Applications of Fluorine in Medicinal Chemistry - ACS Publications

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Applications of Fluorine in Medicinal Chemistry Eric P. Gillis,*,† Kyle J. Eastman,† Matthew D. Hill,† David J. Donnelly,‡ and Nicholas A. Meanwell† †

Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ‡ Discovery Chemistry Platforms, PET Radiochemical Synthesis, Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, United States ABSTRACT: The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties associated with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a molecule can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addition, 18F has been established as a useful positron emitting isotope for use with in vivo imaging technology that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compounds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compounds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug molecules and applications in positron emission tomography.

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INTRODUCTION Fluorine has been exploited extensively in drug design and development, with broader incorporation limited largely by synthetic accessibility.1 As synthetic methodology to introduce fluorine has evolved, allowing deployment in ever increasing and more sophisticated settings, this enigmatic element continues to enchant as we learn more about its unique properties not only within the halogen series but also within the limited number of elements that are commonly incorporated into drug molecules.2 Fluorine is also emerging as one of the most prominent atoms in the application of positron emission tomography (PET) due to the favorable half-life of the 18F isotope (109.8 min) when compared to 11C (20.4 min) and 124I (4.2 days), and new synthetic methodology is considerably enhancing its utility, particularly for central nervous system (CNS) drug discovery.3 In this review, we provide an overview of some of the tactical applications of fluorine in the design and optimization of drug candidates with an emphasis on studies published since the last update in the Journal of Medicinal Chemistry.1f

available for hyperconjugative donation. In aliphatic systems, a combination of these effects produces a strong preference for vicinal functionality to align gauche to fluorine, where this influence is of sufficient magnitude to find practical application in the design of drugs and organocatalysts.5 The calculated energies of stabilization for several fluoroethane derivatives that demonstrate this “gauche effect” are presented in Table 1 with comment on what is considered to be the origin(s) of the phenomenon: hyperconjugative electron donation by an adjacent C−H bond into low lying C−F σ* orbitals, C−F···H−X interactions, and/or dipole and electrostatic interactions.5b The application of these effects as potential probes of conformational preferences in drug design and drug−target interactions is beginning to be more fully appreciated, and a number of examples that take advantage of this phenomenon in a productive fashion have been described. Illustrative examples of each mode of influence are provided below. (a) Fluorine−Fluorine Interactions. Vicinally fluorinated alkanes adopt a gauche conformation stabilized by reinforcing hyperconjugative interactions in which the low-lying C−F σ* orbitals accept electron density from adjacent C−H σ bonds. This phenomenon in isolation is modest in magnitude, calculated to be worth 0.8 kcal/mol, but is still sufficient to influence conformation (Figure 1).4b,5b,6 For example, the (±)-erythro and (±)-threo isomers of 9,10-difluorostearic acid, 1 and 2, respectively, differ only by the absolute configuration at C-9, yet (±)-threo-isomer 2 melts at a temperature almost 20 °C higher than (±)-erythro-isomer 1.6,7 This has been attributed to 2 more

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FLUORINE AS A CONFORMATIONAL CONTROL ELEMENT Fluorine can play an important and unique role in influencing molecular conformation. From the perspective of steric effects, the influence of fluorine is anticipated to be marginal; fluorine is a small atom with a van der Waals radius of 1.47 Å, close to the 1.20 Å value for hydrogen.1a,4 However, the high electronegativity of fluorine (3.98 on the Pauling electronegativity scale compared to 2.20 for H, 3.44 for O, and 2.55 for C) results in a highly polarized C−F bond which presents both a strong dipole moment (μ C−F = 1.41 D) and a low lying C−F σ* orbital © 2015 American Chemical Society

Received: February 13, 2015 Published: July 22, 2015 8315

DOI: 10.1021/acs.jmedchem.5b00258 J. Med. Chem. 2015, 58, 8315−8359

Journal of Medicinal Chemistry

Perspective

Table 1. Calculated Energy Differences between the gauche- and anti-Isomers of Select X-Substituted Fluoroethane Derivatives

X

stabilization energy (kcal/mol)

underlying interaction

F OH NH2 NH3+ OAc NHAc

0.5−1.0 1.0−2.0 0.9−1.0 5.8 1.6 1.8

C(F)−H σ to C−F σ* hyperconjugation hyperconjugation and an intramolecular C−F to O−H electrostatic interaction intramolecular C−F H-bonding electrostatic interaction between F δ− and NH3+ δ+ dipoles hyperconjugation of C(OAc)−H σ bond to C−F σ* electrostatic interaction between C−F δ− and N−H δ+

Figure 1. Conformational preferences of 1,2- and 1,3-difluoroalkanes.

readily adopting a stable and elongated form that is favored by a gauche interaction between the two F atoms. This form deploys the two alkyl moieties in an antiperiplanar arrangement which minimizes unfavorable steric interactions. In contrast, when the fluorine atoms of 1 adopt either of two possible gauche arrangements, the alkyl side chains are also gauche to one another. As a consequence, unfavorable steric interactions between the alkyl side chains destabilize both conformations, while the alternative arrangement places the alkyl groups in a more favorable antiperiplanar relationship but the fluorine atoms are also antiperiplanar, unable to take advantage of the gauche effect. To assess their conformational mobility, the Langmuir isotherms for material deposited from a CHCl3 solution onto ultrapure H2O were measured, with the expanded nature of the curve for 1 indicating conformational disorder in contrast to 2 which behaved analogously to the parent unsubstituted acid.7 The overall result is that 1 exhibits no strong conformational preference, leading to structural disorder that is reflected in the lower melting point. 1,3-Difluoroalkane motifs prefer to adopt a conformation that minimizes dipole−dipole interactions between the C−F bonds, with the lowest energy form depicted in Figure 1 and favored by 2.7−3.3 kcal/mol.4b,5b,8 The combination of 1,2- and 1,3difluoro interactions can exert considerable conformational control, as exemplified by the hexafluoroalkanes 3 and 4. The former adopts a helical conformation in both solution and the solid state which minimizes unfavorable dipole−dipole interactions between the 1,3-difluoro motifs while maximizing gauche interactions between the vicinal fluorine atoms (Figure 2).6,9 The same effects underlie the preference for an extended conformation with 4 stabilized by 3 out of 5 possible gauche F−F arrangements. The judicious deployment of CF2 moieties in alkyl chains can be exploited to bias conformation, as exemplified by palmitic acid derivatives 5−7. These derivatives exhibit considerably different melting points as determined by differential scanning calorimetry. Difluorinated palmitic acid 5 melts at 62.9 °C, comparable to the 62.5 °C melting point of the unsubstituted progenitor and suggestive of limited influence of fluorination on conformation.

Figure 2. Solid state structure of 3 (structure drawn with PyMol).

In contrast, acid 6 failed to crystallize but the amorphous material melted at 69 °C while crystalline 7 exhibited a melting point of 89.9 °C.10 In the solid state, 7 adopted an extended anti-zigzag 8316



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For syn-di-F analogue 9 the coupling constants were consistent with a gauche relationship between the two F atoms but with disorder between the two possible conformers. For the synisomer 10, the J values were of a higher magnitude, indicative of an extended structure for the GABA element in which a gauche arrangement between the two F atoms is strongly preferred. The two anti-isomers 11 and 12, similar to 9, favor a gauche relationship between the two fluorine atoms but with considerable disorder between the two possible conformers. The interaction of fluorine with amide N−H functionality is discussed below; however, in analogs 9−12 this interaction was not detected, presumably because of geometric constraints imposed by the macrocyclic ring structure. Molecular modeling of the five compounds, in which the low energy conformations were aligned with the 1H NMR data, indicated that while 8 preferred an overall flat topography, the four difluorinated analogs 9−12 behaved quite differently. Compound 9 exhibited a fully planar geometry, while 10 demonstrated more pucker than 8 and adopted a conformation that is distinct from 9. In contrast, 11 and 12 adopted highly compressed and puckered structures that, while similar to each other, are distinct relative to 9 and 10. The structural similarity of 9 and 10 parallels the similar effect of 13 and 14 on GABAA receptor activity. An explanation for these findings is that while the two anti-isomers 11 and 12 can populate two similarly low energy conformations, a single low energy conformer is strongly preferred for each of the two syn-isomers 9 and 10.11c

conformation that was attributed to a favorable alignment of the dipoles associated with the carbon to fluorine bonds and a minimization of unfavorable steric interactions between the CF2 moiety.10 The amorphous noncrystalline nature of 6 was considered to be a function of repulsive effects between the dipoles of the 1,3-disposed C−F moieties that cannot adequately be satisfied in a single low energy conformation, resulting in disorder of the alkyl chain such that a preferred conformation could not be determined.

The relevance of the fluorine gauche effect in a more complex setting is exemplified by difluoro-substituted analogs 9−12 of unguisin A (8), a macrocyclic heptapeptide isolated from the marine fungus Emericella unguis that incorporates γ-aminobutyric acid (GABA) as one of the amino acid residues (Figure 3).11 The

Figure 3. Unguisin A and the four macrocycle analogues 9−12 containing difluoro GABA elements.

(b) Fluorine−OH and Fluorine−O Interactions. It has been known for some time that fluoroalcohols such as 2fluoroethanol (17, Figure 4) prefer, by 1−2 kcal/mol, a conformation in which fluorine lies gauche to oxygen, a phenomenon that has largely been attributed to a C−F···H−O hydrogen bond.5e,f,g13 However, the role of a stabilizing C−F··· H−O interaction appears nuanced, and the significance of C−F hydrogen-bonding remains a topic of some debate (vida infra).14,15 Recent density functional theory (DFT) calculations and natural bond order (NBO) analysis performed at a high level of theory suggest that σ CH → σ* CF and σ CH → σ* CO hyperconjugation is the dominant force underlying the fluorohydrin gauche effect and that any contribution from a favorable C−F···H−O interaction is electrostatic in nature.16

four stereoisomers 13−16 of the GABA analog 2,3-difluoro-4aminobutyric acid have been studied in isolation as modulators of the GABA receptors GABAA, GABAB, and GABAC.12 As analyzed by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling, the preferred conformations of 13 and 14 are those in which the gauche preferences of both fluorine atoms are fully realized.12a The GABA element of unguisin A has been shown to exhibit conformational disorder based on analysis of NMR spectral data; thus, GABA analogs 13−16 were incorporated into unguisin A in order to explore the effect of their intrinsic conformational preferences on the overall secondary structure of 8. 1H NMR spectra of the four difluorinated analogs 9−12 were recorded at a range of temperatures, and the 3JH,H and 3JH,F values were measured.11c 8317



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Figure 4. Energy-minimized conformations of 17 and 18.

Accordingly, the preferred conformer remains the gauche form even when the hydroxyl group is replaced by an OMe group. It has also been calculated that for each of the four most populated conformers of 3-fluoro-1,2-propanediol (18) all heteroatoms lie gauche and a hydroxyl proton approaches fluorine.17 As indicated by DFT calculations and multidimensional NMR, in this system hyperconjugation from σCH and σCC into σ*CO and σ*CF appears to be the dominant influence.17 Alternatively, under certain circumstances the C−F···H−O interaction can be of sufficient strength to significantly reduce the ability of an alcohol to act as a H-bond donor. It is notable that this influence more than counteracts the enhanced acidity due to

the proximal electron-withdrawing fluorine atom.18 This is most strikingly exemplified by cis-disposed 3-fluorocyclohexanol 20, one of the series of fluorinated alcohols 19−22 designed to systematically explore this phenomenon. In contrast to 19, fluoro analog 20 weakly donates a H-bond to N-methylpyrrolidinone in CCl4, measured as a negative pKAHY where pKAHY is a measure of the H-bonding potential of a functional group and is distinct from pKa.18 The reduced H-bond donating effect of the alcohol in 20 is strongly dependent on the dihedral angle of the fluorohydrin and is maximal at 180°. The effect of fluorine on the H-bond donating properties of the O−H in vicinal fluorohydrin 22 is more modest compared to the hydrogen analog 21, reflecting both the altered dihedral angle and the less than ideal 1,5 geometric relationship between the fluorine and hydrogen atoms. The importance of the fluorohydrin gauche effect in drug design is highlighted in the design of fluoro analogs of the HIV-1 protease inhibitor indinavir (23) and its hydroxy epimer (24). All four of the possible fluorinated diastereomers (25−28) were prepared and tested for potency in a HIV-1 protease inhibition assay.19 These data revealed profiles that are complex, an anticipated outcome based on the multiple influences of F: an effect on the acidity of the OH, conformational preferences, steric interactions, and solvation. The syn,syn diastereomer 25 fully preserved the potent inhibitory activity of 23 while the anti,anti diastereomer 26 was 10-fold weaker. For the epiindinavir series, the syn,anti analog 27 was 8-fold more potent than its progenitor 24 while the anti,syn analog 28 was 36-fold weaker than 24. A careful analysis of the 1H−1H and 1H−19F coupling constants of this series revealed that the syn,syn and syn,anti analogs 25 and 27, respectively, fully reproduced the extended conformation observed crystallographically with 23 bound to HIV-1 protease, a conformation favored by a gauche relationship between the fluorine and OH substituents.19 Diastereomers 26 and 28 were more conformationally mobile and populated additional conformers, a potential reason for their weaker protease inhibitory potency relative to the progenitors.

