Fluorinated phenylalanines: synthesis and pharmaceutical applications

Recent advances in the chemistry of peptides containing fluorinated phenylalanines (Phe) represents a hot topic in drug research over the last few decades. ᴅ- or ʟ-fluorinated phenylalanines have had considerable industrial and pharmaceutical applications and they have been expanded also to play an important role as potential enzyme inhibitors as well as therapeutic agents and topography imaging of tumor ecosystems using PET. Incorporation of fluorinated aromatic amino acids into proteins increases their catabolic stability especially in therapeutic proteins and peptide-based vaccines. This review seeks to summarize the different synthetic approaches in the literature to prepare ᴅ- or ʟ-fluorinated phenylalanines and their pharmaceutical applications with a focus on published synthetic methods that introduce fluorine into the phenyl, the β-carbon or the α-carbon of ᴅ-or ʟ-phenylalanines.


Introduction
Major efforts have been focused on the synthesis of fluorinated organic molecules particularly for drug development. The replacement of hydrogen by fluorine has been used in the development to improve the biophysical and chemical properties of bioactives. Such tuning in properties arises from the small size of fluorine, the next in size to hydrogen. However, the high electronegativity of the fluorine leads to low polarizability and a strong covalent bond to carbon [1][2][3][4]. Therefore, the introduction of fluorine into phenylalanine (Phe) can modulate the acidity, basicity, hydrophobicity, geometry, conformation, reac-tivity, and moreover the bioavailability of the analogue [1]. Fluorinated amino acids (FAAs) have considerable industrial and pharmaceutical potential [2]. Also, they have played an important role as enzyme inhibitors as well as therapeutic agents [3,4]. Moreover, they modulate the properties of peptides and proteins [5][6][7], influencing aspects such as protein folding, protein-protein interactions, ribosomal translation, lipophilicity, acidity/basicity, optimal pH, stability, thermal stability, and therapeutic properties [8][9][10]. This extends to metabolic properties of membrane permeability and reactivity [11][12][13][14]. The effect of peptide structure and stability has been found to depend on the position and number of fluorine atoms within the amino acid chains [15][16][17]. Incorporation of fluorinated aromatic amino acids into proteins can increase their shelf life, especially in therapeutic proteins and peptide-based vaccines [18]. Enhanced catabolic stability [6] can arise from the role of particular aromatic amino acids in membrane-protein interactions [19]. Furthermore, fluorinated aromatic amino acids can alter enzymatic activity as a result of enhanced protein stability [5]. Also fluorinated aromatic amino acids can destabilize ΙΙ-cation interactions whereas their increased hydrophobicity enhances binding affinity [19]. Moreover, the incorporation of fluorinated amino acids into proteins provides the opportunity for probing structure (by NMR techniques) including protein-protein and protein-ligand interactions and consequently metabolic processes [20,21].
In this review we provide an overview for the various syntheses of FPhes and analogues. Five different categories of FPhe are represented and are classified I-V according to the position of the fluorine(s) (Figure 1). Direct attachment of the fluorine atom to the aryl ring of Phe or fluorinated groups directly attached to a spacer extending from the aryl ring constitute types I and II (Figure 1), accordingly we reported herein different methods for their synthesis.

Negishi cross coupling of aryl halide and organozinc compounds
Jackson and co-workers reported the synthesis of a range of phenylalanine derivatives via Negishi cross-coupling reactions of aryl halides and Zn homoenolates of the protected (R)iodoalanine 2. The reaction was activated using Pd(0) as a catalyst.
The two-inseparable para/meta isomers of all-cis-2,3,5,6-tetrafluorocyclohexylphenyliodide 4 and 5 were subjected to Jackson's methodology for the synthesis of the appropriate amino acid products. Thus, the coupling of the zinc homoenolate of (R)-iodoalanine 2 with a mixture of 4 and 5 in the presence of Pd(dba) 3 and SPhos resulted in an excellent conversion to the fully protected amino acid isomers 6 and 7, which were readily separated from each other by chromatography. Deprotection of isomers 6 and 7 gave the individual free amino acids p-(S)-8 and m-(S)-9 [37,38] (Scheme 2).

