**Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging**

**Masoud Sadeghzadeh 1,\*, Barbara Wenzel 1, Daniel Gündel 1, Winnie Deuther-Conrad 1, Magali Toussaint 1, Rare¸s-Petru Moldovan 1, Ste**ff**en Fischer 1, Friedrich-Alexander Ludwig 1, Rodrigo Teodoro 1, Shirisha Jonnalagadda 2, Sravan K. Jonnalagadda 2, Gerrit Schüürmann 3,4, Venkatram R. Mereddy 2, Lester R. Drewes <sup>5</sup> and Peter Brust <sup>1</sup>**


Academic Editors: Anne Roivainen and Xiang-Guo Li Received: 26 March 2020; Accepted: 11 May 2020; Published: 14 May 2020

**Abstract:** Monocarboxylate transporters 1-4 (MCT1-4) are involved in several metabolism-related diseases, especially cancer, providing the chance to be considered as relevant targets for diagnosis and therapy. [18F]FACH was recently developed and showed very promising preclinical results as a potential positron emission tomography (PET) radiotracer for imaging of MCTs. Given that [18F]FACH did not show high blood-brain barrier permeability, the current work is aimed to investigate whether more lipophilic analogs of FACH could improve brain uptake for imaging of gliomas, while retaining binding to MCTs. The 2-fluoropyridinyl-substituted analogs **1** and **2** were synthesized and their MCT1 inhibition was estimated by [14C]lactate uptake assay on rat brain endothelial-4 (RBE4) cells. While compounds **1** and **2** showed lower MCT1 inhibitory potencies than FACH (IC50 = 11 nM) by factors of 11 and 25, respectively, **1** (IC50 = 118 nM) could still be a suitable PET candidate. Therefore, **1** was selected for radiosynthesis of [18F]**1** and subsequent biological evaluation for imaging of the MCT expression in mouse brain. Regarding lipophilicity, the experimental log D7.4 result for [18F]**1** agrees pretty well with its predicted value. In vivo and in vitro studies revealed high uptake of the new radiotracer in kidney and other peripheral MCT-expressing organs together with significant reduction by using specific MCT1 inhibitor α-cyano-4-hydroxycinnamic acid. Despite a higher lipophilicity of [ 18F]**1** compared to [18F]FACH, the in vivo brain uptake of [18F]**1** was in a similar range, which is reflected by calculated BBB permeabilities as well through similar transport rates by MCTs on RBE4 cells. Further investigation is needed to clarify the MCT-mediated transport mechanism of these radiotracers in brain.

**Keywords:** monocarboxylate transporters (MCTs); FACH; 18F-labeled analog of FACH; α-CCA; blood-brain barrier (BBB); positron emission tomography (PET) imaging

#### **1. Introduction**

Monocarboxylate transporters (MCTs), comprising 14 isoforms, are dedicated to the solute carrier 16 (*SLC16*) gene family [1,2]. Of all the MCT isoforms, MCT1-4 are well characterized and known as membrane-bound carriers that bidirectionally transport short-chain monocarboxylic acids, most notably L-lactate, pyruvate, and ketone bodies along with protons across the plasma membrane of mammalian cells [2]. The tissue distribution of the MCT isoforms is quite variable. Although MCT1 is ubiquitously distributed in the muscles, it is additionally expressed along with MCT4 in the brain and other peripheral organs like small intestine, liver, heart, kidney, and blood cells [1,3]. Aberrant expression such as, upregulation of MCT1 and MCT4 has been reported in a large number of tumors (e.g., neuroblastomas, high-grade gliomas, carcinomas of renal cells, breast epithelium, colorectal and squamous tissues, and cervical and lung cancers) where expression is correlated to poor outcomes. In these tissues, the MCTs serve to facilitate the shuttling of lactate between cells with different metabolic requirements [4–6]. Due to the metabolic reprogramming, considered as a hallmark of cancer, tumor cells indeed switch from glucose to lactate as a crucial energy supply, hence, their metabolism heavily relies on glycolysis and consequently the lactate efflux through MCT1 and MCT4 in order to prevent their own acidosis and to regenerate NAD<sup>+</sup> [7]. Accordingly, both transporters are attractive therapeutic and even diagnostic targets for the treatment and detection of human cancers [4,7,8].

