2-amino-6-fluoropyridine (Sigma) and 2a. ¹H NMR (500 MHz, CDC1₃) δ 8.33 (dd, J = 2.2, 8.1 Hz, 1H), 7.82–7.69 (m, 2H), 7.59 (s, 1H), 6.47 (dd, J = 2.4, 7.8 Hz, 1H), 5.69–5.52 (m, 2H), 4.17 (m J = 11.5 Hz, 1H), 3.78 (td, J = 2.4, 11.6 Hz, 1H), 2.18–2.06 (m, 3H), 1.89–1.62 (m, 3H), 1.54 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 162.28 (d, ¹J_C–F= 237.9 Hz), 154.78, 154.48, 152.22 (d, ³J_C–F = 15.0 Hz), 148.98, 142.42 (d, ³J_C–F = 8.0 Hz), 135.78, 116.40, 108.20, 100.54 (d, ²J_C–F = 35.6 Hz), 81.98, 68.77, 52.25, 31.64, 29.26, 25.15, 23.09. ESI–MS m/z 386.2 [M+H]⁺.

4.3.14. N⁶-(tert-butyl)-N²-(6-fluoropyridin-2-yl)-9H-purine-2,6-diamine 24b

Yield 61%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.62 (s, 1H), 9.35 (s, 1H), 8.25 (d, J = 7.4 Hz, 1H), 7.88 (s, 1H), 7.85 (q, J = 8.2 Hz, 1H), 6.58 (d, J = 7.5 Hz, 1H), 6.50 (s, 1H), 1.51 (s, 9H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.54 (d, ¹J_C–F= 233.6 Hz), 154.20, 153.99, 152.68 (d, ³J_C–F = 15.9 Hz), 149.89, 142.64 (d, ³J_C–F = 7.9 Hz), 136.87, 115.34, 108.52, 99.50 (d, ²J_C–F = 36.2 Hz), 51.32, 28.99. ESI–MS m/z 302.07 [M+H]⁺, 324.06 [M+Na]⁺, 300.12 [M–H]⁻.

4.3.15. N-(6-(2-fluoroethoxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-2-yl)benzo[d]thiazol-6-amine 25a

Starting from 6-aminobenzothiazole and 4. Yield 46%. ¹H NMR (500 MHz, CDCl₃) δ 8.87 (s, 1H), 8.64 (d, J = 2.1 Hz, 1H), 8.06 (d, J= 8.8 Hz, 1H), 7.91 (s, 1H), 7.51 (dd, J = 2.2, 8.8 Hz, 1H), 7.22 (s, 1H), 5.62 (dd, J = 2.3, 10.5 Hz, 1H), 4.98–4.75 (m, 4H), 4.20 (dd, J = 3.0, 10.7 Hz, 1H), 3.79 (td, J = 2.5, 11.6 Hz, 1H), 2.29–2.02 (m, 3H), 1.87–1.60 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 160.60, 155.37, 153.20, 152.01, 148.81, 138.33, 138.04, 135.03, 123.54, 119.00, 116.56, 110.65, 82.41, 81.47 (d, J = 170.9 Hz), 68.84, 65.83 (d, J = 21.6 Hz), 31.34, 25.13, 23.07. ESI–MS m/z 437.2 [M+Na]⁺.

4.3.16. N-(6-(2-fluoroethoxy)-9H-purin-2-yl)benzo[d]thiazol-6-amine 25b

Yield 53%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.92 (s, 1H), 9.68 (s, 1H), 9.17 (s, 1H), 8.77 (s, 1H), 7.97 (d, J = 8.83 Hz, 1H), 7.79 (dd, J = 2.15, 8.88 Hz, 1H), 4.93–4.76 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.48, 155.06, 154.33, 152.80, 147.61, 139.34, 139.03, 134.31, 122.60, 118.63, 114.81, 109.6, 81.91 (d, ¹J_C–F= 166.2 Hz), 65.38 (d, ²J_C–F = 19.2 Hz). ESI–MS m/z 331.06 [M+H]⁺, 353.05 [M+Na]⁺, 328.99 [M–H]⁻.

