Novel Potent Proline-Based Metalloproteinase Inhibitors: Design

Oct 3, 2016 - Novel Potent Proline-Based Metalloproteinase Inhibitors: Design, (Radio)Synthesis, and First in Vivo Evaluation as Radiotracers for Posi...
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Novel Potent Proline-Based Metalloproteinase Inhibitors: Design, (Radio)Synthesis, and First in Vivo Evaluation as Radiotracers for Positron Emission Tomography Dmitrii V. Kalinin,†,‡ Stefan Wagner,§ Burkhard Riemann,§ Sven Hermann,‡,∥ Frederike Schmidt,⊥ Christoph Becker-Pauly,⊥ Stefan Rose-John,⊥ Michael Schaf̈ ers,‡,∥ and Ralph Holl*,†,‡,#,∇ †

Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany ‡ Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Münster, 48149 Münster, Germany § Department of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1, 48149 Münster, Germany ∥ European Institute for Molecular Imaging, University of Münster, Waldeyerstraße 15, 48149 Münster, Germany ⊥ Biochemical Institute, Christian-Albrechts-University Kiel, 24098 Kiel, Germany # German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel, 38124 Braunschweig, Germany S Supporting Information *

ABSTRACT: As dysregulation of matrix metalloproteinase (MMP) activity is associated with a wide range of pathophysiological processes like cancer, atherosclerosis, and arthritis, MMPs represent a valuable target for the development of new therapeutics and diagnostic tools. We herein present the chiral pool syntheses, in vitro evaluation, and SAR studies of a series of D- and L-proline- as well as of (4R)-4hydroxy-L-proline-derived MMP inhibitors possessing general formula 1. Some of the synthesized hydroxamic acids were found to be potent MMP inhibitors with IC50 values in the nanomolar range, also demonstrating no off-target effects toward the other tested Zn2+-dependent metalloproteases (ADAMs and meprins). Utilizing the structure of the (2S,4S)-configured 4-hydroxyproline derivative 4, a selective picomolar inhibitor of MMP-13, the radiolabeled counterpart [18F]4 was successfully synthesized. The radiotracer’s biodistribution in mice as well as its serum stability were evaluated for assessing its potential use as a MMP-13 targeting PET imaging agent.



INTRODUCTION The metzincin superfamily of proteases comprises most of the Zn2+-dependent metalloproteases. The metzincins are mainly involved in the degradation and remodeling of extracellular matrix (ECM) components, such as collagens, but also cleave non-ECM proteins like von Willebrand factor, aggrecan, cytokines, and substance P. Typical representatives of metzincins are the MMPs (matrix metalloproteinase), the ADAMs (a disintegrin and metalloproteinase), and the astacins, including the meprins.1 Despite relatively low amino acid sequence identity, these enzymes contain the conserved HExxHxxG/NxxH/D Zn2+-binding motif in their active site and exhibit an analogous three-dimensional organization of their catalytic domains.2,3 MMPs play an essential role in important remodeling events such as tissue turnover, wound healing, and angiogenesis.4−6 In humans, MMPs currently comprise more than 23 different members.7 Historically MMPs are classified according to their ability to degrade specific substrates, thus distinguishing collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), © 2016 American Chemical Society

stromelysins (MMP-3, -10, -11), matrilysins (MMP-7), metalloelastases (MMP-12), membrane-type MMPs (MT-MMPs: MMP-14−17, MMP-24−25), and other MMPs (MMP-19−23, MMP-26−28).8 MMPs share similar general structural features, typically comprising four distinct domains: a pro-domain, which keeps MMPs inactivated until it is removed; a catalytic domain, where the substrate cleavage takes place and which contains the Zn2+-binding motif HExxHxxGxxH; a linker region of variable length; and a hemopexin-like domain, the so-called substrate specificity site, which forms a propeller blade structure.9 Similar to MMPs, ADAMs, which are transmembrane or secreted metalloproteases, contain a pro-domain and a catalytic domain with corresponding functions. But additionally, they contain a unique disintegrin domain, a cysteine-rich domain, an epidermal growth factor-like domain, and a transmembrane domain; they also contain a C-terminal cytoplasmic tail.10 ADAMs shed (“cut off”) extracellular portions of transReceived: August 29, 2016 Published: October 3, 2016 9541

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Figure 1. General structure (1) of the envisaged proline derivatives and structures of radiofluorinated MMP and ADAM inhibitors for PET. The Zn2+-binding groups are highlighted in red.34−36

pulmonary fibrosis, asthma, emphysema, and the acute respiratory distress syndrome.24 Regarding ADAMs, sheddases ADAM10 and ADAM17 (also known as TACE, tumor necrosis factor-α-converting enzyme) are involved in the generation of amyloid plaques and also shed inflammatory mediators promoting thereby cancer, inflammation, and Alzheimer’s disease.25 Finally, meprins have been reported to be crucial for the development of inflammatory disorders like acute renal failure, urinary tract infections, and inflammatory bowel disease as well as fibrosis and neurodegeneration.12,26 Therefore, metalloproteinases represent valuable targets for the development of new therapeutics and diagnostic tools. Additionally, they can serve as determinants for understanding the pathophysiology of inflammatory conditions. In this respect, the noninvasive imaging and the quantification of MMP activity in vivo using (radio)labeled inhibitors are of great (pre)clinical interest. Despite evident and convincing data, indicating that MMP inhibitors have a high potential for treating different pathologies, their further use in the clinic has been stalled, mainly due to their limited proof of efficacy and their side effects, which might be related to nonselective inhibition of metalloproteases.24,27,28 Therefore, gaining selectivity between the metalloproteases without decreasing inhibitory potency is a primary goal in the development of MMP inhibitors, which would help to improve their efficacy and avoid undesirable side effects. To find new effective inhibitors of MMPs, this work is focused on the development of compounds that mimick structural features of collagen, the main component of the ECM, which is a principal and universal substrate for many metalloproteases. Being the most abundant protein in mammals, collagen exhibits a triple helix (three supercoiled polyproline II-like chains) with a repetitive XaaYaaGly sequence, in which Xaa is often (2S)-proline and Yaa is often

membrane proteins (e.g., receptors), releasing the soluble ectodomains from the cell surface, thereby activating them.11 Finally, meprins, members of the astacin family, represent complex multidomain metalloproteases comprising meprin α and meprin β, which form disulfide-linked homo- or heterodimers. Meprin α and meprin β degrade a variety of biologically active proteins, but exhibit different substrate specificities.12 For example, whereas gastrointestinal peptides like gastrin and cholecystokinin are readily cleaved by meprin β, substance P and cytokines represent substrates for meprin α.3 Moreover, both meprins cleave proteins of the ECM like collagen type IV, laminin-1, nidogen-1, and fibronectin.3 Importantly, meprins together with other members of the astacin family induce collagen deposition by cleaving off the Cand N-terminal prodomains of fibrilar collagens I and III.13,14 Many pathophysiological processes are related to the dysregulation of MMPs, ADAMs, and meprins. For instance, the overexpression of MMPs and ADAMs is related to cardiovascular risk factors and promotes arterial remodeling (intimal-medial thickening, fibrosis, calcification), resulting in atherosclerosis and cardiovascular disease progession.15−17 Increased levels of MMP-9 were found to be associated with neuronal damage and apoptosis in animal models of cerebral ischemia and in human stroke.18 Furthermore, metalloproteases promote hallmarks of cancer such as cell proliferation (MMP1−3, -7, -9, -11, -19, ADAM12 etc.), cell migration (MMP-1−3, -7, -9, -13, -14, ADAM10 etc.), cell invasion (MMP-2, -9, -14 etc.), metastasis (MMP-8), and angiogenesis (MMP-2, -9, -14), thereby playing a key role in the development of human breast, colon, thyroid, lung, and prostate cancers.19,20 Moreover, MMPs have been found to be involved in joint cartilage destruction associated with osteoarthritis. MMP-13 is considered as the main contributor to this pathology due to its ability to degrade type II collagen, the major structural protein of articular cartilage.21−23 Notably, MMP activity is also associated with a wide range of respiratory diseases like idiopathic 9542

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Scheme 1. Synthesis of Phenylacetylene Derivatives 8a−fa

Reagents and conditions: (a) 4-bromobenzyl bromide, ACN, Δ, 6a 69%, 6b 85%, 6c 90%, 6d 98%, 6e 92%, 6f 40%; (b) trimethylsilylacetylene, Pd(PPh3)4, CuI, NEt3, Δ, 7a 88%, 7b 90%, 7c 86%, 7d 71%, 7e 71%, 7f 82%; (c) TBAF, THF, 0 °C, 8a 69%, 8b 70%, 8c 47%, 8d 99%, 8e 82%, 8f 89%; (d) (1) Boc2O, NEt3, CH2Cl2, rt, (2) mCPBA, CH2Cl2, (3) HCl-saturated MeOH, Δ, 75% (3 steps).38,39 a

Scheme 2. Synthesis of L-Proline Derivatives 12a

a Reagents and conditions: (a) 4-iodobenzoyl chloride, NEt3, DMAP, CH2Cl2, 0 °C → rt, 86%; (b) Pd(PPh3)4, CuI, NEt3, ACN, rt, 11b 60%, 11d 92%, 11e 99%, 11f 92%, 11g 99%, 11h 60%; (c) H2NOH·HCl, NaOMe, MeOH, rt, 12b 49%, 12d 49%, 12e 45%, 12f 56%, 12g 48%, 12h 66%.

(2S,4R)-4-hydroxyproline.29,30 In this work, metalloprotease inhibitors were developed which possess proline as well as hydroxyproline scaffolds and display general structure 1 (Figure 1). To ensure the desired selectivity of the designed inhibitors, apart from alterations of the main scaffold, particular emphasis was placed on structural variations of the lipophilic side chain, addressing the S1′ pocket of the enzymes, which is located on their “specificity loop” and which is highly variable among different MMPs.31 X-ray crystallographic data of different MMP inhibitors in complex with various MMPs indicate that some MMPs, especially MMP-13 (but also MMP-3, MMP-8, and MMP-9), are able to accommodate ligands with long lipophilic residues addressing the S1′ pocket and its unique S1″ side pocket.32 In contrast, other MMPs, like for instance MMP-1, MMP-7, and MMP-11, have only a shallow S1′ pocket, so compounds that inhibit these enzymes should have a shorter lipophilic tail.33 Therefore, to achieve selectivity for MMP-13, the envisaged compounds in this study possess a long lipophilic side chain, containing a diphenylacetylene moiety, which was varied in length by attaching substituents on the distal phenyl ring. The activity of the synthesized compounds toward MMP2, -8, -9, and MMP-13 was investigated and their selectivity over other metzincins, such as ADAM10, ADAM17, meprin α, and meprin β, was studied. Besides inhibition of the enzymes, noninvasive imaging of metalloprotease activity in vivo is of enormous interest in basic research and in clinical applications. Therefore, attempts to prepare and evaluate radiolabeled MMP- and ADAM-targeting ligands for positron emission tomography (PET) were undertaken.37 Some selected examples are shown in Figure 1

with the structures of [18F]FB-ML5, [18F]2, and [18F]3.34−36 Utilizing the unique pharmacological profile of the (2S,4S)-4hydroxyproline based compound 4, a selective picomolar inhibitor of MMP-13 (Table 1), which was synthesized in the course of the present study, the radiofluorinated PET tracer [18F]4 was successfully developed (Figure 1) and its first in vivo study was performed.