The conformational bias of alkyl aryl ethers is also influenced by fluorine substitution. The low energy conformation of anisole (29), favored by ∼3 kcal/mol, is that in which the OMe moiety is close to coplanar with the phenyl ring (Figure 5).20 The planar conformation is stabilized by an interaction between the aryl ring π system and oxygen lone pair electrons which rehybridize to facilitate orbital overlap, overcoming the inherent allylic 1,3

Figure 5. Geometry associated with the low energy conformation of 29.

strain.21 However, this conformational preference can be perturbed by introducing ortho substituents that disfavor a planar topology because of increased allylic 1,3 strain.20c,22 α-Fluorination provides access to this same orthogonal arrangement but with a smaller substituent size while also reducing the potential for CYP 450-mediated dealkylation.1a,23 The preference for the CF3O moiety to align orthogonal to the plane of the aromatic ring, calculated to be favored by ∼0.5 kcal/mol, is attributed to weakened oxygen lone pair donation into the aromatic π orbitals;1a,24 this conformational preference is also observed with difluoroalkoxy moieties.23d,e A role for the conformational mobility of fluorinated alkyl phenyl ethers in the expression of biological activity was explored in an analog of the cholesterol ester transfer protein (CETP) inhibitor 30. The tetrafluoro-substituted ether 31 demonstrated an 8-fold increase in inhibitory potency relative to 30, a gain hypothesized to be due, in part, to the ability of this substituent to adopt a more orthogonal projection of the CF2HCF2O moiety.25 8318



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The synthesis of fluorinated analogs of the neurotransmitter GABA (34) designed to take advantage of the fluorineammonium gauche effect was explored as an approach to providing tool molecules with which to probe stereochemical preferences in biological recognition (Figure 7).11,12,27 (R)-3Fluoro-γ-aminobutyric acid (35, (R)-3F-GABA) and its enantiomer 36 ((S)-3F-GABA) were prepared in optically pure form in which the zwitterionic nature of the progenitor is maintained at neutral pH, a consequence of reduced amine basicity combined with a more acidic carboxylic acid moiety.27a Analogous to 34, both 35 and 36 adopt an extended conformation in solution and the more stable conformers are those in which the fluorine and NH3+ are in a gauche relationship (Figure 7).27a This analysis suggests that conformer A is disfavored for 35, while conformer C is disfavored for 36. Although less potent than 34, both 35 and 36 activated a cloned human GABAA receptor with comparable potency, a result that suggested that conformer B, in which the NH3+ and CH2CO2− moieties are in an antiperiplanar relationship, is recognized by the GABAA receptor.27a,b Neither compound was a substrate for GABA aminotransferase, but 35 was a 10-fold more potent inhibitor of this enzyme than 36.27b Moreover, GABA aminotransferase catalyzed the elimination of HF from 35 with over 10-fold greater efficiency than for 36. These data, in conjunction with modeling studies, suggested that conformer C is preferentially recognized by this enzyme.27b Fluorine analogs of the glutamate mimic N-methyl-D-aspartate (NMDA, 37) also provide an illustration of the influence of the fluorine−ammonium gauche effect on biological activity. NMDA is an agonist at the GluN2A and GluN2B receptors that are activated by glutamic acid (38). The preferred conformational relationship between the amine and acid moieties that is

This hypothesis stimulated the design of a series of compounds in which alternative substituents were examined. Improved potency was observed with the furan analog 32, with modeling studies suggesting a nonplanar confirmation analogous to that hypothesized for 31. However, this may not provide a definitive explanation, since differences between the available torsion angles and electron distribution between the two substituents were noted.25

(c) Conformational Bias Induced by Fluorine− Ammonium Interactions. Fluorine interacts strongly with ammonium species to favor a gauche stereochemical relationship. Of potential importance in drug design, this interaction is believed to be governed by a favorable electrostatic interaction between the electronegative fluorine atom and the positively charged ammonium moiety.4a,5b,g For 2-fluoroethylammonium species, the gauche conformation is preferred by 5.8 kcal/mol; for the neutral amine form this bias is only ∼1 kcal/mol.4a,5b,g In 3-fluoro-N-methylpiperidinium (33) these effects are manifested as an overwhelming conformational bias that populates only the two conformers in which fluorine is axial, with the equatorial N−CH3 strongly preferred over the more sterically congested axial N−CH3 (Figure 6).26

recognized by these proteins was studied using the two 3-F substituted diastereomers 39 and 40.27e The 1H and 19F NMR spectra of 39 were consistent with this molecule adopting conformer A in solution (Figure 8). In contrast, data for 40 indicated that conformer C was not appreciably populated, but the experimental data failed to discriminate between conformers A and B.27e DFT calculations indicated that conformer B, found in the single crystal X-ray structure, is more stable than conformer A. In Xenopus laevis oocytes expressing GluN2A and GluN2B glutamate receptors, 39 evoked currents indicative of

Figure 6. Structure and conformational preferences of 33.

Figure 7. Structures and conformational preferences of 35 and 36. 8319



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Figure 8. Structures and conformational analysis of 39 and 40.

agonist activity, although the effects of this molecule were less than that observed with NMDA, while 40 was inactive. Since only 39 was capable of activating these two glutamate receptors, the preferred conformation was concluded to be that represented by conformer A in Figure 8. This result is consistent with the X-ray cocrystal structure of 37 bound to the highly homologous GluN2D receptor.27e (d) Conformational Effects between Fluorine and Amides. The introduction of a fluorine atom proximal to an amide moiety can influence molecular conformation in a topologically dependent fashion. Capsaicin (41), a constituent of peppers that is an agonist of the transient receptor potential cation channel subfamily V member 1 (TRPV1) protein, is a potentially useful vehicle to study this phenomenon in relation to receptor recognition.28 The two enantiomers of α-fluoro capsaicin (42) were prepared in optically pure form and evaluated for agonist activity at the TRPV1 receptor. It was predicted based on the calculated rotamer energies summarized in Figure 10 that the trans conformation would be favored by 6 kcal/mol over the gauche conformation and by 8 kcal/mol over the cis conformer.28,29 The trans conformer is stabilized by both a favorable dipole−dipole interaction between the C−F and CO bonds and a productive electrostatic interaction between the electronegative fluorine atom and the electropositive amide N−H. Through-space interactions involving fluorine, denoted herein with a red arrow (Figure 9), are a topic of ongoing debate and may generally be described as multipolar in nature (vida infra). In practice, both enantiomers performed similarly as agonists of the TRPV1 receptor, leading to the conclusion that the receptor-bound form is that in which the side chains extend in the same plane as the amide and the aryl ring (conformation A, Figure 9) rather than projecting orthogonally (conformation B, Figure 9). The conformation of anilides can be influenced by introduction of a fluorine atom ortho to the anilide N−H. This phenomenon has been used to understand the preferred topology of 43, a potent spiroindane-based antagonist of the calcitonin gene-related peptide (CGRP) receptor that holds potential as a therapeutic for the prevention of migraine

Figure 9. Potential bound conformers of the two enantiomers of 42.

Figure 10. Calculated relative energy levels of conformational isomers of α-fluoroamides.

attacks.30 Although 43 exhibited reasonable oral bioavailability, the pharmacokinetic profile of more potent derivatives was less than optimal, with poor oral absorption attributed to a high polar surface area (PSA). In an attempt to address this problem, attention was focused on the design of isosteres of the central amide moiety that would offer reduced PSA as a means of facilitating oral absorption.30a A torsion plot indicated a preference for the amide and spiroindane core moieties to be coplanar with the lowest calculated energies observed at 0° and 180°, leading to two topologically distinct conformations. In order to investigate the optimal binding topology, a fluorine atom was introduced independently at each of the two positions ortho to the N−H. In this way, complementary topologies that are preferred by ∼3 kcal/mol are underpinned by a combination of repulsive steric and electrostatic interactions between the CO and fluorine atom and an attractive electrostatic association between the N−H and fluorine. The isomers 44 and 45 demonstrated a 10-fold difference in CGRP receptor affinity, where the extended conformation represented by 44 is favored by the receptor, confirmed by the synthesis of fused ring compounds constrained to unambiguous topology.31 The role of 4-(R)-hydroxyproline (4R-Hyp, 47) in enhancing the thermal stability of collagen, the major structural protein in connective tissue, has been clarified through studies based on the 8320



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Figure 11. Conformational preferences of N-acetylproline derivatives 51−54.

proline analogs 4-(R)-fluoroproline (48) and 4-(S)-fluoroproline (49).13f,g,32 The thermal stability of the characteristic triple helix polypeptide structure of collagen, where each peptide chain comprises repetitive sequences of X-Y-Gly (where X is often proline (46) and Y is frequently either 46 or 47), was originally attributed to a network of H-bond interactions between the 4R-Hyp hydroxyl moiety and bridging water molecules.13f,33 However, studies with 48 and 49 have led to the conclusion that the effect of hydroxylation of 46 on the structure of collagen has its origin in a stereoelectronic phenomenon associated with the electron-withdrawing properties of the hydroxyl substituent and the effect that this modification exerts on the conformational preference of the pyrrolidine ring.14,32

the conformation of the amide bond. The Cγ-exo pucker that is favored by 53 allows the amide CO lone pair of electrons to donate into the π* orbital of the ester carbonyl (n → π* interaction). This stabilizes the trans (Z) isomer and pyramidalizes the ester moiety, thereby inducing incipient chirality in a functionality that is typically planar and achiral.34,35 In contrast, the Cγ-endo conformation preferred by 54 weakens this interaction considerably, leading to a higher population of the cis (E) amide topology. This effect is observed in the single crystal X-ray structures of N-acetyl-4R-Hyp and N-acetyl-4S-Hyp and related compounds.35 The structure−activity relationships (SARs) associated with inhibitors of the serine proteases dipeptidyl dipeptidase IV (DPP-4) and fibroblast activation protein (FAP) provide striking examples of the importance of fluorine absolute stereochemistry on inhibitory potency in the context of proline derivatives.39 In the DPP-4 inhibitor series 55−58, 4-(S)-isomer 56 is over 450-fold more potent than 4-(R)-isomer 57, while the difluoro homologue 58 demonstrated potency comparable to both 56 and the unsubstituted parent 55 (Table 2).38a This

The circular dichroism (CD) spectrum of (46-48-Gly)10 is identical to the CD spectra of the collagen-based polypeptides (46-46-Gly)10 and (46-47-Gly)10, consistent with this polypeptide also adopting a triple-helix structure where both substituted proline moieties adopt a Cγ-exo pucker.32f However, molecular modeling studies of (46-46-Gly)10 have suggested that the first proline prefers to adopt a Cγ-endo pucker, leading to the hypothesis that installing 49 at this position would promote triple helix formation while 48 would not.32h,34 This postulate has been confirmed experimentally with the preparation of (49-46-Gly)10, which assembles into a triple helix structure, and (48-46-Gly)10, which does not.32h,i 4,4-Difluoroproline (50) does not substitute for 47 in collagen-related peptides, since it does not induce conformational bias.14 DFT calculations indicate that N-acetylproline methyl ester (51) inherently favors the Cγ-endo conformation by 0.41 kcal/mol, resulting in a 2:1 ratio at room temperature, while the 4R-Hyp derivative 52 prefers the Cγ-exo conformation by 0.48 kcal/mol (Figure 11).32c,d For N-acetyl-4-(R)-fluoroproline methyl ester (53) the Cγ-exo conformation is preferred by 0.48 kcal/mol, mimicking 4R-Hyp; for N-acetyl-4-(S)-fluoroproline methyl ester (54) the Cγ-endo conformation is more stable by 0.61 kcal/mol.32c,d,j The Cγ-endo conformation is also the preferred conformer of proline and is observed in the single crystal X-ray structure of Boc-4-(S)fluoroproline.32j The effects of fluorine on the conformation of proline can include the establishment of a favorable gauche interaction between the fluorine and the N−CO moiety that also influences

Table 2. SAR Associated with Isoleucine-Based Inhibitors of DPP-4

compd

R

R′

DPP-4 IC50 (nM)

55 56 57 58

H H F F

H F H F

1.5 0.6 290 0.8

structure−activity pattern is reproduced in the series of FAP inhibitors 59−62 where the 4-(S)-isomer 60 is over 300-fold more potent than its 4-(R)-enantiomer 61 (Table 3).38b The significant inhibitory activity in each series associated with the difluoro analogs 58 and 62 indicates an absence of fluorinederived steric interference in the S1 pocket of the enzymes to which the pyrrolidine moiety binds.38b It is hypothesized that the 4-(R)- and 4-(S)-fluorine atoms preferentially stabilize a single conformational pucker of the pyrrolidine ring, presumably the Cγ-endo by analogy with 53 and 54, since both proline and its 8321



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With a 2′-fluoro α-configuration, the fluorinated nucleoside analog 65 (Figure 13) is unable to sample the south conformation

Table 3. SAR Associated with FAP Inhibitors

compd

R

R′

FAP IC50 (nM)

59 60 61 62

H H F F

H F H F

10.3 3.3 1000 3.2

4,4-difluoro analog exhibit only a limited conformational preference for an Cγ-exo- or Cγ-endo-pucker.13g Studies of 3-fluoroprolinamide derivatives 63 and 64 also revealed conformational bias as a consequence of the 3-fluorine substitution and which are the reverse of those preferred by 4-fluorine substitution (Figure 12). In the solid state both amide moieties adopt the (Z)-trans configuration; however, the conformation of the pyrrolidine rings is distinct.36 In each case the pyrrolidine ring is stabilized by a gauche interaction between the F atom, which adopts an axial orientation, and the amide moiety (Figure 12). For 63 the Cγ-exo:Cγ-endo pucker ratio was predicted to be 2:1 and, consistent with this, 63 crystallized in the Cγ-exo conformation. In this case the preference for the Cγ-exo conformation is influenced by unfavorable steric and electronic interactions between the F atom and the ester moiety. For 64 the Cγ-endo conformation prevailed in solution to the extent of 97% based on the ≤1 Hz coupling constant measured between the C-2 and C-3 protons in the 1H NMR spectrum. These conformational preferences were expressed when 63 and 64 were incorporated into short polypeptides.32e,36,37 (e) Conformational Bias Induced by Fluorine in Nucleoside Analogs. The small size of fluorine has encouraged its introduction into the ribose ring of nucleoside analogs where its effects on conformational preferences have been extensively explored.23b,c,39 These effects are prominent but complex, dictated by the interplay of several influences: anomeric effects, gauche interactions, dipole−dipole interactions, the antiperiplanar effect, and interactions between the F atom and the base.40 As a consequence, the outcome on conformation is dependent on the particular substitution pattern of an individual nucleoside analog. Ribose analogs with 2′-fluoro substitution in an α-configuration favor a north conformation, while conformational bias with a 2′-fluoro-β configuration is more varied and less stringent.40

Figure 13. Preferred conformations of fluorinated nucleoside analogs 65−67.

that is thought to be predominantly recognized by HIV-1 reverse transcriptase.4b,40−42 Consistent with this, the adenosine derivative 65 is inactive as an inhibitor of HIV-1 replication in cell culture, although poor phosphorylation to the triphosphate cannot be ruled out as a contributory factor. In contrast, the β-configured 2′-fluoro isomer 66 exhibits a strong preference for the south pucker, and this is reinforced by the 3′α-F substituent in 67. Both compounds inhibit HIV-1 replication in cell culture, with measured EC50 values of 4.4 μM for 66 and 0.72 μM for 67.4b,41c,43 The 2′-α-fluororibose derivatives 68 and 69 also strongly favor a north conformation, with both gauche and anomeric effects contributing to the conformational stability of 68.44 On the basis of DFT calculations, the C-4′ aminomethyl derivative 69 is predicted to adopt a north conformation that is stabilized by both a gauche effect and an intramolecular C−H to fluorine interaction, suggested to be a H-bond, from one of the protons of the aminomethylene moiety (Figure 14). In contrast, the 2′-OMe analog of 69 is predicted to prefer a south conformation, while the 2′-OH variant prefers the north pucker but is flexible enough to adopt the south conformation.44b The introduction of a 2′-β-fluoro substituent to both 70 and 72 enhances the preference for a north pucker; the population of this conformation for 70, 71, 72, and 73 is 58%, 100%, 28%, and

Figure 12. Structure and solid state conformation of 3-fluoroproline derivatives 63 and 64 and the predicted equilibria. 8322



Perspective

more readily than 77 has been attributed to an unfavorable interaction between the C-6 aza and 3′-fluorine atoms.41c The effects of fluorine substitution on ribose conformational preferences in nucleoside analogs may be muted in more complex settings, as illustrated by a study of clevudine (79, L-FMAU), a 2′-β-fluoro nucleoside analog in the L-ribose series (Figure 15). Clevudine (79) potently inhibits hepatitis B virus (HBV) replication in cell culture (EC50 = 100 nM) by virtue of being metabolized in cells to the 5′-triphosphate that is a potent inhibitor of the HBV DNA polymerase.47 In the single crystal X-ray structure, 79 adopts a south pucker due to favorable gauche effects between the 2′-fluorine and ring oxygen atoms, as well as between the 3′-OH and ring oxygen atom, a conformation that places the fluorine and OH in an antiperiplanar relationship. Although this conformation is thought to be the more stable, docking of the triphosphate of 79 in a model of the HBV polymerase active site suggested that the north pucker would be preferred by the enzyme. This observation prompted a computational study using Monte Carlo methodology which revealed that the south and north conformations of the triphosphate of 78 have similar energies and that in this case altering the ribose ring pucker would incur only a minimal energetic cost.48 While this change in conformation was postulated to be able to occur when bound to the acitve site of HBV polymerase because of an inherent flexibility, the modeling studies suggested that the unique conformational aspects of the triphosphate of 78 would prevent it from being incorporated into the growing DNA chain because the 5′-triphosphate moiety would not be presented in a geometry that would allow an efficient reaction with the 3′-OH of the primer strand. Thus, the triphosphate of 78 was predicted to bind to the active site in conjunction with a conformational change that allows it to effectively interfere with access of the natural substrate.