4.3.17. N⁶-(tert-butyl)-8-ethyl-N²-(2-(2-morpholinoethoxy) quinolin-6-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 26

Starting from 17 and 6b. Yield 43%. ¹H NMR (500 MHz, CDCl₃) δ 8.29 (s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.77–7.67 (m, 2H), 6.88 (d, J = 8.8 Hz, 1H), 5.75 (s, 1H), 5.54 (d, J = 11.0 Hz, 1H), 4.82–4.60 (m, 2H), 4.21 (d, J = 10.4 Hz, 1H), 3.87–3.79 (m, 4H), 3.71 (t, J = 11.7 Hz, 1H), 3.18–3.06 (m, 2H), 3.04–2.80 (m, 6H), 2.78–2.64 (m, 2H), 2.11 (d, J = 11.5 Hz, 1H), 1.89 (d, J = 13.0 Hz, 1H), 1.84–1.70 (m, 2H), 1.66 (d, J = 12.3 Hz, 1H), 1.57 (s, 9H), 1.40 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 160.25, 151.05, 148.15, 144.25, 142.20, 138.33, 137.01, 133.84, 131.44, 127.37, 125.82, 123.87, 114.49, 113.14, 83.27, 69.25, 65.96, 61.70, 57.21, 53.45, 52.25, 30.15, 29.38, 25.23, 23.65, 22.30, 12.44. ESI–MS m/z 575.45 [M+H]⁺, 597.42 [M+Na]⁺.

4.3.18. N⁶-(tert-butyl)-8-ethyl-N²-(2-(2-morpholinoethoxy) quinolin-6-yl)-9H-purine-2,6-diamine QAP1

Yield 82%. ¹H NMR(600 MHz, CDCl₃) δ 11.07 (bs, 1H), 8.22 (s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.65 (dd, J = 2.4, 9.0 Hz, 1H), 7.02 (bs, 1H), 6.84 (d, J = 8.8 Hz, 1H), 5.63 (bs, 1H), 4.60 (t, J = 5.8 Hz, 2H), 3.76 (t, J = 4.7 Hz, 4H), 2.85 (t, J = 5.8 Hz, 2H), 2.66–2.58 (m, 4H), 2.48 (q, J = 7.7 Hz, 2H), 1.57 (s, 9H), 1.18 (t, J = 7.6 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 160.96, 155.44, 154.16, 150.76, 150.56, 142.44, 137.97, 136.52, 127.65, 125.67, 123.64, 115.06, 114.72, 113.63, 67.07, 62.95, 57.77, 54.13, 52.15, 29.41, 22.57, 12.42. ESI–MS m/z 491.35 [M+H]⁺, 489.38 [M–H]⁻.

4.4. Synthetic procedures for radiolabeling precursors

4.4.1. 2-(4-((6-(tert-butylamino)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-2-yl)amino)phenyl)ethanol 28

Starting from 4-aminophenethyl alcohol 27 (74 mg) and 2a (140 mg), compound 28 (155 mg, 83%) was synthesized according to the general procedure described for compounds 19a-26 (method A) and was isolated as an off-white solid. ¹H NMR (500 MHz, CDC1₃) δ 7.66 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 6.85 (s, 1H), 5.69 (s, 1H), 5.59–5.50 (m, 1H), 4.23–4.08 (m, 1H), 3.85 (t, J = 6.8 Hz, 2H), 3.75 (td, J = 2.6, 11.6 Hz, 1H), 2.84 (t, J = 6.5 Hz, 2H), 2.04 (m, 2H), 1.96 (s, 1H), 1.86 (s, 1H), 1.78–1.59 (m, 3H), 1.54 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 156.36, 154.77, 149.42, 139.29, 134.97, 131.39, 129.36, 119.27, 115.57, 81.67, 68.72, 63.87, 52.02, 38.76, 31.65, 29.34, 25.14, 23.10. ESI–MS m/z 410.9 [M+H]⁺.