RESULTS AND DISCUSSION Chemistry. The envisaged proline derivatives exhibit a lipophilic side chain comprising a diphenylacetylene moiety, which should be built up via a Sonogashira coupling reaction. To obtain compounds with various substituents at the terminal phenyl ring, a series of substituted phenylacetylene derivatives was synthesized. On the basis of the described synthesis of morpholinomethyl-substituted phenylacetylene derivative 8d,38 pyrrolidine (5a), piperidine (5b), 4-hydroxypiperidine (5c), and thiomorpholine (5e) were alkylated with 4-bromobenzyl bromide to yield tertiary amines 6a−c,e (Scheme 1). Subsequently, a Sonogashira coupling with trimethylsilylacetylene was performed to give phenylacetylene derivatives 7a− c,e. Finally the trimethylsilyl protective group of amines 7a−c,e was cleaved with tetrabutylammonium fluoride to yield terminal alkynes 8a−c,e. Additionally, sulphone 5f was synthesized from thiomorpholine according to a described procedure39 and converted analogously to the corresponding phenylacetylene derivative 8f (Scheme 1). The desired proline-derived hydroxamic acids were synthesized in chiral pool syntheses. Starting from L-proline methyl 9543

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Scheme 3. Synthesis of 4-Substituted L-Proline Derivatives 16, 20, and 23a

Reagents and conditions: (a) 4-iodobenzoyl chloride, NEt3, DMAP, CH2Cl2, 0 °C → rt, 97%; (b) Pd(PPh3)4, CuI, NEt3, ACN, rt, 15d 99%, 15g 75%, 15h 79%, 15i 83%, 15j 100%, 15k 100%; (c) H2NOH·HCl, NaOMe, MeOH, rt, 16d 26%, 16g 66%, 16h 54%, 16i 41%, 16j 38%, 16k 51%; (d) p-nitrobenzoic acid, DIAD, PPh3, THF, 0 °C → rt, 80%; (e) K2CO3, MeOH, rt, 84%; (f) Pd(PPh3)4, CuI, NEt3, ACN, rt, 19a 92%, 19b 94%, 19c 98%, 19d 69%, 19e 96%, 19g 99%, 19h 78%, 19i 93%, 19j 98%, 19k 100%; (g) H2NOH·HCl, NaOMe, MeOH, rt, 20a 22%, 20b 47%, 20c 20%, 20d 44%, 20e 44%, 20g 61%, 20h 35%, 20i 52%, 20j 49%, 20k 57%; (h) phthalimide, DIAD, PPh3, THF, 0 °C → rt, 61%; (i) Pd(PPh3)4, CuI, NEt3, ACN, rt, 22d 85%, 22g 90%; (j) H2NOH·HCl, NaOMe, MeOH, rt, 23d 55%, 23g 79%. a

Scheme 4. Synthesis of Tertiary Amines 28d,e and 29da

a

Reagents and conditions: (a) 4-iodobenzyl bromide, NEt3, 24 47%, 25 64%; (b) Pd(PPh3)4, CuI, NEt3, ACN, rt, 26d 85%, 26e 99%, 27d 93%; (c) H2NOH·HCl, NaOMe, MeOH, rt, 28d 53%, 28e 38%, 29d 40%.

ester hydrochloride (9), the 3,4-unsubstituted pyrrolidine derivatives were obtained (Scheme 2). The acylation of secondary amine 9 with 4-iodobenzoyl chloride yielded amide 10, which was subsequently subjected to Sonogashira couplings with several of the previously synthesized terminal alkynes as

well as some commercially available phenylacetylene derivatives. The C−C coupling reactions gave diphenylacetylene derivatives 11b,d−h, which were finally transformed into hydroxamic acids 12b,d−h by reacting the esters with hydroxylamine. The (R)-configured hydroxamic acid ent-12d, 9544

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Scheme 5. Synthesis of the Fluorinated Proline and 4-Hydroxyproline Derivatives 34, 35, and 37a

a Reagents and conditions: (a) DAST, CH2Cl2, −78 °C → rt, 30 65%, 31 49%; (b) phenylacetylene, Pd(PPh3)4, CuI, NEt3, ACN, 32 98%, 33 80%; (c) H2NOH·HCl, NaOMe, MeOH, rt, 34 67%, 35 78%; (d) 4-fluorophenylacetylene, Pd(PPh3)4, CuI, NEt3, ACN, 95%; (e) H2NOH·HCl, NaOMe, MeOH, rt, 27%.

Scheme 6. Synthesis of Fluorinated 4-Hydroxyproline Derivative 4a

Reagents and conditions: (a) trimethylsilylacetylene, Pd(PPh3)4, CuI, NEt3, Δ, 85%; (b) TBAF, THF, 0 °C, 90%; (c) TsCl, NEt3, CH2Cl2/pyridine (4:1), 0 °C → rt, 73%; (d) TBAF, 65 °C, 70%; (e) 18, Pd(PPh3)4, CuI, NEt3, ACN, rt, 90%; (f) H2NOH·HCl, NaOMe, MeOH, rt, 54%. a

the enantiomer of morpholinomethyl-substituted diphenylacetylene derivative 12d was synthesized in the same way, starting from D-proline methyl ester hydrochloride. The 4-substituted pyrrolidine derivatives could be accessed from (2S,4R)-configured 4-hydroxyproline derivative 13 (Scheme 3). Its reaction with 4-iodobenzoyl chloride gave benzamide 14, which was coupled with various phenylacetylene derivatives and transformed into hydroxamic acids 16d,g−k as described for the 3,4-unsubstituted pyrrolidine derivatives. To obtain (4S)-configured 4-hydroxyproline derivatives, a Mitsunobu reaction was performed. The transformation of (4R)-configured 4-hydroxyproline derivative 14 with p-nitrobenzoic acid in the presence of DIAD and triphenylphosphine yielded the (4S)-configured ester 17, which was subsequently

cleaved to yield 4-hydroxyproline derivative 18 (Scheme 3). Analogous to its (4R)-configured diastereomer 13, aryl iodide 18 was subjected to Sonogashira couplings with miscellaneous phenylacetylene derivatives and subsequently transformed into hydroxamic acids 20a−e,g−k. Additionally, the hydroxy group in position 4 of the pyrrolidine ring should be replaced by an amino group. Therefore, 4-hydroxyproline derivative 14 was reacted with phthalimide under Mitsunobu conditions (Scheme 3). Subsequently, the resulting (4S)-configured pyrrolidine derivative 21 was coupled with phenylacetylene and the morpholinomethyl-substituted phenylacetylene derivative 8d, yielding esters 22g and 22d, respectively. To obtain the desired hydroxamic acids, esters 22g and 22d were reacted with 9545

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Table 1. MMP Inhibitory Activity of the Synthesized Proline-Based Hydroxamates 12, 16, 20, 23, 28, and 29 Compared to 44a

a

n.d.: not determinable.

hydroxyproline derivative 13 were N-alkylated with 4iodobenzyl bromide, yielding amines 24 and 25, respectively (Scheme 4). Sonogashira couplings of aryl iodides 24 and 25 with phenylacetylene derivatives 8d and 8e gave diphenylacetylene derivatives 26d, 26e, and 27d, which were finally

hydroxylamine. Under these conditions, besides the aminolysis of the ester group, the cleavage of the phthalimide moiety occurred, giving access to primary amines 23g and 23d. Additionally, a series of tertiary amines was envisaged. To obtain these compounds, proline derivative 9 and 49546

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Figure 2. (A) Compound 20g (in yellow), 16g (in cyan), and 12g (in magenta) docked in the active site of MMP-13 (PDB: 3KRY).46 The Zn2+-ion is shown as a gray sphere; amino acid residues in proximity to the surface are depicted as white stick models (Leu184 is depicted as yellow stick model). Oxygen and nitrogen atoms are colored in red and blue, respectively. The surface is colored as follows: lipophilic regions are in green, hydrophilic in magenta, and neutral in white. (B) LigPlot-generated two-dimensional schematic diagram of MMP-13−hydroxamic acid 20g interactions. Hydrogen bonds are dashed orange lines, hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms. The Zn2+-ion is shown as a green sphere.



BIOLOGICAL EVALUATION In Vitro MMPs Assays and SAR. The synthesized proline hydroxamates were assayed in vitro against the activated collagenases MMP-8 and MMP-13 as well as against gelatinases MMP-2 and MMP-9 using synthetic fluorogenic substrates according to a previously reported procedure.43 The potent MMP inhibitor (R)-N-hydroxy-2-{[4-methoxy-N-(pyridin-3ylmethyl)phenyl]sulfonamide}-3-methylbutanamide (44, CGS 27023A),44,45 possessing proven in vivo efficacy, was used as a positive control. The IC50 values obtained in the performed in vitro inhibition assays of hydroxamates 12, 16, 20, 23, 28, and 29 are summarized in Table 1. As shown in Table 1, the newly synthesized proline derivatives are potent MMP inhibitors, with IC50 values mostly in the nanomolar range. Notably, when comparing the inhibitory activities of the different hydroxamic acids toward the investigated MMPs, most of the compounds were found to preferentially inhibit MMP-13. Among them, the (2S,4S)configured 4-hydroxyproline derivative 20k, bearing a long and flexible lipophilic tail, effectively inhibited MMP-13 in the picomolar range (IC50(MMP-13) = 0.7 nM), with selectivities over the other tested MMPs ranging from 4 to 170. It was established that the structure of the lipophilic tail as well as the configuration of the pyrrolidine scaffold significantly affected MMP inhibitory activity and selectivity. Thus, (2S,4R)-4hydroxyproline derivative 16g showed weak inhibition against MMP-2, -9, and -13 in the micromolar range, with a slightly more pronounced activity toward MMP-8 (IC50(MMP-8) = 392 nM). In contrast, its diastereomer 20g, possessing an unnatural (2S,4S)-4-hydroxyproline scaffold, was strikingly 14− 69-fold more active toward all four tested MMPs with a comparable selectivity toward MMP-8 (IC50(MMP-8) = 11 nM). Irrespective of the substituent at the terminal phenyl ring

transformed into hydroxamic acids 28d, 28e, and 29d via an aminolysis with hydroxylamine. Synthesis of Fluorinated Compounds. To evaluate potential radiofluorinated MMP tracers based on the proline and 4-hydroxyproline lead structure in vitro and in vivo, the nonradioactive counterparts were synthesized according to Scheme 5 and 6 for initial in vitro evaluation. First, utilizing key intermediate 14, via a Sonogashira coupling reaction with 4-fluorophenylacetylene, diphenylacetylene derivative 36 was obtained, which was subjected to an aminolysis with hydroxylamine to yield the desired fluorinated hydroxamic acid 37 (Scheme 5). Then, the 4-fluorine substituted proline derivatives 34 and 35 were synthesized (Scheme 5). At first, alcohols 14 and 25 were fluorinated by treating the compounds with DAST.40 Subsequently, the obtained alkyl fluorides 30 and 31, which exhibit (S)configuration in position 4 of the pyrrolidine ring, were coupled with phenylacetylene and subjected to an aminolysis with hydroxylamine, yielding hydroxamic acids 34 and 35 (Scheme 5). Finally, 4-hydroxyproline derivative 4, possessing a 5fluoropentoxy substituent in the para-position of the terminal phenyl ring, was synthesized (Scheme 6). To build up the desired lipophilic tail, aryl bromide 3841 was subjected to a Sonogashira coupling reaction with trimethylsilylacetylene and a subsequent desylilation to give phenylacetylene derivative 40. The hydroxy group of 40 was then tosylated and the resulting tosylate 41 was subjected to a SN2 reaction with tetra-nbutylammonium fluoride (TBAF)42 to give 5-fluoropentoxy phenylacetylene derivative 42. Lastly, using a Sonogashira reaction, 42 was coupled with 4-hydroxyproline derivative 18 to give ester 43, which was transformed into the desired hydroxamic acid 4 by performing an aminolysis with hydroxylamine. 9547

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Figure 3. Compound 20i (cyan, left) and 20d (magenta, right) docked in the active site of MMP-13 (PDB: 3KRY).46 The Zn2+-ion is shown as a gray sphere; amino acid residues in proximity to the surface are depicted as white stick models (Pro255, Phe252, Leu218, and Lys249 are depicted as yellow stick models). Oxygen and nitrogen atoms are colored in red and blue, respectively. The surface is colored as follows: lipophilic regions are in green, hydrophilic in magenta, and neutral in white.