Alkylations of fluorinated aryl halides with a chiral auxiliary
Alternatively, the coupling of the bis(dimethoxybenzyl)-protected sulfonamide 14, instead of the esters 10a and 10b with zincate 11 using a variety of catalysts and different reaction conditions, was unsuccessful. However, coupling of 4-[bis(dimethoxybenzyl)difluoromethyl]benzyl bromide (14) with the lithium enolate of William`s lactone 15 gave the pro- tected amino acid 16 in 80% yield. The desired protected amino acid 17 was readily obtained after reduction of 16 using PdCl 2 as a catalyst, followed by treating the product with Fmoc-OSu in dioxane/aq Na 2 CO 3 (99%, two steps) [40] as illustrated in Scheme 4.
Alternatively, phenylalanines 29a,b were also synthesized by alkylation of 26a,b with the chiral auxiliary 31, which was obtained by reaction of the cyclic dipeptide 30 with triethyloxonium tetrafluoroborate. The alkylation reaction of 26a,b was carried out with n-BuLi in THF at −78 °C to give 32a,b. Acid hydrolysis of the alkylated product 32a,b afforded the ethyl esters of tetrafluorophenylalanine 33a,b, which by alkaline hydrolysis afforded the tetrafluoro derivatives 29a and 29b, respectively [43] (Scheme 7).
A one-pot double alkylation of the chiral auxiliary 39 with benzyl iodides 40a,b gave cis-dialkyl derivatives 41a,b in 70-72% yield. The subsequent removal of the auxiliary followed by treatment with Fmoc-OSu gave the N-protected 2-fluoro-and 2,6-difluorophenylalanine derivatives 42a,b in quantitative yields [45] (Scheme 9). The radiolabeled 2-[ 18 F]-fluoro-ʟ-phenylalanine 46 was synthesized as a promising radiopharmaceutical agent for molecular imaging by positron emission tomography (PET

Hydrolysis of Erlenmeyer's azalactone
A multistep Erlenmeyer azalactone synthesis was reported as an important method for the synthesis of fluorinated α-amino acids 53a-h. Thus, a three-component condensation of a series of fluorinated benzaldehydes 50a-h, N-acetyl-or N-benzoylglycine 51a or 51b, respectively, and an excess of acetic anhydride in the presence of sodium acetate afforded the oxazolones 52a-h. The subsequent reductive ring cleavage of 52a-h without isolation, was carried out with red phosphorus in hydroiodic acid to give the fluorinated phenyl- alanine analogues 53a-h. Alternatively, a two-step sequence to generate amino acids 53a-h was attempted by first hydrolysis of 52a-h to form acids 54a-h which then were reduced with P/HI to the desired products 53a-h [48]. The free amino acid 53i was prepared by the same protocol [49] (Scheme 12). 2,5-Difluorophenylalanines with either R or S configuration were synthesized also via the Erlenmeyer azalactone method. The synthesis started with the multicomponent reaction of aldehyde 55, acetylglycine 51a and acetic anhydride to give the azalactone 56. The subsequent basic hydrolysis of 56 gave 57 that, on catalytic hydrogenation, afforded racemic difluorinated Phe 58. The isomers were separated by selective hydrolysis using a protease from Bacillius sp to generate the (S)-N-acetyl acid 59 with >99.5% ee and the corresponding (R)-N-acetyl ester 60 with >99.5% ee [50] (Scheme 13).
Interestingly substitution of Phe by either 77b or 77a in the proteasome inhibitors bortezomib or epoxymicin, led to an increase in the efficiency as anticancer proteasome inhibitors. The fluorinated amino acids 77a and 77b were used mainly for two reasons, i.e., the ready availability and hydrophobicity [55].
Further, (S)-pentafluorophenylalanine (Pff, 77a) was used to stabilize proteins for potential applications in various proteinbased biotechnologies. To improve protein stability, natural hydrocarbon amino acids were replaced with Pff 77a. The effect of enhanced protein stability upon this replacement is referred as to 'fluoro-stabilization effect' [56].
Cross-coupling reactions with boronic acids were found to be successful only for the synthesis of para and meta-deriva-tives. Several attempts were made to prepare the ortho-substituted derivatives 93 and 95. The synthesis of ᴅ,ʟ-93 or ʟ-95 was achieved by vinylation of the protected ᴅ,ʟ-N-Boc-2bromophenylalanine (89) using a Stille coupling reaction to give the o-vinyl derivative 90 as key intermediate. A hydroboration reaction of compound 90 afforded the primary alcohol 91, which was directly fluorinated and deprotected to give the free amino acids 93 (ᴅ and ʟ). Alternatively, alcohol 91 was activated by tosylation to give 94 as a precursor for radiofluorination that was achieved to give 2-[ 18 F]FELP ʟ-95 using [ 18 F]fluoride complexed with Kryptofix ® /K + followed by deprotection with HCl and purification. This product emerged as promising new PET tracer for brain tumor imaging [58] (Scheme 21).