4.4.2. 4-((6-(tert-butylamino)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-2-yl)amino)phenethyl 4-methylbenzenesulfonate 29

To a solution of compound 28 (50 mg, 0.12 mmol) and Et₃N (75 µL, 0.54 mmol) in dry DCM (10 mL) at 0 °C under argon was added TsCl (116 mg, 0.60 mmol). The mixture was allowed to warm up and stirred at RT for 72 h before water (15 mL) was added. The layers were separated and the aqueous layer was extracted with DCM (2 × 10 mL). The combined organic layers were dried over MgS04 and concentrated under vacuum to a yellow oil. Purification by gradient flash chromatography (SiO₂, DCM/MeOH, 98/2) yielded compound 29 (46 mg, 68%) as an off-white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.71 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 8.2 Hz, 2H), 6.83 (s, 1H), 5.59 (s, 1H), 5.56 (dd, J = 3.7, 8.9 Hz, 1H), 4.19 (t, J = 7.2 Hz, 2H), 4.18–4.14 (m, 1H), 3.8 (td, J = 2.4,11.6 Hz, 1H), 2.92 (t, J = 7.2 Hz, 2H), 2.41 (s, 3H), 2.10–2.05 (m, 2H), 1.80–1.72 (m, 3H), 1.67–1.63 (m, 1H), 1.54 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ 156.19, 154.75, 149.38, 144.72, 139.66, 135.08, 133.10, 129.89, 129.27, 128.87, 127.95, 118.98, 115.65, 81.65, 71.12, 68.76, 52.04, 34.88, 31.64, 29.33, 25.13, 23.10, 21.76. ESI–MS m/z 587.2 [M+Na]⁺.

4.4.3. 2-(2-((triisopropylsilyl)oxy)ethoxy)benzo[d]thiazol-6-amine 31

To a solution of 2-((triisopropylsilyl)oxy)ethanol (303 mg, 1.39 mmol) in a mixture of dry THF (1 mL) and dry DMF (128 µL) at 0 °C under argon was added a 60% dispersion of NaH in mineral oil (67 mg, 2.77 mmol). The mixture was allowed to react for 1 h at 0 °C before a solution of compound 30 (150 mg, 0.82 mmol) in a mixture of dry THF (1 mL) and dry DMF (128 µL) was added. The reaction mixture allowed to reach RT and refluxed for 48 h before water (20 mL) was added. The aqueous layer was extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with brine (15 mL), dried over MgSO₄, and concentrated under vacuum to a brown oil. Purification by column chromatography (SiO₂, Hexane/EtOAc, 9/1) yielded compound 31 (169 mg, 56%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 7.44 (d, J = 8.5 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.70 (dd, J = 2.4, 8.5 Hz, 1H), 4.58 (d, J = 4.9 Hz, 2H), 4.06 (d, J = 4.9 Hz, 2H), 3.58 (s, 2H), 1.24–0.96 (m, 21H). ¹³C NMR (126 MHz, CDCl₃) δ 170.25, 142.99, 142.23, 133.34, 121.25, 114.71, 106.80, 72.80, 61.74, 18.04, 12.11. ESI–MS m/z 367.15 [M+H]⁺, 389.30 [M+Na]⁺.

4.4.4. N⁶-(tert-butyl)-9-(tetrahydro-2H-pyran-2-yl)-N²-(2-(2-((triisopropylsilyl)oxy)ethoxy)benzo[d]thiazol-6-yl)-9H-purine-2,6-diamine 32

Starting from compounds 31 (189 mg) and 2a (133 mg), compound 32 (187 mg, 68%) was synthesized according to the general procedure described for compounds 19a–26 (Method A) and was isolated as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.34 (d, J = 2.3 Hz, 1H), 7.71 (s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.31 (dd, J = 2.3, 8.7 Hz, 1H), 7.07 (s, 1H), 5.60 (s, 1H), 5.54 (dd, J = 2.5, 10.3 Hz, 1H), 4.63 (t, J = 5.1 Hz, 2H), 4.19–4.12 (m, 1H), 4.09 (t, J = 5.1 Hz, 2H), 3.74 (td, J = 2.5, 11.3 Hz, 1H), 2.19–2.00 (m, 3H), 1.79–1.70 (m, 2H), 1.66–1.61 (m, 1H), 1.52 (s, 9H), 1.08 (m, 21H). ¹³C NMR (126 MHz, CDCl₃) δ 171.50, 156.31, 154.75, 149.41, 144.09, 136.87, 135.12, 132.55, 120.47, 118.47, 115.75, 111.78, 81.86, 72.96, 68.70, 61.75, 52.04, 31.47, 29.28, 25.14, 23.08, 18.04, 12.11. ESI–MS m/z 638.42 [M–H]⁻, 640.50 [M+H]⁺.