Figure 14. 2′-α-F ribose derivatives 68 and 69 and the intramolecular C−F···H interaction considered to contribute to the stability of the north conformation of 69.

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APPLICATIONS OF THE ELECTRON-WITHDRAWING PROPERTIES OF FLUORINE Because of its strong electronegativity, fluorine is a powerful tool for modulating the pKa of proximal functionality and the electron density of aromatic and heteroaromatic rings.1c,i,49 The pKa of an ionizable center in a drug molecule changes the lipophilicity profile (because of the pH dependence of the distribution coefficient, D), which influences solubility, permeability, and protein binding. Changes in pKa can manifest as changes in potency, selectivity, toxicity, and pharmacokinetic (PK) properties including absorption, distribution, metabolism, and excretion (ADME).50 Strategic deployment of fluorine can be guided by a generalized set of rules used to predict the effect of fluorine on the pKa of proximal functionality. (a) Fluorine in Aliphatic Systems. In simple linear systems the influence of successive fluorine substitution is additive, as summarized by the data presented in Table 4. For example, each fluorine substitution in ethylamine reduces the pKa by 1.6−1.7 units. CF3CH2NH2 is so weakly basic that this moiety has been exploited as an amide isostere in both peptidometics and small molecules.51 The influence of fluorine on amine basicity decreases as carbon homologation increases the distance between the fluorine atom and the amine, with estimates of the effect summarized in Table 5.51b,c There is a −1.7 pKa unit change for each fluorine atom introduced to the β-carbon while the effect is muted to differences of −0.7, −0.3, and −0.1 pKa units at the γ-, δ-, and ε-carbon atoms, respectively, which provides for a precise means of modulating the basicity of an alkylamine.

Figure 15. Compound 79 and its north and south conformers.

58%, respectively.41b,c It is postulated that for 73, an intramolecular interaction between the 2′-flourine atom and the C−H of the purine C-8 atom may influence the ribose ring conformation.45 The 2′,3′-difluorouridine derivative 74 exhibits a bias toward the south conformation such that in solution this pucker represents ∼80% of the equilibrium conformer population.41b,c The effect of 3′-fluoro substitution on ribose ring conformation has also been studied in the context of 75−78. The 3′-α-fluorothymidine derivative 75 adopts the south conformation in solution to the extent of 95% and 97% in CD3OD and DMSO, respectively. This preference also applies to the 2′-H, 2′-OH, 2′-NH2, and 2′-N3 homologues with alternative base moieties, where this pucker is preferred to an extent of ≥94%.46 3′-β-Fluorouridine 76 prefers the north pucker almost exclusively based on NMR analysis, although ab initio calculations suggest that the south conformation should be more stable. Difluorinated nucleoside analogs 77 and 78 prefer a north conformation in both the single crystal X-ray structures and in solution. Analog 77 is considered to be stabilized by a weak F···H interaction between the C-3′-F and the C-6 H of the base. The observation that 78 samples the south conformation 8323



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Table 4. Effect of Proximal Fluorine Substitution on the pKa of Acids, Alcohols, and Amines acid

pKa

alcohol

pKa

amine

pKa

CH3CO2H CH2FCO2H CHF2CO2H CF3CO2H CH3CH2CO2H CF3CH2CO2H C6H5CO2H C6F5CO2H

4.8 2.6 1.3 0.5 4.9 3.1 4.2 1.7

CH3CH2OH (CH3)2CHOH (CH3)3COH CF3CH2OH (CF3)2CHOH (CF3)3COH C6H5OH C6F5OH

15.9 17.1 19.0 12.4 9.3 5.4 10.0 5.5

CH3CH2NH2 CH2FCH2NH2 CHF2CH2NH2 CF3CH2NH2

10.7 9.0 7.3 5.7

Table 5. Effect of Carbon Chain Homologation on Basicity of Fluorinated Amines

n

position

ΔpKa

1 2 3 4

β-F γ-F δ-F ε-F

−1.7 −0.7 −0.3 −0.1

Analogous to aliphatic amines, the influence of fluorine on the basicity of unstrained cyclic amines is generally additive in nature but with the added consideration that the electron-withdrawing effect of the σ-acceptor is exerted in both directions around the connectivity of the ring. The predicted and experimental effects for piperidine are summarized in Table 6.51a Thus, Table 6. Calculated and Experimental Effects of Fluorine Substitution on the pKa of Piperidine

minimized if the pKa of the piperidine was between 6.5 and 8.0, prompting an examination of electron-withdrawing substituents designed to reduce the basicity of the N atom to within the targeted range. The cyclopropyl- and the β-fluoroethylsubstituted compounds 81 and 82, respectively, met the targeted basicity criteria but 81 was associated with time-dependent CYP 450 inhibition, a known liability of cyclopropylamines, while 82 liberated the highly toxic fluoroacetic acid in vivo as the result of metabolic N-dealkylation.53 The solution to this problem was to install the fluorine atom in the piperidine ring, and the more basic (pKa = 7.6) axial isomer MK-0731 (83) was ultimately selected for clinical evaluation over the less basic (pKa = 6.6) equatorial homologue 84.52b In a related series of compounds, in which the amine moiety was appended to the C-2 carbon of the dihydropyrrole ring, the problem of P-gp efflux was also resolved by attenuating the basic nature of the molecule (Table 7). In this case, two fluorine atoms β to the amine provided the optimal combination of properties, demonstrated by the SAR associated with 85−88.54 A pKa of 7 minimized P-gp efflux while maintaining potency, a compromise exemplified by the two complementary topologies represented by 86 and 88. A similar strategy was adopted in a quest for selective inhibitors of platelet-derived growth factor receptor (PDGFR), a member of the receptor tyrosine kinase inhibitor class 3 family.55 The enzyme exists in two forms, PDGFRα and PDGFRβ, encoded by different genes. Inhibitors of PDGFRβ were sought in an effort to provide tools to illuminate the role of the enzyme in tumor growth, angiogenesis, and fibrosis. Guided by a homology model of the kinase, the piperidine 89 was identified as a refined lead with an IC50 of 3 nM against PDGFRβ in a cell-based assay, 10-fold selectivity over KDR and >100-fold over cKit and cFMS. However, despite apparently good passive

3-fluoropiperidine is predicted to be a weaker base than piperidine by 2.0 pKa units based on Δ of −1.7 exerted via the proximal β-fluorine atom bonding network and an additional Δ of −0.3 induced by the δ-disposed F atom; this predicted value is close to the measured difference of −1.8 pKa units. In this case, the difference between predicted and experimental values may be attributed to the conformational preferences discussed earlier in which the fluorine atom adopts an axial disposition in the protonated form but prefers an equatorial projection when the amine is unprotonated.26a In the axial orientation, the dipole of the C−F bond is aligned in an antiparallel fashion with the N+−H bond, stabilizing the protonated state and resulting in a higher measured pKa than predicted; equatorially oriented fluorine atoms in a piperidine ring lower amine pKa more significantly. This phenomenon has been exploited in drug design.52 An example of the effects of F substitution on basicity is provided by the kinesin spindle protein (KSP) inhibitor 80.52 This KSP inhibitor was explored as a potential treatment for taxane-refractory solid tumors, but P-glycoprotein (P-gp) efflux of the compound limited its efficacy.52b Structure−activity studies revealed that recognition by the transporter was 8324



Perspective

quinuclidine ring was accomplished with fluorinated analog 93; this structural modification altered the efflux ratio in the desired direction but at the expense of markedly reduced intrinsic potency toward the receptor.

Table 7. Potency, Physical Properties, and Off-Target Activities of the KSP Inhibitors 85−88

As part of an examination of the SARs associated with piperazine 94 as a 5HT1D agonist with potential utility for the relief of migraine attacks, piperidine 95 was prepared and found to possess a similar in vitro profile (Figure 16A).52a However, while 94 was rapidly absorbed in rats, 95 was associated with poor oral bioavailability attributed to increased basicity, which prompted synthesis of 4-F-piperidine 96. This compound (Ymax = 58%) largely recapitulated the in vitro profile of 95, which is a partial agonist at 5HT1D receptor (Ymax = 63%). The pKa of 96 is similar to that of piperazine 94, and this compound exhibited much improved absorption in the rat compared to 95. Interestingly, on the basis of the similarity of calculated electrostatic potential contours of 4-fluoro-1,4-dimethylpiperidine and 1,4-dimethylpiperazine, an isosteric relationship between the rings was suggested a priori in which the electron density of fluorine mimics the lone pair of electrons on the piperazine N atom (Figure 16B). However, other factors appear to underlie the SARs associated with this series of 5HT1D agonists. Attenuating the basicity of nitrogen atoms is also a useful tactic for reducing inhibition of the cardiac K+ channel encoded by the human ether-a-go-go-related gene (hERG). This has been demonstrated in a series of 4-aminopiperidine-based bacterial topoisomerase inhibitors, exemplified by 97, that demonstrated activity toward Staphylococcus aureus (S. aureus) and Mycobacterium tuberculosis (Mtb).57 In this exercise, attention was focused on modulating the more basic secondary amine (pKa = 8.27) by introducing fluorine at the 3 position of the piperidine ring. Introduction of the fluorine at the 3 position would also affect the tertiary amine which has a measured pKa of 5.75. This was indeed the case, with the axial fluorinecontaining cis-3R,4S isomer 98 exhibiting much reduced basicity at both amines and attenuated hERG inhibition. The cis-3S,4R isomer 99 presented a similar profile. One enantiomer of the trans isomer, in which the fluorine is equatorially disposed, showed a further reduction in the pKa values for both amines (pKa of 6.66 and 4.07) and a 3-fold reduced affinity for hERG compared to the progenitor 97. The 3-fluoropiperidine moiety was subsequently incorporated into the related compound 100 which focused on amplifying the Mtb inhibition observed with 97.57c Replacing the 4′-OH of neomycin (101) with fluorine was explored as an approach to avoiding 4′-OH modification by O-adenyltransferase (ANT(4′)) enzymes as well as for its potential to protect the adjacent 3′-OH against modification by O-phosphotransferase (APH(3′)) enzymes.58 These pathways have developed in bacteria to negate the activity of the aminoglycoside class of antibiotic. The synthetic approach was designed to access the equatorial and axial isomers 102 and 103, respectively, both of which demonstrated activity comparable to

permeability and aqueous solubility (>1 mg/mL), the compound showed poor exposure in the rat following oral dosing, which was attributed to a combination of a moderate metabolic clearance rate (clearance of 33 mL min−1 kg−1 following a 1 mpk intravenous (iv) dose) and P-gp efflux, with a ratio of 5.7 determined in vitro. Attention was focused on attenuating the basicity of the piperidine to reduce P-gp recognition, and trans (equatorial fluorine) and cis (axial fluorine) derivatives 90 and 91, respectively, were prepared in racemic form. Interestingly, in this example there was little difference between the measured pKa values of the equatorial and axially disposed isomers. In contrast to predictions based on precedent, the passive permeability of both 90 and 91 was reduced but both exhibited a reduced efflux ratio and slightly lower iv clearance (24 and 28 mL min−1 kg−1, respectively) in the rat. The trans isomer 90 offered a 2- to 3-fold improved area under the curve (AUC) following oral dosing to rats, while the AUC for the cis isomer was similar to the parent molecule.55 Poor brain penetration attributed to P-gp efflux was found with the α7 nicotinic acetylcholine receptor agonist 92, a compound of interest for treating the cognitive deficits and negative symptoms of schizophrenia.56 Attenuation of the basicity of the 8325



Perspective

Figure 16. (A) Structure−activity relationships and physical property data associated with 5HT1D agonists 94−96. (B) Proposed structural mimicry between N-benzylpiperazine and N-benzyl-4-fluoropiperidine.

101 in susceptible S. aureus, Klebsiella pneumonia (K. pneumonia), and Escherichia coli (E. coli). This result indicates that substitution was accommodated by the A-site of the bacterial 30S ribosomal subunit. As anticipated from the structural change, protection against two forms of ANT(4′) was observed, with axial F isomer 103 retaining activity toward two strains of Pseudomonas aeruginosa expressing APH(3′), while both 101 and 102 were inactive.58 This observation was attributed to the lower nucleophilicity of the 4′-OH in 103 which reduced the propensity for phosphate transfer, a strategy that was preserved in the amide derivative 104. An X-ray cocrystal structure of 104 bound to the bacterial ribosome 30S subunit A site revealed a binding mode analogous to that of the 4′-equatorial hydroxyl compound. Similar to the contacts made by the 4′-H in the 4′-OH prototype, the axial fluorine atom of 104 was in close contact with C-2, N-3, and C-4 of guanosine 1491.58 The distances between the fluorine atom and C-2, N-3, and C-4 are 2.7 Å, 2.6 Å, and 2.8 Å, respectively. These are less than the sum of the van der Waals radii which for the individual atoms are 1.47 Å for F, 1.7 Å for C, and 1.55 Å for N. It is believed that the kidney toxicity associated with aminoglycosides is related to the number of basic amine groups that mediate binding to the ribosomal A site; however, these basic amines are an essential element of the antibacterial pharmacophore.59 The modified aminoglycoside antibiotic 3′,4′,3‴,4‴-tetradeoxyneomycin (105) and its N1-(S)-hydroxyaminobutyric amide derivative 106 exhibit improved antibacterial activity compared to the hydroxylated progenitor. This benefit is due to an absence of hydroxyl substituents which, as part of resistance pathways discussed above, are targeted by modifying enzymes for acetylation.59 The introduction of electronwithdrawing substituents proximal to the hydroxylated butyramide provided an opportunity to modulate the pKa of the terminal amine in an effort to achieve a balance between antibacterial activity and avoidance of renal toxicity. Hydroxylated analog 107, monofluoro analog 108, and difluoro analog 109 provided a successive reduction of the amine pKa of up to 3 log10. All three compounds demonstrated minimum inhibitory concentration (MIC) levels against resistant strains which were superior to 105 but comparable to 106.59 However, difluoro derivative 109 was 2-fold less toxic toward the HK2 human kidney cell line, suggestive of improved tolerance, while 107 and 108 were comparable to 106 in this assay. (b) Fluorine in Aromatic/Heteroaromatic Systems. Substitution of aromatic and heteroaromatic rings with electronwithdrawing substituents is a common practice in drug discovery

that can provide important insight into SAR, modulate the physical properties of other substituents, and potentially interfere with oxidative metabolic processes.60−62 Although fluorine is frequently 8326



