Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual

Article pubs.acs.org/jmc

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting DualAction Prodrug for the Treatment of Osteoporosis Haibo Xie,† Gang Chen, and Robert N. Young* Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada S Supporting Information *

ABSTRACT: A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonylleucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

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such as odanicatib2,3 are in late stages of development for the treatment of osteoporosis. BPs bind avidly and almost irreversibly to hydroxyapatite but are slowly liberated during the resorption process and inhibit the ability of osteoclasts to attach to bone and release the acid necessary for resorption. Because of their avidity for bone, BPs have been used as a vehicle to target other bioactive molecules to bone where they may exert a local effect and/or be released in situ by the action of resident enzymes or due to acid instability.4−8 Although BPs such as alendronic acid (ALN) (1) are effective drugs, osteoporosis is often not treated until substantial bone loss has already occurred, and although they may halt the progression, they do not significantly reverse the bone loss. Therefore, there remains an important medical need to restore bone lost in osteoporosis through stimulation of the bone-forming process. Parathyroid hormone can stimulate bone formation,9 and a stabilized analogue, teriparatide, has been developed and is marketed for this purpose. However,

INTRODUCTION Healthy bones constantly turnover throughout the body in a natural homeostatic process with bone resorption mediated by specialized cells known as osteoclasts and bone formation mediated by osteoblasts. In healthy individuals, these activities are in balance, but as we age, the balance is often disrupted and resorption can begin to outstrip formation, leading to net bone loss and eventually to osteoporosis. Osteoporosis is one of the most common diseases associated with aging and affects >25% of women and >10% of men in their lifetimes. Morbidity associated with osteoporosis and fractures in the elderly have been estimated to cost $22B in 2008 in the US alone.1 Bisphosphonates (BPs) form one of the most important classes of current drug treatments for osteoporosis and act by suppressing the bone resorption process. During resorption, osteoclasts travel on the bone surface and, at sites of resorption, form a seal to form a space (lacuna) and release acid into this lacuna to dissolve the hydroxyapatite mineral component of bone. Osteoclasts also release the protease enzyme cathepsin K (Cat K) to cleave and process the collagen matrix, which is the other major structural component of bone. Inhibitors of Cat K are effective antiresorptive agents, and some Cat K inhibitors © 2017 American Chemical Society

Received: April 28, 2017 Published: July 12, 2017 7012

DOI: 10.1021/acs.jmedchem.6b00951 J. Med. Chem. 2017, 60, 7012−7028

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formation effects and inhibition of resorption, leading to an additive or synergistic effect (Figure 1). In previous studies, we have shown that PGE2 can be conjugated with bisphosphonates20 and that these conjugates were well tolerated when dosed intravenously (IV) to rodents. Radiolabeling confirmed that the conjugates targeted bone after dosing and that the unbound portion of the dose was rapidly cleared from systemic circulation. Initially, conjugate 3 with PGE2 carboxyl function bound as an amide to the amino function of ALN was prepared and shown to be efficiently taken up into the bones of rats after IV administration (∼15% of dose). However, the bound conjugate was too stable and did not release free PGE2 (or ALN). Another conjugate, 4, where PGE2 was bonded through the C-15 hydroxyl group of PGE2 via an ester bond to a “custom” bisphosphonate, (4(carboxythio)butane-1,1-diyl)-bis(phosphonic acid) was found to be quite stable and to effectively target bone after IV dosing and to release PGE2 in situ with a 4−5 day half-time. This conjugate was well-tolerated even at high doses and showed good efficacy when tested in the ovariectomized (OVX) rat model of osteoporosis when dosed once weekly with bone growth efficacy equivalent to that obtained for a similar dose of PGE2 administered daily at its maximum tolerated dose. However, the bisphosphonate portion of 4 was found to be inactive (as an inhibitor of bone resorption) at the administered dose, and thus, 4 did not test the potential of the hoped-for synergism. Chemical instability of PGE2 made it difficult to develop chemistry to link to a known, active bisphosphonate such as alendronic acid. Subsequently, with the identification of potent, selective, and more chemically stable EP4 receptor agonists (such as 5a), it became feasible to contemplate the preparation of conjugates designed to liberate both the agonist and alendronate. Thus, we prepared a third class of conjugates exemplified by structure 6 where the protected EP4 agonist 5b was linked to 4-hydroxyphenylacetic acid via an ester bond to the carboxylic acid function and ALN to the phenol function via a carbamate group.21 Ester 5b was used for ease of synthesis and was shown to be rapidly hydrolyzed in blood to yield active carboxylic acid 5a. Conjugate 6 was radiolabeled (with 3H on 5 and 14C on the linker) and was shown to bind largely intact to bones (3H/14C ratio maintained) after IV dosing to rats. Following the loss of the labels from bones over time indicated that agonist 5a was liberated with a 4−5 day half-time. The alendronate component was shown to be “liberated” (while still attached to the bone) with a 22 day half-time by measuring the loss of 14C label associated with the carbamate linker group.22 Conjugate 6 demonstrated robust stimulation of bone formation when dosed once weekly over 6 and 12 weeks at 5 or 15 mg/kg in the OVX rat model, but the antiresorptive effects expected from such a dose of alendronate were not evident.23,24 It is possible that a component of the loss of 14Clabel we observed could also be attributed to simple bone turnover and release of the entire bound half of the conjugate with the carbamate bond still intact. The half-time of release of alendronate from skeletal bones in vivo has been reported to be approximately 200 days in the rat,25 but more recent studies measuring the loss of tetracycline from the rat bone surface have indicated half-times of approximately 3 weeks in female rats.26 If the latter measure was more representative, we would conclude that conjugate 6 did not release the free ALN component at a sufficient rate to have truly tested the potential

teriparatide is dosed daily via intramuscular injection and has been associated with an increase in cancer in rodents.10 The prostaglandin hormone prostaglandin E2 (PGE2) (2) has been shown to have anabolic activity in vivo and to stimulate bone growth in animals and in humans.11−13 PGE2 interacts with four distinct receptors, EP1, 2, 3, and 4, and the bone-forming activity has been associated with activation of the EP4 receptor subtype.14 EP4 is the predominant PGE2 receptor found in adult bone cells,15 and a number of potent and very selective EP4 receptor agonists have been reported in recent years; some have been shown to mimic the anabolic effects of PGE2 on bone16−18 when dosed in vivo. EP4 receptor activation has been shown to suppress apoptosis in boneforming osteoblast cells and osteoid precursor cells (such as preosteoblasts) found in bone marrow.14 Preosteoblasts undergo a high rate of apoptosis during maturation, and thus, even a small suppression of apoptosis could lead to an important increase the surviving mature osteoblast population and thus to enhanced bone formation. Unfortunately, both PGE2- and EP4-selective agonists exert side effects in vivo19 (including vasodilation and gastrointestinal disturbance) that render their direct use as bone anabolic drugs impractical.

To avoid such side effects and provide a sustained and localized effect, we conceptualized the use of conjugate drugs wherein PGE2 or other EP4-selective agonists were linked to bone-targeting bisphosphonates via hydrolyzable linker groups. Such conjugates would need to be relatively stable in the bloodstream, allowing them time to reach the bone compartment and then bind to bone as an intact entity. Ideally, any conjugate that did not bind to bone would be eliminated from the body rapidly without liberating the EP4 agonist (EP4a). Optimally, conjugate that adhered to bone would undergo slow hydrolysis to locally liberate active EP4 agonist and active bisphosphonate (BP) and would therefore exert both bone 7013

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Figure 1. Bisphosphonate targeting of conjugate drug to bone and slow release of active bisphosphonate (BP) and EP4 receptor agonist (EP4a) in situ by the action of local hydrolytic enzymes.