4.4.5. 2-((6-((6-(tert-Butylamino)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-2-yl)amino)benzo[d]thiazol-2-yl)oxy)ethanol 33

To a solution of compound 32 (187 mg, 0.29 mmol) in THF (7 mL) at RT was added TBAF (880 µL, 1 M solution in THF). After stirring for 1 h at RT, the mixture was concentrated under vacuum and the crude residue was directly purified by gradient flash chromatography (SiO₂, Hexane/EtOAc) to yield compound 33 (127 mg, 91%) as a white solid. ¹H NMR (500 MHz, CDC1₃) δ 8.33 (d, J = 2.3 Hz, 1H), 7.71 (s, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.32 (dd, J = 2.3, 8.7 Hz, 1H), 7.00 (s, 1H), 5.70 (s, 1H), 5.54 (dd, J = 2.4, 10.3 Hz, 1H), 4.7 (t, J = 4.1 Hz, 2H), 4.18–4.13 (m, 1H), 4.03 (t, J = 4.2 Hz, 2H), 3.91 (s, 1H), 3.75 (td, J = 2.6, 11.4 Hz, 1H), 2.16–2.00 (m, 3H), 1.79–1.68 (m, 2H), 1.67–1.60 (m, 1H), 1.53 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 171.83, 156.28, 154.73, 149.39, 143.48, 137.14, 135.13, 132.52, 120.54, 118.61, 115.70, 111.71, 81.86, 73.95, 68.72, 61.60, 52.05, 31.45, 29.27, 25.14, 23.09. ESI–MS m/z 484.28 [M+H]⁺.

4.4.6. 2-((6-((6-(tert-Butylamino)-93-(tetrahydro-2H-pyran-2-yl)-9H-purin-2-yl)amino)benzo[d]thiazol-2-yl)oxy)ethyl 4-methylbenzenesulfonate 34

To a solution of compound 33 (73 mg, 0.15 mmol) in dry pyridine (3 mL) at 0 °C under argon was added TsCl (43 mg, 0.23 mmol). The mixture was allowed to reach RT and stirred for 12 h under argon. The reaction mixture was neutralized with a 2% aqueous HC1 solution and extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with brine (15 mL), dried over MgSO₄, and concentrated under vacuum. Purification by gradient flash chromatography (SiO₂, Hexane/EtOAc, 9/1) yielded compound 34 (65 mg, 68%) as a solid. ¹H NMR (500 MHz, CDCl₃) δ 8.36 (d, J = 2.2 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.71 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.32–7.25 (m, 3H), 6.98 (s, 1H), 5.59 (s, 1H), 5.55 (dd, J = 2.3, 10.4 Hz, 1H), 4.69 (t, J = 4.5 Hz, 2H), 4.45 (t, J = 4.4 Hz, 2H), 4.19–4.14 (m, 1H), 3.76 (td, J = 4.9, 8.8, 9.7 Hz, 1H), 2.38 (s, 3H), 2.19–2.05 (m, 3H), 1.79–1.71 (m, 2H), 1.68–1.63 (m, 1H), 1.54 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 156.22, 154.77, 149.43, 145.13, 143.51, 137.21, 135.23, 132.80, 132.67, 130.06, 129.97, 128.10, 120.64, 118.47, 115.85, 111.55, 81.90, 68.74, 68.30, 67.52, 52.06, 31.45, 29.31, 25.16, 23.12, 21.74. ESI–MS m/z 636.22 [M–H]⁻, 638.19 [M+H]⁺, 660.19 [M+Na]⁺.