From enamine intermediates
The synthesis (R)-2,5-difluorophenylalanine derivative  logues of type I and II. The most convenient method involved a Negishi cross coupling of an aryl halide and the Zn homoenolate of the protected (R)-iodoalanine 2 using a Pd(0) catalyst. This method provided a versatile range of fluorinated phenylalanine products with high enantioselectivities and in acceptable yields.

Multistep synthesis from ethyl trifluoropyruvate hemiketal
The reaction of ethyl trifluoropyruvate hemiketal 130 with thionyl chloride in pyridine afforded the chlorinated derivative 131, which upon treatment with zinc powder in DMF, afforded the dihalogenated olefin 132. The substitution of one fluorine atom in 132 with a tributylstannyl group to give 133 was accomplished by the reaction with (Bu 3 Sn) 2 CuLi in THF at −78 °C. The reaction took place following an addition-elimination mechanism. Then, coupling of 133 with iodobenzene in the presence of Pd(PPh 3 ) 4 and CuI as the co-catalyst afforded ethyl (E)-3-phenyl-3-fluoro-2-methoxypropenoate (134) which was converted into the corresponding α-ketoacid 135 by treatment with trimethylsilyl iodide. Finally, the reaction of 135 with aqueous ammonia followed by reduction with sodium borohy-

Ring opening of aziridine derivatives by HF/Py
The ring opening reaction of aziridines 138a,b by treatment with hydrogen fluoride in pyridine afforded 3-fluorophenylalanine esters 139a,b. The subsequent enzymatic hydrolysis of

Fluorination and reductive amination of phenylpyruvate
A direct fluorination of the ester derivatives of phenylpyruvic acids 140a,b with F 2 followed by hydrolysis of the resulting fluoropyruvates in 50% isopropanol in the presence of NaHCO 3 gave 3-fluoro-3-phenylpyruvate 141 in 40-50% yields [68]. The direct reductive amination gave a partially racemized mixture of threo and erythro-136 with the erythro stereoisomer 136 as the major product (Scheme 32).
Alternatively, a visible light (14 Watt CFL) mediated benzylic fluorination of a series of N-and C-terminally protected phenylalanines 147 using Selectfluor and dibenzosuberenone in acetonitrile, afforded the β-fluorophenylalanine derivatives 148 in variable yields with partial racemization. Phthalimido and trifluoroacetyl N-terminal protecting groups (R 1 = Phth or TFA) and unprotected C-terminal derivatives (R 2 = H) provided the most efficient outcomes (80 and 67% yield, respectively). An N-acetyl group was also suitable as protecting group for the reaction providing the desired product with 57% yield. Also, methyl and ethyl esters as C-terminal protecting groups in combination with phthalimino as the N-terminal protecting group, were both successfully explored. However, when the trifluoroacetyl amide was used as a substrate the methyl ester performed better than the ethyl ester (74% versus 60% yield). However, N-protecting groups such as Boc, Fmoc, and Cbz were not compatible with the fluorination (0-10% yield). Moreover, when tert-butyl, trityl, and adamantyl protecting groups were installed for C-terminal protection additional fluorination, decomposition, and consequently low yields of the β-fluorinated derivatives 148 were observed [73] (Scheme 35).