2 of the pyrrolidine ring from (S) to (R), leading to compound ent-12d, has ambiguous effects. Whereas no alteration of MMP2 inhibitory potency was observed, MMP-8 and MMP-9 were significantly more susceptible to inhibition by (R)-proline derivative ent-12d (Table 1). By contrast, in the case of MMP13, compound 12d, possessing a L-proline scaffold, showed a lower IC50 value (280 nM) compared to its (R)-configured enantiomer ent-12d (IC50(MMP-13) = 636 nM). Whereas the synthesized amides are generally able to strongly inhibit the proteolytic activity of the examined MMPs, the tertiary amines 28d, 29d, and 28e exhibit little to no inhibitory activity. This finding can be attributed to the absence of the carbonyl oxygen atom in these compounds, which in the case of the amides undergoes strong interactions with Leu185 or Ala186. Similar interactions with Leu185 or Ala186 can be observed in X-ray crystal structures of MMP-13 in complex with sulfonamide-based inhibitors like N-hydroxy-1(2-methoxyethyl)-4-({4-[4-(trifluoromethoxy)phenoxy]phenyl}sulfonyl)piperidine-4-carboxamide (SC-78080).46 The substituent in the para-position of the distal phenyl ring of the synthesized diphenylacetylene derivatives was found to influence the inhibitory potency of the compounds. For Lproline derived hydroxamic acids 12g, 12h, 12d, 12e, 12f, and 12b, the introduction of a substituent at the terminal phenyl ring generally leads to a decrease in inhibitory potency against all tested MMPs. Whereas for the primary amine 12h the observed loss in potency is most pronounced against MMP-2 and MMP-13, the piperidine derivatives 12d, 12e, 12f, and 12b are particularly less active against MMP-8 and MMP-9 compared to the unsubstituted diphenylacetylene derivative 12g. For the 4-hydroxyproline derivatives, contrary observations were made. For the (2S,4R)-configured diastereomer, a substituent in the para-position of the distal phenyl ring, generally led to an increase in inhibitory activity against all tested MMPs compared to the unsubstituted compound 16g. Whereas for hydroxamic acid 16h the introduction of the amino group led to a 3- to 4-fold enhancement in inhibitory potency against all tested MMPs, compound 16i, bearing a more lipophilic N,N-dimethylamino group, showed a distinct increase in inhibitory activity against MMP-13. The trend is

of the lipophilic side chain, a similar trend was apparent for all pairs of diastereomers that were compared (e.g., 16h and 20h, 16i and 20i as well as 16k and 20k, Table 1). The removal of the hydroxy group of the (2S,4S)-configured 4-hydroxyproline derivatives leads to a loss of inhibitory activity, resulting in (2S)-proline hydroxamates, with inhibitory activities generally ranging between those of the respective diastereomeric 4-hydroxyproline derivatives. Thus, for instance, poorly active (2S,4R)-configured compound 16g (IC50 values ranging from 0.39 to 2 μM against the four tested MMPs) can be transformed into the more potent hydroxamic acid 12g, with IC50 values from 26 to 250 nM, by removing its hydroxy group (Table 1). These data confirm that the synthesized (2S,4S)-4-hydroxyproline derivatives are more potent inhibitors of the investigated MMPs than compounds with a (2S,4R)-4hydroxyproline or a (2S)-proline scaffold. To rationalize these observations, the compounds were docked into the available crystal structure of MMP-13 (PDB: 1KRY).46 As the hydroxy groups of both diastereomeric 4-hydroxyproline scaffolds point out from the active site and are highly exposed to the solvent (Figure 2A) without directly interacting with the enzyme, no clear explanation for that phenomenon can be given based on the obtained docking scores and poses. Additionally, no obvious clashes were observed between the hydroxy groups of the (2S,4R)-4-hydroxyproline derivatives and the lipophilic residue Leu184 of the MMP-13 enzyme. However, the close proximity of these two groups (ca. 3.2 Å for the (4R)configured diastereomer compared to ca. 4.5 Å for the (4S)configured stereoisomer, Figure 2) in the active site is unfavorable and might result in the decreased potency of the (2S,4R)-4-hydroxyproline-based compounds. This could also explain the partial recovery of inhibitory activity in case of the (2S)-proline derivatives, which lack the 4-hydroxy group. The exchange of the hydroxy group in position 4 of the pyrrolidine ring with an amino group leading to (2S,4S)configured primary amines 23g and 23d was found to be detrimental for MMP inhibitory activity. In case of the L-proline derivative 12d, possessing a morpholinomethyl-substituted diphenylacetylene moiety as lipophilic fragment, the inversion of configuration in position 9548

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Table 2. MMP Inhibitory Activity of the Synthesized Fluorinated Proline-Based Hydroxamates 4, 34, 35, and 37 Compared to 44

IC50 ± SD [nM] 1

compd

X

R

R

stereo isomer

MMP-2

MMP-8

MMP-9

MMP-13

4 34 35 37 44

CO CO CH2 CO

O(CH2)4CH2F H H F

OH F F OH

(2S,4S) (2S,4S) (2S,4S) (2S,4R)

5±1 33 ± 14 >10000 600 ± 170 4 ± 0.001

21 ± 8 25 ± 11 >10000 200 ± 25 11 ± 2

108 ± 11 184 ± 10 >10000 796 ± 89 11 ± 2

0.07 ± 0.02 159 ± 22 >10000 394 ± 32 16 ± 4

Figure 4. Overall structure of the catalytic domain with compound 4 (in cyan) docked in the active site of MMP-13 (PDB: 3KRY).46 The Zn2+-ion is shown as a gray sphere; amino acid residues in proximity to the surface are depicted as white stick models. Oxygen and nitrogen atoms are colored in red and blue, respectively. The surface is colored as follows: lipophilic regions are in green, hydrophilic in magenta, and neutral in white.

Further variations of the amino group in the para-position of the diphenylacetylene moiety led to heterocyclic compounds 20d, 20b, 20e, and 20a. Whereas pyrrolidine derivative 20a and piperidine derivative 20b are weak MMP inhibitors, their more polar morpholine (20d) and thiomorpholine (20e) analogues were found to be more active, especially against MMP-2 and MMP-13. This latter finding is in agreement with docking studies showing that the morpholine moiety of compound 20d is located in proximity to Lys249 at the bottom of the S1′ pocket of MMP-13 and that the morpholine oxygen atom is able to form a hydrogen bond with this amino acid (Figure 3, right). Again, the introduction of a flexible lipophilic n-pentyl moiety was found to be most beneficial in increasing inhibitory activity and, more importantly, in improving selectivity toward MMP-13. (2S,4S)-4-Hydroxyproline derivative 20k demonstrates an over 15-fold enhanced inhibition of MMP-2 (IC50(MMP-2) = 3 nM) and an over 40-fold increased inhibition of MMP-13 (IC50(MMP-13) = 0.7 nM) compared to the unsubstituted analogue 20g. The extraordinarily high

even more pronounced for the n-pentyl-substituted compound 16k, resulting in a 13-fold increase in inhibitory potency against MMP-2, a 2.4-fold increased inhibitory activity against MMP-9 and a 181-fold increased affinity toward MMP-13 when compared to unsubstituted compound 16g. In this series of compounds, only the morpholinomethyl substituted hydroxamic acid 16d was equally or even less active than compound 16g. For the (2S,4S)-configured 4-hydroxyproline derivatives, the introduction of a relatively small substituent, like an amino or a methoxy group, in the para-position of the distal phenyl ring has relatively little effect on inhibitory activity. Like its diastereomer 16i, N,N-dimethylamino-substituted compound 20i was found to exhibit selectivity toward MMP-13 over the other tested MMPs. The potency increase of the N,Ndimethylamino derivatives toward MMP-13 can be explained by the fact that, in comparison to the respective primary amines, their two methyl groups form additional lipophilic interactions with hydrophobic residues Pro255, Phe252, and Leu218 in the S1′ pocket of MMP-13 (Figure 3, left). 9549

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Scheme 7. Synthesis of PET Tracer [18F]4a

a

Reagents and conditions: (a) 41, Pd(PPh3)4, CuI, NEt3, ACN, rt, 52%; (b) H2NOH·HCl, NaOMe, MeOH, rt, 29%; (c) K(K222)[18F]F, K2CO3, ACN, 110°C, 20 min, rcy: 46.3 ± 9.3% (d.c.).

fluoropentoxy substituent. The terminal fluorine of this substituent is able to form a hydrogen bond with Lys249, which is located at the bottom of the S1′ pocket and represents another important pharmacophoric element, which additionally contributes to inhibitory activity and to the desired selectivity (Figure 4). Selectivity over Other Metzincins (ADAM10, ADAM17, Meprin α, and Meprin β). To investigate the inhibitory potential of the proline-derived hydroxamate inhibitors against other metzincins, fluorogenic peptide cleavage assays were performed using recombinant ectodomains of ADAM10, ADAM17, meprin α, and meprin β. For this, 11 compounds (20e, 20j, 20k, 4, 34, 12g, ent-12d, 16j, 23g, 16k, and 35) were tested in two different concentrations (5 and 50 μM, reconstituted in DMSO). ADAM10, ADAM17 as well as meprin β showed no inhibition upon incubation with any of these compounds (Supporting Information). Meprin α was inhibited by some hydroxamates, showing diminished activity at 5 μM inhibitor concentration up to complete inhibition at 50 μM. Because of this observation, 16 additional compounds (16g, 16i, 16d, 20i, 20h, 20d, 12h, 12d, 12b, 20b, 12e, 20a, 12f, 28d, 29d, and 37; 1−50 μM) were tested against meprin α. Again, some inhibitors exhibited inhibitory capacity toward meprin α activity, with estimated IC50 values in the micromolar range (Supporting Information). Thus, these compounds were much weaker in inhibiting meprin α than they were inhibiting MMPs and they were also less potent inhibitors of meprin α than the hydroxamate inhibitor actinonin. Overall, the proline-derived hydroxamate compounds display little inhibitory potential against meprin α and no inhibition of the other tested metzincins ADAM10, ADAM17, and meprin β. Therefore, these inhibitors seem to be specific for MMPs. Radiochemistry. Because of its unique biological profile, compound 4 was chosen for the radiosynthesis of its 18Flabeled analogue [18F]4. At first, precursor 46 was prepared, which possesses a tosylate leaving group (Scheme 7). For this purpose, aryl iodide 18 was subjected to a Sonogashira coupling with phenylacetylene derivative 41 (Scheme 5) to give ester 45, which was transformed into the desired precursor 46 via an aminolysis with hydroxylamine. Then radiolabeled [18F]4 was prepared in one step. Nucleophilic substitution (SN2) of the tosylate group in the precursor compound 46 with [18F]fluoride provided the desired product [18F]4 in radiochemical yields (rcy) of 46.3 ± 9.3% (n = 5) (decay-corrected based on cyclotron-derived [18F]fluoride ions (d.c.)) in 105 ± 12 min from the end of radionuclide production. The target compound was isolated with radiochemical purities of >99%

inhibitory activity of 20k toward MMP-13 can be explained by greasy interactions of the lipophilic n-pentyl chain, protruding deeply into the S1′ pocket, where it interacts with a number of lipophilic residues like Ile243, Ile218, Pro255, and Ala238. In Vitro MMP Inhibitory Activity of Fluorinated Derivatives. To select radiofluorinated candidates for potentially visualizing in vivo MMP-13 activity, the synthesized series of fluorinated proline and 4-hydroxyproline derivatives was also assayed for their inhibitory activity toward MMP-2, MMP-8, MMP-9, and MMP-13. As shown in Table 2, all synthesized amides, possessing a fluorine atom at different positions, inhibit MMP-13 with different selectivities over the other tested MMPs, whereas the fluorinated tertiary amine 35 exhibits no inhibition toward the studied enzymes. Among these amides, the (2S,4R)-configured 4-hydroxyproline derivative 37, possessing a fluorine-substituted distal phenyl ring, shows only moderate inhibitory activity toward the investigated MMPs while (2S,4S)-4-fluoroproline derivative 34 is a significantly more potent MMP-inhibitor. Nevertheless, both compounds demonstrated a lack of selectivity toward MMP-13 and were not considered as candidates for the development of a MMP-13 targeting PET tracer. By contrast, the (2S,4S)-configured 4-hydroxyproline derivative 4 containing a 5-fluoropentoxy moiety, mimicking the long lipophilic tail of the potent and selective MMP-13 inhibitor 20k (Table 1), showed a MMP binding profile suitable for the development of a PET tracer (Table 2). 4 excellently inhibits MMP-13 in the picomolar range (IC50(MMP-13) = 0.07 nM), with selectivities over the other tested MMPs ranging from 71× to 1543×. The synthesized 4hydroxyproline-based derivative 4 represents a good example for an inhibitor, which overcomes to a certain extent the challenging task of obtaining selectivity for MMP-13 over other MMPs that possess a deep S1′ pocket without losing inhibitory potency. The excellent MMP-13 inhibitory activity of 4 can be explained by the fact that it comprises structural features which were found to be the most beneficial in terms of inhibitory activity within the synthesized series of proline-derived hydroxamates 12, 16, 20, 23, 28, 29, 34, 35, and 37. Besides a Zn2+-chelating hydroxamate moiety, these structural elements include a (2S,4S)-configured 4-hydroxyproline scaffold and a lipophilic side chain, which is attached to the scaffold via an amide moiety whose carbonyl group can undergo interactions with, e.g., Leu185 and Ala186 near the entrance of the S1′ pocket (Figure 4). Moreover, 4 possesses a diphenylacetylene moiety, which is directed straight into the S1′ cleft of MMP-13 to form greasy interactions with, e.g., Pro242, Ile243, and Thr245 and which is elongated by the flexible and lipophilic 59550

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Figure 5. Radio-HPLC chromatograms of a typical quality control (QC) of a produced [18F]4 batch (left) and of the in vitro stability of [18F]4 (tR = 12.7 min) after incubation in human blood serum at 37 °C after 10 min (middle) and 90 min (right) measured at analytical HPLC-system B, method B.