Positron emission tomography (PET) is known as a powerful tool for non-invasive molecular detection of early metabolic changes in cancer progression [9]. [18F]Fluorodeoxyglucose ([18F]FDG), a radiolabeled glucose analog, is well known as a standard PET tracer used for diagnosis, staging and treatment monitoring in clinical oncology [10,11]. Considering the lack of specificity and sensitivity of [18F]FDG for several types of tumors [10], there is still an unmet clinical need for cancer detection and therapy. Thus, the complementary concept based on components of the aerobic glycolysis and metabolism by malignant cells is more intriguing and potentially rewarding [2,12–14]. In this regard, [ 18F]DASA-23, recently developed as a potent radiotracer for imaging tumor glycolysis by targeting pyruvate kinase M2, is currently in phase I clinical trials (ClinicalTrials.gov, NCT03539731) [15]. Because the metabolic reprogramming in cancer cells may also result in the overexpression of MCT1/MCT4 in many cancers [4,7], MCT-targeting PET studies provide an opportunity to achieve more accurate and useful understanding of certain aspects of the tumor-specific metabolism [8,16].

During the last decade, only a few 11C- or 18F-labeled substrates of MCTs such as [11C]lactate, [ 11C]pyruvate as well as their 18F-labeled analogs were investigated for imaging of MCTs by PET [17–20]. To the best of our knowledge, only limited examples of the MCTs inhibitors were investigated as PET radiotracers for in vivo applications [21]. Although one of the best characterized inhibitors, α-cyano-4-hydroxycinnamic acid (α-CCA), possesses a 10-fold selectivity for MCT1 compared to other subtypes (Figure 1) [22], it also shows significant inhibitory potency towards the mitochondrial pyruvate carrier in isolated mitochondria [23]. Accordingly, very potent and more specific MCT1/MCT4 inhibitors have been developed based on a comprehensive structure-activity relationship study on a series of α-CCA derivatives [24,25]. Based on this approach, we recently developed and evaluated [ 18F]FACH as the first 18F-labeled inhibitor of MCTs (Figure 1) [26]. Along with a high inhibitory potency towards MCT1 (11.0 nM) and MCT4 (6.5 nM) [26], [18F]FACH showed very promising pharmacokinetics in healthy mice, in particular in the kidneys, as organ with a high physiological expression of MCT1 [1,3,27].

**Figure 1.** Chemical structures of α-CCA and its novel derivatives as potent monocarboxylate transporters 1/4 (MCT1/MCT4) inhibitors; <sup>a</sup> Inhibition of [14C]lactate uptake was determined in RBE4 cells (for MCT1) and in MDA-MB-231 cells (for MCT4) by measuring intracellular radioactivity after 60 min incubation without and with the respective inhibitor at 37 ◦C [25].

Nonetheless, [18F]FACH showed only moderate brain uptake, which might be related to the rather hydrophilic features (log D7.4 = 0.42) [28], assuming that [18F]FACH could only passively enter the brain. This assumption would be in accordance with the fact that for the structural analog α-CCA, the site of its MCT inhibition has been demonstrated to be the extracellular surface [29,30]. Moreover, it is well established that MCT1 is also the prominent monocarboxylic acid transporter in the cerebral microvascular endothelium, facilitating the bidirectional transport of lactate through brain endothelial cells and the blood-brain barrier (BBB) [31]. Many brain tumors, such as gliomas and neuroblastomas, produce high amounts of lactic acid and consequently up-regulate MCT1, thus, inducing acidosis in the tumor microenvironment [5]. MCT1 is therefore proposed as a most likely therapeutic target for neuroblastomas and gliomas, and α-CCA has been able to suppress tumor growth via inhibition of MCT1 [5,32,33]. Accordingly, the development of MCT1-targeting radiotracers possessing sufficiently high brain permeability would be an important step forward toward brain imaging.