Fluorination of aziridinium derivatives
The N,N-dibenzylated 3-fluorophenylalanine derivative 151 was prepared with excellent diastereoisomeric ratio (dr > 99:1) from α-hydroxy-β-amino ester 142. In this case, XtalFluor-E was used to activate the OH group in the substrate and displaced by neighboring amino-group participation creating an aziridinium intermediate 150. The latter then was opened stereo-and regioselectively by fluoride to give 151 in good yield and high diastereoisomeric purity (Scheme 36). The subsequent deprotection of 151 had to be achieved with BrO 3 − , because hydrogenolysis resulted in defluorination [74].
Alternatively, a series of substituted anti-β-fluorophenylalanine derivatives 154a-d was obtained from the corresponding enantiopure α-hydroxy-β-aminophenylalanine esters [75,76]  or to other fluorination reagents such as fluoropyridinium tetrafluoroborate, 2,4,6-trimethylfluoropyridinium tetrafluoroborate, or NFSI. The fluorination process was explored with a broad range of substituted Phe derivatives. The removal of the PIP auxiliary group without affecting the newly introduced fluorine atom was attempted by a two-step, one-pot protocol involving an in situ esterification of a highly electrophilic pyridinium triflate intermediate [77] and afforded the anti-β-fluoro-α-amino acid methyl ester 160a in 52% yield and with 98.8% ee (Scheme 39).
On the other hand, when the quinoline-based ligand 162 was used, it was shown to promote the palladium-catalyzed direct electrophilic fluorination of β-methylene C(sp 3 )-H bonds. Thus, fluorinations of ʟ-phenylalanine 4-trifluoromethylphenylamides 161a-l carrying a range of functional groups such as fluoro, chloro, bromo, methoxy, acetyl, cyano, nitro, and trifluoromethyl, were well-tolerated and afforded the corresponding anti-β-fluoro-α-amino acids 163a-l in moderate to excellent yields [78] (Scheme 40).
An alternative approach to the difluorinated compound 168a was achieved by the condensation of 164a with (S)-1-phenylethylamine (169) immediately hydrolyzed to provide the racemic carboxamide 172. The subsequent removal of the chiral auxiliary by catalytic hydrogenation then afforded the carboxamide 173. Finally, an acid-mediated hydrolysis of the carboxamide 173 to generate the free amino acids ʟ-or ᴅ-168a, was carried out with aqueous H 2 SO 4 . However, the acid hydrolysis step was accompanied with extensive racemization [81,83] (Scheme 42). Comparative studies demonstrated that using ligand 175 rather than other ligands gave higher yields and no β-elimination products. The effective removal of the auxiliary using triflic anhydride with LiOH as nucleophile, gave product 177a in good yield. Alternative nucleophiles such as EtOH or methyl esters of amino acids, in the presence of catalytic amounts of CoCl 2 , afforded product 177b or fluorinated Phe dipeptides [84] 177c-g as racemic mixtures (Scheme 43).

Pharmaceutical applications of fluorinated phenylalanine derivatives
Peptides and proteins containing FPhe are important tools to identify enzyme-substrate complexes, mechanisms of protein aggregation, and modifying the chemical and thermal stabilities of proteins. The properties of protein were preserved, when low levels of fluorine are incorporated into the constituent amino acids, and were comparable with that of the original proteins. Helpfully, fluorine incorporation may favorably adjust protein function including improved stability and substrate selectivity.

Applications of FPhe derivatives in positron emission tomography (PET)
The molecular imaging technique positron emission tomography (PET) provides information on tumor metabolism, which allows for a more accurate diagnostic and therapy response in neuro-oncology, compared to, for example, magnetic resonance imaging (MRI). PET is particularly well-suited to differentiate neoplastic tissue from non-specific changes induced by chemotherapy treatments [85]. PET is particularly used for the early detection of tumors and metastases, and is an established tool for the diagnosis, staging, and the treatment planning of various malignancies. The selective imaging of tumors using PET exploits radiotracers that target aberrant cellular metabolism or increased protein expression [86,87]. Here, the 18 F isotope is particularly useful for the preparation of radiotracers to be used in PET due to its relatively long half-life (109 min). In this section we highlight two selected 18 [53,[88][89][90][91][92]. This compound was used for PET imaging of melanoma in animal models.
The low affinity of 178 for the ʟ-type amino acid transporter1 (LAT1), however, limited the use of this compound as PET radiotracer for brain tumor imaging [93][94][95][96] (Figure 2). anticancer drug currently being in clinical trials for the treatment of relapsed and refractory multiple myeloma (RRMM) [102,103]. Melflufen is a next generation form of the more historical drug, melphalan 180 ( Figure 3).