elimination of the tracer and/or potential metabolites. In the first minutes after injection, accumulation of radioactivity was observed in large vessels and well perfused tissues such as liver, lung, kidneys, heart, and the spleen followed by a continuously decreasing signal until the end of the study. Moderate activity accumulation was found in harderian glands behind the eyes and submandibular glands. Visual inspection did not reveal further regions of interest presenting with an increased uptake of radioactivity. Accumulation of radioactivity in the bones, indicating defluorination of [18F]4, was not observed. PET image data was analyzed quantitatively for selected regions of interest (Figure 7). Time curves of regional radioactivity concentrations expressed as the percentage of injected dose per volume (%ID/mL) reveal a rapid washout of [18F]4 from blood. In fact, blood levels reach a peak at 38 s pi (27% ID/mL) with a subsequent decrease (50% decline from peak activity within 18 s). [18F]4 is taken up by the liver rapidly with a peak at 6 min pi (43%ID/mL). Thereafter, a slow and steady decrease of radioactivity in the liver to 25%ID/mL at 90 min pi was observed. The kidneys present with an uptake of radioactivity to a level of 11%ID/mL at 3 min pi, which is maintained until the end of the imaging study. Concurrently, only very low levels of radioactivity accumulated in the urinary bladder until 90 min pi, excluding the renal route as the major elimination pathway of [18F]4. Further regional analysis of brain, lung, heart, spleen, and muscle tissue reveal a similar distribution kinetic where after the initial perfusion phase in the first 5 min the radioactivity concentration stays more or less unchanged in the observed time window of 90 min.

and with specific activities of 0.7−33.2 GBq/μmol at the end of the synthesis. Partition Coefficient (log D (exp)). [18F]4 represents a lipophilic compound with a log D (exp) of 2.50 ± 0.01. In comparison to the calculated log D value (clogD) of 1.18*, the real solubility of [18F]4 in 1-octanol in the biphasic 1-octanol/ PBS system is increased by factor 21 (*, calculated log D value (clogD) was calculated by ACD/Chemsketch version ACD/ Laboratories 6.00 (log D = log P at physiological pH (7.4)). Stability in Human and Mouse Serum. An in vitro stability study was carried out using human and mouse blood serum, respectively. During incubation for up to 90 min at 37 °C, compound [18F]4 exhibited high serum stability. As shown in Figure 5, only the parent compound [18F]4 was detected by radio-HPLC in human serum. Significant radiometabolites or decomposition products could not be observed. The same results were obtained from the experiments with mouse serum. In Vivo Biodistribution Study. An in vivo biodistribution study in adult C57/Bl6 mice after intravenous injection of 400 kBq/g bodyweight of [18F]4 was performed as a 90 min dynamic PET scan. Representative maximum intensity projections (MIP) of whole body images at selected time points after tracer injection show notable and early accumulation of radioactivity in the liver followed by transport of radioactivity into the intestines (Figure 6). In contrast, only a very low activity concentration was observed in the kidneys and the urinary bladder, reflecting that renal elimination of [18F]4 is only playing a minor role as compared to hepatobiliary



CONCLUSION This study experimentally proves the feasibility of an approach, aiming to identify potent and selective MMP inhibitors that exhibit structural elements of collagen (substrate). The envisaged proline-based MMP inhibitors possessing general formula 1 could be accessed in chiral pool syntheses starting from the methyl esters of D- and L-proline as well as of (4R)-4hydroxy-L-proline. The synthesized compounds comprise potent MMP inhibitors, with IC50 values mostly in the nanomolar range. Their inhibitory activity varies upon altering the structure of the lipophilic side chain and the proline scaffold and also upon changing the configuration of the corresponding stereocenters. For example, whereas the hydroxamates derived from (4R)-4-hydroxy-L-proline, L-proline, and D-proline show low to moderate MMP inhibition, their counterparts derived from the unusual amino acid (4S)-4-hydroxy-L-proline were found to be the most potent inhibitors in these series. When

Figure 6. Maximum intensity projection of the in vivo biodistribution of tracer-associated radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]4. 9551

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Figure 7. In vivo biodistribution of radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]4. Time−activity curves illustrate tracer dynamics in selected regions of interests (ROI). % ID is percentage injected dose.



the hydroxy group in position 4 of the pyrrolidine ring was replaced by an amino group, or when the carbonyl group of the amide moiety was replaced by a methylene group, thus leading to tertiary amines, the inhibitory activity of the compounds was dramatically reduced. Whereas for the (4S)-4-hydroxy-L-proline derivatives the introduction of an amino group or a methoxy group at the distal phenyl ring of the diphenylacetylene moiety has only little effect on the inhibitory activity of these compounds, the introduction of a dimethylamino group in the same location leads to a pronounced selectivity toward MMP-13. Among the cyclic tertiary amines investigated, the morpholinomethyl and thiomorpholinomethyl derivatives, possessing a heteroatom in position 4 of their heterocycles, were relatively active against MMP-13. Of note, compounds possessing long and lipophilic substituents at the distal phenyl ring, like 4 and 20k, were found to demonstrate excellent MMP inhibitory activity in the nanomolar (MMP-2, -8, -9) and picomolar (MMP-13) range as well as noticeable selectivity, exceeding by these parameters reference compound 44. On the basis of computer-aided docking studies, the improved biological profile of these compounds was mainly attributed to interactions between their lipophilic side chains and the S1′ pocket of the enzymes, which is located on their “specificity loop”. Notably, the synthesized compounds demonstrated no off-target effects toward the other tested Zn2+-dependent metalloproteases. The proline derivatives show no inhibition of ADAMs and meprins. This is a promising finding in terms of their future use as therapeutic and diagnostic tools. Therefore, utilizing the structure of the (2S,4S)-configured 4-hydroxyproline derivative 4, a selective picomolar inhibitor of MMP-13, the radiotracer [18F]4 was successfully synthesized in good radiochemical yields of 46.3 ± 9.3%. Representing a new class of radiofluorinated proline-based hydroxamates, ligand [18F]4 exhibited excellent serum stability in vitro and relatively rapid clearance in mice with a clear preference for the hepatobiliary over the urinary excretion pathway. Also, defluorination and nonspecific binding of [18F]4 in nonexcretory organs were not observed in vivo. Thus, the developed radiolabeled compound [18 F]4 represents a promising tool for noninvasive PET imaging and the quantification of MMP activity in vivo, especially in pathologies associated with overexpression of MMP-13, such as human carcinomas, rheumatoid arthritis, and osteoarthritis.

EXPERIMENTAL SECTION

Chemistry, General. Unless otherwise mentioned, THF was dried with sodium/benzophenone and was freshly distilled before use. Microwave assisted synthesis: adjustable parameters are given in brackets: irradiation power, maximum pressure, temperature, hold time. Microwave 1: CEM Discover LabMate (CEM Corp., NC); glass vessel (capacity 10 mL), sealed with Teflon septa; stirring, on; ramp time, 5 min; piezoelectric pressure sensor; external infrared temperature sensor. Microwave 2: MARS 240/50 (CEM Corp., NC); Teflon vessels (capacity 100 mL), sealed with Teflon cap; stirring, on; ramp time, 5 min; continuous mode turntable system; cavity exhaust fan; pressure and temperature control. Thin layer chromatography (TLC): Silica Gel 60 F254 plates (Merck). Reversed phase thin layer chromatography (RP-TLC): Silica Gel 60 RP-18 F254S plates (Merck). Flash chromatography (FC): Silica gel 60, 40−64 μm (Macherey-Nagel); brackets include eluent, diameter of the column, fraction size, Rf value. Automatic flash column chromatography: Isolera One (Biotage); brackets include eluent, cartridge-type. Melting point: Melting point apparatus SMP 3 (Stuart Scientific), uncorrected. Optical rotation α [deg] was determined with a Polarimeter 341 (PerkinElmer); path length 1 dm, wavelength 589 nm (sodium D −1 −1 line); the unit of the specific rotation [α]20 D [deg·mL·dm ·g ] is −1 omitted; the concentration of the sample c [mg · mL ] and the solvent used are given in brackets. 1H NMR (400 MHz), 13C NMR (100 MHz): Agilent DD2 400 MHz spectrometer; δ in ppm related to tetramethylsilane. IR: IR Prestige-21 (Shimadzu). APCI/LC-MS: MicrOTOF-QII (Bruker). HPLC methods for the determination of product purity. Method 1: Merck Hitachi equipment; UV detector, L7400; autosampler, L-7200; pump, L-7100; degasser, L-7614; column, LiChrospher 60 RP-select B (5 μm); LiCroCART 250−4 mm cartridge; flow rate, 1.00 mL/min; injection volume, 5.0 μL; detection at λ = 210 nm for 30 min. Solvents: A, water with 0.05% (V/V) trifluoroacetic acid; B, acetonitrile with 0.05% (V/V) trifluoroacetic acid. Gradient elution (A %): 0−4 min, 90%, 4−29 min: gradient from 90% to 0%, 29−31 min; 0%, 31−31.5 min; gradient from 0% to 90%, 31.5−40 min, 90%. Method 2: Merck Hitachi equipment; UV detector, L-7400; pump, L-6200A; column, Phenomenex Gemini 5 μm C6-Phenyl 110 Å; LC Column 250 mm × 4.6 mm; flow rate, 1.00 mL/min; injection volume, 5.0 μL; detection at λ = 254 nm for 20 min. Solvents: A, acetonitrile:10 mM ammonium formate =1 0:90 with 0.1% formic acid; B, acetonitrile:10 mM ammonium formate = 90:10 with 0.1% formic acid. Gradient elution (A %): 0−5 min, 100%, 5−15 min; gradient from 100% to 0%, 15−20 min; 0%, 20−22 min; gradient from 0% to 100%, 22−30 min, 100%. With the exception of compounds 12e (92.2%) and ent-12e (94.5%), the purities of the final products were ≥95% as determined by HPLC. Synthetic Procedures. (2S,4S)-1-[4-({4-[(5-Fluoropentyl)oxy]phenyl}ethynyl)benzoyl]-N,4-dihydroxypyrrolidine-2-carboxamide (4). Hydroxylamine hydrochloride (53 mg, 0.76 mmol) and a 5.4 M solution of sodium methoxide in methanol (0.20 mL, 1.1 mmol) were 9552