4.5. Topoisomerase IIα inhibition assays

Topoisomerase inhibition assay were performed using a eukaryotic topoisomerase IIα drug screening kit purchased from TopoGen (Port Orange, FL). All complementary materials (including topoisomerase inhibitor etoposide) were also purchased from TopoGen, and all experiments were performed according to the manufacturer’s instructions and specifications. In a representative inhibition experiment, 14 µL of H₂O, 4 µL of topoisomerase reaction buffer, 1 µL of supercoiled DNA substrate, and 1 µL of eukaryotic topoisomerase-IIα (∼2 units of activity, as described by the manufacturer) were combined in a 1.7 mL microcentrifuge tube. In experiments involving added drug, 2 µL of DMSO stock solution (10% v/v) of compound were added, and the initial volume of water was adjusted to allow for a final reaction volume of 20 µL The reaction mixture was vortexed gently, centrifuged briefly, and incubated at 37 °C for 30 min. After 30 min, 2 µL of 10% SDS was added to quench the reaction, 2 µL of proteinase K was added to digest remaining proteins, and the reaction mixture was again incubated at 37 °C for 15 min. After this incubation, 2.5 µL of 10× loading dye was added to the reaction mixture, and the tube was subsequently vortexed lightly and centrifuged. The sample was then loaded onto a 1% agarose gel and electrophoresed for 5 h at 5 V/cm. Gels were stained using incubation in a 1 × TAE solution containing 5 µg/mL ethidium bromide. Gels were visualized with a BioRad chemiluminescence detector, and the data were processed with Quantity One software (Bio-Rad, version 4.3.0).

4.6. Cell culture

Human breast cancer cell lines SK-BR-3 and MCF-7 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA) and maintained by weekly serial passage in a 5% CO₂(g) atmosphere at 37 °C. Cells were harvested using a formulation of 0.25% trypsin and 0.53 mM EDTA in Hank’s buffered salt solution (HBSS) without calcium or magnesium. SK-BR-3 cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle medium: F-12 medium, supplemented with 10% fetal calf serum, 2 mM l-glutamine, nonessential amino acids, and 100 units/mL penicillin and streptomycin. MCF-7 cells were grown in minimum essential medium, supplemented with 10% fetal calf serum, 0.01 mg/mL bovine insulin (Sigma Aldrich, St. Louis, MO), non-essential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and 100 units/mL penicillin and streptomycin.

4.7. Cell proliferation assay

The cellular proliferation of MCF-7 and SK-BR-3 cells upon treatment drugs was quantified using an MTT assay obtained from ATCC. For the assay, MCF-7 (50 000 cells/well) and SK-BR-3 (20 000 cells/well) were seeded in 96-well plates (Costar, 3596) overnight (18–24 h). Then, after overnight incubation, the old media was aspirated and discarded, and new media mixed (via 1:20 dilution) with different drug concentrations were added to each well in 100 µL aliquots. The drug-treated plates were then incubated for 72 h at 37 °C/5% CO₂. After drug treatment, 10 µL of MTT reagent was added to each well to yield a final concentration of 5 mg MTT/mL and the plates were then incubated at 37 °C/5% CO₂ for 3 h more. After this incubation, cells were solubilized overnight (18–24 h) at room temperature with the kit-provided detergent reagent. After this solubilization step, the absorbance of each well at 570 nm was measured using a SpectraMax M5 plate reader (Molecular Devices, New Orleans, LA), and the amount of proliferation in each well was normalized to 1 using the untreated, control well.