Although α-CCA as well as its new analog FACH (Figure 1) contain a Michael acceptor unit, their highly predominating carboxylate form under physiological conditions (ACD-calculated p*K*<sup>a</sup> < 1 vs. pH 7.4; see Table 1 below) masks a respective electrophilic reactivity. This implies that these compounds are most likely not active as protein-attacking electrophiles. Moreover and as mentioned above, α-CCA has been reported to remain extracellular, inhibiting MCT from the outside in a competitive manner and without adverse effects under therapeutic concentrations [29,30].

In this context, it is interesting that for all respective cinnamic acid derivatives, the electron-withdrawing α-CN substituent is required for their MCT inhibition potency, which holds also for FACH [26]. As a possible explanation, we hypothesize that their non-covalent interaction with the MCT protein at the cellular surface may include an electrostatic Arg-carboxylate binding motif as primary anchor. This may facilitate a further complex stabilization through approaching a Cys-thiol by the Michael acceptor β-carbon that is activated further through the α-CN substitution (Figure 2, left). To balance the carboxylate anionic charge, a separate extracellular proton, required for the MCT action as respective symporter, could be attached temporarily at a His-nitrogen (not shown in Figure 2). In case of a successful carboxylate transport such as for the lactate efflux, a respective (additional) His-proton could be liberated to the interstitial compartment.

**Figure 2.** Hypothetical MCT-inhibitor binding at the extracellular surface (left) and at an interior protein site with no aqueous solvation (right). Michael addition may become active for the neutral carboxylic acid form (right), but would be reversible due to the α-CN substitution that enhances the retro-Michael reaction significantly (see, e.g., [34,35]).

In case the Arg-carboxylate interaction would result in a charge compensation sufficient to unmask the Michael acceptor reactivity, the Cys-thiol might add to the α,β-unsaturated unit, possibly following a proton transfer from Arg to the carboxylate (right part of Figure 2). In this case, however, the α-CN substituent enhances both the Michael and the retro-Michael reactivity, making this covalent reaction reversible through stabilization of the carbanion intermediate [34,35]. In conclusion, we hypothesize that despite the Michael acceptor unit common to all cinnamic acid derivatives, their mode of action is probably non-covalent or under water-poor/free conditions at least only temporarily covalent, with a correspondingly negligible risk to form permanent covalent bonds to nucleophilic protein sites. Results from a respective toxicity study will be reported in due course.

With the goal to improve the brain uptake by passive diffusion, we designed new analogs of FACH by replacing the less lipophilic propyl groups with more lipophilic aryl and heteroaryl moieties (Figure 3). Notably, the structurally modified analogs yet need to retain an acceptable inhibitory potency towards MCT1. On the basis of compound A, which was reported to exhibit high MCT1 inhibition (IC50 = 8.0 nM, Figure 1) [25], two fluorinated analogs were developed by introducing 2-fluoropyridinyl and phenyl groups (compounds **1** and **2**, Figure 3). Herein we describe the organic synthesis of the new compounds and their inhibitory potency for MCT1-mediated lactate transport. Furthermore, radiofluorination of **1** was performed and the resulting new radiotracer [18F]**1** was investigated in mice to assess the impact of higher lipophilicity on the in vivo features compared to [ 18F]FACH for imaging of MCT1 in mouse brain.