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Article

376.0040, found 376.0054. HPLC (method 1): tR = 14.0 min, purity 98.7%. Methyl (2S,4S)-1-(4-Iodobenzoyl)-4-[(4-nitrobenzoyl)oxy]pyrrolidine-2-carboxylate (17). Under N2 atmosphere, DIAD (0.35 mL, 360 mg, 1.8 mmol) was added dropwise to an ice-cooled solution of 14 (300 mg, 0.80 mmol), p-nitrobenzoic acid (270 mg, 1.6 mmol), and triphenylphosphine (460 mg, 1.8 mmol) in dry THF (20 mL). Then the reaction mixture was stirred at ambient temperature for 4.5 h. Thereafter, the solvent was removed in vacuo and the residue was purified by flash column chromatography (Ø = 3 cm, h = 15 cm, V = 20 mL, cyclohexane/ethyl acetate = 4/1 → 3/1) to give 17 (330 mg, 0.64 mmol, 80%) as colorless solid; mp = 78−80 °C. TLC: Rf = 0.27 (cyclohexane/ethyl acetate = 3/1). Specific rotation: [α]20 D = +19.3 (c = 1.2, CH3OH). 1H NMR (DMSO-d6): δ [ppm] = 2.28−2.38 (m, 0.65H, CHCH2CH), 2.39−2.47 (m, 0.35H, CHCH2CH), 2.65−2.81 (m, 1H, CHCH2CH), 3.48 (s, 3 × 0.35H, CO2CH3), 3.67 (s, 3 × 0.65H, CO2CH3), 3.72 (d, J = 12.0 Hz, 0.65H, NCH2), 3.81 (d, J = 14.0 Hz, 0.35H, NCH2), 3.95 (dd, J = 12.0/5.3 Hz, 0.65H, NCH2), 4.02 (dd, J = 14.1/5.0 Hz, 0.35H, NCH2), 4.72 (d, J = 8.9 Hz, 0.35H, NCH), 4.85 (dd, J = 9.4/2.1 Hz, 0.65H, NCH), 5.49−4.56 (m, 0.65H, CHO), 5.57−4.63 (m, 0.35H, CHO), 7.21−7.29 (m, 2 × 0.35H, 2′H4‑iodobenzoyl, 6′-H4‑iodobenzoyl), 7.30−7.39 (m, 2 × 0.65H, 2′-H4‑iodobenzoyl, 6′-H4‑iodobenzoyl), 7.77−7.83 (m, 2 × 0.35H, 3′-H4‑iodobenzoyl, 5′H4‑iodobenzoyl), 7.83−7.89 (m, 2 × 0.65H, 3′-H4‑iodobenzoyl , 5′H4‑iodobenzoyl), 8.04−8.15 (m, 2H, 2″-H4‑nitrobenzoyl, 6″-H4‑nitrobenzoyl), 8.34−8.42 (m, 2H, 3″-H4‑nitrobenzoyl, 5″-H4‑nitrobenzoyl); two rotamers exist in the ratio 65:35. 13C NMR (DMSO-d6): δ [ppm] = 34.1 (1C, CHCH2CH), 52.2 (1C, CO2CH3), 54.1 (1C, NCH2), 57.4 (1C, NCH), 74.4 (1C, CHO), 97.3 (1C, C-4′4‑iodobenzoyl), 123.9 (2C, C3″4‑nitrobenzoyl, 5″4‑nitrobenzoyl), 129.0 (2C, C-2′4‑iodobenzoyl, C-6′4‑iodobenzoyl), 130.6 (2C, C-2″ 4‑nitrobenzoyl , C-6″ 4‑nitrobenzoyl ), 134.7 (1C, C1″4‑nitrobenzoyl), 135.2 (1C, C-1′4‑iodobenzoyl), 137.3 (2C, C-3′4‑iodobenzoyl, C-5′4‑iodobenzoyl), 150.4 (1C, C-4″4‑nitrobenzoyl), 163.5 (1C, O C4‑nitrobenzoyl), 167.8 (1C, OC4‑iodobenzoyl), 171.2 (1C, CO2CH3); two rotamers exist in the ratio 65:35, the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3109, 2951, 1724, 1635, 1523, 1408, 1342, 1269, 1199, 1103, 1056, 1006, 833, 717. HRMS (m/z): [M + H]+ calcd for C20H18IN2O7 525.0153, found 525.0159. HPLC (method 1): tR = 19.8 min, purity 97.8%. Methyl (2S,4S)-4-Hydroxy-1-(4-iodobenzoyl)pyrrolidine-2-carboxylate (18). At ambient temperature, potassium carbonate (1.0 g, 7.2 mmol) was added to a solution of 17 (330 mg, 0.63 mmol) in dry methanol (20 mL). After stirring the reaction mixture for 30 min, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography (Ø = 3 cm, h = 15 cm, V = 20 mL, ethyl acetate, Rf = 0.28) to give 18 (200 mg, 0.53 mmol, 84%) as colorless solid; mp = 153−154 °C. Specific rotation: 1 [α]20 D = −62.2 (c = 2.4, CH3OH). H NMR (DMSO-d6): δ [ppm] = 1.82 (dt, J = 12.5/6.5 Hz, 0.75H, CHCH2CH), 2.00−2.08 (m, 0.25H, CHCH2CH), 2.31−2.46 (m, 1H, CHCH2CH), 3.29−3.35 (m, 0.75H, NCH2), 3.35−3.41 (m, 0.25H, NCH2), 3.47 (s, 3 × 0.25H, CO2CH3), 3.56 (dd, J = 10.3/5.9 Hz, 0.65H, NCH2), 3.65 (s, 3 × 0.75H, CO2CH3), 3.71 (dd, J = 12.6/5.2 Hz, 0.25H, NCH2), 4.19−4.27 (m, 0.75H, CHOH), 4.28−4.34 (m, 0.25H, CHOH), 4.44−4.49 (m, 0.25H, NCH), 4.55 (dd, J = 8.5/6.7 Hz, 0.75H, NCH), 5.00 (d, J = 2.6 Hz, 0.25H, CHOH), 5.16 (d, J = 4.1 Hz, 0.75H, CHOH), 7.16−7.22 (m, 2 × 0.25H, 2′-H4‑iodobenzoyl, 6′-H4‑iodobenzoyl), 7.28−7.35 (m, 2 × 0.75H, 2′-H4‑iodobenzoyl, 6′-H4‑iodobenzoyl), 7.73−7.80 (m, 2× 0.25H, 3′H4‑iodobenzoyl, 5′-H4‑iodobenzoyl), 7.81−7.87 (m, 2 × 0.75H, 3′-H4‑iodobenzoyl, 5′-H4‑iodobenzoyl); two rotamers exist in the ratio 75:25. 13C NMR (DMSO-d6): δ [ppm] = 36.8 (1C, CHCH2CH), 51.9 (1C, CO2CH3), 56.2 (1C, NCH2), 57.2 (1C, NCH), 68.5 (1C, CHOH), 97.2 (1C, C4′4‑iodobenzoyl), 129.1 (2C, C-2′4‑iodobenzoyl, C-6′4‑iodobenzoyl), 135.3 (1C, C1′4‑iodobenzoyl), 137.2 (2C, C-3′4‑iodobenzoyl, C-5′4‑iodobenzoyl), 167.6 (1C, OC4‑iodobenzoyl), 171.7 (1C, CO2CH3); two rotamers exist in the ratio 75:25; the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3271, 3001, 2947, 2920, 1735, 1589, 1435, 1327, 1207, 1168, 1080, 1010, 813, 748, 709. HRMS (m/z): [M + H]+ calcd for C13H15INO4 376.0040, found 376.0054. HPLC (method 1): tR = 13.7 min, purity 98.5%.

added to a solution of 43 (87 mg, 0.19 mmol) in methanol (5 mL), and the reaction mixture was stirred at ambient temperature overnight. Then the solvent was removed in vacuo, and the residue was dissolved in water (15 mL) and extracted with ethyl acetate (6×). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (Ø = 1 cm, h = 15 cm, V = 5 mL, ethyl acetate → dichloromethane/methanol = 9.8/0.2 → 9.3/0.7) to give 4 (47 mg, 0.10 mmol, 54%) as colorless solid; mp = 182−184 °C. TLC: Rf = 0.36 (CH2Cl2/CH3OH = 9/1). Specific rotation: [α]20 D = +22.3 (c = 1.6, CH3OH). 1H NMR (DMSO-d6): δ [ppm] = 1.45−1.55 (m, 2H, O(CH2)2CH2(CH2)2F), 1.63−1.82 (m, 5H, OCH2CH2CH2CH2CH2F, CHCH2CH (1H)), 2.36 (ddd, J = 12.6/ 8.5/6.0 Hz, 1H, CHCH2CH), 3.38 (dd, J = 10.2/5.7 Hz, 1H, NCH2), 3.57 (dd, J = 10.2/5.8 Hz, 1H, NCH2), 4.03 (t, J = 6.4 Hz, 2H, ArOCH2), 4.11−4.22 (m, 1H, CHOH), 4.34−4.40 (m, 1H, NCH), 4.46 (dt, J = 47.5/6.1 Hz, 2H, CH2F), 5.40 (d, J = 6.5 Hz, 1H, CHOH), 6.95−7.04 (m, 2H, 3″-H 4‑[(5‑fluoropentyl)oxy]phenyl , 5″H4‑[(5‑fluoropentyl)oxy]phenyl), 7.46−7.54 (m, 2H, 2″-H4-[(5-fluoropentyl)oxy]phenyl, 6″-H4‑[(5‑fluoropentyl)oxy]phenyl), 7.54−7.65 (m, 4H, 3′-Hbenzoyl, 5′-Hbenzoyl, 2′-Hbenzoyl, 6′-Hbenzoyl) 8.96 (br s, 1H, CONHOH), 10.81 (s, 1H, CONHOH); two rotamers exist in the ratio 85:15; the signals of the major rotamer are given. 13C NMR (DMSO-d6): δ [ppm] = 21.4 (d, J = 5.4 Hz, 1C, O(CH2)2CH2(CH2)2F), 28.2 (1C, OCH2CH2(CH2)3F), 29.5 (d, J = 19.2 Hz, 1C, O(CH2)3CH2CH2F), 37.2 (1C, CHCH2CH), 57.0 (1C, NCH), 57.1 (1C, NCH2), 67.5 (1C, ArOCH2), 68.7 (1C, CHOH), 83.7 (d, J = 161.6 Hz, 1C, O(CH2)4CH2F), 87.4 (1C, C C), 91.1 (1C, CC), 113.7 (1C, C-1″4‑[(5‑fluoropentyl)oxy]phenyl), 114.9 (2C, C-3″4‑[(5‑fluoropentyl)oxy]phenyl, C-5″4‑[(5‑fluoropentyl)oxy]phenyl), 124.4 (1C, C-4′benzoyl), 127.8 (2C, C-2′benzoyl, C-6′benzoyl), 130.9 (2C, C-3′benzoyl, C5′benzoyl), 133.1 (2C, C-2″4‑[(5‑fluoropentyl)oxy]phenyl, C6″4‑[(5‑fluoropentyl)oxy]phenyl), 135.6 (1C, C-1′benzoyl), 159.2 (1C, C4″4‑[(5‑fluoropentyl)oxy]phenyl), 168.0 (1C, OCbenzoyl), 168.7 (1C, CONHOH); the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3178, 3105, 2943, 2904, 1600, 1500, 1431, 1249, 1068, 1049, 848, 833, 763, 640. HRMS (m/z): [M + H]+ calcd for C25H28FN2O5 455.1977, found 455.1985. HPLC (method 2): tR = 16.2 min, purity 97.8%. Methyl (2S,4R)-4-Hydroxy-1-(4-iodobenzoyl)pyrrolidine-2-carboxylate (14). Triethylamine (0.76 mL, 0.56 g, 5.5 mmol) was added to solution of (2S,4R)-4-hdroxyproline methyl ester hydrochloride (500 mg, 2.8 mmol) in dichloromethane (25 mL), and the mixture was stirred for 15 min at 0 °C. After the addition of 4dimethylaminopyridine (34 mg, 0.28 mmol), a solution of 4iodobenzoyl chloride (730 mg, 2.8 mmol) in dichloromethane (15 mL) was added dropwise over a period of 10 min at 0 °C. Then the mixture was stirred for 2 h at ambient temperature. Afterward, the reaction mixture was washed with a saturated aqueous solution of NaHCO3 and the aqueous phase was extracted with dichloromethane (3×). The combined organic phases were dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography (Ø = 3 cm, h = 15 cm, V = 20 mL, ethyl acetate, Rf = 0.28) to give 14 (1.0 g, 2.7 mmol, 97%) as colorless solid; mp = 146− 1 147 °C. Specific rotation: [α]20 D = −109.4 (c = 1.9, CH3OH). H NMR (DMSO-d6): δ [ppm] = 1.96 (ddd, J = 13.1/9.3/4.3 Hz, 1H, CHCH2CH), 2.20 (ddt, J = 13.1/7.9/2.0 Hz, 1H, CHCH2CH), 3.27− 3.30 (m, 1H, NCH2), 3.66 (s, 3H, CO2CH3), 3.72 (dd, J = 11.0/3.8 Hz, 1H, NCH2), 4.26−4.30 (m, 1H, CHOH), 4.53−4.57 (m, 1H, NCH), 5.11 (d, J = 3.4 Hz, 1H, CHOH), 7.31−7.35 (m, 2H, 2′H4‑iodobenzoyl, 6′-H4‑iodobenzoyl), 7.83−7.86 (m, 2H, 3′-H4‑iodobenzoyl, 5′H4‑iodobenzoyl); two rotamers exist in the ratio 85:15; the signals of the major rotamer are given. 13C NMR (DMSO-d6): δ [ppm] = 37.1 (1C, CHCH2CH), 51.9 (1C, CO2CH3), 57.7 (1C, NCH), 57.8 (1C, NCH2), 68.9 (1C, CHOH), 97.6 (1C, C-4′4‑iodobenzoyl), 129.4 (2C, C2′4‑iodobenzoyl, C-6′4‑iodobenzoyl), 135.0 (1C, C-1′4‑iodobenzoyl), 137.2 (2C, C3′4‑iodobenzoyl, C-5′4‑iodobenzoyl), 168.0 (1C, OC4‑iodobenzoyl), 172.3 (1C, CO2CH3); two rotamers exist in the ratio 85:15; the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3437, 3194, 2916, 1755, 1735, 1604, 1435, 1346, 1280, 1234, 1203, 1180, 1083, 1056, 1006, 829, 756. HRMS (m/z): [M + H]+ calcd for C13H15INO4 9553