4.8. General radiochemistry

QMA light ion-exchange cartridges and C-18 light Sep-Pak^® cartridges were obtained from Waters (Milford, MA). High-performance liquid chromatography (HPLC) was performed on a Shimadzu system equipped with a DGU-20A degasser, two LC-20AB pumps, a SPD-M20A photodiode array detector, and a Flow Count PIN diode radiodetector from BioScan. HPLC solvents (A: H₂O with 0.1% v/v TFA, and B: MeCN with 0.1% v/v TFA) were filtered before use. Semi-preparative HPLC purification was performed on a C-18 Phenomenex Luna reversed-phase column (10 mm × 250 mm, 5 µm; 5 mL/min) employing gradient A (0–13 min, 20–80% B; 13–17 min, 80% B; 17–20 min, 80–20% B; 20–30 min, 20%) or gradient B (0–15 min, 20–80% B; 15–17 min, 80% B; 17–22 min, 80–20% B; 22–25 min, 20%). Analytical HPLC was performed on a C-18 Phenomenex Luna reversed-phase column (4.6 mm × 250 mm, 5 µm; 1 mL/min) employing gradients A or B. Radioactivity was assayed using a Capintec CRC1243 Dose Calibrator (Capintec, Ramsey, NJ). No-carrier-added ¹⁸F-fluoride was obtained via the ¹⁸O(p,n)¹⁸F nuclear reaction of 11-MeV protons in an EBCO TR-19/9 cyclotron using enriched ¹⁸O-water.

4.9. Radiosynthesis of [¹⁸F]-18

[¹⁸F]-fluoride in water was trapped on a QMA light cartridge preconditioned with 0.5 M K₂CO₃ (5 mL) and water (5 mL). The activity was eluted from the cartridge into a sealed reaction vial with 1 mL of a solution of Kryptofix K₂₂₂ (2.5 mg) and K₂CO₃ (0.5 mg) in MeCN (5 mL) and water (2.5 mL). Water was removed azeotropically with acetonitrile (3 × 1 mL) at 110 °C under a slow stream of argon. To the dry K[¹⁸F]F–K₂₂₂ complex was added a solution of the tosylate precursor 29 (4.0 mg) in dry MeCN (400 µL), and the mixture was heated at 110 °C for 15 min. The reaction vial was then cooled to RT and a 10 µL aliquot was removed and diluted with water for HPLC analysis (gradient A). The radio-chromatogram showed one peak corresponding to the [¹⁸F]THP-protected intermediate (t_R = 14.9 min). The reaction mixture was transferred to a vial containing 0.5 N HCl in MeOH (120 µL). The mixture was allowed to react for 15 min at RT and a 10 µL aliquot was removed and diluted with water for HPLC analysis (gradient A). The radio-chromatogram showed one peak corresponding to [¹⁸F]-18 (t_R = 12.7 min). The reaction mixture was injected into semi-prep HPLC for purification (gradient A). The fraction containing [¹⁸F]-18 was collected, MeCN was evaporated under vacuum, and the residual solution was diluted with water (5 mL). The solution was passed through a C18 light Sep-Pak^® cartridge preconditioned with EtOH (5 mL) and water (5 mL). The cartridge was washed with water (3 mL) and [¹⁸F]-18 was eluted with EtOH (1 mL). EtOH was evaporated at 70 °C with a gentle flow of argon and the product was formulated in 0.9% saline for PET studies. The final isolated decay corrected radiochemical yield (dcRCY) after formulation was around 26% with radiochemical purity (RCP) superior to 98% as determined by analytical HPLC (gradient A; t_R = 12.7 min). The total radiosynthesis time was about 125 min.