Figure 2. Conceptual differential enzymatic release of active EP4 agonist (5a) and bound bisphosphonate from conjugate 7 after binding to the bone surface.

methacrylamide copolymer-based conjugates designed to release prostaglandin E1 (PGE1)28,29 have been previously described in the literature for the treatment of osteoporosis and bone diseases. Cat K efficiently cleaves small peptide substrates such as Cbz-Gly-Pro-Arg-AMC or Cbz-Leu-Arg-AMC,30 and thus, such peptides could be incorporated into our linking moiety. The reliance on Cat K as a release mechanism offered an ability to extrapolate observations in the rat to humans with some confidence. We proposed to elaborate the benzyl function of the Nterminal carboxybenzyl (Cbz) group of these peptide Cat K substrates by addition of an acetic acid moiety on the Cbz group, which could be linked to 5b via an ester in a manner

of the dual antiresorption/growth stimulation as originally proposed. Therefore, to better explore the potential of dual action conjugates, we designed a new class of differentially cleavable conjugate prodrugs exemplified by compound 7 (Figure 2), wherein the EP4 agonist 5b and ALN are linked via a phenylacetic acid-peptide linker such that in situ esterase activity in the bone compartment should liberate 5a (as shown to be effective with conjugate 6) and the alendronate component would be unambiguously liberated by cleavage of the peptide linker via the action of Cat K. Cat K is the major peptidase released by osteoclasts at the bone surface.27 Cat Kcleavable conjugates including N-(2-hydroxypropyl)7014

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Figure 3. Synthetic strategy for preparation of conjugate 7.

Scheme 1. Synthesis of Model Peptide-Bisphosphonate Conjugate 10a

a

Reagents and conditions: (i) 4-aminobenzyl alcohol, HOBt, EDCI, DMF, rt, 89%; (ii) Pd/C, H2, rt; (iii) Cbz-Pro-OH, HOBt, EDCI, DMF, rt, 95% for 2 steps; (iv) CDI, DMF, rt; (v) alendronic acid, nBu4NOH, DMF, rt, 55% for 2 steps.

assume a 5 mg/kg dose (the dose found to be effective for conjugate 6 and for the generic conjugate 7 equivalent to approximately 1 mg/kg of alendronate) and that approximately 5% of the dose was taken up into bones (as was the case for conjugate 6) and that the release half-time was 1 week, we can estimate that the dose of active alendronate from a conjugate such as 7 would be approximately 0.025 mg kg−1 week−1, which is very similar to the safe and effective human dose. To quantify uptake of the conjugates into bone and the rates of release of the active components, we decided to prepare doubly radiolabeled conjugates where the EP4 agonist component 5b was labeled with tritium and the linking PABA element was labeled with 14C. We would thus expect uptake of both labels into the bones and then be able to follow the loss of labels found in the bones over time as demonstration of release of the components. If the desired half-times for liberation of the active components were observed, then the successful conjugate would be a candidate for the much more demanding efficacy studies in the OVX rat model of osteoporosis.33 Thus, our task was to design and devise a synthesis of the requisite conjugates, demonstrate that the peptide linker unit(s) were sufficiently stable in plasma or serum and were substrates for Cat K. Once this had been achieved, we would then prepare the optimal conjugate in dual [3H/14C]-labeled form and confirm uptake of the intact conjugate into bone after IV administration and to quantify biodistribution and rates of release of the two active components by following the fates of the 14C and 3H labels in bone and other tissues over time.

similar to that used in 6, and to replace the 7-amino-4methylcoumarin (AMC) group with a para-aminophenylbenyzl alcohol (PABA) moiety, which would be linked to ALN via a carbamate function to form prototypic conjugate 7 (Figure 2). There are a number of examples in the literature where the PABA self-immolative linker has been employed in prodrugs to liberate amino-functionalized drugs (for a review, see TranoyOpalinski et al).31 Thus, if our conjugate was a substrate for Cat K and was sufficiently stable to peptidases and esterases in the bloodstream to reach and bind intact to bone, then subsequent hydrolysis of the ester would liberate 5a, and cleavage of the peptide would liberate ALN (which would remain bound to bone). We anticipated that, given the similarity of structures at the point of attachment of 5a, the ester hydrolysis should proceed at a rate similar to that we had observed for 6 (halftime of approximately 5 days). Our goal was to develop a conjugate drug compatible with an infrequent dosing regimen (once weekly or longer) in that, given the poor absorption of bisphosphonate drugs, we anticipated the need to dose such a conjugate by the intravenous route. Ideally, ALN would be released at a similar or somewhat slower rate with half-times compatible with once weekly dosing. The usual dose of alendronate is 70 mg (∼1.4 mg/kg), which is dosed orally once weekly. Notably, however, the oral bioavailability of alendronate was very low: 1.7% in rats and 0.64% in women.32 Given that approximately 50% of absorbed alendronate is taken up into bones, the actual amount of alendronate reaching the bones is approximately 0.01 mg kg−1 week−1. The effective dose of alendronate from our conjugate would depend on the actual dose, the degree of bone uptake and the rate of release. If we 7015

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Article

RESULTS AND DISCUSSION

Table 1. Comparison of Rates of Cleavage of Peptide Substrates with Rat and Human Cathepsin K

Synthetic Strategy. The prototypic conjugate was designed and is depicted as generic structure 7 (Figure 2). The EP4 agonist (5b) (EP4a) would be conjugated via the 15hydroxyl function by esterification with 4-hydroxymethylphenylacetic acid; the peptide component would be prepared by standard peptide synthesis, and the C-terminus would react with p-aminobenzyl alcohol. The N-terminus would then be liberated and coupled to the alcohol (8) via a carbamate linkage to derive the penultimate precursor (9) (Figure 3). Because of the extreme polarity and poor nonaqueous solubility of alendronic acid (ALN), we envisaged attaching ALN via a carbamate as the last step in the synthesis. Evaluation of Model Peptide−Alendronate Conjugates. At the outset and before committing to the total synthesis of 7, it was necessary to confirm that the peptidePABA-ALN component was a substrate for both rat and human Cat K and that the peptide would be stable enough in the bloodstream to allow efficient partitioning to the bone. The rat and human Cat K enzymes are known to have quite different substrate specificities.34 Although our aim was ultimately to treat humans, most animal models of osteoporosis, such the OVX model, are performed in rodents, and demonstration of efficacy in an animal model would be a necessary first step in any future development plan. Although Cbz-Gly-Pro-Arg-AMC is reported to be an excellent substrate for human Cat K, it is a poor substrate for the rat enzyme. Other peptides where the Pro is replaced by less constrained lipophilic amino acids (such as Phe and Leu) have been reported to be better substrates for rat Cat K.34 For these requirements to be evaluated, model peptidePABA-ALN conjugates Cbz-Pro-Arg-PABA-ALN (10), CbzLeu-Arg-PABA-ALN (11), and Cbz-Gly-Pro-Arg-PABA-ALN (12) were synthesized. For the preparation of 10, Cbz-arginine hydrochloride was first coupled with p-aminobenyl alcohol under standard amide formation conditions to provide amide 13a (Scheme 1). The Cbz was removed by hydrogenation, and the liberated amine was coupled with Cbz-proline to form dipeptide 14. We then planned to couple the benzyl alcohol with alendronic acid (ALN) using reagents such as carbonyldiimidazole or p-nitrophenyl chloroformate, but it was anticipated that the unprotected guanidine group on the Arg might interfere with this coupling. However, attempts to protect the arginine by trifluoroacetylation (by reaction with ethyl trifluoroactate in DMF) led to acylation of the benzyl alcohol rather than the guanidine group, suggesting that protection was unnecessary. An attempt to react 14 with pnitrophenyl chloroformate was unsuccessful, but carbonyldiimidazole reacted smoothly with 14 in DMF to give hemi-imidazolide 15 in good yield. Reaction of 15 with the tetra-n-butylammonium salt of alendronic acid in DMF gave desired carbamate 10 in excellent yield. Cbz-Leu-Arg-PABAALN (11) and Cbz-Gly-Pro-Arg-PABA-ALN (12) were readily prepared in a similar manner. Cleavage of the Peptide Bisphosphonates 10, 11, and 12 by Cathepsin K and Stability in Rat Plasma. The peptide conjugates were evaluated as putative substrates for human and rat Cat K, and rates of cleavage were compared to the standard substrate Cbz-Leu-Arg-AMC. The results are presented in Table 1. The standard substrate was cleaved by 63 and 71% after 6 min incubation with human and rat Cat K, respectively, in keeping with observations in the literature,34