Antidiabetes drugs, sitagliptin:
(R)-2,4,5-Trifluorophenylalanine 38b is a constituent of sitagliptin (183, Figure 6). Sitagliptin is used to decrease the level of blood sugar  in patients with type 2 diabetes and belongs to the dipeptidyl peptidase-4 (DPP-4) class of inhibitors [114,115]. This enzyme breaks down the incretins GLP-1 and GIP, gastrointestinal hormones released in response to a meal. By preventing the breakdown of GLP-1 and GIP, they are able to increase the secretion of insulin by the pancreas that modulates blood sugar level when it is high. Sitagliptin was granted FDA approval in October, 2006 [116]. Retagliptin phosphate: Retagliptin phosphate (184) is under investigation as a DPP-4 inhibitor for treating type-2 diabetes. It is an analogue of sitagliptin which was developed for the same application [109,117], but compound 184 appears to have an improved activity [118]. Retagliptin showed efficacy in clinical trials and is now entering phase III studies. (R)-2,4,5-Trifluorophenylalanine 38b is used as a building block in the synthesis of compound 184 [119,120] (Figure 7).     (187) is a small cyclic peptide containing ᴅ-4-FPhe. Ulimorelin acts as a selective agonist of the ghrelin/growth hormone secretagogue receptor (GHSR-1A), which is currently being developed by Tranzyme Pharma (code name TZP-101) as a first-in-class treatment for both, postoperative ileus (POI) and diabetic gastroparesis. POI describes a deceleration or arrest in intestinal motility following surgery [109,[125][126][127] (Figure 10).

The glucagon-like peptide-1 receptor (GLP1R):
3'-Fluorophenylalanine is a key motif in the structure of the glucagon-like peptide-1 receptor (GLP1R, 188, Figure 11). GLP1R is a receptor protein found on beta cells of the pancreas and on neurons of the brain. It is participating in the modulation of blood sugar levels by increasing insulin secretion. Consequently, GLP1R plays a key role in the development of drugs to treat diabetes mellitus [128][129][130].

Sodium channel blockers (benzazepinone Nav1.7 blocker):
Sodium channel blockers are used in the treatment of neuropathic pain. This is a chronic, debilitating condition that results from injury of the peripheral or central nervous system and can be triggered by a variety of events or conditions, including diabetes, shingles and chemotherapy [131]. Merck reported [132] the discovery of a structurally novel class of benzazepinone hNav1.7 voltage-gated sodium channel blockers containing 2-trifluoromethoxy-ʟ-phenylalanine derivative 189 and 3-ʟ-FPhe 190 ( Figure 12) [133]. Compounds 189 and 190 were investigated as potential drugs for the treatment of neuro-pathic pain because they inhibited action potential firing. It was suggested, based on genetic studies, that a selective Nav1.7 inhibition, will produce robust inhibition of pain without significant side effects [134,135].

Conclusion
In view of the increased significance of FAAs in the development of bioactive compounds, considerable efforts were dedicated to the development of efficient synthetic protocols to FAAs. Among them, a range of fluorinated phenylalanines emerged, that have enhanced the biophysical, chemical and biological properties of bioactives. Accordingly, synthetic approaches to five distinct classes of fluorinated analogues were reviewed here. Synthetic protocols and strategies varied according to the position of the fluorine substituent. Also included were 18 FPhe derivatives, some of which emerged as promising radiotracers in positron emission tomography (PET). Finally, it is notable that there are a significant number of FPhe derivatives which are nowadays incorporated into drug scaffolds of compounds either licensed or currently being studied in clinical trials.