**Figure 3.** New analogs of FACH investigated in the current study.

#### **2. Results and Discussion**

#### *2.1. Organic Chemistry and Monocarboxylate Transporter Inhibition*

For developing the compounds **1** and **2,** the di-arylamine intermediate **5** was synthesized via the Buchwald-Hartwig aryl amination according to the previously reported procedures (Scheme 1) [36,37]. Alkylation of 6-fluoro-*N*-(3-methoxyphenyl)pyridin-2-amine **5** using 1-iodopropane and sodium hydride afforded **6** in 95% yield [38]. Compound **7** was obtained via a second Buchwald–Hartwig amination of **5** with phenyl bromide in negligible yield (< 10%). However, a stepwise addition of the palladium (Pd) catalyst and the phosphine ligand together with a longer reaction time led to the formation of **7** in moderate yield (46%). This might be related to the decreased electron density of the nitrogen atom due to the 2-fluoropyridinyl substituent and/or the steric hindrance effect. Both **6** and **7** were afterwards subjected to Vilsmeier–Haack formylation [39] to afford **8** and **9** with yields of 57% and 68%, respectively. Finally, Knoevenagel condensation of aldehydes **8** and **9** with cyanoacetic acid generated **1** and **2** in nearly quantitative yields (Scheme 1) [26].

**Scheme 1.** Synthesis of **1** and **2**; reagents and reaction conditions: (a) Pd(OAc)2 (5 mol %), Xantphos (5 mol %), Cs2CO3, 1,4-dioxane, Ar, 105 ◦C, 50 min, 96%; (b) 1-iodopropane, NaH (60% oil dispersion), DMF, Ar, r.t., 1.5 h, 95%; (c) PhBr, Pd(OAc)2 (15 mol %), Xantphos (15 mol %), Cs2CO3, 1,4-dioxane, Ar, 105 ◦C, 24 h, 46%; (d) POCl3, DMF, Ar, 80 ◦C, 2–4 h, 57% (for **8**) and 68% (for **9**); (e) *i.* cyanoacetic acid, piperidine, ACN, reflux; *ii.* HCl (6 M), r.t. 30 min, 95% (for **1**) and 98% (for **2**).

Inhibition of MCT1-mediated lactate transport of **1** and **2** was investigated by [14C]lactate uptake assays using immortalized rat brain endothelial-4 cells (RBE4) [40] which express mainly MCT1 [24,25]. Both compounds dose-dependently inhibited the lactate uptake, with IC50 values of 118 nM (**1**) and 274 nM (**2**). Accordingly, replacing the 1-fluoropropyl group of FACH by a 2-fluoropyridinyl group in **1** resulted in a 10-fold decrease of the inhibitory potency. When comparing **2** with compound A, the substitution of the phenyl ring by a 2-fluoropyridinyl ring in **2** (Figure 1) caused an even stronger reduction of the inhibitory potency [25]. We therefore decided to proceed with **1** for radiofluorination and biological evaluation.

In order to develop the new MCT1-targeting radiotracer [18F]**1**, a precursor including a suitable leaving group was required for the nucleophilic aromatic substitution (SNAr) with [18F]fluoride. A nitro precursor (**15**) with an unprotected carboxylic acid function (Scheme 2) was synthesized considering the good results obtained for the aliphatic nucleophilic substitution with unprotected precursor in the one-step radiosynthesis of [18F]FACH [28]. Initial Buchwald–Hartwig aryl amination between 2-amino-6-nitropyridine and 3-bromoanisole provided the *N*-substituted anisidine **12** in 80% yield (Scheme 2) [36]. Alkylation of **12** under basic condition provided **13** with a yield of 93% [38]. Vilsmeier–Haack formylation of **13** gave aldehyde **14** in 92% yield [39]. It was followed by Knoevenagel condensation with cyanoacetic acid to provide **15** in 67% overall yield [26]. The chemical purity of the precursor **15** was > 98%, according to NMR and HPLC analyses.

**Scheme 2.** Synthesis of the nitro precursor (**15**): Reagents and reaction conditions: (a) Pd(OAc)2 (5 mol %), Xantphos (5 mol %), Cs2CO3, 1,4-dioxane, Ar, 105 ◦C, 2 h, 80%; (b) 1-iodopropane, NaH (60% oil dispersion), DMF, Ar, r.t., 1.5 h, 93%; (c) POCl3, DMF, Ar, 80 ◦C, 1.5 h, 92%; (d) i. cyanoacetic acid, piperidine, ACN, reflux, 5 h; ii. HCl (6 M), r.t. 30 min, above 98%.