DOI: 10.1021/acs.jmedchem.6b01291 J. Med. Chem. 2016, 59, 9541−9559

Journal of Medicinal Chemistry

Article

O(CH2)2CH2(CH2)2OTs), 1.58−1.66 (m, 4H, OCH2CH2CH2CH2CH2OTs), 2.40 (s, 3H, ArCH3), 3.91 (t, J = 6.4 Hz, 2H, ArOCH2), 4.00 (s, 1H, CCH), 4.03 (t, J = 6.3 Hz, 2H, CH2OTs), 6.87−6.92 (m, 2H, 2-H4‑ethynylphenyl, 6-H4‑ethynylphenyl), 7.36− 7.41 (m, 2H, 3-H4‑ethynylphenyl, 5-H4‑ethynylphenyl), 7.44−7.49 (m, 2H, 3′H4‑methylbenzenesulfonate, 5′-H4‑methylbenzenesulfonate), 7.76−7.80 (m, 2H, 2′H4‑methylbenzenesulfonate, 6′-H4‑methylbenzenesulfonate). 13C NMR (DMSO-d6): δ [ppm] = 21.1 (1C, ArCH3), 21.5 (1C, O(CH2)2CH2(CH2)2OTs), 27.8 (1C, OCH2CH2(CH2)3OTs), 27.9 (1C, O(CH2)3CH2CH2OTs), 67.3 (1C, ArOCH2,), 70.8 (1C, O(CH2)4CH2OTs), 79.1 (1C, C CH), 83.5 (1C, CCH), 113.5 (1C, C-44‑ethynylphenyl), 114.7 (2C, C24‑ethynylphenyl, C-64‑ethynylphenyl), 127.5 (2C, C-2′4‑methylbenzenesulfonate, C6′ 4‑methylbenzenesulfonate ), 130.1 (2C, C-3′ 4‑methylbenzenesulfonate , C5′4‑methylbenzenesulfonate), 132.5 (1C, C-1′4‑methylbenzenesulfonate), 133.2 (2C, C-34‑ethynylphenyl, C-54‑ethynylphenyl), 144.8 (1C, C-4′4‑methylbenzenesulfonate); 158.9 (1C, C-14‑ethynylphenyl). IR (neat): ν̃ [cm−1] = 3275, 2978, 2924, 1604, 1508, 1342, 1292, 1246, 1165, 1018, 952, 914, 833, 806, 771, 663. HRMS (m/z): [M + H]+ calcd for C20H23O4S 359.1312, found 359.1328. HPLC (method 1): tR = 24.6 min, purity 94.0%. 1-Ethynyl-4-[(5-fluoropentyl)oxy]benzene (42). Tetrabutylammonium fluoride trihydrate (460 mg, 1.5 mmol) was added to 41 (240 mg, 0.66 mmol), and the reaction mixture was stirred at 65 °C for 30 min. Then water was added, and the mixture was extracted with ethyl acetate (3×). The combined organic layers were washed with water and brine, dried (Na2SO4), and filtered, and the solvent was removed in vacuo. The residue was purified by flash column chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, cyclohexane/ethyl acetate = 1/0 → 9/1) to give 42 as colorless solid (95 mg, 0.46 mmol, 70%); mp = 43 °C. TLC: Rf = 0.57 (cyclohexane/ethyl acetate = 9/1). 1H NMR (DMSO-d6): δ [ppm] = 1.45−1.52 (m, 2H, O(CH2)2CH2(CH2)2F), 1.65−1.78 (m, 4H, OCH2CH2CH2CH2CH2F), 3.98−4.01 (m, 3H, OCH2(CH2)4OH, CCH), 4.45 (dt, J = 47.5/6.1 Hz, 2H, CH2F), 6.91−6.94 (m, 2H, 3-H1‑ethynylphenyl, 5-H1‑ethynylphenyl), 7.37−7.41 (m, 2H, 2-H1‑ethynylphenyl, 6-H1‑ethynylphenyl). 13C NMR (DMSO-d6): δ [ppm] = 21.4 (d, J = 5.6 Hz, 1C, O(CH2)2CH2(CH2)2F), 28.1 (1C, OCH2CH2(CH2)3F), 29.5 (d, J = 19.2 Hz, 1C, O(CH2)3CH2CH2F), 67.4 (1C, OCH2(CH2)4F) 79.1 (1C, CCH), 83.5 (1C, CCH), 83.7 (d, J = 161.7 Hz, 1C, O(CH2)4CH2F), 113.5 (1C, C11‑ethynylphenyl), 114.7 (2C, C-31‑ethynylphenyl, C-51‑ethynylphenyl), 133.2 (2C, C-21‑ethynylphenyl, C-61‑ethynylphenyl), 159.0 (1C, C-41‑ethynylphenyl). IR (neat): ν̃ [cm−1] = 3275, 2943, 2870, 1600, 1504, 1473, 1288, 1242, 1172, 1029, 1002, 968, 833, 686, 644. HRMS (m/z): [M + H]+ calcd for C13H16FO 207.1180, found 207.1178. HPLC (method 1): tR = 24.0 min, purity 91.3%. Methyl (2S,4S)-1-[4-({4-[(5-Fluoropentyl)oxy]phenyl}ethynyl)benzoyl]-4-hydroxypyrrolidine-2-carboxylate (43). Under N2 atmosphere, copper(I) iodide (9 mg, 0.05 mmol), tetrakis(triphenylphosphine)palladium(0) (28 mg, 0.024 mmol), and triethylamine (0.27 mL, 0.20 g, 2.0 mmol) were added to a solution of 18 (91 mg, 0.24 mmol) in acetonitrile (5 mL) at ambient temperature. Then a solution of 42 (75 mg, 0.36 mmol) in acetonitrile (5 mL) was added dropwise over a period of 2 h. After stirring, the reaction mixture for additional 3 h, the solvent was removed in vacuo, and the residue was purified twice by flash column chromatography ((1) Ø = 2 cm, h = 15 cm, V = 10 mL, cyclohexane/ethyl acetate = 3/1 → 0/1; (2) Ø = 2 cm, h = 15 cm, V = 10 mL, acetonitrile) to give 43 (99 mg, 0.22 mmol, 90%) as colorless solid; mp = 110 °C. TLC: Rf = 0.18 (CH2Cl2/ CH3OH = 9.8/0.2). Specific rotation: [α]20 D = −24.4 (c = 1.7, CH3OH). 1H NMR (DMSO-d6): δ [ppm] = 1.47−1.54 (m, 2H, O(CH2)2CH2(CH2)2F), 1.66−1.80 (m, 4H, OCH 2 CH 2 CH 2 CH 2 CH 2 F), 1.83 (dt, J = 12.5/6.5 Hz, 1H, CHCH2CH), 2.43 (ddd, J = 12.5/8.7/5.7 Hz, 1H, CHCH2CH), 3.36 (dd, J = 10.3/5.7 Hz, 1H, NCH2), 3.60 (dd, J = 10.3/5.9 Hz, 1H, NCH2), 3.66 (s, 3H, CO2CH3), 4.03 (t, J = 6.4 Hz, 2H, ArOCH2), 4.22−4.28 (m, 1H, CHOH), 4.46 (dt, J = 47.5/6.1 Hz, 2H, CH2F), 4.58 (dd, J = 8.6/6.5 Hz, 1H, NCH), 5.18 (d, J = 4.2 Hz, 1H, CHOH), 6.97−7.01 (m, 2H, 3″-H4‑[(5‑fluoropentyl)oxy]phenyl, 5″H4‑[(5‑fluoropentyl)oxy]phenyl), 7.47−7.53 (m, 2H, 2″-H4-[(5-fluoropentyl)oxy]phenyl, 6″-H4‑[(5‑fluoropentyl)oxy]phenyl), 7.53−7.57 (m, 2H, 2′-Hbenzoyl, 6′-Hbenzoyl), 7.58−7.62 (m, 2H, 3′-Hbenzoyl, 5′-Hbenzoyl); two rotamers exist in the

5-{4-[(Trimethylsilyl)ethynyl]phenoxy}pentan-1-ol (39). Under N2 atmosphere, copper(I) iodide (116 mg, 0.61 mmol), tetrakis(triphenylphosphine)palladium(0) (350 mg, 0.30 mmol), and trimethylsilylacetylene (1.4 mL, 1.0 g, 10 mmol) were added to a solution of 5-(4-bromophenoxy)pentan-1-ol (1.3 g, 5.1 mmol) in triethylamine (35 mL). The mixture was heated to reflux for 18 h. After evaporation of the solvent, the residue was purified by flash column chromatography (Ø = 3 cm, h = 15 cm, V = 20 mL, cyclohexane/ethyl acetate = 9/1 → 1/2) to give 39 (1.2 g, 4.3 mmol, 85%) as yellowish oil. TLC: Rf = 0.26 (cyclohexane/ethyl acetate = 2/ 1). 1H NMR (DMSO-d6): δ [ppm] = 0.21 (s, 9H, Si(CH3)3), 1.36− 1.51 (m, 4H, O(CH2)2CH2CH2CH2OH), 1.66−1.75 (m, 2H, OCH2CH2(CH2)3OH), 3.40 (t, J = 6.2 Hz, 2H, O(CH2)4CH2OH), 3.97 (t, J = 6.5 Hz, 2H, OCH2(CH2)4OH), 4.34 (br s, 1H, CH2OH), 6.87−6.93 (m, 2H, 2-H4‑[(trimethylsilyl)ethynyl]phenyl, 6H4‑[(trimethylsilyl)ethynyl]phenyl), 7.34−7.40 (m, 2H, 3H4‑[(trimethylsilyl)ethynyl]phenyl, 5-H4‑[(trimethylsilyl)ethynyl]phenyl). 13C NMR (DMSO-d6): δ [ppm] = 0.02 (3C, Si(CH3)3), 22.1 (1C, O(CH2)2CH2(CH2)2OH), 28.5 (1C, OCH2CH2(CH2)3OH), 32.2 (1C, O(CH2)3CH2CH2OH), 60.6 (1C, O(CH2)4CH2OH), 67.6 (1C, OCH2(CH2)3CH2OH), 92.2 (1C, SiCC), 105.5 (1C, SiC C), 114.0 (1C, C-4 4‑[(trimethylsilyl)ethynyl]phenyl ), 114.7 (2C, C24‑[(trimethylsilyl)ethynyl]phenyl, C-64‑[(trimethylsilyl)ethynyl]phenyl), 133.2 (2C, C34‑[(trimethylsilyl)ethynyl]phenyl, C-54‑[(trimethylsilyl)ethynyl]phenyl), 159.1 (1C, C14‑[(trimethylsilyl)ethynyl]phenyl). IR (neat): ν̃ [cm−1] = 3309, 2939, 2866, 2152, 1604, 1504, 1469, 1246, 1168, 1026, 860, 833, 759. HRMS (m/ z): [M + H]+ calcd for C16H25O2Si 277.1618, found 277.1624. HPLC (method 1): tR = 24.1 min, purity 96.1%. 5-(4-Ethynylphenoxy)pentan-1-ol (40). A solution of tetrabutylammonium fluoride trihydrate (1.6 g, 5.1 mmol) in THF (15 mL) was added dropwise to an ice-cooled solution of 39 (1.2 g, 4.3 mmol) in THF (50 mL). The mixture was stirred at 0 °C for 40 min. Then a saturated aqueous solution of ammonium chloride (55 mL) was added and the mixture was extracted with ethyl acetate (3×). The combined organic layers were washed with water and brine, dried (Na2SO4), and filtered, and the solvent was removed in vacuo. The residue was purified by flash column chromatography (Ø = 4 cm, h = 15 cm, V = 30 mL, cyclohexane/ethyl acetate = 9/1 → 1/1) to give 40 as colorless solid (800 mg, 3.9 mmol, 90%); mp = 43 °C. TLC: Rf = 0.17 (cyclohexane/ethyl acetate = 2/1). 1H NMR (DMSO-d6): δ [ppm] = 1.37−1.51 (m, 4H, O(CH2)2CH2CH2CH2OH), 1.66−1.75 (m, 2H, OCH2CH2(CH2)3OH), 3.37−3.43 (m, 2H, O(CH2)4CH2OH), 3.97 (t, J = 6.5 Hz, 2H, OCH2(CH2)4OH), 3.99 (s, 1H, CCH), 4.36 (t, J = 5.1 Hz, 1H, CH2OH), 6.89−6.94 (m, 2H, 2-H4‑ethynylphenyl, 6H4‑ethynylphenyl), 7.36−7.42 (m, 2H, 3-H4‑ethynylphenyl, 5-H4‑ethynylphenyl). 13 C NMR (DMSO-d6): δ [ppm] = 22.1 (1C, O(CH2)2CH2(CH2)2OH), 28.5 (1C, OCH2CH2(CH2)3OH), 32.2 (1C, O(CH2)3CH2CH2OH), 60.6 (1C, O(CH2)4CH2OH), 67.6 (1C, OCH2(CH2)4OH), 79.1 (1C, CCH), 83.6 (1C, CCH), 113.5 (1C, C-4 4‑ethynylphenyl ), 114.7 (2C, C-2 4‑ethynylphenyl , C64‑ethynylphenyl), 133.2 (2C, C-34‑ethynylphenyl, C-54‑ethynylphenyl), 159.0 (1C, C-14‑ethynylphenyl). IR (neat): ν̃ [cm−1] = 3251, 2943, 1608, 1504, 1469, 1288, 1253, 1168, 1111, 1033, 1006, 991, 833, 806, 709. HRMS (m/ z): [M + H]+ calcd for C13H17O2 205.1223, found 205.1220. HPLC (method 1): tR = 19.8 min, purity 95.2%. 5-(4-Ethynylphenoxy)pentyl 4-Methylbenzenesulfonate (41). A solution of 4-toluenesulfonyl chloride (1.1 g, 5.8 mmol) in dry dichloromethane (10 mL) was added dropwise to an ice-cooled solution of 40 (590 mg, 2.9 mmol) and triethylamine (0.40 mL, 0.29 g, 2.9 mmol) in a mixture of dichloromethane and pyridine (4:1, 40 mL). After stirring the mixture for 30 min at 0 °C, the reaction mixture was allowed to warm to room temperature and stirring was continued overnight. Then water was added and the mixture was extracted with ethyl acetate (3×). The combined organic layers were washed with water and brine, dried (Na2SO4), and filtered, and the solvent was removed in vacuo. The residue was purified by flash column chromatography (Ø = 3 cm, h = 15 cm, V = 20 mL, cyclohexane/ ethyl acetate = 9/1 → 2/1) to give 41 as colorless solid (760 mg, 2.1 mmol, 73%); mp = 58−60 °C. TLC: Rf = 0.50 (cyclohexane/ethyl acetate = 3/1). 1H NMR (DMSO-d6): δ [ppm] = 1.31−1.41 (m, 2H, 9554