Cbz-Pro-Arg -PABA-ALN (10) (% cleaved)a time (h)

Cbz-Leu-Arg-PABAALN (11) (% cleaved)a

Cbz-Gly-Pro-Arg -PABA-ALN (12) (% cleaved)a

Hum Cat K

Rat Cat K

Hum Cat K

Rat Cat K

Hum Cat K

Rat Cat K

90 98

5 10 52

66 75 79 (3 h)

73 80 95

91 100

11 20 80

1 2 20

Compounds were incubated with human or rat Cat K at 37 °C and sampled after various times for loss of starting conjugate. Standard substrate Cbz-Leu-Arg-AMC with rat Cat K showed 71% cleavage in 6 min and with human Cat K showed 63% cleavage in 6 min.

a

and 10 and 12 were efficiently cleaved by human Cat K (90% after 1 h) but not by the rat enzyme. Gratifyingly, 11 was processed by both enzymes at a similar rate with 66 and 71% cleavage observed after 1 h incubation with human and rat Cat K, respectively. To demonstrate that these peptide conjugates could survive in the bloodstream long enough to reach and bind intact to bones, we evaluated their stability in rat plasma and serum and compared to inactivated controls. Compounds were incubated at 37 °C, and aliquots were sampled at various time points monitoring the disappearance of the conjugate by HPLC. The results are presented in Table 2. Both 10 and 11 were stable for Table 2. Stability of Model Peptide Conjugates in Rat Plasma and Serum Cbz-Pro-Arg -PABA-ALN (10) (% remaining)a

Cbz-Leu-Arg -PABA-ALN (11) (% remaining)a

Cbz-Gly-Pro-Arg -PABA-ALN (12) (% remaining)a

time (h)

plasma

serum

plasma

serum

plasma

serum

0 1 2 4 24 24 (control)

96 95 90 90 65 86

93 89 78 77 69 97

82 78 78 64 59 83

99 97 95 86 70 95

95 51 38 17 16 49

91 82 72 68 64 93

Compounds were incubated in rat plasma or serum at 37 °C and sampled at various times and compared to inactivated control plasma or serum. a

at least 2 h (with 80% or more remaining), long enough to allow efficient uptake into bones. Somewhat surprisingly, 12 was much less stable with only approximately 40% remaining in serum after 2 h. It is not clear what mechanism is responsible for this loss, nor did we determine the point of cleavage, but presumably the compound is a substrate for another protease found in blood. Thus, the leucine-arginine dipeptide was chosen as the basis for our linker unit, and conjugate 16 became our synthetic goal. Of course it is also possible that 16 might be cleaved in the liver, and only an in vivo bone uptake experiment could confirm the ultimate utility of this peptide as a metabolically stable but in situ cleavable linker. Synthesis of EP4a-Leu-Arg-PABA-ALN Conjugate 16. The synthesis of the desired conjugate proceeded largely as planned and with the use of carbonyldiimidazole as coupling agent for the final attachment of the alendronate to PABAalcohol (Scheme 2). Thus, Cbz-Leu-Arg-PABA (17a) was 7016

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Scheme 2. Synthesis of Conjugate 16a

a Reagents and conditions: (i) HOBt, EDCI, DMF, rt, 89%; (ii) Pd/C, H2; (iii) Cbz-Leu-OH, HOBt, EDCI, DMF, rt, 85% for 2 steps; (iv) (1) TBSCl, imidazole, THF, 0 °C to rt; (2) K2CO3, MeOH/THF = 1:2, 68% for 2 steps; (v) DMAP, DCC, DCM, RT, 99%; (vi) TBAF, THF, 100%; (vii) COCl2, PhNMe2, THF, rt; then 17b, Et3N, DMF; 84%; (viii) CDI, THF, rt; (ix) (1) alendronic acid bis-tetra-n-butylammonium salt, DMAc, rt; (2) ion exchange; (3) C-18 desalting, 47% for 2 steps.

deblocked by hydrogenation (over Pd/C) to provide the free amino compound 17b. 4-Hydroxymethylphenylacetic acid was exhaustively silyl protected with t-butyldimethylsilyl chloride, and the carboxylsilyl group was then removed selectively with potassium carbonate to give free 4-t-butyldimethylsilyloxyphenylacetic acid 18. DCC-mediated esterification of 18 with the 15-hydroxyl function of 5b proceeded smoothly to give ester 19, and then removal of the silyl protecting group with tetra-nbutylammonium fluoride gave alcohol 8, which was reacted with an equivalent of phosgene to give the intermediate chlorocarbonate that was then reacted with 17b to form

carbamate 20. Finally, 20 was reacted with an equivalent of carbonyldiimidazole (CDI) to form the hemi-imidazole adduct 21, which reacted smoothly with ALN in dimethylacetamide. Alendronate was reacted as its bis-tetra-n-butylammonium salt as this allowed the reaction to be carried out in anhydrous dimethylacetamide (alendronic acid is virtually insoluble in any solvent other than water either as the free acid or as a disodium salt). The product tetra-n-butylammonium salt form of 16 was converted to the potassium salt and purified by chromatography on a strong anion exchange column with elution with pH 8 potassium hydrogen phosphate buffer followed by removal of 7017

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Scheme 3. Synthesis of a p-Iodo Analogue of 5ba

a Reagents and conditions: (i) PhI, AlCl3, DCM, rt, 34%; (ii) DAST, toluene, rt, 34%; (iii) MePO(OMe)2, n-BuLi, THF, −78 °C, 72%; (iv) TBAF, THF; (v) (COCl)2, DMSO, Et3N, DCM, −78 °C; (vi) 24, NaH, ZnCl2, THF, 73%; (vii) Ru-TsDPEN-Cy, HCOOH, Et3N, DCM, −5 °C, 24 h, 74%.