4.10. Radiosynthesis of [¹⁸F]-19b

[¹⁸F]-19b was synthesized in two steps from tosylate precursor 34 using the same radiolabelling conditions as for compound [¹⁸F]-18. Briefly, to anhydrous K[¹⁸F]F–K₂₂₂ complex was added a solution of the tosylate precursor 34 (3.8 mg) in dry MeCN (400 µL), and the mixture was heated at 110 °C for 15 min and then allowed to cool to RT and a 10 µL aliquot was removed and diluted with water for HPLC analysis (gradient B). The radio-chromatogram showed single peak corresponding to the [¹⁸F]THP-protected intermediate (t_R = 19.9 min). For the removal of the THP group, the reaction mixture was transferred to a vial containing a mix of MeOH and TFA (120 µL, 50% v/v MeOH) and incubated at room temperature for 15 min and a 10 µL aliquot was removed and diluted with water for HPLC analysis (gradient B). The radio-chromatogram showed a major peak corresponding to [¹⁸F]-19b (t_R = 15.5 min). The reaction mixture was injected into semi-prep HPLC for purification (gradient A). The fraction containing [¹⁸F]-19b was collected, MeCN was evaporated under vacuum, and the residual solution was diluted with water (5 mL). The solution was passed through a C18 light Sep-Pak® cartridge preconditioned with EtOH (5 mL) and water (5 mL). The cartridge was washed with water (3 mL) and [¹⁸F]-19b was eluted with EtOH (1 mL). EtOH was evaporated at 70 °C with a gentle flow of argon and the product was formulated in 0.9% saline for PET studies. The final isolated dcRCY after formulation was around 6% with radiochemical purity (RCP) greater than 98% as determined by analytical HPLC (gradient B; t_R = 15.6 min). The total radiosynthesis time was about 135 min.

4.11. Xenografts models

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines. Female severe combined immunodeficient (SCID, Restricted Flora, CB17SC-F) mice (6–8 weeks old) were obtained from Taconic Farms, Inc. (Hudson, NY, USA) and were allowed to acclimatize at the MSKCC vivarium for 1 week prior to implanting tumors. Mice were provided with food and water ad libitum. SK-BR-3 tumors were induced on the right shoulder via subcutaneous injection of 1.0 × 10⁶ cells in a 200-αL cell suspension of a 1:1 v/v mixture of media with reconstituted basement membrane (BD Matrigel, Collaborative Biomedical Products, Inc., Bedford, MA, USA) under 2% isoflurane anesthesia. Palpable tumors developed after a period of 25–30 days.

4.12. Small-animal positron emission tomography imaging

PET imaging experiments were conducted either on a microPET Focus 120 or on a microPET R4 rodent scanner (Concorde Microsystems). Mice bearing subcutaneous SK-BR-3 tumors (100–200 mm³) (n = 4) were administered [¹⁸F]-Topo IIα inhibitors formulations [5.2–7.2 MBq (140–194 µCi) in 200 µL 0.9% sterile saline] via tail vein injection. Approximately 5 min prior to the PET images, mice were anesthetized by inhalation of 2% isoflurane (Baxter Healthcare, Deerfield, IL, USA)/oxygen gas mixture and placed on the scanner bed; anesthesia was maintained using 1% isoflurane/gas mixture. PET data for each mouse were recorded via a 15-min static scans at 30 min, 1 h, 2 h and 4 h post-injection. A minimum of 20 million coincident events were recorded. An energy window of 350–700 keV and a coincidence timing window of 6 ns were used. Data were sorted into two-dimensional histograms by Fourier re-binning, and transverse images were reconstructed by filtered back-projection into a 128 × 128 × 63 (0.72 × 0.72 × 1.3 mm) matrix. The image data were normalized to correct for non-uniformity of response of the PET, dead-time count losses, positron branching ratio and physical decay to the time of injection, but no attenuation, scatter or partial-volume averaging correction was applied. The measured reconstructed spatial resolution for the Focus 120 is approximately 1.6 mm in full width at half maximum at the center of the field of view.

Abbreviations

DAST

diethylaminosulfur trifluoride

DCM

dichloromethane

DHP

dihydroyrane

DMF

dimethylformamide

DMSO

dimethylsulfoxide

ESI

electrospray ionization

GI

gastrointestinal

MS

mass spectrometry

NMR

nuclear magnetic resonance

PET

positron emission tomography

p.i

post-injection

PTSA

para-toluenesulfonic acid

QAP1

quinoline aminopurine 1

RP-HPLC

reversed phase high-performance liquid chromatography

RT

room temperature

SARs

structure–activity relationships

SNAr

aromatic nucleophilic substitution

TBAF

tetra-n-butylammonium fluoride

TFA

trifluoroacetic acid

THF

tetrahydrofurane

THP

tetrahydropyrane

TLC

thin layer chromatography

Topo-II

type II topoisomerase

UV

ultra-violet