DOI: 10.1021/acs.jmedchem.6b01291 J. Med. Chem. 2016, 59, 9541−9559

Journal of Medicinal Chemistry

Article

ratio 75:25; the signals of the major rotamer are given. 13C NMR (DMSO-d 6 ): δ [ppm] = 21.4 (d, J = 5.2 Hz, 1C, O(CH2)2CH2(CH2)2F), 28.1 (1C, OCH2CH2(CH2)3F), 29.5 (d, J = 19.5 Hz, 1C, O(CH2)3CH2CH2F), 36.8 (1C, CHCH2CH), 51.9 (1C, CO2CH3), 56.2 (1C, NCH2), 57.2 (1C, NCH), 67.5 (1C, ArOCH2), 68.5 (1C, CHOH), 83.7 (d, J = 161.6 Hz, 1C, O(CH2)4CH2F), 87.4 (1C, CC), 91.1 (1C, CC), 113.7 (1C, C-1″4‑[(5‑fluoropentyl)oxy]phenyl), 114.9 (2C, C-3″4‑[(5‑fluoropentyl)oxy]phenyl, C-5″4‑[(5‑fluoropentyl)oxy]phenyl), 124.5 (1C, C-4′benzoyl), 127.5 (2C, C-2′benzoyl, C-6′benzoyl), 131.0 (2C, C-3′benzoyl, C-5′benzoyl), 133.1 (2C, C-2″4‑[(5‑fluoropentyl)oxy]phenyl, C6″4‑[(5‑fluoropentyl)oxy]phenyl), 135.4 (1C, C-1′benzoyl), 159.2 (1C, C4″ 4‑[(5‑fluoropentyl)oxy]phenyl ), 167.7 (1C, O = CN), 171.7 (1C, CO2CH3); the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3371, 2954, 1743, 1600, 1500, 1431, 1249, 1211, 1172, 1087, 1018, 829, 759, 682. HRMS (m/z): [M + H]+ calcd for C26H29FNO5 454.2024, found 454.2022. HPLC (method 1): tR = 21.6 min, purity 97.8%. Methyl (2S,4S)-4-Hydroxy-1-{4-[(4-[{5-(tosyloxy)pentyl]oxy}phenyl)ethynyl]benzoyl}pyrrolidine-2-carboxylate (45). Under N2 atmosphere, copper(I) iodide (10 mg, 0.05 mmol), tetrakis(triphenylphosphine)palladium(0) (63 mg, 0.05 mmol), and triethylamine (0.61 mL, 0.44 g, 4.4 mmol) were added to a solution of 18 (210 mg, 0.55 mmol) in acetonitrile (7 mL) at ambient temperature. Then a solution of 41 (300 mg, 0.82 mmol) in acetonitrile (7 mL) was added dropwise over a period of 2 h. After stirring, the reaction mixture for additional 3 h, the solvent was removed in vacuo, and the residue was purified twice by flash column chromatography ((1) Ø = 3 cm, h = 15 cm, V = 20 mL, cyclohexane/ethyl acetate = 2/1 → 0/1; (2) automatic flash column chromatography, 5% → 100% ACN in H2O, Biotage SNAP KP-C18-HS 30 g) to give 45 (170 mg, 0.28 mmol, 52%) as yellowish oil. TLC: Rf = 0.24 (dichloromethane/ methanol = 9.5/0.5). Specific rotation: [α]20 D = −15.6 (c = 1.5, CH3OH). 1H NMR (DMSO-d6): δ [ppm] = 1.33−1.43 (m, 2H, O(CH2)2CH2(CH2)2OTs), 1.59−1.69 (m, 4H, OCH2CH2CH2CH2CH2OTs), 1.79−1.88 (m, 1H, CHCH2CH), 2.40 (s, 3H, ArCH3), 2.40−2.48 (m, 1H, CHCH2CH), 3.31−3.40 (m, 1H, NCH2), 3.60 (dd, J = 10.2/5.9 Hz, 1H, NCH2), 3.66 (s, 3H, CO2CH3), 3.95 (t, J = 6.4 Hz, 2H, ArOCH2), 4.04 (t, J = 6.3 Hz, 2H, CH2OTs), 4.20−4.30 (m, 1H, CHOH), 4.58 (dd, J = 8.5/6.8 Hz, 1H, NCH), 5.18 (d, J = 3.9 Hz, 1H, CHOH), 6.93−7.00 (m, 2H, 3″H 4‑alkyloxyphenyl , 5″-H 4‑alkyloxyphenyl ), 7.44−7.63 (m, 8H, 3‴H4‑methylbenzenesulfonate, 5‴-H4‑methylbenzenesulfonate, 2″-H4‑alkyloxyphenyl, 6″H4‑alkyloxyphenyl, 2′-Hbenzoyl, 6′-Hbenzoyl, 3′-Hbenzoyl, 5′-Hbenzoyl), 7.76− 7.82 (m, 2H, 2‴-H4‑methylbenzenesulfonate, 6‴-H4‑methylbenzenesulfonate); two rotamers exist in the ratio 80:20; the signals of the major rotamer are given. 13C NMR (DMSO-d6): δ [ppm] = 21.1 (1C, ArCH3), 21.5 (1C, O(CH2)2CH2(CH2)2OTs), 27.8 (1C, OCH2CH2(CH2)3OTs), 27.9 (1C, O(CH2)3CH2CH2OTs), 36.8 (1C, CHCH2CH), 51.9 (1C, CO2CH3), 56.2 (1C, NCH2), 57.2 (1C, NCH), 67.4 (1C, ArOCH2), 68.5 (1C, CHOH), 70.8 (1C, CH2OTs), 87.4 (1C, CC), 91.1 (1C, CC), 113.7 (1C, C-1″4‑alkyloxyphenyl), 114.9 (2C, C-3″4‑alkyloxyphenyl, C5″4‑alkyloxyphenyl), 125.5 (1C, C-4′benzoyl), 127.5 (4C, C-2′benzoyl, C6′benzoyl, C-2‴4‑methylbenzenesulfonate, C-6‴4‑methylbenzenesulfonate), 130.1 (2C, C-3‴4‑methylbenzenesulfonate, C-5‴4‑methylbenzenesulfonate), 131.0 (2C, C-3′benzoyl, C-5′benzoyl), 132.5 (1C, C-1‴4‑methylbenzenesulfonate), 133.1 (2C, C2″4‑alkyloxyphenyl, C-6″4‑alkyloxyphenyl), 135.4 (1C, C-1′benzoyl), 144.8 (1C, C-4‴4‑methylbenzenesulfonate), 159.1 (1C, C-4″4‑alkyloxyphenyl), 167.7 (1C, O CN), 171.7 (1C, CO2CH3); the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3414, 2947, 1743, 1600, 1419, 1354, 1246, 1172, 1091, 956, 914, 833, 763, 663. HRMS (m/z): [M + H]+ calcd for C33H36NO8S 606.2156, found 606.2118. HPLC (method 1): tR = 23.2 min, purity 97.8%. 5-[4-({4-[(2S,4S)-4-Hydroxy-2-(hydroxycarbamoyl)pyrrolidine-1carbonyl]phenyl}ethynyl)phenoxy]pentyl 4-methylbenzenesulfonate (46). Hydroxylamine hydrochloride (180 mg, 2.6 mmol) and a 5.4 M solution of sodium methoxide in methanol (0.48 mL, 2.6 mmol) were added to a solution of 45 (390 mg, 0.65 mmol) in methanol (10 mL), and the reaction mixture was stirred at ambient temperature overnight. Afterward, hydroxylamine hydrochloride (180 mg, 2.6 mmol) and a 5.4 M solution of sodium methoxide in methanol (0.48

mL, 2.6 mmol) were added and reaction mixture was stirred for 4 h. Then the solvent was removed in vacuo, and the residue was purified twice by flash column chromatography ((1) automatic flash column chromatography, 5% → 100% ACN in H2O, Biotage SNAP KP-C18HS 60 g; (2) Ø = 2 cm, h = 15 cm, V = 10 mL, dichloromethane/ methanol = 9.5/0.5, Rf = 0.44) to give 46 (110 mg, 0.19 mmol, 29%) as colorless solid; mp = 163 °C (decomposition). Specific rotation: 1 [α]20 D = +16.0 (c = 1.4, CH3OH). H NMR (DMSO-d6): δ [ppm] = 1.35−1.41 (m, 2H, O(CH2)2CH2(CH2)2OTs), 1.60−1.67 (m, 4H, OCH2CH2CH2CH2CH2OTs), 1.71−1.79 (m, 1H, CHCH2CH), 2.33−2.39 (m, 1H, CHCH2CH), 2.40 (s, 3H, ArCH3), 3.38 (dd, J = 10.2/5.8 Hz, 1H, NCH2), 3.57 (dd, J = 10.2/5.8 Hz, 1H, NCH2), 3.94 (t, J = 6.4 Hz, 2H, ArOCH2), 4.04 (t, J = 6.3 Hz, 2H, CH2OTs), 4.13− 4.19 (m, 1H, CHOH), 4.37 (dd, J = 8.2/6.9 Hz, 1H, 1H, NCH), 5.39 (br s, 1H, CHOH), 6.94−6.99 (m, 2H, 2″-H4‑ethynylphenoxy, 6″H4‑ethynylphenoxy), 7.45−7.48 (m, 2H, 3‴-H4‑methylbenzenesulfonate, 5‴H4‑methylbenzenesulfonate), 7.48−7.53 (m, 2H, 3″-H4‑ethynylphenoxy, 5″H4‑ethynylphenoxy), 7.55−7.61 (m, 4H, 2′-H4‑(pyrrolidine‑1‑carbonyl)phenyl, 6′H 4‑(pyrrolidi ne‑1‑carbonyl)phe nyl , 3′-H 4‑(pyr rolidine‑1‑carbonyl)phenyl , 5′H4‑(pyrrolidine‑1‑carbonyl)phenyl), 7.76−7.81 (m, 2H, 2‴-H4‑methylbenzenesulfonate, 6‴-H4‑methylbenzenesulfonate), 8.96 (br s, 1H, CONHOH), 10.81 (s, 1H, CONHOH); two rotamers exist in the ratio 90:10; the signals of the major rotamer are given. 13C NMR (DMSO-d6): δ [ppm] = 21.1 (1C, ArCH 3 ), 21.5 (1C, O(CH 2 ) 2 CH 2 (CH 2 ) 2 OTs), 27.8 (1C, OCH2CH2(CH2)3OTs), 27.9 (1C, O(CH2)3CH2CH2OTs), 37.2 (1C, CHCH2CH), 57.0 (1C, NCH), 57.1 (1C, NCH2), 67.4 (1C, ArOCH2), 68.7 (1C, CHOH), 70.8 (1C,CH2OTs), 87.4 (1C, CC), 91.1 (1C, CC), 113.7 (1C, C-4″4‑ethynylphenoxy), 114.9 (2C, C2″4‑ethynylphenoxy, C-6″4‑ethynylphenoxy), 124.4 (1C, C1′4‑(pyrrolidine‑1‑carbonyl)phenyl), 127.5 (2C, C-2‴4‑methylbenzenesulfonate, C6‴4‑methylbenzenesulfonate), 127.8 (2C, C-3′4‑(pyrrolidine‑1‑carbonyl)phenyl, C5′4‑(pyrrolidine‑1‑carbonyl)phenyl), 130.1 (2C, C-3‴4‑methylbenzenesulfonate, C5‴4‑methylbenzenesulfonate), 130.9 (2C, C-2′4‑(pyrrolidine‑1‑carbonyl)phenyl, C6′4‑(pyrrolidine‑1‑carbonyl)phenyl), 132.5 (1C, C-1‴4‑methylbenzenesulfonate), 133.1 (2C, C-3″ 4‑ethynylphenoxy , C-5″ 4‑ethynylphenoxy ), 135.6 (1C, C4′4‑(pyrrolidine‑1‑carbonyl)phenyl), 144.8 (1C, C-4‴4‑methylbenzenesulfonate), 159.1 (1C, C-1″4‑ethynylphenoxy), 167.9 (1C, OCbenzoyl), 168.7 (1C, CONHOH); the signals of the major rotamer are given. IR (neat): ν̃ [cm−1] = 3190, 2931, 1597, 1419, 1354, 1246, 1172, 1087, 956, 914, 829, 810, 763, 663. HRMS (m/z): [M + H]+ calcd for C32H35N2O8S 607.2109, found 607.2113. HPLC (method 2): tR = 17.6 min, purity 92.2%. Radiochemistry: General Methods. The radiosyntheses were carried out on a modified PET tracer radiosynthesiser (TRACERLab FxFDG, GE Healthcare). The recorded data were processed by the TRACERLab Fx software (GE Healthcare). Separation and purification of the radiosynthesized compounds were performed on the following semipreparative radio-HPLC system A: K-500 and K501 pump, K-2000 UV detector (Herbert Knauer GmbH), NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec) and an ACE 5 AQ column (250 mm × 10 mm). Method A started with a linear gradient from 10% to 90% CH3CN in water (0.1% TFA) over 30 min, holding for 5 min, and followed by a linear gradient from 90% to 10% CH3CN in water (0.1% TFA) over 5 min, with λ = 254 nm and a flow rate of 5.5 mL·min−1. Radiochemical purities were determined using the analytical radio-HPLC system B: Two Smartline 1000 pumps and a Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γdetector (Raytest Isotopenmessgeräte GmbH), and a Nucleosil 100−5 C-18 column (250 mm × 4 mm). Method B started with a linear gradient from 10% to 100% CH3CN in water (0.1% TFA) over 15 min, holding for 3 min, followed by a linear gradient from 100% to 10% CH3CN in water (0.1% TFA) over 2 min, with λ = 254 nm and a flow rate of 1.0 mL·min−1. The recorded data of both HPLC-systems were processed by the GINA Star software (Raytest Isotopenmessgeräte GmbH). No-carrier-added aqueous [18F]fluoride was produced on a RDS 111e cyclotron (CTI-Siemens) by irradiation of a 2.8 mL water target using 10 MeV proton beams on 97.0% enriched [18O]H2O by the 18O(p,n)18F nuclear reaction. After unloading the target, it was rinsed with 2.0 mL of ultrapure water. This rinsed water containing [18F]fluoride ions was used for the radiosyntheses. 9555