Perspective

date and are referred to as super acceptor groups.61 The utility of the SF5 moiety (entry 10), stereochemically distinct from CF3, is increasing as new synthetic methods address its installation (vide infra).61b,c The inductive effects of fluorine on the acidity of phenol (pKa = 9.81) are significant. For example, 2,6-difluorophenol (pKa = 7.12) performs as a lipophilic isostere of a carboxylic acid in analogs of GABA (34), where additional functional mimicry was anticipated because of similarity between C−F and the CO moiety of the acid.63 Both 110 and 111 are competitive inhibitors of GABA aminotransferase with measured Ki values of 11 and 6.3 mM, respectively.63 This concept was subsequently extended to inhibitors of rat lens aldose reductase where a 2,6difluorophenol effectively substituted for an acetic acid moiety. This is exemplified by comparison of the prototype 112 with 113, and 114 with 115−119. Potency was increased in each matched pair and could be further enhanced in the sulfonamide series 115−119.64

deployed as an aryl substituent, its high inductive electronwithdrawing effect (σF = 0.45) is counterbalanced by a resonance component (σR = −0.39), resulting in a net para-effect (σP) of 0.06 (Table 8, entry 1). As a result, fluorine as an aryl substituent demonstrates an overall electron-withdrawing effect that is less than chlorine (Table 8, entry 2).60 However, in systems such as CF3 where π-resonance is not possible the inductive electronwithdrawing effect of fluorine can exert a powerful influence on substituents. This is exemplified by comparison of the matched pairs CH3/CF3 (Table 8, entries 1 and 6), P(O)(CH2CH2CH3)2/ P(O)(CF2CF2CF3)2 (Table 8, entries 4 and 23), SO2CH3/ SO2CF3 (Table 8, entries 11 and 17), and SO2CH2CH3/ SO2CF2CF3 (Table 8, entries 13 and 18) where the σP values differ by 0.71, 0.60, 0.24, and 0.35, respectively. These modifications are accompanied by significant increases in lipophilicity in the cases of CF3 (0.74) and SO2CF3 (2.18). Interestingly, the poly-fluorinated sulfonated sulfilimine (Table 8, entry 24) and sulfoximine (Table 8, entry 25) are the most powerful electron-withdrawing substituents described to

The acidity of phenolic functionality in drug candidates can be optimized for target binding through incorporation of fluorine. The introduction of a fluorine atom ortho to the para-disposed phenol of 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) inhibitor 120, which emerged from a systematic study of bisphenols, afforded inhibitor 121 (Table 9).65 This compound demonstrated enhanced potency and selectivity for the enzyme versus 17β-HSD2, an enzyme that catalyzes the reverse reaction, the oxidation of estradiol. This phenol moiety is considered to mimic the phenol of estrogen which forms H-bonding interactions with Glu282 and His221 in the active site of the enzyme. An alternative binding mode for 121 was also postulated based on a docking experiment, one in which 121 binds to a site between the steroid and cofactor binding sites and interacts with the nicotinamide moiety.65c In this series, the introduction of a second fluorine atom at the other ortho position gave 122 which 8327



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Table 8. Hammett Substituent Constants Describing the Electron-Withdrawing Properties of Fluorinated Substituents in Aromatic/Heteroaromatic Rings1e,60,61

IMPACT OF FLUORINATION ON DRUG POTENCY, PERMEABILITY, METABOLISM, AND PHARMACOKINETIC PROPERTIES The strategic incorporation of fluorine to improve drug potency has become increasingly prevalent in recent decades.1a,b,f,66 Fluorine substitution can enhance potency and impact target selectivity by affecting pKa, modulating conformation, hydrophobic interactions and lipophilicity, or a combination of these properties.1i Fluorine has also been used as a tool to address issues associated with drug metabolism.62 Fluorine substitution as a direct replacement for a labile hydrogen atom/s (e.g., Ar−H → Ar−F, CH3 → CF3) can reduce metabolism including when installed on an aromatic group that is prone to oxidation (e.g., electron-rich phenyl rings). Fluorine is frequently included in bioisosteres of carbonyl-containing moieties. Additionally, fluorine can modulate lipophilicity and restrict conformation, which may afford improved metabolic stability. Although the strategic introduction of fluorine has been used to overcome issues associated with metabolic stability, there are occasions where it may be detrimental. These include changes in primary pharmacology due to increased lipophilicity and adjustments of pharmacokinetic properties. In order to overcome these limitations, pharmacological activity and metabolic stability are typically optimized in parallel. (a) Influence of Fluorine on Potency. Human kinesin Eg5 is an attractive target for the treatment of cancer due to its role in establishing bipolar spindles. Inhibition of this enzyme causes mitotic arrest, which triggers cell apoptosis in certain tumors. The dihydropyrimidine-2(1H)-thione derivative monastrol (123) was identified as an allosteric inhibitor of the ATPase activity of Eg5, with activity residing in the (S)-enantiomer and drug−target interactions confirmed by solving an X-ray structure of the cocrystal.67 Efforts toward expanding the SAR associated with 123 led to the identification of a closely related series of 5-aroyl dihydydropyrimidine derivatives demonstrating superior potency for which enzyme inhibitory activity surprisingly resided in the (R)-enantiomer. The prototype of this series was (R)mon97 (124), and optimization afforded fluorastrol (125) as a compound with 5-fold improved growth inhibition toward five tumor and nontumor cell lines.68 X-ray cocrystal structures obtained from racemic 124 and 125 with human Eg5 contained only the (R)-isomers bound to the enzyme.69 Some differences in the dispositions between 123 and 124 in the binding pocket of the enzyme were noted. For 124, the key interactions are established by the N-3-H of the dihydropyrimidine ring which forms a H-bond with the main chain carbonyl moiety of Gly117, the thioxo moiety which interacts with Glu118, and the phenol O−H which engages the protein via H-bonding to the main chain carbonyl of Glu118, the main chain amino moiety of Ala133 and side chain nitrogen atom of Arg119.68,69 Difluoro homologue 125 binds in the same orientation, but the 2-thioxo moiety projects into the solvent-exposed region of the protein and interacts with two H2O molecules (Figure 17).68a The pyrimidine N-3-H engages the carbonyl oxygen atom of Gly117, and the phenol interacts with Glu118, Arg119, Ala133 in a similar fashion to 124. The enhanced potency of 125 appears to be a function of three multipolar interactions involving the aryl-4-fluoro atom: one to the guanidinium side chain of Arg221, one with the backbone carbonyl moiety of Gly217, and one with the amide of Ala218.68a Furthermore, it was hypothesized that sandwich π−π stacking of the fluorinated phenyl ring with the salt bridge formed between Glu116 and Arg221 (not shown in Figure 17) is

Table 9. SARs Associated with Potency and Selectivity for 17β-HSD1 Inhibitors 120−122

compd

R

R′

17β-HSD1 IC50 (nM)

17β-HSD2 IC50 (nM)

selectivity factor

120 121 122

H F F

H H F

69 8 56

1950 940 312

28 118 6

Perspective

exhibited reduced potency and selectivity, suggesting that the acidity of the phenol may have an optimal range for binding to the enzyme. 8328



Perspective

enhanced because of the electron-withdrawing effect of the two fluorine atoms and the interactions of the 4′-fluorine with the protein.

Figure 18. Interactions of the 4-fluorobenzyl moiety of 128 with thrombin.

A survey of X-ray crystal structures in the Cambridge Structural Database (CSD) and the Protein Data Bank (PDB) found that a close interaction of fluorine with an amide carbonyl is not uncommon; the C−F to CO angle typically lies between 100° and 140°, where a smaller angle is associated with a closer contact distance. The energy of this interaction has been estimated to range from 0.2 to 0.38 kcal mol−1.70c In the context of a related but intrinsically more potent series evaluated in racemic form, the introduction of a 4-fluoro substituent provided 131 as the most potent thrombin inhibitor (Ki = 5 nM). However, although 131 is 13-fold more potent than its unsubstituted hydrogen analog 130, the 4-chloro (132) and 4-methoxy (133) homologues also demonstrate enhanced potency. X-ray cocrystals of 132 and 133 indicate that these substituents are not sufficiently close to the Asn98 CO to establish productive interactions, suggesting that the enhancement in potency observed with 131 is not the result of a productive fluorine to CO interaction.70d

A systematic fluorine scan was used to establish SARs associated with 126, an inhibitor of thrombin probed as a racemate. Specifically, aryl substituents of the N-benzylimide moiety were surveyed, with the data on select compounds presented in Table 10.70 The results highlighted the positive effect of a 4-fluoro substituent, with 128 exhibiting 5-fold enhanced potency over progenitor 126 and the 3-fluoro isomer 127. An X-ray cocrystal of 128 with the enzyme showed a close contact between the fluorine atom and both the CO carbon of Asn98 (3.5 Å, F approaches CO at an angle of 96°) and the α-C-H of this residue (2.1 Å at an angle of 157°) (Figure 18).

The capacity of covalently bound fluorine to act as a H-bond acceptor is controversial and an issue of continuing debate.14,15 Although this type of interaction is considered to be weak in nature, numerous instances of C−F···H−X close contacts have been observed crystallographically, and searches of the PDB and CSD have played a prominent role in efforts to describe the character of C−F···H−X contacts.1b,14,15,71 However, a predictive model for F···H contact distance has remained elusive. Model systems have also been used to gain insight into the underlying mechanics of the C−F···H−X interaction, with some cases supportive of a role for fluorine as a weak hydrogen-bond acceptor (HBA).15,72 Nuances in the interpretation of NMR 1H JF,H(X) coupling, infrared (IR) vibrational shifts, and computational modeling complicate study of this weak interaction.71d,73 It is generally agreed that the HBA capacity of fluorine is significantly weaker than that of oxygen, and it is often difficult to separate the role of C−F···H−X bonding from other more dominant forces. Fluorine to hydrogen interactions are described in the literature as either hydrogen bonding or multipolar in nature. In deference to the ambiguity around fluorine and H-bonding, we have chosen to depict fluorine to hydrogen contacts as multipolar interactions, marked as a red arrow.

Figure 17. Key drug−target interactions between 125 and Eg5.

Table 10. SARs Associated with Substituted N-BenzylimideBased Thrombin Inhibitors

compd

R

R′

R″

Ki (μM)

126 127 128 129

H F H F

H H F H

H H H F

0.27 0.360 0.057 0.590 8329



Perspective

Figure 19. Factor VIIa inhibitor 134, noting interactions observed in the X-ray cocrystal structure, and its progenitor 135.

the pKa of the aniline N−H by an estimated 3 units, thereby strengthening the interaction with Gly216. The gem-difluoro substituents may be regarded as mimics of the shape and dipole of the sulfone oxygen atoms.75b Similar SARs were observed in the analogous series of aminopyrazinone-based thrombin inhibitors summarized in Table 11.75d

An aryl C−F moiety has been explored as a lipophilic isostere of a pyridone carbonyl moiety in the context of factor VIIa and thrombin inhibitors (Figure 19).74 The fluorophenyl derivative 134 was designed as a factor VIIa inhibitor based on the potent activity associated with the pyridone 135. A cocrystal structure revealed the anticipated close contact between the fluorine atom of 134 and the N−H of Gly216.74,75 The distance between the fluorine atom of 134 and the nitrogen atom of Gly216 in the X-ray cocrystal structure was measured to be 3.4 Å, an identical distance to that between the aniline nitrogen atom of 134 and the carbonyl oxygen of Gly216.74a A similar tactic was successful in the design of thrombin inhibitors based on the prototype 136 (RWJ-445167), a potent compound (Ki = 4 nM) that advanced into clinical evaluation but suffered from unacceptably low exposure following oral dosing.75 This compound engages Gly216 of thrombin via the sulfonamide N−H (H-bond donor (HBD)) and the pyridone CO (HBA). The fluorophenyl derivatives 137 and 138 were designed as more lipophilic inhibitors in an effort to address the poor bioavailability observed with 136. Both compounds are potent inhibitors of thrombin, with Ki values measured as 8.6 nM for 137 and 1.2 nM for 138. X-ray cocrystal data for these compounds indicated a close contact between the aryl fluorine atom and the backbone nitrogen atom of Gly216, in both cases measured as 3.17 Å; based on a N−H bond length of 1.03 Å, a F···H distance of 2.14 Å was calculated.75b,c

Table 11. SARs Associated with the Series of Thrombin Inhibitors 141−145

compd

X

R

thrombin Ki (nM)

141 142 143 144 145

CH CH CH N N

H F CH3 H F

0.8 0.1 1.1 0.27 0.042

The principle of engaging the N−H of Gly216 of factor Xa by a fluorine atom in an inhibitor was extended to the design of fluoro-substituted indazole derivatives, represented by 147 as an optimized compound (Ki = 15.9 nM).76 In this molecule, the fused fluorophenyl ring substitutes for the pyrazole carboxamide moiety found in razaxaban (146).76 The importance of the fluorine substituent was established via the matched-pairs analysis compiled in Table 12. A 60-fold improvement in potency was measured for 149 and 151 compared to 148 and 150, respectively, which represents an energy difference of ∼2.4 kcal mol−1. An X-ray cocrystal of factor Xa-bound 147 confirmed a close interaction between the C-7 fluorine atom and the backbone N−H of Gly216. The measured F···N distance

The gem-difluoroethyl moiety markedly influences potency in the chloro-substituted series of thrombin inhibitors represented by 137. Replacing the gem-difluoroethyl moiety with either a gem-dimethyl or a cyclopropyl ring, compounds 139 and 140, respectively, resulted in a 7-fold decrease in thrombin inhibition. Replacing the pyridine of 137 with a phenyl reduced potency by a similar amount.75 In this context, the gem-difluoroethyl moiety not only blocks metabolism at the benzylic position but functions as a lipophilic isostere of the sulfonamide moiety of 136. The inductive electron withdrawal by the fluorine atoms reduces 8330



Perspective

of 2.90 Å is within H-bonding range and is comparable to the 3.04 Å contact observed between the Gly216 N−H and the carboxamide oxygen of 146.76

Table 12. SARs Associated with the Series of Indazole-Based Factor Xa Inhibitors 148−151

compd

X

n

factor Xa Ki (nM)

148 149 150 151

H F H F

1 1 2 2

>14400 223 6850 124

difference in potency. Although this initial survey failed to identify compounds that were significantly more potent, the observation of the F···H−N interaction inspired the design of a series of compounds in which C−F was replaced by a carbonyl moiety, a powerful H-bond acceptor. Pyridone 155 inhibited HCV NS5B with an IC50 of 17 nM and prevented replication of an HCV genotype 1b replicon with EC50 = 300 nM. X-ray cocrystal data for an analogue confirmed an H-bond between the pyridone CO and the backbone N−H of Tyr448.77 The MAP kinase 1 (MEK1) inhibitor 156 is noncompetitive with ATP, and in the X-ray cocrystal structure of the ternary complex the molecule occupied a pocket adjacent to the adenosine triphosphate (ATP) binding site. In this structure the glycol moiety lies close to the MgATP phosphates.78 The conformation of the molecule was oriented via an intramolecular H-bond between the aniline N−H and the adjacent amide carbonyl. This arrangement projected the iodophenyl moiety orthogonal to the plane of the core and established a halogen bond between the iodine atom and the carbonyl of Val127. The 4-fluoro atom of the benzamide established close contacts with the N−H moieties of Ser212 and Val211, interactions described as H-bonds (Figure 21).78 Optimization of 156 led to CH4987655 (RO4987655, 157), a selective MEK1 inhibitor (IC50 = 5.2 nM), in which the steric bulk added at the 5 position of the benzamide is designed to block hydrolysis of the amide moiety and improve orally bioavailability. Compound 157 demonstrated slow dissociation kinetics from the enzyme and potent antitumor activity in vivo, alone or in combination, and was selected for clinical evaluation.78d The X-ray cocrystal of the ternary complex of 157 with MEK1 and adenylylimidodiphosphate revealed binding interactions similar to 156; the benzamide 4-fluoro atom is proximal to the backbone NHs of Val211 and Ser212. An alternative path of optimization explored replacement of the C−F bond with an amide carbonyl. This ultimately afforded 158, which inhibits MEK1 with IC50 = 38 nM. X-ray cocrystal data with a prototype molecule suggested inhibitor interaction with the NHs of Val211 and Ser212.78a,b Aromatic fluorides are common structural elements in inhibitors of DPP-4, the serine protease that degrades glucaogon-like peptide

A high-throughput screening campaign identified indole-2carboxylic acid derivative 152 as a moderately potent inhibitor of hepatitis C virus (HCV) NS5B polymerase (IC50 = 0.9 μM) that was, however, inactive in a cell-based replicon assay.77 A cocrystal of 152 with the polymerase revealed that drug−target association was dominated by hydrophobic interactions and that the carboxylic acid did not establish specific contacts with the enzyme. Interestingly, the fluorine atom appeared to engage the N−H of Tyr448 with a 2.6 Å distance between the fluorine atom of 152 and the nitrogen atom (Figure 20). The carbon atom adjacent to fluorine approached the carbonyl oxygen of Ile447 with a distance of just 2.9 Å, less than the sum of the van der Waals radii and suggestive of a productive interaction. The importance of the F···H−N interaction was underscored by an extensive SAR survey that established the importance of the fluorine atom and its regiochemistry. This is emphasized by comparing the matched pairs 153 and 154 which reflect a 40-fold

Figure 20. Key interactions between 152 and HCV NS5B polymerase. 8331



Perspective

Figure 21. Key drug−target interactions between 156 and MEK1 and the structures of analogs 157 and 158.