Scheme 4. Model Studies on Reduction p-Iodo Precursors to Provide Labeled 5ba

a Reagents and conditions: (i) Pd(OAc)2 (10 mol %), NaBH4; (1.0 equiv), DMF, 70 °C, 30 min; (ii) Pd(OAc)2 (5 mol %), NaBH4 (1.2 equiv), NaBD4 (1.2 equiv), DMF, rt, 30 min.

catalyzed reduction of aryl iodides by sodium borohydride (or tritiide), and this methodology seemed to meet to our needs as well as being a procedure operable on a lab scale in house. This approach required an iodo-substituted analogue of 5b. Thus, the analogue of 5 (compound 22) bearing a para-iodo group on the pendant phenyl ring was synthesized. Freidel-Crafts acylation of iodobenzene with ethyl chlorooxylate followed by fluorination with DAST gave difluorinated ester 23, that was converted to Wittig reagent 24 by reaction with the anion derived from dimethyl methylphosphonate. Compound 24 was then reacted with aldehyde 25 followed by selective reduction of the resulting ketone 26 to give 22 following the same

excess salts by C-18 chromatography and lyophilization to give final conjugate 16. Synthesis of 3H-Labeled 16. To track the fate of the EP4 agonist in the conjugate, we required a radiolabel on the EP4 agonist component (5) of conjugate 16. We used 5 labeled with tritium at the 15-position in the past, but the use of this labeled compound in the synthesis developed for 16 (Scheme 2) would require carrying the label through multiple steps, which is not optimal. It is preferred to introduce the label at as late a step as possible, and therefore, we contemplated tritiation via the reduction of an aryl halide as the last step to give the final conjugate. A recent publication35 has described palladium7018

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Scheme 5. Synthesis of Tritium-Labeled Conjugate 16a

a

Reagents and conditions: (i) 18, DMAP, DCC, DCM, rt, 64%; (ii) TBAF, THF; (iii) (1) COCl2, PhNMe2; (2) 17b, Et3N, DMF, 73% for 3 steps; (iv) (1) CDI, THF, rt; 2) alendronic acid bis-tetra-n-butylammonium salt, DMAc, rt; (2) ion exchange; (3) C-18 desalting column, 44%; (v) Pd(OAc)2, 5 mol %, [3H]-NaBH4, DMF, rt, 30 min, 100% chemical yield, 10.6% radiochemical yield.

procedure as previously described for the synthesis of 513,36 (Scheme 3). We then evaluated the reduction of 22 to provide 5b and optimized the reduction for efficient introduction of a tritium atom. For the efficiency of the reduction to be confirmed, model reactions were carried out first on intermediate 23. These studies showed it was possible to reduce the amount of reagent (NaBH4) to one equivalent (effectively four equivalents of H) and still achieve essentially complete conversion. Thus, 23 was smoothly reduced to give 27 with 10% Pd(OAc)2 and one equivalent of NaBH4 in DMF at 70 °C in 90% yield (Scheme 4A). Compound 22 was then shown to also be reduced smoothly with NaBH4/Pd(OAc)2 under the Nagasaki conditions to provide 5b in excellent yields. Commercial [3H]-NaBH4 was available with specific activity of 10.9 Ci/mmol, but this reagent, although highly radioactive, contains less than 10% tritium atoms and more than 90% protons. Thus, the possibility existed that if there were a significant isotope effect in the reduction, the radiochemical yield could be drastically reduced even if “undiluted” [3H]NaBH4 was used. For this possibility to be evaluated, model studies were performed in which reductions were carried out on 22 in DMF using 5% Pd(OAc)2 and a mixture of NaBH4 (1.2 equiv) and NaBD4 (1.2 equiv). The reaction proceeded efficiently (95% yield), and the H/D ratio of the product was shown to be 1.43:1, indicating at most a small isotope effect (Scheme 4B).

However, to further maximize the potential radiochemical yield in the preparation of [3H]-16, we envisaged a reaction where the iodo precursor would first be reacted with less than one equivalent of sodium borotritiide, and then the reaction would be driven to completion with the addition of excess NaBH4. We required that such a reduction be viable on small (milligram) scale. This was anticipated to provide maximal incorporation of tritium and to facilitate purification by completely converting the iodo precursor. Thus, a further model reaction was carried out where 22 (6.6 μmol) was reacted with [3H]-NaBH4 (12.1 μCi, 1.1 μmol) for 30 min and then followed by excess NaBH4 to complete the reduction. This procedure yielded tritium-labeled 5b in good chemical and radiochemical yield (26−40% radiochemical yield) and free of unreacted 22. It then remained to demonstrate that the same chemistry could work with the corresponding iodo analogue of conjugate 16. Thus, iodo-EP4a (22) was converted in a sequence identical to that used before for the proteo conjugate to provide the 4-iodophenyl analogue of conjugate 16 (compound 31) (Scheme 5). Model reduction of 31 under the same conditions of Pd(OAc)2 (5%) and NaBH4 (1 equiv in DMF) gave clean and complete conversion to proteo-product 16. Finally, the reaction was repeated using 31 (4.0 mg, 2.9 μmol) and [3H]-NaBH4 (5.3 mCi, 1.53 μmol) and followed with excess NaBH4 to complete the conversion to give [3H]-16 (3.6 mg, 100% chemical yield) in 10.6% radiochemical yield. 7019

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Figure 4. Pharmacokinetics of elimination over 6 h (panel A) and tissue distribution (panel B) of radiolabel 6 h after a single IV dose of [3H]-16 in rats.

Scheme 6. Synthesis of 14C-Labeled Conjugate 16a

Reagents and conditions: (i) CDI [carbonyl-14C] (1.0 equiv), 1.6 mg in 50 μL of THF, rt, 1 h; (ii) alendronic acid bis-tetra-n-butylammonium salt (2.4 equiv), DMAc, rt, 52% radiochemical yield for 2 steps. a

cleaved bisphosphonate, and the supernatant solution was counted to quantify any unbound radioactivity in the supernatant. Compound 16 was shown to liberate 27% of the label after 30 min, 44% after 4 h, and 53% over 24 h. When [3H]-16 was allowed to bind to bone powder for 30 min in buffer, and then the bone powder was filtered, resuspended, and then incubated in fresh rat serum, buffer, or inactivated serum; only approximately 95%) to bone powder (bovine) within 30 min when stirred in PBS buffer and in rat plasma. The conjugate was then incubated in rat plasma at 37 °C, and after a fixed amount of time, the incubate was stirred with bone powder to adsorb the intact conjugate and any 7020

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of 5 mg/kg to rats, and blood samples were drawn at intervals over 6 h. Compound 16 was not sufficiently soluble at the concentrations desired for dosing (2 mg/mL) in PBS alone and was therefore evaluated further in formulations of PBS with the addition of a variety of surfactants such as PEG400, Tween, and Cremaphor, but these all gave slightly cloudy solutions. A suitable vehicle (95% PBS, pH 7.4, 5% poloxamer 188) was eventually found that yielded a homogeneous solution, and this was used for subsequent in vivo experiments. Thus, three rats were injected with [3H]-16 (5 mg/kg, 31 μCi per rat), and blood samples were taken at intervals over 6 h. The dose was well-tolerated, and the experiment was terminated after 6 h; tissue samples, including samples of long bones, were taken and analyzed for radioactivity. Processed samples showed that the conjugate was rapidly cleared from the bloodstream (Figure 4A) with most radioactivity found in the liver at 6 h and approximately 2.5% of the administered label found in bone (Figure 4B). This was somewhat less than we had observed in similar experiments with other conjugates such as 6, where approximately 5% was found in bone after 6 h21,22 but similar to that observed for conjugate 4 (3.5%)20 and indicated that at least a portion of the conjugate had survived intact to bind to bone and at a level potentially adequate to justify testing the concept in a subsequent dual-label experiment. With this result in hand, we proceeded to prepare 16 radiolabeled with 14C at the carbamate group, which attaches alendronate to the peptide linker (Scheme 6). [14C]-16 would then be mixed with [3H]-16 to carry out the dual-label experiment to quantify the overall in vivo stability and uptake into bone and to monitor the release of both the EP4a (5a) and alendronate in the same experiment. Synthesis of 14C-Labeled Conjugate 16. Incorporation 14 of C into the PABA-ALN carbamate linker was achieved using commercially available [14C]-carbonyldiimidazole (CDI) in the final coupling step (Scheme 6). The reaction sequence proceeded smoothly on a milligram scale, and 0.5 mCi of CDI provided 0.26 mCi of [14C]-16 for a 52% radiochemical yield. Biodistribution, Uptake, and Release of [3H,14C]-16 in rats. For these experiments, a mixture of [3H]-16 and [14C]-16 (8.9 and 1.8 μCi/rat, respectively) was diluted with unlabeled 16 and dosed by IV administration to 15 rats at a total dose of 5 mg kg−1 rat−1. Groups of three were terminated after 6 h and 1, 7, 14, and 28 days, and the bones and tissues (liver, spleen, kidney, heart, and brain) were collected and analyzed quantitatively for both 3H and 14C labels. The radioactivity observed was corrected for whole body/organ weight, and the results are depicted in Figure 5 as a percentage of the original total administered dose. It was immediately apparent that the uptake of 14C into bones was at least 2-fold higher than that of 3 H, and this difference remained constant over 28 days, whereas the levels of both activities fell. However, relative levels of radioactivity found in liver and other organs were roughly equal throughout the time course (Table 3). Notably, the levels of both 3H and 14C fell in bones with half-times of 4.4 days for 3H and 5 days for 14C over the first two weeks, but both to appeared plateau between two and four weeks postdosing. Levels in other tissues decayed exponentially and did not plateau over 28 days when residual levels were very low (Table 3). These data indicate that significant loss of the tritiated EP4 agonist component occurred prior to attachment to the bones, whereas thereafter, the rate of release of the bound tritium was very similar to what had been observed in previous studies with