DOI: 10.1021/acs.jmedchem.6b01291 J. Med. Chem. 2016, 59, 9541−9559

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Computational Methods. To perform molecular docking studies, the crystal structure of human MMP-13 in complex with the inhibitor SC-78080, which has structural similarities with the compounds under study, was used as protein model (PDB: 3KRY).46 The protein was prepared using the LigX module of MOE (version 2014.09; Chemical Computing Group, Montreal, Canada) according to the following procedure. Briefly, the structure of the hydroxamate moiety of SC78080 was corrected and its hydroxy group was deprotonated. All solvent particles were removed, except one conserved water molecule (H2O299) located near the Zn2+-ion. Hydrogen atoms were automatically added, and tautomeric forms and protonation states of the amino acid residues were automatically assigned, using the “Protonate 3D” module47 at pH 7.0 (asparagine, glycine, and histidine were allowed to “flip” in “Protonate 3D”). Issues related with missing atoms in several amino acids were automatically corrected, and the model was subjected to restrained energy minimization. Energy minimized structures of the ligands were generated in MOE modeling software using the MMFF94x force field within the RMS gradient of 0.1 kcal/mol/Å2. In all ligands, strong acids were deprotonated and strong bases protonated. Docking studies were performed in the active site of the prepared MMP-13 enzyme without any constrains, using the default “Rigid Receptor” protocol of MOE, which comprises several steps which were described previously.48,49 The placement, rescoring, and refinement mode were Triangle Matcher, London ΔG, and Force Field, respectively. Only those docking poses were taken into consideration, where both oxygen atoms of the hydroxamate moiety were located within the sphere (1 Å radius) around the same atoms of the original ligand SC-78080. The filtered top-ranked docking poses of each ligand were subjected to postdocking energy minimization in the active site, using the MMFF94x force field within the RMS gradient of 0.1 kcal/ mol/Å2. Finally, rescoring of the resulting poses was performed, using the Affinity ΔG scoring methodology. The obtained top-ranked docking pose of original ligand SC-78080 from 3KRY gives RMSD values lower than 1.0 Å, indicating reliability of the docking protocol, which is able to correctly reproduce the X-ray structure of the MMP-13−inhibitor complexes. Visual inspection of the docking pose of the (2S,4S)-configured 4-hydroxyproline derivative 4 indicates that the hydroxamate moiety is coordinating the Zn2+-ion and hydrogen bonding with Glu223, Ala186, His222, His232, and His226. The carbonyl oxygen atom forms strong interactions with Leu185 or Ala186 (backbone) and lipophilic interactions with Leu184. The carbon atoms of the 4-hydroxyproline ring are involved in lipophilic interactions with Leu184, whereas its hydroxy group is highly exposed to the solvent. The diphenylacetylene moiety is interacting with a number of hydrophobic residues of the S1′ pocket of MMP-13, including Ile243, Leu239, Phe241, Tyr244, and Thr245, whereas its 5-fluoropentoxy tail, protruding even deeper to the pocket, forms additional greasy interactions with Gly237, Ala238, and a hydrogen bond with Lys249. Radiosyntheses of [18F]4. In a computer controlled TRACERLab FxFDG Synthesizer, the batch of aqueous [18F]fluoride ions (655−3895 MBq) from the cyclotron target was passed through an anion exchange resin (Sep-Pak Light Waters Accell Plus QMA cartridge, preconditioned with 5 mL of 1 M K2CO3 and 10 mL of water for injection). [18F]Fluoride ions were eluted from the resin with a mixture of 40 μL of 1 M K2CO3, 200 μL of water for injection, and 800 μL of DNAgrade CH3CN containing 20 mg (53 μmol) of Kryptofix2.2.2 (K222) in the reactor. Subsequently, the aqueous K(K222)[18F]F solution was carefully evaporated to dryness in vacuo. An amount of 5.0 mg (8.2 μmol) of precursor 46 in 1.0 mL of DNA-grade CH3CN was added, and the mixture was heated at 110 °C for 20 min. Then the solution was cooled to 55 °C, 10 mL of water for injection was added, and the mixture was passed through a Waters Sep-Pak C18 Light cartridge (preconditioned with 10 mL of ethanol and 10 mL of water). The cartridge was washed with 10 mL of water for injection and eluted with a 0.5 mL of DMF that was preheated to 120 °C. The eluate was diluted with 0.5 mL of water for injection, and this raw product solution was purified by gradient-radio-HPLC system A (method A). The product fraction of compound [18F]4 (retention time tR ([18F]4)

= 22.1 min) was evaporated to dryness in vacuo and formulated in 1.00 mL of water for injection:EtOH (9:1 (v/v)). Product compound [18F]4 was obtained in rcy of 46.3 ± 9.3% (n = 5) (d.c.) in 105 ± 12 min from the end of radionuclide production. The target compound was isolated in radiochemical purities of >99% and with specific activities of 0.7−33.2 GBq/μmol at the end of the synthesis. Radiochemical purities and specific radioactivities of [18F]4 (retention time tR ([18F]4) = 12.7 min) were determined by analytical radio-HPLC B (method B). Radiochemical identity of [18F]4 was determined by coelution of this compound with the nonradioactive counterpart 4 that was added to a preparation of [18F]4 at the analytical radio-HPLC B (method B) detected by the γ-detector ([18F] 4) and the UV-detector (4), respectively. Determination of the Partition Coefficient (log D (exp)). The lipophilicity of radioligand [18F]4 was assessed by determination of the water−octanol partition coefficient following a published procedure.50 In brief, approximately 408 kBq of [18F]4 were mixed with equal amounts (0.6 mL) of PBS (pH 7.4) and 1-octanol and the resulting biphasic system was mixed vigorously for 3 min at rt. The tubes were centrifuged (5 min). Then 400 μL of the 1-octanol layer was separated and 400 μL of PBS were added. The mixture was mixed vigorously for 10 min at rt followed by centrifugation of the tubes for 5 min. Two samples of 100 μL of each layer were counted in a γ-counter (Wallac Wizard, PerkinElmer Life Science). The partition coefficient was determined by calculating the ratio cpm(octanol)/cpm(PBS) and expressed as log D (exp) (log(cpmoctanol/cpmPBS)). Two independent experiments were performed in duplicate, and data were provided as mean values ± standard deviation. Stability in Human and Mouse Serum. The serum stability of radioligand [18F]4 was evaluated by incubation in human serum at 37 °C for up to 90 min. An aliquot of formulated [18F]4 (20 μL, 6.7 MBq) was added to a sample of human serum (200 μL), and the mixture was incubated at 37 °C. Samples of 20 μL each were taken after periods of 10, 30, 60, and 90 min and quenched in 100 μL of icecold DNA-grade CH3CN followed by centrifugation for ≥5 min. The supernatant was analyzed by analytical radio-HPLC B (method B, tR ([18F]4) = 12.7 min). Serum stability investigations in mouse serum were performed analogously. In Vitro MMP Inhibition Assays. The inhibition potencies of proline-based hydroxamates 12, 16, 20, 23, 28, 29, 34, 35, and 37 against activated MMP-2, -8, -9, and -13 were assayed using the synthetic fluorogenic substrate (7-methoxycoumarin-4-yl)acetyl-ProLeu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl)Ala-ArgNH2 (R&D Systems) as described previously.50 Briefly, MMP-2, -8, -9, or -13 (each at 2 nM) and test compounds at varying concentrations (10 pM to 1 mM) in Tris-HCl (50 mM), pH 7.5, containing NaCl (0.2 M), CaCl2 (5 mM), ZnSO4 (20 μM), and 0.05% Brij 35, were preincubated at 37 °C for 30 min. An aliquot of substrate (10 μL of a 50 μM solution) was added to the enzyme−inhibitor mixture (90 μL), and the fluorescence changes were monitored using a Fusion Universal microplate analyzer (Packard Bioscience) with excitation and emission wavelengths of 330 and 390 nm, respectively. Reaction rates were measured from the initial 10 min and plotted as a function of inhibitor concentration. From the resulting inhibition curves, the IC50 values were calculated by nonlinear regression analysis using the Grace 5.1.8 software (Linux). In Vitro ADAM and Meprin Inhibition Assay. The Bac-to-Bac system (Gibco, Thermo Fisher Scientific) was used for heterologous expression of recombinant protease ectodomains in insect cells. Expression, purification, and activation of recombinant human meprins was performed as described previously.51,52 Sequences for murine ectodomains of ADAM10 (aa 20-673) and ADAM17 (aa 18-617) were cloned into pFastBac expression plasmid with addition of meprin β signal peptide and N-terminal His-tag. Expression and purification of ADAM ectodomains was performed according to the protocol used for meprin β.51 For determination of protease activity, quenched fluorogenic peptide cleavage assays were performed. For this, recombinant ectodomains were incubated with hydroxamate inhibitors (1−50 μM, reconstituted in DMSO) in HEPES buffer for 30 min at RT (for 9556

DOI: 10.1021/acs.jmedchem.6b01291 J. Med. Chem. 2016, 59, 9541−9559

Journal of Medicinal Chemistry

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detailed information, see table in Supporting Information). Changes in fluorescence intensity were measured every 30 s for 2 h at 37 °C using the fluorescent spectrometer Infinite F200 PRO (Tecan). In Vivo Biodistribution. All animal experiments were approved by the North Rhine−Westphalia Agency for Nature, Environment, and Consumer Protection (Landesamt für Natur, Umwelt and Verbraucherschutz Nordrhein−Westfalen , LANUV). Adult C57/BL6 mice (n = 4, female, 18−21 g body weight) were anaesthetized by isoflurane/ O2, and one lateral tail vein was cannulated using a 27 G needle connected to 15 cm of polyethylene catheter tubing. [18F]4 (400 kBq/ g bodyweight) was injected as a bolus (50 μL compound flushed with 100 μL saline) via the tail vein, and subsequent PET scanning was performed. Small Animal PET Scanning. PET experiments were carried out using a 32 module quadHIDAC scanner (Oxford Positron Systems Ltd., Oxford, UK) dedicated for small animal imaging with uniform spatial resolution (