Figure 22. Key interactions between 159 and DPP-4.

1 (GLP-1), and cocrystal structures have revealed close interactions between the fluorine atoms and H-bond donors of the enyzme.79,80 The 2,4,5-trifluorophenyl moiety of sitagliptin (159) is an important structural element that conferred an improved safety profile compared to the 2,5-difluoro homologue.80b The 2,4,5-trifluorophenyl ring occupies the S1 subsite of the enzyme, and the 2-fluoro atom is close to the side chain N-Hs of Arg125 and Asn710, with the distance between this fluorine atom and the nitrogen atom of Asn710 measured as 3.2 Å (Figure 22).80a,k The primary amine of 159 forms salt bridges with Glu205 and Glu206 in the S2 pocket, the amide oxygen atom engages Tyr547 via the intermediacy of a water molecule, and the pyrazolopyridine heterocycle occupies an extended S2 site, stacking against Phe347 with both of the vicinal triazole nitrogen atoms interacting with H2O molecules. The SARs around the fluorophenyl moiety are consistent with a role in drug−target interactions based on the imperfect comparison of 160 and 161 where the absence of the 2-fluorine results in an approximate 5-fold erosion of potency.80a The 2,5-difluorophenyl moiety is preserved in more refined molecules including omarigliptin (163), a long acting compound with PK properties suitable for once-weekly dosing in humans, and its predecessor 162.80k,l An X-ray cocrystal structure of fluoroomarigliptin (164) revealed similar interactions between the 2-fluoro atom and the enzyme.80l That the 2-fluorophenyl moiety can function as a surrogate for a more conventional H-bond acceptor is illustrated by the comparable inhibitory potency exhibited by 165 and the amide 166, one of a series of three matched pairs where the DPP-4

inhibitory constants were similar.80h An X-ray cocrystal structure of an analogue of 165 confirmed the proximity of the aryl fluorine atom to the side chains of Asn710 and Arg125.80h

Interestingly, 167 is representative of a related series of DPP-4 inhibitors based on α-amino acid derivatives that bound to the enzyme with a reversed binding mode compared to the β-amino derivatives described above with the pyrrolidine ring occupying the S1 pocket.80b,h The fluoroolefin 168 was examined as a potential isostere of the amide moiety of 167 as part of a probe of the selectivity and PK properties of this molecule.80e The matched pair of analogues 167 and 168 exhibit similar DPP-4 inhibitory 8332



Perspective

activity, establishing an effective isosteric relationship between the fluoroolefin and the amide, with a cocrystal of the more potent homologue 169 revealing that both series bound to the enzyme in similar modes. In the X-ray cocrystal structure, the fluorine atom of 169 interacted with the side chain N-Hs of both Asn710 and Arg125, interactions described as H-bonding in nature.80e aconitase, a component of the tricarboxylic acid cycle.62a,83 A dose of 2−10 mg/kg in humans is lethal, with a lowest observed effect level of 0.1 mg/kg.62a As a defensive posture to prevent the entry of fluoroacetate into its own citric acid (Krebs) cycle, S. cattleya expresses a fluoroacetyl-CoA-specific thioesterase (FIK) that hydrolyzes fluoroacetyl-CoA with a one-million-fold selectivity over acetyl-CoA. The differences in rate for the two substrates (Kcat/KM = 5 × x107 vs 30 M−1 s−1) represent a remarkable discrimination between substrates that differ by a single fluorine for hydrogen substitution in such a small molecule. Initial explanations for the selectivity were derived from X-ray cocrystal data which suggested several influencing factors: enzyme recognition via close interactions of the fluorine atom with the N-Hs of Gly69 and Arg120; enhanced electrophilicity of fluoroacetate compared to acetate; restricted access of H2O to the active site; and reduced interaction between the CO moiety and the enzyme (Figure 23). However, more recent studies of the process have observed a kinetic isotope effect only for the (R)-H atom of fluoroacetic acid. This suggests that the basis for selectivity resides in the catalytic activity of the enzyme, where abstraction of the (R)-H to afford a ketene intermediate occurs 104-fold more rapidly in fluoroacetate than acetate.84

The introduction of an R-methyl substituent at C-8 of the triazolopyrazine moiety of 159 afforded 170 which demonstrated 4-fold improved DPP-4 inhibitory potency with activity sensitive to the absolute configuration at this center, since the S-isomer was 20-fold weaker.80c Expansion of this substituent to benzyl (171) and 4-fluorobenzyl (172) moieties resulted in further gains in potency that exhibited heightened sensitivity to the chirality at C-8. The high potency of 172 was attributed to an interaction between the 4-fluorophenyl and Ser630 mediated by a water molecule and described as H-bonding in nature based on an X-ray cocrystal structure of 172 with DPP-4.80c

(b) Influence of Fluorine on Permeability. Permeability (Pe), the ability of a molecule to pass through a cell or cellular membrane, is a major consideration during the process of lead optimization of orally bioavailable compounds.85 Drug and metabolite permeability plays a significant role in ADME, in addition to affecting other considerations such as toxicity, clinical dosing, and formulations. Permeability is typically measured as the rate (10−6 cm/s) at which a molecule is passively diffused or actively transported (uptake/efflux) across membranes. Passive permeability is most affected by two parameters: molecular size (increase correlates with decreased Pe) and lipophilicity (increase correlates with increased Pe). However, active transport is organ specific and considerably more difficult to predict. Fluorine is often used to influence permeability by way of modulation of lipophilicity, association with pendent H-bond donors, or reduction of amine basicity. Most orally administered drugs have log P values, a common measure of lipophilicity of between 1 and 5.86 The log P of a neutral molecule is typically increased upon the addition of an aryl or vinyl fluorine but, conversely, often decreases with alkyl fluorination.87 Monofluorination of a terminal alkyl methyl group typically leads to a larger reduction in lipophilicity than trifluorination, while difluorination is predicted to have a similar effect on log P as monofluorination.87b These effects are primarily due to participation of arylfluorines in resonance electron donation and the large dipole associated with carbon−fluorine bonds in fluoroalkanes.87b

A gem-difluoromethyl moiety was conceived as a functional replacement for the ketone carbonyl in V-10,367 (173), a compound that mimics the FKBP12-binding portion of FK506 and exhibits neurotrophic activity in vitro.81 The difluoroamide 174 inhibits FKBP12-mediated rotamase activity with a Ki of 19 nM, comparable in potency to 173. An X-ray cocrystal of 173 with FKBP12 indicated that the ketone oxygen atom engaged the Tyr26 hydroxyl group through an electrostatic interaction at a distance of 3.4 Å. With 174, this ketone functionality is absent but one of the F atoms approximates the carbonyl-Tyr26 hydroxyl interaction with the distance between the F and phenol oxygen atom determined to be 3.18 Å. This was described as at least an electrostatic and possibly a H-bonding interaction, while the other fluorine atom was within van der Waals contact of two hydrogen atoms of Phe36, with distances measured as 3.0 and 3.3 Å, respectively.81 Fluoroacetic acid (175) is a naturally occurring toxin produced predominantly in the plant kingdom but that is also synthesized by the soil microbe Streptomyces cattleya (S. cattleya).82 Conversion of fluoroacetate to (−)-erythro-2-fluorocitrate (2R,3R) (176) in mammals leads to potent inhibition of the enzyme 8333



Perspective

Figure 23. Interactions of 175 in the fluoroacetyl-CoA-specific thioesterase active site.

For amine-containing molecules, the log D may increase upon the introduction of proximal fluorine atoms if basicity is reduced. Permeability across a confluent Caco-2 cell layer for the two closely related series of factor Xa inhibitors 177−180 and 181−183 was found to be improved via fluorine substitution of the hydrogen atom ortho to the anilide N−H, although the differences between 179 and 180 are small (Table 13).88

was not affected, while a reduction was observed in 3 of 42 cases. For the remaining 12 examples, permeability was increased by only 0.1 or 0.2 log10 cm s−1, not considered significant by the authors for the purpose of discussion. Since this study was conducted on a relatively small number of examples, it was suggested that although it cannot be concluded definitively that an ortho-fluorine atom will enhance permeability, it would nevertheless be a useful strategy to explore. A useful strategy to address a range of developability problems, including membrane permeability and CNS penetration, is to introduce intramolecular H-bonds or electrostatic interactions. This approach was explored in the context of improving the delivery of β-site amyloid precursor protein cleaving enzyme 1 (BACE-1) inhibitors (Table 14).90,91 Prototype BACE-1

Table 13. Effect on Caco-2 Permeability of Substituents Ortho to an Anilide in Two Series of Factor Xa Inhibitors

Table 14. Enzyme Inhibition, Porcine Renal Epithelial (LLC-PK1) Cell Permeability, and Efflux Ratios for the Series of BACE-1 Inhibitors 184−187

The increase in permeability observed for fluoro-substituted compounds 178, 180, and 182, compared to their matched partners 177, 179, and 181, may be due to an electrostatic interaction between the fluorine atom and pendent N−H that effectively masks its H-bond donor properties, thereby enhancing membrane permeability.15a,89 Lending support to this hypothesis, the permeability of the ortho-nitrile 183 is significantly reduced compared to both 181 and 182. This is presumably a reflection of the increased H-bond donor capacity of the N−H that cannot be satisfied in an intramolecular fashion because of the geometrical constraints associated with the linear nitrile substituent. However, an effect of the increased PSA associated with this substituent may be a contributory factor. Anilides and benzamides are common motifs in drug design and are particularly prevalent in kinase inhibitors. Fluorine substitution has been studied as a tool to improve the permeability of these chemotypes. In a matched-pairs analysis of this phenomenon, 12 of 27 pairs of anilide derivatives (Figure 24A) exhibited effective parallel artificial membrane permeability assay (PAMPA) permeability that improved by ≥0.3 log10 cm s−1 when the orthohydrogen atom was replaced by a fluorine atom. The same substitution in benzamides (Figure 24B) improved permeability in 9 of 15 cases.15a This effect was not considered to be a function of lipophilicity since the meta-fluoro isomers did not demonstrate enhanced permeability. In 6 of the 42 cases examined, permeability

inhibitor 184 was subject to efficient efflux by cell lines expressing either rat or human P-gp which contributed to the low brain levels attained by this compound following oral administration to rats.91 Hydrogen-bonding plays a role in compound recognition by P-gp, and to address this problem focus was placed on perturbing the electronics of the acetamide N−H.91,92 Analogues 185−187 all demonstrated improved efflux ratios, where the two fluorinated derivatives 186 and 187 performed as effectively as MeO-substituted 184.91 Passive membrane permeability and P-gp efflux ratios were improved by an analogous tactical approach applied to a series of CNS-penetrant bradykinin B1 antagonists that were explored as potential agents for the relief of pain (Table 15).93 The introduction of fluorine atoms into the acetamide moiety of 188 resulted in compounds 189−191 which demonstrated improved profiles. 8334



Perspective

Table 15. Bradykinin B1 Binding, Passive Permeability, P-gp Efflux Ratios in LLC-PK1 Cells, and H-Bond Strength for the Series of Aminocyclopropanecarboxamide-Based Antagonists 188−191

compd

R

hBK1 Ki (nM)

passive permeability Papp (10−6 cm/s)

P-gp efflux ratio in LLC-PK1 cells expressing human MDR1

HBA (log) strength of CO

188 189 190 191

CH3 CHF2 CF3 CF3CF2

0.93 0.40 0.57 1.6

21 311 28 31

8.6 3.2 2.3 1.4

2.12 1.63 1.39 1.35

This was attributed to a reduction in the strength of the terminal amide carbonyl to function as an H-bond acceptor, although an intramolecular interaction between the fluorine atoms and the N−H may also contribute. The N-terminal amide N−H of the tachykinin hNK2 receptor antagonist 192 was deemed critical for potency but was believed to contribute to the observed poor membrane permeability assessed in Caco-2 cells (Table 16).94 The introduction of a

Caco-2 cell layer assay. Although chloro-substituted analog 193 offered an advantage over the prototype, fluoro derivative 194 was found to be superior. By way of comparison, the orthofluorine substituent in 194 improved permeability similarly to pyridine 196, which exhibits 2- to 3-fold higher transit in both assays compared to its matched pair, the phenyl derivative 195.94 The principle of introducing a fluorine proximal to an N−H to improve permeability was also effective in a series of aminoisoindole-based BACE-1 inhibitors exemplified by prototype 197 (Table 17).95 The physical properties associated with the embedded amidine moiety of this chemotype impeded effective CNS penetration, resulting in poor reduction of β-amyloid peptides in mouse brain. The introduction of a fluorine atom at C-4 afforded 198, which exhibited modestly improved potency toward BACE-1 but demonstrated markedly changed physical properties. While the pKa of 198 is 8.4, this is reduced to 7.1 for the prototype 197, which is associated with an increase in the log D (determined by liquid chromatography). These changes were associated with increased Caco-2 cell permeability and a reduced efflux ratio, effects attributed to the formation of a weak interaction between the amidine NH2 and fluorine moieties characterized as a H-bond. Calculations indicated a less negative solvation energy for 198 compared to isomers where the fluorine is located meta or para to the amidine moiety. This results in an overall effect that was interpreted as shielding of the polar amine moiety from solvent. Interestingly, the resolved S-isomer of 198 displayed improved permeability over the racemate (Papp = 22 × 10−6 cm/s) and an efflux ratio of 1.9. This compound demonstrated robust lowering of β-amyloid peptides in the brain of C57BL/6 mice following oral administration. (c) Influence of Fluorine on Metabolism and PK Properties. In part because of the strength of the C−F bond, fluorine is often used to overcome issues associated with poor metabolic stability, where it may be deployed as the direct