Figure 5. Radioactivity found in long bones at various times after dosing doubly labeled [3H/14C]-16 in rats (expressed as a percentage of the administered dose).

Table 3. Levels of Radioactivity Found in Various Tissues after Dosing Doubly Labeled [3H/14C]-16 percentage of dosea bone

3

H C 3 H 14 C 3 H 14 C 3 H 14 C 3 H 14 C 3 H 14 C 14

liver spleen kidney heart brain

6h

1 day

7 days

14 days

28 days

2.10 5.52 8.95 7.03 0.35 0.34 0.21 0.11 0.025 0.017 0.022 0.014

2.12 4.63 2.33 2.50 0.22 0.23 0.11 0.047 0.021 0.009 0.025 0.012

1.30 3.51 0.48 0.61 0.076 0.086 0.053 0.020 0.014 LOD LOD LOD

0.80 2.52 0.30 0.27 0.039 0.050 0.026 0.013 LOD LOD LOD LOD

0.74 2.44 0.08 0.10 0.037 0.039 0.017 0.0092 LOD LOD LOD LOD

a

Levels are expressed as a percentage of the originally administered radioactivity and are the average of data from three rats. Bone data were calculated for long bone samples assuming total bone represented 8% of the body weight. LOD, limit of detection.

conjugate 6. The release of the bound 14C was surprisingly fast as it has been estimated that between 37 and 66% of bone surface is resorbed in female rats within three weeks,26 a rate more consistent with the 22 day half-time for loss of 14C label in our previous in vivo studies with conjugate 6.22 This may indicate that the conjugate can be cleaved by peptidase enzymes other than cathepsin K, which can access the conjugate on the bone surface, or alternatively, that because the conjugate is bound and presented on the exposed surface of the bones, its cleavage may be faster than the global rate of resorption (measured by loss of a mineral matrix bound marker such as tetracycline), where resorption is driven both by dissolution of mineralization by acid and the cleavage of revealed collagen matrix by cathepsin K. The plateauing of release of both 3H and 14C after two weeks may suggest that overlay of new bone has trapped some conjugate where it is no longer accessible. 7021

DOI: 10.1021/acs.jmedchem.6b00951 J. Med. Chem. 2017, 60, 7012−7028

Journal of Medicinal Chemistry

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Article

CONCLUSIONS Novel dual action prodrug 16 has been designed and synthesized wherein a potent bone anabolic EP4 receptor agonist (5) and a potent bone resorption inhibitor, alendronic acid, are conjugated via a differentially cleavable dipeptide-paraaminobenylalcohol (Leu-Arg-PABA) linker. We have also developed syntheses of radiolabeled analogues of 16 designed to interrogate the pharmacokinetic tissue distribution uptake into bones and release of the EP4 agonist and alendronate components in vivo. A tritium-labeled analogue ([3H]-16) was prepared where the EP4 agonist was labeled via tritiation of an aryl iodide as the last step using palladium-catalyzed reduction with sodium borotritiide. This efficient labeling allowed us to evaluate the stability of 16 in rat plasma where we found some degree of hydrolysis in plasma (∼55% over 24 h). This stability was considered sufficient to warrant further in vivo evaluation, and thus, a second labeled conjugate ([14 C]-16) was synthesized using 14C-carbonyldiimidazole such that the carbonyl carbon of the carbamate group joining the linker to alendronic acid was labeled with 14C. This allowed dosing of a double-labeled conjugate to show that 16 targets and binds to bone after IV administration. However, there was significant loss of the tritium label prior to bone binding as indicated by the ratio of radioactivity found in bones, suggesting that approximately 50% of the EP4 agonist component was lost prior to binding to bones. Nonetheless, analysis of the loss of radiolabels over time indicated that the bound conjugate releases both active constituents at a similar sustained rate. It is assumed that the EP4 agonist (5) is liberated in situ through the action of esterases as the release rate (half-time of 4.4 days) is similar to that observed for other conjugate prodrugs such as 6 in earlier studies,21,22 and based on the 5 mg/kg dose and 2.2% bone uptake, we calculate that the active EP4 agonist 5a would be released at a rate of approximately 4 μg kg−1 day−1. This is somewhat lower than the calculated daily release of 5a (∼18 μg kg−1 day−1)23 for the efficacious dose24 of conjugate 6. The alendronate component was released with a roughly 5 day half-time, which is much faster than previously observed for conjugate 6. On the basis of the 5% bone uptake of 14C, this would allow calculation of the release of active alendronate at a rate of approximately 5 μg kg−1 day−1. Compound 16 was designed so that the alendronate component would be released through protease cleavage of the peptide-NH-PABA bond, which leads to self-immolation and liberation of free amino function of alendronate (which is itself bound to the bone surface). This cleavage was designed to be mediated by cathepsin K, an important protease released by osteoclasts in the bone compartment in vivo and thus would concentrate release at sites of higher bone resorption. However, we cannot rule out a possible role for other proteases that may be present on or near the bone surface. Given the very similar half times for loss of 3H and 14C, we also cannot rule out the possibility that some of the observed tritium loss may be due to peptidase cleavage (prior to esterase cleavage) as this would lead to the loss of both labels at the same time. Only an in vivo efficacy study in the OVX rat model of osteoporosis can confirm that the anabolic activity of the free EP4 receptor agonist is expressed in the bone compartment. Such studies are now being considered. Optimally, further stabilization of the ester linkage (possibly via α-substitution) could enhance systemic stability of the conjugate (but also possibly slow the hydrolysis of the bone-bound conjugate ester). Such options are now

being evaluated and will be reported on in due course. Notably, this work has established the viability of dipeptide-PABA linker as a release mechanism for bone-targeted bisphosphonate prodrugs in vivo.