Table 16. Human NK2 Receptor Binding Affinity and Permeability in Caco-2 and PAMPA Assays for the Series of Antagonists 192−196 Derived from Phenylalanine

halogen atom at the ortho-position, designed to interact with the N−H, resulted in improved permeability in both a PAMPA and

Table 17. Enzyme Inhibition, Physical Property Attributes, and Caco-2 Cell Permeability Properties of BACE-1 Inhibitors 197 and 198

compd

R

BACE-1 IC50 (nM)

pKa

log D

Papp apical to basolateral through Caco-2 cells (10−6 cm/s)

efflux ratio Papp(B−A)/Papp(A−B) in Caco-2 cells

197 198

H F

500 158

8.4 7.1

0.7 0.9

3.4 12

12 3.1

8335



Perspective

replacement for a metabolically labile H atom in both aromatic (Ar−H → Ar−F) and aliphatic settings (CH3 → CHF2, CF3).62a,96 In addition, electron-rich phenyl and heterocyclic rings or olefins that are prone to oxidation may exhibit enhanced metabolic stability after the installation of fluorine atoms or fluorine-containing substituents. Fluorine has been used as an isostere of the carbonyl moiety that eliminates susceptibility to reductive processes and may improve metabolic stability due to the effect of fluorine on modulating lipophilicity and restricting conformation. The judicious deployment of a fluorinated substituent in the triazolopyrazine moiety of the DPP-4 inhibitor 159 was instrumental in the identification of compounds with oral bioavailability (Table 18).80a,b,m The ethyl-substituted analog

Table 19. Effects on Potency and Metabolic Stability of Replacing CH3 by CF3 in the tert-Butyl Substituents of NK1 (202, 203) and TRPV1 (204, 205) Receptor Antagonists

Table 18. SARs and PK Associated with Analogues of 159

compd

R′

IC50 for inhibition of DPP-4 (nM)

199 200 201 159

Et CF2H CF2CF3 CF3

37 29 71 18

a

Cl in the rat (mLmin−1 kg−1)a

oral bioavailability in the rat (%)a

70 66 58 60

2 39 61 76

The same structural modification was examined in the context of the endothelin antagonist bosentan (210) and the CCR9 antagonist vercirnon (212).99b In each case, the metabolic stability of the Cp-CF3 derivatives 211 and 213 in RLM was increased, with clearance declining from 37 to 100-fold more potent in mouse distal-site tumor models.112 Fluorination of a benzodioxole methylene was also used to probe the role of metabolites of MDMA (238), a commonly abused drug known as ecstasy, in the expression of psychotomimetic effects and toxicity.114 The difluoro analogues of 238 and MDA (240), 239 and 241, respectively, were designed to avoid oxidation of the benzodioxole methylene. However, while DFMDA (239) exhibited serotonin transporter (SERT) affinity between that of 238 and 240, neither 239 (dosed up to 120 mg) nor 241 (dosed up to 250 mg) showed a significant physiological effect in humans at doses that both 238 and 240 showed activity.114 The lactone moiety of the quinoline-based alkaloid camptothecin (242), considered to be critical for antitumor activity, is subject to hydrolytic ring opening under physiological conditions which limits its therapeutic utility.115 Replacing the lactone CO

context, since detailed studies of the 3,3-difluoropyrrolidine derivatives 229 and 230 (PF-00734200) do not appear to undergo metabolic activation in this fashion.105 However, the HIV-1 nucleotide-competing reverse transcriptase inhibitor 231 exhibits a short half-life in rat and human liver microsomes, with the 3,3-difluoropyrrolidine identified as the metabolically labile site, although the degradation pathway was not explicitly determined.106 The metabolic lability of aryl alkyl ethers can also be improved by fluorination, exemplified by the robust stability of some 2,2,2trifluoroethoxy ethers in RLM, although the antiarrhythmic agent flecainide (232) is subject to oxidative dealkylation at the least sterically hindered ether.107,108 An interesting but perhaps underutilized application of fluorine to resolve problematic metabolism is in the context of the benzodioxole moiety. This is a prevalent structural element in natural products that presents challenges in drug discovery due to the potential for mechanismdependent inhibition of CYP 450 enzymes and subsequent generation of catechols.109 CYP 450 enzymes oxidize the methylene to a carbene that binds tightly to the Fe atom and forms a slowly dissociating metabolic-intermediate (MI) complex that exhibits a characteristic absorption maximum at 455 nm.110 This phenomenon is a potential source of drug−drug interactions and is exemplified by paroxetine (233), a compound

Figure 25. Metabolic activation pathway for the 3-fluoropyrrolidine element of 228. 8338



Perspective

natural product. The β-fluoro-substituted derivative 244 demonstrated greatly improved hydrolytic stability following incubation in H2O at 37 °C and pH 7.4 (96% of 244 remained after 6 h compared to just 43% of 242).115

■

SF5 FUNCTIONAL GROUP The SF5 moiety offers interesting properties and an unusual shape that are of potential advantage in drug design, but applications have been limited, largely a function of poor synthetic accessibility.116 Lack of awareness may also play a role despite the fact that SF5 data were included in the Craig plot of σ constants versus π values for aromatic ring substituents.117 Recent advances in the introduction of SF5 into building blocks and molecules are beginning to change both perceptions and awareness of this moiety, and SF5 is being exploited more frequently in drug optimization campaigns. DSM-265 (246), an inhibitor of Plasmodium falciparum (Pf) dihydroorotate dehydrogenase (DHODH), is the first SF5-containing molecule to enter clinical trials, where it is undergoing evaluation for its potential as an antimalarial agent.118

The lipophilicity and electron-withdrawing properties of the SF5 moiety are both characterized as higher than those of CF3 but but with lipophilicity less than tert-butyl.99a For example, in the context of 210 which has a measured log D of 0.99, the log D values of the SF5 and CF3 analogues are 0.37 and 0.14, respectively, a trend recapitulated with 212.99a However, the shape of SF5 is markedly different, offering octahedral geometry and a larger size (>2-fold) when compared to tetrahedral CF3, although it is still two-fold smaller than a tert-butyl substituent (Table 23).99b The electron density patterns of the two moieties also differ, with SF5 presenting a pyramidal shape while CF3 displays an inverted cone.

with an isosteric C−F moiety was explored as a potential solution to this problem with both the α-fluoro (243) and β-fluoro (244) derivatives prepared. The potency of 244 toward a series of tumor cell lines in vitro was superior to 243 but inferior to 242 (Table 22). This was remedied by the cyclohexyl-substituted homologue 245 that demonstrated in vitro antitumor properties comparable to the

Table 23. Comparison of the Physical Chemistry Attributes of CF3 and SF5

Table 22. In Vitro Antitumor Activity of 242−245 in Three Cell Lines

compd

A549 IC50 (μM)

242 243 244 245

0.65 46.2 9.95 0.71

CF3

SF5

σP σR σI π electronegativity pKa of anilinium ion van der Waals volume (cm3 mol−1)

0.54 0.12 1.09 1.09 3.36 2.94 20.5

0.68 0.11 0.55 1.51 3.65 2.37 49.2 (calcd)

These properties can translate into differences in biological activity as illustrated by the matched pairs of cannabinoid receptor B1 ligands 247 and 248 where the SF5 derivative is 2-fold more potent than the CF3 analogue, a consistent observation in this series.61c,119 Direct comparisons of SF5 with CF3 have also been made in the serotonin reuptake inhibitors and receptor modulators fluoxetine (249), fenfluramine (251), and norfenfluramine (253).120 In the matched pair 249 and 250, the latter demonstrated reduced binding to 5HT2a and 5HT2c receptors compared to 249 with no effect on the affinity for the 5HT2b

MDA-MB-435 IC50 (μM) HCT-116 IC50 (μM) 0.45 >100 58.33 0.41

attribute

0.07 50.91 6.35 0.07 8339



Perspective

cytotoxicity compared to 262 and other analogues, while retaining good membrane permeability.122

subtype. However, substitution of CF3 by SF5 in the fenfluramine series produced more pronounced effects with 252 exhibiting an almost 10-fold increased affinity for 5HT2b and 5HT6 receptors compared to 251 while affinity for the 5HT2c receptor was also increased but more modestly.120 Interestingly, this observation did not extend to the norfenfluramine matched pair, since the receptor binding profile of 254 was similar to 253.

A matched pairs analysis compared the effect on biological potency and developability parameters of analogues of the endothelin antagonist 210 and the CCR9 antagonist 212 in which the tert-butyl substituent of each was replaced with several moieties including the SF5 and CF3, compounds 264−267.99b In the series based on 210, the CF3 (264) and SF5 (265) analogues performed similarly as endothelin A antagonists but were 10-fold less potent than 210. In the CCR-9 antagonists, biological assay at a concentration of 50 nM revealed comparabale activity for 210, 266, and 267. In both series, the SF5 and CF3 substituents conferred improved metabolic stability compared to the tertbutyl prototype.

In a series of thymidine phosphorylase inhibitors, the SF5 analog 256 exhibited a modest 3-fold potency advantage over the CF3 homologue 255. However, in a series of dopamine D3 antagonists based on a triazole chemotype, compounds 257−260 which are racemic, no significant differences were seen in hERG inhibition nor the overall profiles at the D2 and D3 receptors (Table 24).121 The calculated PSA for the four Table 24. Functional Dopamine D2 and D3 pKi, hERG IC50, PSA, and ACD log D Data for 257−260

compd

R

dopamine D2 functional pKi

dopamine D3 functional pKi

hERG pIC50

PSA (Å2)

ACD log D

257 258 259 260

3-CF3 3-SF5 4-CF3 4-SF5

7.5 6.9 6.9 6.8

9.3 8.9 9.3 9.1

5.5 6.0 6.0 6.3

60 60 60 60

1.8 2.5 21 2.5

Although not yet explored in the context of drug design, alkyl SF5 derivatives have been shown by experimental and theoretical methods to exert an effect on conformational preferences that has been attributed to a combination of steric and stereoelectronic effects.123 In alcohols substituted at the β-position with fluorinated moieties, an SF5 showed a higher barrier to rotation than fluorinated methyl substituents. An SF5 substituent also influences the conformation of alkyl chains in a fashion that reflects a volume that is smaller than that of a tertbutyl moiety.

■

FLUORINE IN POSITRON EMISSION TOMOGRAPHY The three pillars of survival for a drug in phase 2 clinical trials have been defined as (1) the demonstration of exposure of a drug candidate at the target site of action over a desired period of time, (2) the determination of the binding of a drug to its pharmacological target based on its mode of action, and (3) the expression of a pharmacological effect that is commensurate with the demonstrated target exposure and target binding.124 Positron emission tomography (PET) imaging has been viewed as a useful, noninvasive translational technique that has the potential to assess target engagement, particularly in the CNS, providing insight into the first two pillars described above, in cases where a suitable and effective labeled ligand can be developed.3e,f,125

compounds was identical, but the calculated log D of the SF5 derivatives was higher than the CF3 analogues, reflecting their individual π coefficients. In an analysis of the effects of aryl substituent variation in the series of trypanothione reductase inhibitors 261−263, explored as potential antiprotozoal agents, the SF5 derivative 263 performed similarly to the CF3 analogue, and both offered a modest advantage over tert-butyl analog 261.122 This SAR was rationalized by the modeling of 263 in the active site of the enzyme and attributed to a combination of size and electronic effects on the aryl ring. However, 263 demonstrated 2-fold reduced 8340



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particularly with respect to the labeling element. Given that the PET detector does not distinguish the source of the signal, in vivo generation of labeled metabolites has the potential to confuse the interpretation of results. For CNS applications, the PET tracer must offer good blood−brain barrier penetration with low nonspecific binding if the signal-to-noise ratio is to be practically useful. For the latter, physical properties play a significant role with less lipophilic compounds associated with lower nonspecific binding. For good CNS penetration a log P between 1.5 and 2.5 or a log D of 1−3 appears to be optimal. However, predicting the performance of PET tracers remains a challenge despite the development of methods to assess the nonspecific affinity of candidate molecules.3g,128 [18F]-Fluorine is produced most commonly in a cyclotron, and although it has become the most widely used PET imaging radionuclide, generating [18F]-labeled radioligands in high specific activity can present significant synthetic chemistry challenges. One factor is the microscale nature of the synthesis, where [18F]-fluoride is the limiting reagent, typically present in only pico- to nanomolar concentrations while the labeling precursor is used in large excess. This can lead to low radiochemical yields due to competing side reactions, which are not always seen with the corresponding nonradioactive (19F) model reaction. The term radiochemical yield (RCY) commonly refers to the isolated amount of purified labeled compound divided by the amount of radioactivity originally present at the start of the synthesis. Preparative expediency is of the essence given the challenge associated with the half-life of [18F]. Successful synthesis of a useful PET radioligand requires generation of the [18F] source, most typically fluoride, followed by its incorporation into the molecule under investigation, purification, and formulation for iv injection into a living animal. The entire preparative processes must be completed in less than 3 h. With these unique challenges, methodologies that incorporate [18F]-fluorine into a molecule rapidly, efficiently, and as late in the synthetic pathway as possible have generated a significant amount of published research over the past decade.129 This section will summarize some the recent advances in radiochemical synthetic approaches to incorporate [18F]-fluorine into important drugs, druglike molecules, and PET radioligands used to answer key questions in drug discovery and development. [18F]-Fluorine is generated in a cyclotron yielding either 18 [ F]-fluoride or [18F]-fluorine gas ([18F]-F2). [18F]-Fluoride is produced by proton bombardment of liquid isotopically enriched [18O]-H2O via the 18O(p,n)18F nuclear reaction.127 The [18F]fluoride produced in this fashion must be further processed to provide a species capable of incorporating 18F into molecules. Since fluoride is poorly nucleophilic because of its high solvation in water, it is separated from the [18O]-H2O and any metal impurities generated from the cyclotron target by use of a quaternary ammonium chloride polymer (QMA) or Chromafix PS-HCO3 (filled with quaternary ammonium bicarbonate polymers) anion exchange resin cartridge. The [18F]-fluoride is eluted from the resin cartridge with an alkali carbonate in CH3CN/H2O and mixed with a phase transfer catalyst like Kryptofix 2.2.2 (K.2.2.2) or a crown ether. Alternatively, [18F]-fluoride can be eluted from the resin cartridge with tetrabutylammonium hydroxide to generate [18F]-tetrabutylammonium fluoride ([18F]-TBAF). The final step is an azeotropic drying process to remove any residual water from the elution mixture. A recently developed method reduces byproducts and preparation time, thereby increasing the overall radiochemical

There is considerable value in determining that a specific molecular target is being adequately interrogated by a drug candidate in vivo in situations where measuring a pharmacodynamic effect is not straightforward. Determining target engagement allows a level of rational decision making with respect to identifying doses to explore in efficacy studies or assessing the potential of a molecule and its intrinsic mechanism to affect a particular disease state. For example, the demonstration of target engagement in the absence of the anticipated pharmacological effect may invalidate the underlying hypothesis allowing early termination of a drug candidate. Molecular imaging tools may increase the speed at which a drug reaches clinical trials, provide information about the safety profile, and enrich the potential treatment population, all issues of considerable interest in the drug discovery and development process.126 Molecular imaging is a term used for the combination of imaging technologies that provide both anatomic and functional information around a biological target. By combining techniques like X-ray or magnetic resonance imaging (MRI), which provide anatomical information, with functional imaging techniques such as PET, we now have the ability to monitor biological processes in living systems at the molecular level. PET has been described as a new kind of “precision pharmacology”.126 PET ligands confer the ability to quantitatively monitor in vivo molecular events in real time, which can address several questions that are critical to the drug discovery process in humans.126 This includes target engagement, dose−receptor occupancy relationships, metabolism, biodistribution, and the use of PET radioligands as biomarkers for patient enrichment strategies, thereby enabling personalized medicine treatment and offering more effective clinical trials.126 PET imaging involves the synthetic incorporation of shortlived radionuclides into biologically active molecules, and once inside the body, decay of the radioisotope emits a positron that travels a short distance until it combines with an electron in an event described as annihilation. This generates two photons each with an energy of 511 keV that travel collinearly in opposite directions. Detection of the γ radiation generated during the annihilation allows for well-defined images at the molecular level as a function of time. The radioactive isotope of fluorine, [18F]fluorine, is particularly suited to the development of PET ligands due in part to its 109.7 min half-life. This half-life permits time for multistep syntheses with incorporation of 18F into complex molecules while offering the convenience of same-day imaging. [18F]-Fluorine has low positron energy, traveling only short distances before annihilation occurs, which affords better spatial resolution for imaging than other PET radionuclides while offering lower overall radiation exposure to a patient.127 Several factors are of importance to the design of effective PET ligands in general, and some are associated more specifically with the use of [18F]-fluorine.3g,128 Fundamentally, the ligand under consideration must have functionality that facilitates incorporation of a labeled atom. Syntheses are typically designed specifically for late-stage introduction in deference to the short half-lives of PET radionuclides. Introduction of fluorine often presents a significant synthetic challenge, although the demand for [18F]-fluorinated ligands has, in part, stimulated the development of new methodology and the design of prosthetic moieties for label incorporation as a frequently utilized option.129 With respect to ligand pharmacology, high potency and selectivity (typically >100-fold) toward the target of interest are paramount, with affinity constants in the low to subnanomolar range preferred. Adequate metabolic stability is also of importance, 8341



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Scheme 1. Synthesis of 269

yields by using ionic liquids (tetrabutylammonium methanesulfonate or 1-butyl-3-methylimidazolium triflate) dissolved in MeOH to release [18F]-fluoride from a Chromafix PS-HCO3 polymer cartridge.130 In this process, azeotropic drying requires only 1 min, resulting in a 10% increase of available radioactivity for subsequent [18F]-fluorine labeling. A second approach relies upon trapping aqueous [18F]-fluoride on a strong anionexchange cartridge and then eluting the radionuclide with an anhydrous solution of [K.2.2.2]OH− in CH3CN which can be used directly for nucleophilic fluorination reactions without azeotropic drying, facilitating the automated production of [18F]-labeled products.131 [18F]-F2(g) is obtained from the nuclear reaction of 18O(p,n)18F through the addition of F2 (0.2%) to an enriched [18O]O2 gas target.127 The specific activity (SA) for the “intarget” protocol is in the range of 27 mCi/mmol (1 GBq/mmol), which is significantly lower than the specific activities reached using [18F]-fluoride which can be as high as 150 Ci/μmol (5500 GBq/μmol).132 SA is defined as the amount of isolated activity of a purified [18F] PET tracer divided by the mass (or molar amount) of the total sum of all radioactive and nonradioactive species present within that isolated PET tracer.133 When PET imaging studies are performed, the amount of radioactivity to be administered is fixed within a range to ensure high quality images are obtained. Thus, the SA determines the molar amount of PET radioligand that has been delivered during these imaging studies. In some applications, the [18F] radiopharmaceutical may be a toxic molecule or is being used to visualize a low density receptors in the brain.134 In these cases, obtaining a [18F] product in high SA is key to obtaining a high quality PET image without producing a pharmacodynamic effect, toxicological event, or saturating the target of interest. Improvements that allow the generation of [18F]-F2(g) with a higher SA have focused on a “post-target” method using [18F]-fluoromethane to produce [18F]-F2(g) via an electrical discharge chamber using a 18F/19F exchange reaction and a low amount of the F2 carrier gas. High SA [18F]-fluoromethane can be produced by nucleophilic substitution of CH3I with [18F]-fluoride, and this “post-target” synthesis generates [18F]-F2(g) with specific activities up to 15 Ci/μmol (555 GBq/μmol).135 (a) Aromatic Nucleophilic Fluorination Using [18F]Fluoride. The generation of high specific activity [18F]-fluoride and [18F]-F2(g) has facilitated an increase in the design and application of 18F-labeled PET radioligands procured via either nucleophilic or electrophilic fluorination processes. Fluorinated arenes are a widely used motif in drug design, and methods to label aromatic rings with [18F] offer convenient access to PET ligands. Nucleophilic fluorination of aromatic rings typically involves the displacement of a leaving group facilitated either by an electron-deficient substituent attached to the aromatic ring or, more commonly, by an exogenous catalyst. Numerous important [18F]-PET radioligands and [18F]labeled drugs have been produced using the latter approach, and these methodologies dominate the literature with good progress made recently toward developing processes to label unactivated and electron-rich aromatic systems.129,136−144

The most common procedures are summarized in Figure 26, although not all of these have been exemplified using [18F]fluoride, and some may require additional optimization for successful application.129 (b) Nucleophilic Fluorination of Alkyl Groups. The incorporation of [18F]-fluorine at a specific aliphatic position on a molecule has traditionally been accomplished via a nucleophilic displacement of a halide or sulfonate using [18F]-fluoride in a polar aprotic solvent such as DMSO, DMF, or acetonitrile.129e,145 Numerous useful [18F]-PET radioligands have been generated using this approach, with the clinically approved [18F]-florbetapir (269), marketed as Amyvid, a representative example (Scheme 1). [18F]Florbetapir is the first Food and Drug Administration (FDA) approved PET tracer for quantifying amyloid plaque burden in humans during therapy or as a patient enrichment biomarker for designing more effective clinical trials.146 The synthesis of 269 involves a two-step process in which the tosyl group of 268 is displaced by [18F]-flouride at elevated temperature using K.2.2.2/[18F]KF followed by removal of the Boc protecting group.147 Improved methods for aliphatic nucleophilic fluorination with [18F]-fluoride include the use of microwave, ionic liquids, the inclusion of tertiary alcohols, and the use of fluorous solid-phase methods that allows rapid purification of [18F]labeled products.148 The enantioslective preparation of [18F]-fluorohydrins, via epoxide ring opening using the chiral catalysts [18F]-(R,R)(salen)CoF and [18F]-(R,R,R,R)-(linked salen)Co2OTsF, comprises an effective approach to several clinically validated PET radioligands that can be prepared under mild conditions (MeCN, 50 °C, 20 min).149 These conditions are compatible with basesensitive functional groups, epimerizable stereocenters, and nitrogen-rich motifs, as exemplified by the enantioselective [18F]-labeling of [18F]-(S)-THK-5105 (270), a potential PET ligand for assessing tau pathology, and [18F]-FETNIM (271), a promising PET radioligand for quantifying tumor hypoxia in vivo.150 This type of process opens up an avenue to explore the relationship between stereochemistry and imaging properties of important PET radioligands, an area unexplored because of the difficulty of chiral [18F]-fluorination reactions.

A late-stage process for the direct [18F]-labeling of benzylic C−H bonds using [18F]-fluoride exploits a Mn(salen)OTs species as a fluoride transfer catalyst. This process has enabled the radiolabeling of drug molecules and common chemical intermediates that can be used as building blocks to generate [18F]-labeled tracers.150c [18F]-labeled compounds can be prepared in radiochemical yields (RCY) ranging from 20% to 72% in 10 min without the need for preactivation of the labeling precursor. The facility of this process provides a translational 8342



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Figure 26. Processes for the nucleophilic fluorination of arenes and heteroarenes.

potential tau imaging agent (Scheme 3b). Axn alternative procedure with broad application to the preparation of aryl and heteroaryl [18F]-trifluoromethyl derivatives relies upon the Cucatalyzed cross-coupling reaction of aryl and heteroaryl iodides with methyl 2-chloro-2,2-difluoroacetate and [18F]-fluoride.153 The preparation of [18F]-labeled fluoxetine (285) from the iodide 284 depicted in Scheme 4 is representative of the process. A related process utilizes CHF2I as the source of the 18FCF2− and converts iodophenyl derivatives 286 into labeled CF3 compounds 287 (Scheme 5). This process is tolerant of a wide range of functionality. Although 4-iodophenol gave a poor yield, this problem is solved by protection as the benzyloxy ether.154 (c) Electrophilic Fluorination of Arenes. Although many sources of nucleophilic fluorine exist, fewer sources of electrophilic fluorine are available. Selectfluor (288) is one of the most reactive electrophilic fluorination reagents and is safe, nontoxic, and easy to handle.155 As such, 288 represents a significant advance in preparative organofluorine chemistry. The preparation

method to label PET radioligands for human use. Eight druglike molecules have been derivatized with an [18F]-label including ibuprofen methyl ester (272), an analog of celecoxib (274), and a protected form of enalaprilat (276) to afford 273, 275, and 277, respectively (Scheme 2).150c A simple one step nucleophilic radiosynthesis of monolabeled [18F]-trifluoromethyl derivatives with high specific activity has been developed based on the reaction of difluorovinyl-functionalized precursors 278 with [18F]-fluoride using standard K.2.2.2 conditions (Scheme 3a).151 This procedure affords a mixture of two 18F-labeled products, an isotopic exchange with the 2,2,difluorovinylethyltosylate precursor 279, and the desired [18F]2-fluoro-2-2-trifluoroethyltosylate (280) which predominates by a ratio of 10:1. This method has been used to synthesize [18F]lansoprazole (283), a promising PET ligand for imaging the tau pathway in Alzheimer’s disease.152 The difluorovinyl precursor 281 was treated with [18F]-fluoride to afford 283 along with the [18F]-labeled starting material 282 in a combined 14% radiochemical yield, sufficient for exploration of 283 as a 8343



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Scheme 2. Direct [18F]-Labeling of Benzylic C−H Bonds

Scheme 3. Conversion of Difluorovinyl Functionality to the [18F]-Trifluoromethyl Group

Scheme 4. Preparation of 285

Scheme 6. Preparation of 290 from 289

Scheme 5. Cu-Catalyzed Cross Coupling of Phenyl Iodides (286) Employing CHF2I as the [18F]CF2− Source of [18F]-Selectfluor bis(triflate) (290) from 289 has been accomplished using high specific activity [18F]-F2g providing a useful electrophilic [18F]-fluorination agent (Scheme 6).156 This reagent was used to convert 291 to [18F]-6-fluoro-L-DOPA (292) of high specific activity, an important PET radioligand of use in understanding the dopaminergic pathway in human 8344



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Scheme 8. Palladium-Mediated Synthesis of 296

Scheme 9. Nickel-Mediated Synthesis of 300

imaging studies in patients with Parkinson’s disease, schizophrenia, and brain tumors (Scheme 7).157 An alternative approach to the use of 290 for electrophilic [18F]-fluorinations exploits palladium(IV) and nickel(II) complexes where the reactivity of [18F]-fluoride is inverted, allowing it to react in an electrophilic manifold.158 The first step of the palladium process is capture of the [18F]-fluoride as the [18F]-palladium(IV) species 294 derived from 293. This species


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Scheme 11. Preparation of the [18F]-Labeled Exendin Analog 305

synthesis of [18F]-MDL-100907 (300, volinanserin), a promising new PET radioligand for labeling the 5HT2a receptor, from 297 through the intermediacy of 298 which is used to alkylate the piperidine 299 (Scheme 9).160 (d) Synthetic Methods for the [18F]-Labeling of Biologics. The increase in the application of biologics-based drugs has stimulated considerable interest in methods for the introduction of 18F into these molecules with a focus on the use of chemical reactions that are bioorthogonal in nature.161 Compared to more typical 124I-labeling, the use of 18F offers the advantage of same day imaging and reduced dosimetry. However, the direct incorporation of [18F]-fluoride into biologic constructs suffers from a major limitation based on the harsh reaction conditions (high organic solvent concentrations, high temperatures, high pH, etc.) that are required for currently available fluorination processes. To overcome this limitation, several [18F]-labeled prosthetic groups have been designed that take advantage of either random lysine conjugation or site-specific conjugation using [18F]-substituted maleimides, oximes, fluoroproprionates, or click chemistry approaches.162 One highly efficient bioorthogonal ligation approach relies upon an inverse electron demand Diels−Alder reaction (IEDDA) between 1,2,4,5-tetrazines and strained cycloalkenes, including transcyclooctenes and cyclopropene derivatives.163 These reactions exhibit very fast kinetics with rate constants of

Scheme 12. Preparation of 306 for IEDDA Conjugation

acts as a source of electrophilic [ 18 F]-fluorine, which subsequently undergoes oxidative transfer of fluorine to a palladium(II) aryl complex 295. This yields a [18F]-Pd(IV) fluoride species that undergoes reductive elimination of C−18F, thereby generating the [18F]-labeled aryl fluoride derivative, exemplified by the synthesis of [18F]-paroxetine 296 (Scheme 8). This methodology has been translated onto a commercially available automated synthesis unit that has produced quantities of [18F]-labeled PET radioligands suitable for use in imaging studies. This process allows [18F]-labeling at positions of a molecule that have typically been difficult to access.159 In the nickel-based process, a one-step oxidative [18F]-fluorination of aromatic rings is accomplished using [18F]-fluoride, 18-crown-6, an activated nickel complex, and a hypervalent iodine oxidant. This process can be carried out at ambient temperature and pressure, is typically complete after only 1 min, and generates the targeted aryl fluoride products in modest radiochemical yields (13−58%).158 A translational application of this chemistry is exemplified by the

Scheme 13. Labeling of a Bombesin Derivative 310 Mediated by 309

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Scheme 14. Staudinger Ligation Using 313

approximately 20 000 M−1 s−1 in MeOH at 25 °C and have been used successfully in live cell imaging. [18F]-trans-Cyclooctene 302 ([18F]-TCO), prepared from 301, has been developed for use in bioorthogonal ligations (Scheme 10). Several biologics agents have been labeled with 18F by reacting [18F]-TCO with biomolecules that have been preassembled to contain a tetrazine. The preparation of the exendin analog 304 from 303 by an IEDDA with 302 is illustrative of this process which provides a useful agent for imaging pancreatic β-cells (Scheme 11).164 The poor metabolic stability of 302 prevents its application toward in vivo labeling of tetrazine-derived macromolecules. This problem is addressed by the design of a [18F]-labeled tetrazine with favorable pharmacokinetics that has application as a versatile tool for pretargeted PET imaging using in vivo click chemistry derivatization.165 The design relies upon the use of [18F]-3,6-dialkyltetrazine 306, prepared from 305 as depicted in Scheme 12, which was generated in 18% radiochemical yield. Tetrazine 306 was found to be stable in vitro in human plasma at 37 °C for 12 h and in vivo in rodents, with approximately 85% of the molecule remaining intact 120 min after injection.165 This reagent offers considerable potential to conduct an IEDDA reaction with a pretargeted biologic containing either transcyclooctene or cyclopropene in a living animal.