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EXPERIMENTAL SECTION

General Chemistry Methods. 1H and 13C NMR spectra were recorded with a Bruker Avance II 600 MHz spectrometer using a TCI cryoprobe, an Avance III 500 MHz spectrometer using a TXI inverse probe, or an Avance III 400 MHz spectrometer using a BBOF + ATM probe. NMR data processing was performed with MestReNova software (MestreLab Research, ver. 6.0.4-5850). The spectra were referenced to the corresponding solvent signals.39 NMR spectra for all compounds containing a bisphosphonate group were run in DMSO-d6 with 20 μL (35% by weight) of DCl in D2O (Sigma-Aldrich). LC−MS were recorded with an ESI ion source on an Agilent 6200 Time-ofFlight spectrometer coupled with Agilent 1200 series HPLC. Analytical thin-layer chromatography (TLC) was performed on aluminum plates precoated with silica gel 60F-254 as the adsorbent (EMD). The developed plates were air-dried, exposed to UV light and/or dipped in KMnO4 solution, and heated. Flash chromatography was performed on a BioTage Isolera instrument using HP-silica cartridges from BioTage or SiliCycle Inc. Derivatized silica was obtained from SiliCycle Inc. Tetrahydrofuran (THF) was distilled from Na and benzophenone under nitrogen. Dichloromethane (DCM) was distilled from CaH2 under nitrogen. Pyridine, triethylamine (TEA), and diisopropylethylamine (DIPEA) were distilled from CaH2 under nitrogen. Other reagents and solvents were obtained from commercial vendors and used as received. The purities of all final products were 95% or higher as determined by HPLC analysis. Nonradioactive compounds were analyzed using an Agilent 1100 HPLC and PDA detector at 254 nm with conditions 1: Agilent Zorbax SB-C8 column (3.0 × 150 mm, 5 μm) with a gradient of acetonitrile:0.1% formic acid from 5:95 to 95:5 over 5 min with a flow rate of 2 mL/min or conditions 2: Advanced Materials Technology Halo C18 column (4.6 × 50 mm, 5 μm) with gradient methanol:0.1% formic acid from 5:95 to 95:5 over 5 min with a flow rate of 2 mL/min or conditions 3: Halo C18 column with gradient acetonitrile:5 mM ammonium acetate, pH 7, from 5:95 to 95:5 over 5 min with a flow rate of 2 mL/min. Radioactive 16 was analyzed using a Waters Alliance 2695 HPLC with a Radiomatic FSA 150 TR radioactivity detector and Waters 996 PDA at 254 nm with conditions 3. Sodium 7-((R)-2-((R,E)-4,4-Difluoro-3-hydroxy-4-(phenyl-4-t)but1-en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate ([3H]-5a). Compound 22 (3.6 mg, 6.56 μmol) in 100 μL of DMF was treated with Pd(OAc)2 (0.33 μmol in 66 μL of DMF). After 15 min, NaB3H4 (specific activity: 15.6 Ci/mmol) (12.13 mCi in 100 μL of DMF) was added, and the mixture was stirred for 1 h. Then, NaBH4 (6.0 μmol in 131 μL of DMF) was added, and the mixture was stirred for 1 h. Water (3 mL) was added to quench the reaction. The mixture was directly loaded onto a C18 cartridge (0.5 g, Silicycle SPE-R31030B-03P). After washing with water (6 column volumes), MeOH was used to elute the product. The product in MeOH was treated with NaOH (1.0 M, 2.0 mL) and stirred for 30 min. The MeOH was removed under reduced pressure, and the residue was dissolved in water and directly loaded onto a C18 cartridge. After washing with water (6 column volumes), MeOH was used to elute the hydrolysis product, [3H]-5a (3.8 mCi; 31% radiochemical yield). (4-((((4-((S)-2-((S)-1-((Benzyloxy)carbonyl)pyrrolidine-2-carboxamido)-5-guanidino-pentanamido)benzyl)oxy)carbonyl)amino)-1hydroxybutane-1,1-diyl)bis(phosphonic acid) (10). Compound 14 (51 mg, 0.10 mmol) and CDI (25 mg, 0.15 mmol) were dissolved in DMF (0.5 mL). The mixture was stirred at room temperature, and 2 h later, HPLC indicated complete consumption of starting material. A solution of alendronic acid bis-tetra-n-butyl ammonium salt in DMF (0.5 mL) (230 mg, 0.160 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction mixture was reduced to dryness in vacuo, taken up in ACN/H2O (1:1), and loaded on an 7022