Another approach to introducing 18F into biomolecules uses strain-promoted, copper-free click chemistry to conjugate [ 18 F]-norbornene derivative 309 ([18F]-NFB), prepared from the established prosthetic peptide labeling compound [ 18 F]-N-succinimidyl-4-fluorobenzoate (308, [ 18 F]-SFB), which in turn is obtained from 307, to a tetrazine species (Scheme 13).166,167 Compound 309 was cyclized with a tetrazinefunctionalized derivative of bombesin (TT-BBN, 310), a 14residue neurotransmitter that targets the gastrin-releasing peptide receptor (GRPR) that is overexpressed in human prostate cancer (Scheme 13).168 Compound 309 is frequently employed in rapid and high-yielding click chemistry reactions conducted under mild conditions in the absence of copper, making it an attractive option for radiolabeling of protein and antibody fragments with 18F. One mild and effective procedure for introducing 18F into both biomolecules and small molecules relies upon a traceless Staudinger ligation using [18F]-fluoroethylazide (313) which reacts with a diphenylphosphine derivative 312 to afford a β-fluoroamide derivative 316 and the thiol side product 317 via the intermediacy of 314 and 315, as depicted in Scheme 14.169 This process is attractive, since it results in a native amide bond without inclusion of the phosphine oxide in the final product and can be used to introduce a [18F]-fluoroethylamide under catalyst-free conditions in short reaction times and with high radiochemical yields. Commercially available 2-[18F]-fluoro-2-deoxy-D-glucose (318) is widely used in assessing the metabolic status of a range of organs, including the brain, lungs, heart, and tumor cells. Tumor cells, having high metabolic demand, accumulate 318 which is recognized as glucose by the transporter.170 In its acyclic form, 318 presents an aldehyde that readily reacts with

Scheme 15. Derivatization of an O-Alkylated Hydroxylamine Derivative with 318

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Scheme 16. Silicon-, Boron-, and Aluminum-Based Reagents for [18F]-Fluorine Incorporation

utility in both a preclinical and clinical setting where labeled ligands provide useful tools for understanding drug disposition and evaluating drug−target engagement. Taken together, these developments are providing a strong impetus to develop new synthetic methodologies that are facilitating a broader deployment of fluorine in drug design. This in turn is providing opportunity to further clarify the nuanced role of this element in ever more complex settings. We anticipate that our understanding of the organic and medicinal chemistry of fluorine will continue to evolve and will contribute to the design and development of important future drugs to address the considerable unmet medical need that remains in human health.

O-alkylated hydroxylamines to afford an oxime. This reactivity has been exploited to label biologics, exemplified by the conjugation of the cyclic RGD peptide 319 in a simple, single step radiosynthesis to afford 320 (Scheme 15).171 This represents a novel and useful method for the labeling of biologics without the need for an on-site cyclotron. Several prosthetic moieties based on silicon-, boron-, and aluminum-based reagents that take advantage of [18F]-fluoride reactivity have been devised (Scheme 16).172 These reagents allow [18F]-fluorine to be introduced under mild conditions in a single step using preassembled precursors, an attractive feature because they offer a final “kitlike” procedure for radiolabeling biologic products via a simple GMP synthesis.173 By use of this methodology, a prepackaged sterile, lyophilized protein kit can be mixed with [18F]-fluoride to generate the final PET radioligands suitable for human use.173

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

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Notes

CONCLUSION Over the past two decades our understanding of the unique and enigmatic properties of fluorine has deepened considerably. This has led to more sophisticated and creative approaches to the deployment of this element in the design of drug candidates. The introduction of fluorine into a molecule can affect a range of properties of critical importance to drug design. Mastering when and how to install this element in the context of a complex organic molecule, where the resulting effects may be somewhat cryptic in nature rather than simply additive, has the potential to lead to refined drug candidates. This may offer greater probability of compound success in an arena where failure in development is a far too common event. In this review we have captured some of the creative applications of fluorine in drug design, many of which have been made possible by the emerging understanding of the fundamental attributes of this element. Although fluorine is a prominent element in marketed drugs and development candidates, its prevalence is very likely limited by issues associated with an incomplete understanding of how to productively deploy this atom to the greatest effect and the difficulty of synthetic access to fluorinated building blocks. This is particularly the case for prosthetic groups like the SF5 moiety which is of contemporary interest. In addition to the importance of fluorine in drug design, the 18F isotope is established as an appealing and useful positron emitter that offers considerable

The authors declare no competing financial interest. Biographies Eric P. Gillis received his Ph.D. degree from the University of Illinois at UrbanaChampaign under the supervision of Professor Martin Burke. His graduate studies focused on the development of MIDA boronates for iterative cross-coupling, slow-release cross-coupling, and automated small molecule synthesis. In 2010 he joined Bristol-Myers Squibb where he is a member of the Department of Discovery Chemistry. Kyle J. Eastman received his Ph.D. degree from The Pennsylvania State University under the tutelage of Professor Ken Feldman. His graduate studies focused on mechanism of action studies of a diazoparaquinone family of natural products as well as natural product synthesis. Kyle subsequently joined the laboratories of Professor Phil Baran at The Scripps Research Institute in La Jolla, CA, where he directed efforts toward method development and natural product synthesis of indole containing architectures. He joined Bristol-Myers Squibb in 2008 where he is a medicinal chemist in the Department of Discovery Chemistry. Matthew D. Hill received his Ph.D. in Organic Chemistry in 2008 from The Massachusetts Institute of Technology (MIT) under the supervision of Professor Mohammad Movassaghi. In the course of his graduate studies Matthew developed several new methodologies for the preparation of azaheterocycles. Prior research includes the synthesis of phorbol analogues under the supervision of Professor Mark McMills at Ohio University and work on age-related macular degeneration with Professor Koji Nakanishi at Columbia University, NY. Currently a 8348



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ChemBioChem 2004, 5, 637−643. (b) Müller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007, 317, 1881−1886. (c) Shah, P.; Westwell, A. D. The role of fluorine in medicinal chemistry. J. Enzyme Inhib. Med. Chem. 2007, 22, 527−540. (d) Bégué, J. P.; Bonnet-Delpon, D. Bioorganic and Medicinal Chemistry of Fluorine; Wiley: Hoboken, NJ, 2007; pp 1−365. (e) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320−330. (f) Hagmann, W. K. The many roles for fluorine in medicinal chemistry. J. Med. Chem. 2008, 51, 4359−4369. (g) Yamazaki, T.; Taguchi, T.; Ojima, I. Unique properties of fluorine and their relevance to medicinal chemistry and chemical biology. In Fluorine in Medicinal Chemistry and Chemical Biology; Ojima, I., Ed.; Wiley-Blackwell, Chichester, U.K., 2009; pp 1−46. (h) Wang, J.; Sánchez-Roselló, M.; Aceña, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Fluorine in pharmaceutical industry: fluorine-containing drugs introduced to the market in the last decade (2001−2011). Chem. Rev. 2014, 114, 2432−2506. (i) Eastman, K. J.; Gillis, E. P.; Meanwell, N. A. Tactical applications of fluorine in drug design and development. Fluorine in Heterocyclic Chemistry, Volume 1, 5-Membered Heterocycles and Macrocycles; Nenajdenko, V., Ed.; Springer International: Cham, Switzerland, 2014; pp 1−54. (j) Nenajdenko, V. G.; Muzalevskiy, V. M.; Shastin, A. V. Polyfluorinated ethanes as versatile fluorinated C2-building blocks for organic synthesis. Chem. Rev. 2015, 115, 973−1050. (k) Alonso, C.; Martínez de Marigorta, E.; Rubiales, G.; Palacios, F. Carbon trifluoromethylation reactions of hydrocarbon derivatives and heteroarenes. Chem. Rev. 2015, 115, 1847− 1935. (2) (a) Ahrens, T.; Kohlmann, J.; Ahrens, M.; Braun, T. Functionalization of fluorinated molecules by transition-metal-mediated C−F bond activation to access fluorinated building blocks. Chem. Rev. 2015, 115, 931−972. (b) Yang, X.; Wu, T.; Phipps, R. J.; Toste, F. D. Advances in catalytic enantioselective fluorination, mono-, di-, and trifluoromethylation, and trifluoromethylthiolation reactions. Chem. Rev. 2015, 115, 826−870. (c) Liang, T.; Neumann, C. N.; Ritter, T. Introduction of fluorine and fluorine-containing functional groups. Angew. Chem., Int. Ed. 2013, 52, 8214−8264. (d) Champagne, P. A.; Desroches, J.; Hamel, J.-D.; Vandamme, M.; Paquin, J.-F. Monofluorination of organic compounds: 10 years of innovation. Chem. Rev. 2015, 115 DOI: 10.1021/cr500706a. (3) (a) Le Bars, D. Fluorine-18 and medical imaging: radiopharmaceuticals for positron emission tomography. J. Fluorine Chem. 2006, 127, 1488−1493. (b) Miller, P. W.; Long, N. J.; Vilar, R.; Gee, A. D. Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography. Angew. Chem., Int. Ed. 2008, 47, 8998−9033. (c) Alauddin, M. M. Positron emission tomography (PET) imaging with 18F-based radiotracers. Am. J. Nucl. Med. Mol. Imaging 2012, 2, 55−76. (d) Ametamey, S. M.; Honer, M.; Schubiger, P. A. Molecular imaging with PET. Chem. Rev. 2008, 108, 1501−1516. (e) Piel, M.; Vernaleken, I.; Rösch, F. Positron emission tomography in CNS drug discovery and drug monitoring. J. Med. Chem. 2014, 57, 9232−9258. (f) Honer, M.; Gobbi, L.; Martarello, L.; Comley, R. A. Radioligand development for molecular imaging of the central nervous system with positron emission tomography. Drug Discovery Today 2014, 19, 1936−1944. (g) Zhang, L.; Villalobos, A. Recent advances in the development of PET and SPECT tracers for brain imaging. Annu. Rep. Med. Chem. 2012, 47, 105− 119. (h) Li, Z.; Conti, P. S. Radiopharmaceutical chemistry for positron emission tomography. Adv. Drug Delivery Rev. 2010, 62, 1031−1051. (4) (a) O’Hagan, D. Understanding organofluorine chemistry. An introduction to the C-F bond. Chem. Soc. Rev. 2008, 37, 308−319. (b) Hunter, L. The C−F bond as a conformational tool in organic and biological chemistry. Beilstein J. Org. Chem. 2010, 6, 38. (5) (a) Zimmer, L. E.; Sparr, C.; Gilmour, R. Fluorine conformational effects in organocatalysis: an emerging strategy for molecular design. Angew. Chem., Int. Ed. 2011, 50, 11860−11871. (b) Buissonneaud, D. Y.; van Mourik, T.; O’Hagan, D. A DFT study on the origin of the fluorine gauche effect in substituted fluoroethanes. Tetrahedron 2010, 66, 2196− 2202. (c) Abraham, R. J.; Chambers, E. J.; Thomas, W. A. Conformational analysis. Part 22. An NMR and theoretical investigation of the gauche effect in fluoroethanols. J. Chem. Soc., Perkin Trans. 2 1994,

member of Discovery Chemistry at Bristol-Myers Squibb, Matthew has experience in neuroscience and oncology-focused medicinal chemistry. David J. Donnelly received his Ph.D. degree from the State University of New York at Buffalo under the supervision of Professor Michael Detty and conducted postdoctoral studies in radiopharmaceutical production at the University of Michigan in collaboration with Professor Michael Kilbourn. He joined Bristol-Myers Squibb in 2007 where he is a member of the PET radiochemistry synthesis group within the Discovery Chemistry Platforms department. Nicholas A. Meanwell received his Ph.D. degree from the University of Sheffield, Sheffield, England, under the supervision of Dr. D. Neville Jones and conducted postdoctoral studies at Wayne State University, Detroit, MI, in collaboration with Professor Carl R. Johnson. He joined Bristol-Myers Squibb in 1982 where he has supervised teams that have advanced clinical candidates in several areas of antiviral drug discovery, including BMY-433771, an inhibitor of respiratory syncytial virus fusion, the HIV-1 attachment inhibitor BMS-626529 that is being developed as the prodrug BMS-663068, the HCV NS3 protease inhibitor asunaprevir and the HCV NS5A inhibitor daclatasvir, both of which are approved in Japan for the treatment HCV genotype 1b infection.

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ACKNOWLEDGMENTS We thank Carolyn Weigelt for stimulating discussions and Brett R. Beno for assistance with some of the graphics.

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ABBREVIATIONS USED 17β-HSD, 17β-hydroxysteroid dehydrogenase; ADME, absorption, distribution, metabolism, and excretion; ATP, adenosine triphosphate; AUC, area under the curve; BACE, β-site amyloid precursor protein cleaving enzyme; CD, circular dichroism; CETP, cholesterol ester transfer protein; CGRP, calcitonin generelated peptide; CNS, central nervous system; Cp-CF3, trifluoromethylcyclopropyl; CSD, Cambridge Structural Database; CYP 450, cytochrome P450; DHODH, dihydroorotate dehydrogenase; FAP, fibroblast activation protein; DFT, density functional theory; DPP-4, dipeptidy peptidase IV; FDA, Food and Drug Administration; GABA, γ-aminobutyric acid; GLP-1, glucagon-like peptide 1; GSH, glutathione; HBV, hepatitis B virus; HIV-1, human immunodeficiency virus 1; hERG, human ether-a-go-go-related gene; HLM, human liver microsome; IR, infrared; iv, intravenous; HBA, hydrogen-bond acceptor; HBD, H-bond donor; HCV, hepatitis C virus; 4R-Hyp, 4-(R)hydroxyproline; IEDDA, inverse electron demand Diels−Alder reaction; iv, intravenous; KSP, kinesin spindle protein; MEK1, MAP kinase 1; MIC, minimum inhibitory concentration; MRI, magnetic resonance imaging; Mtb, Mycobacterium tuberculosis; NBO, natural bond order; NK1, neurokinin 1 or substance P; NMDA, N-methyl-D-aspartate; NMR, nuclear magnetic resonance; PAMPA, parallel artificial membrane permeability assay; PDGFR, platelet-derived growth factor receptor; PDB, Protein Data Bank; Pe, permeability; PET, positron emission tomography; P-gp, P-glycoprotein; phos-HH3, histone H3 phosphorylation; PK, pharmacokinetic; PSA, polar surface area; QMA, quaternary ammonium chloride polymer; RCY, radiochemical yield; RLM, rat liver microsome; rt, room temperature; SA, specific activity; SAR, structure−activity relationship; SERT, serotonin transporter; TBAF, tetrabutylammonium fluoride; TCO, trans-cyclooctene; TRPV1, transient receptor potential cation channel subfamily V member 1

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