DOI: 10.1021/acs.jmedchem.6b00951 J. Med. Chem. 2017, 60, 7012−7028

Journal of Medicinal Chemistry

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7.67 (d, J = 8.4 Hz, 2H), 7.43−7.17 (m, 7H), 4.97 (ddd, J = 39.4, 19.7, 9.6 Hz, 4H), 4.33 (dd, J = 8.8, 4.1 Hz, 2H), 3.86 (s, 2H), 3.66−3.33 (m, 2H), 3.12 (t, J = 6.5 Hz, 2H), 2.92 (t, J = 6.9 Hz, 2H), 2.06 (dt, J = 16.3, 8.0 Hz, 1H), 1.98−1.41 (m, 11H). HRMS (ESI+)(m/z) calcd for C33H49N8O14P2+ ([M + H]+) 843.2838, found 843.2876. HPLC purity 99% (conditions 1). Benzyl (S)-(5-Guanidino-1-((4-(hydroxymethyl)phenyl)amino)-1oxopentan-2-yl)carbamate (13a). N-Cbz-Arginine (3.45 g, 10 mmol), HOBT (1.53 g, 10 mmol), 4-aminobenzylalcohol (1.23 g, 10 mmol), and EDCI (2.30 g, 10 mmol) were dissolved in 50 mL of DMF. Four hours later, the solvent was removed by high vacuum. The residue was separated by column chromatography (120 g of silica gel; gradient: 5 to 34% methanol/dichloromethane) to give 13a (3.99 g, 89% yield) as a light brown solid. 1H NMR (500 MHz, DMSO) δ 10.20 (s, 1H), 7.87 (t, J = 5.4 Hz, 1H), 7.61 (dd, J = 14.4, 8.1 Hz, 3H), 7.39−7.29 (m, 5H), 7.24 (d, J = 8.4 Hz, 2H), 5.14 (t, J = 5.7 Hz, 1H), 5.08−4.95 (m, 2H), 4.43 (d, J = 5.6 Hz, 2H), 4.19 (dd, J = 13.7, 8.3 Hz, 1H), 3.12 (dd, J = 12.8, 6.5 Hz, 2H), 1.84−1.70 (m, 1H), 1.69− 1.41 (m, 3H). 13C NMR (126 MHz, DMSO) δ 170.65, 156.92, 156.03, 137.54, 137.46, 136.96, 128.35, 127.80, 127.71, 126.85, 118.97, 65.46, 62.57, 54.92, 40.13, 28.94, 25.21. HRMS (ESI+) (m/z) calcd for C21H28N5O4+ ([M + H]+) 414.2136, found 414.2149. Benzyl (S)-2-(((S)-5-Guanidino-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (14). Compound 13a (0.83 g, 2 mmol) was dissolved in 20 mL of methanol, and palladium on carbon (10%) (0.083 g) was added to the solution. With uniform stirring, atmospheric pressure hydrogen was passed through the solution, and 2 h later, TLC indicated complete consumption of starting material. The reaction mixture was filtered through Celite, and the filtrate methanol was removed in vacuo to provide the free amino compound 13b, which was used directly in the next reaction. The residue 13b, N-Cbz-Proline (0.50 g, 2 mmol), HOBT (0.31g, 2 mmol), and EDCI (0.46 g, 2.4 mmol) were dissolved in 10 mL of DMF, and 4 h later, the DMF was removed under high vacuum. The residue was separated by column chromatography (50 g of silica gel; gradient: 5 to 34% methanol/dichloromethane) to yield compound 14 (0.97 g, 95% yield) as an off-white solid. 1H NMR (600 MHz, DMSO) δ 10.15 (d, J = 130.0 Hz, 1H), 8.86 (d, J = 46.9 Hz, 1H), 8.55−8.35 (m, 2H), 7.71 (s, 3H), 7.55 (dd, J = 19.9, 8.3 Hz, 2H), 7.44−7.16 (m, 7H), 5.04 (ddd, J = 42.0, 41.2, 12.9 Hz, 2H), 4.43 (s, 2H), 4.40−4.24 (m, 2H), 3.09 (ddd, J = 17.7, 12.3, 5.9 Hz, 1H), 3.00 (dt, J = 12.5, 6.8 Hz, 1H), 2.23−2.07 (m, 1H), 1.91−1.32 (m, 7H). 13C NMR (151 MHz, DMSO) δ 172.35, 172.08, 170.41, 170.38, 167.13, 157.36, 154.26, 153.82, 137.55, 137.53, 137.48, 136.94, 136.93, 128.41, 128.22, 127.80, 127.53, 127.47, 126.99, 126.92, 118.97, 118.96, 65.99, 65.82, 62.60, 59.90, 59.03, 52.87, 52.82, 47.16, 46.61, 40.06, 31.17, 30.08, 29.04, 28.80, 24.99, 24.91, 23.95, 23.06. HRMS (ESI+) (m/z) calcd for C26H35N6O5+ ([M + H]+) 511.2663, found 511.2674. (4-((((4-((S)-2-((S)-2-((((4-(2-(((R,E)-4-((R)-1-(7-ethoxy-7-oxoheptyl)-5-oxopyrrolidin-2-yl)-1,1-difluoro-1-phenylbut-3-en-2-yl)oxy)-2oxoethyl)benzyl)oxy)carbonyl)amino)-4-methylpentanamido)-5guanidinopentanamido)benzyl)oxy)carbonyl)amino)-1-hydroxybutane-1,1-diyl)-bis(phosphonic acid) (16). CDI (2.2 mg, 0.013 mmol) was dissolved in 50 μL of THF; then, compound 20 (10 mg, 0.011 mmol) was added. The mixture was stirred at room temperature for 1 h, after which HPLC analysis indicated complete consumption of the starting material. Alendronic acid bis-tetra-n-butylammonium salt stock solution in DMAc (40 μL, 0.026 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with AcOH (3.0 μL, 0.052 mmol) and then diluted with 300 μL of 50% acetonitrile−water before loading on an anion-exchange cartridge (strong anion exchange, Silicycle R66430B, Lot 53470). The column was first washed with 1:1 ACN/H2O (5 column volumes), and then the product was eluted with KH2PO4−K2HPO4 buffer (0.08M, pH 8.0, in 2:3 ACN/H2O). The salts were then removed by C18 column, and lyophilization of selected fractions gave 16 as a white power (6 mg, 47% yield). 1H NMR (600 MHz, DMSO) δ 7.55 (d, J = 8.3 Hz, 2H), 7.49 (t, J = 7.2 Hz, 1H), 7.42 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 7.4 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H),

anion-exchange column (Strong anion exchange, Silicycle R66430B, lot 53470). First, it was washed with 1:1 ACN/H2O (5.0 column volume) and then with water (2.0 column volume), and finally, the product was eluted with NaCl solution (5.0%). The NaCl was then removed by desalting on a C18 column. After lyophilization, compound 10 was obtained as white power (40 mg, 55%). 1H NMR (500 MHz, DMSO) δ 7.61 (t, J = 8.4 Hz, 2H), 7.45−7.20 (m, 7H), 5.09−4.95 (m, 2H), 4.90 (s, 1H), 4.40−4.22 (m, 2H), 3.48−3.29 (m, 2H), 3.03 (d, J = 6.3 Hz, 1H), 2.91 (t, J = 6.9 Hz, 2H), 2.14 (d, J = 8.5 Hz, 1H), 1.74 (dd, J = 33.2, 27.3 Hz, 8H), 1.60−1.33 (m, 3H). HRMS (ESI+) (m/z) calcd for C31H46N7O13P2+ ([M + H]+) 786.2623, found 786.2650. HPLC purity 97% (conditions 2). 4-((((4-((S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-4-methylpentanamido)-5-guanidino-pentanamido)benzyl)oxy)carbonyl)amino)1-hydroxybutane-1,1-diyl)bis(phosphonic acid) (11). Compound 17a (54 mg, 0.10 mmol) and CDI (25 mg, 0.15 mmol) were dissolved in DMF (0.5 mL), and the mixture was stirred at room temperature. Two hours later, HPLC indicated complete consumption of starting material. A solution of alendronic acid bis-tetra-n-butyl ammonium salt in DMF (0.5 mL) (230 mg, 0.160 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction mixture was reduced to dryness in vacuo and taken up in 1:1 ACN/ H2O and loaded on an anion-exchange column (strong anion exchange, Silicycle R66430B, lot 53470). First, it was washed with 1:1 ACN/H2O (5.0 column volume) and then with water (2.0 column volume), and finally, the product was eluted with NaCl solution (5.0%). The NaCl then was removed by desalting on a C18 column. After lyophilization, 11 was obtained as a white power (40 mg, 52% yield). 1H NMR (500 MHz, DMSO) δ 7.60 (d, J = 8.3 Hz, 2H), 7.28 (dd, J = 34.4, 6.8 Hz, 7H), 5.03−4.95 (m, 2H), 4.90 (s, 2H), 4.40 (dd, J = 8.9, 5.2 Hz, 1H), 4.05 (dd, J = 9.5, 5.4 Hz, 1H), 3.11 (t, J = 6.5 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H), 1.87−1.71 (m, 3H), 1.71−1.50 (m, 5H), 1.49−1.35 (m, 3H), 0.89−0.74 (m, 6H). HRMS (ESI+) (m/z) calcd for C32H50N7O13P2+ ([M + H]+) 802.2936, found 802.2964. HPLC purity 97% (conditions 1). (4-((((4-((S)-2-((S)-1-(((Benzyloxy)carbonyl)glycyl)pyrrolidine-2carboxamido)-5-guanidino-pentanamido)benzyl)oxy)carbonyl)amino)-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) (12). Compound 14 (0.300 g, 0.587 mmol) was dissolved in 10 mL of methanol; palladium on carbon (10%) (0.030g) was added to the solution. With uniform stirring, atmospheric pressure hydrogen was passed through the solution. Two hours later, TLC indicated completed consumption of the starting material. The reaction mixture was filtered through Celite, and the solvent was removed to give crude pyrrolidine-2carboxylic acid [4-guanidino-1-(4-hydroxymethyl-phenylcarbamoyl)butyl]-amide, which was used directly in the next step. The residue (theoretically 0.587 mmol), Cbz-Gly-OH (0.133 g, 0.636 mmol), HOBT (0.091 g, 0.594 mmol), and EDCI (0.13 g, 0.69 mmol) were dissolved in 10 mL of DMF. Four hours later, the solvent was removed under high vacuum. The residue was separated by column chromatography on silica gel (gradient: 5 to 34% methanol/ dichloromethane), and then the solvent was removed in vacuo to provide benzyl ((S)-2-(((S)-5-guanidino-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)glycinate (0.333 g, 94% yield) as an off-white solid. HRMS (ESI+) (m/ z) calcd for C28H38N7O6+ ([M + H]+) 568.2878, found 568.2888. The glycinate from the reaction above (76 mg, 0.13 mmol) and CDI (46 mg, 0.28 mmol) were dissolved in 0.8 mL of DMF. The mixture was stirred at room temperature. Two hours later, HPLC indicated complete consumption of the starting material. A solution of alendronic acid bis-tetra-n-butyl ammonium salt (230 mg, 0.160 mmol) in DMF (0.5 mL) was added, and the mixture was stirred at room temperature for 4.0 h. The reaction mixture was reduced to dryness in vacuo, taken up in 1:1 ACN/H2O, and loaded on an anionexchange column (Strong Anion Exchange, Silicycle R66430B, lot 53470). First, it was washed with 1:1 ACN/H2O (5.0 column volume) and then with water (2.0 column volumes), and finally, the product was eluted with NaCl solution (5.0%). The NaCl was then removed by desalting on a C18 column. After lyophilization, compound 12 was obtained as a white power (60 mg). 1H NMR (500 MHz, DMSO) δ 7023

DOI: 10.1021/acs.jmedchem.6b00951 J. Med. Chem. 2017, 60, 7012−7028

Journal of Medicinal Chemistry

Article

7.09 (d, J = 8.0 Hz, 2H), 5.75 (m, 1H), 5.69−5.50 (m, 2H), 5.11−4.86 (m, 4H), 4.42−4.36 (m, 1H), 4.03 (m, 4H), 3.65 (q, J = 15.3 Hz, 2H), 3.18−3.08 (m, 3H), 2.93 (t, J = 6.8 Hz, 2H), 2.46−2.41 (m, 1H), 2.22 (t, J = 7.4 Hz, 2H), 2.18−2.11 (m, 2H), 2.06 (dd, J = 12.6, 7.7 Hz, 1H), 1.86−1.78 (m, 2H), 1.75 (s, 1H), 1.71−1.58 (m, 4H), 1.57−1.40 (m, 7H), 1.25 (s, 1H), 1.18 (dd, J = 14.7, 7.4 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H), 1.07 (dd, J = 15.2, 7.2 Hz, 2H), 0.83 (m, 6H). HRMS (ESI+) (m/z) calcd for C57H81F2N8O18P2+ ([M + H]+) 1265.5107, found 1265.5088. HPLC purity 96% (conditions 3) Preparation of [3H]-16. Compound 31 (4.0 mg, 2.88 μmol) in 100 μL of DMF was treated with Pd(OAc)2 (0.15 μmol in 30 μL of DMF) at room temperature. After 15 min, NaB3H4 (5.29 mCi, 1.53 μmol, 10.9 Ci/mmol specific activity) in 50 μL of DMF was added, and after 1 h, NaBH4 (2.0 μmol in 20 μL DMF) was added. After 1 h, the reaction mixture was directly loaded on a C18 column. After washing with water (6 column volume), the product was eluted an acetonitrile/ water mixture (1:1 ACN/H2O) and counted 0.56 mCi. Radiochemical yield 10.6%. HPLC purity 96% (conditions 1). Preparation of [14C]-16. CDI [carbonyl-14C] (8.9 μmol, 0.5 mCi, 1.45 mg, 56 mCi/mmol specific activity) was dissolved in 50 μL of THF, and then compound 20 (11 mg, 10 μmol) was added. The mixture was stirred at room temperature for 1 h. Alendronic acid bistetra-n-butylammonium salt in DMAc (40 μL, 0.024 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with CH3COOH (3.0 μL, 0.052 mmol) and then diluted with 300 μL of 50% acetonitrile/water before being loaded on an anion-exchange column (Strong anion exchange, Silicycle R66430B, lot 53470). The column was washed with 1:1 ACN/H2O (5.0 column volume). Then, product was eluted by KH2PO4−K2HPO4 solution (0.08 M pH 8.0, 2:3 ACN/H2O). The salts were then removed by C18 column. The product was dissolved in 4.0 mL of 1:1 water/i-PrOH mixture and counted 0.26 mCi. Radiochemical yield 52%. (5S,8S)-13-Amino-8-((4-(hydroxymethyl)phenyl)carbamoyl)-5isobutyl-3,6-dioxo-1-phenyl-2-oxa-4,7,12-triazatridecan-13-iminium chloride (17a). Compound 13a (0.625 g, 1.58 mmol) was dissolved in 20 mL of methanol; Pd/C (10%) (0.063 g) was added to the solution. With uniform stirring, atmospheric pressure hydrogen was passed through the solution, and 2 h later, TLC indicated complete consumption of starting material. The reaction mixture was filtered through Celite, and the filtrate methanol was removed under reduced pressure to provide crude 13b that was used directly in the next step. Crude 13b (theoretically 1.58 mmol) was dissolved in 45 mL of DMF. Cbz-Leu-OH (0.421 g, 1.587 mmol), HOBT (0.303 g, 1.979 mmol), and EDCI (0.303 g, 1.581 mmol) were added, and the mixture was stirred for 12 h at room temperature. The solvent was removed under high vacuum, and the residue was purified by column chromatography (50 g of silica gel; gradient: 5 to 34% methanol/ dichloromethane) to yield compound 17a (0.760 g, 85% yield) as an off-white solid. 1H NMR (500 MHz, DMSO) δ 10.19 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.91 (t, J = 5.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.2 Hz, 1H), 7.44 (s, 1H), 7.33 (dt, J = 19.6, 4.2 Hz, 7H), 7.26− 7.21 (m, 3H), 5.06−4.93 (m, 2H), 4.53−4.31 (m, 3H), 4.09 (dt, J = 9.1, 5.6 Hz, 1H), 3.14 (dd, J = 12.3, 6.1 Hz, 2H), 1.86−1.72 (m, 1H), 1.69−1.32 (m, 6H), 0.86 (t, J = 7.2 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 172.46, 170.17, 156.96, 155.96, 137.54, 137.46, 137.05, 128.33, 127.74, 127.58, 126.86, 118.95, 65.37, 62.56, 53.24, 52.76, 40.65, 40.08, 29.23, 25.07, 24.22, 23.10, 21.41. HRMS (ESI+) (m/z) calcd for C27H39N6O5+ ([M + H]+) 527.2976, found 527.2994. 2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)acetic Acid (18). 4-(Hydroxymethyl)phenylacetic acid (3.33 g, 20 mmol) and imidazole (3.40 g, 50 mmol) were dissolved in THF (50 mL) and cooled in an ice−water bath. TBSCl (6.34 g, 42 mmol) was dissolved in THF (10 mL) and added dropwise. After 2 h, TLC indicated complete consumption of starting material. The cold bath was removed, and 200 mL of hexanes was added, after which the mixture was filtered and the solvents were removed under reduced pressure. The residue was dissolved in MeOH (20 mL) and THF (40 mL). K2CO3 (10 g, excess) was added as a solid, and 2 h later, the mixture was filtered and the

solvents were removed under reduced pressure. The residue was dissolved in 150 mL of water, and the pH was adjusted to 2.0 with HCl. The solution was extracted with MTBE (50 mL × 2), and the combined MTBE extracts were dried over MgSO4 and the solvents were removed under reduced pressure to provide 18 (5.52 g, 68% yield) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.29 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 4.72 (s, 2H), 3.64 (s, 2H), 0.94 (s, 9H), 0.09 (s, 6H). Ethyl 7-((R)-2-((R,E)-3-(2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)-2-oxoethoxy)-4,4-difluoro-4-phenylbut-1-en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (19). Compound 18 (0.65 g, 2.33 mmol), compound 5b (0.85 g, 2.0 mmol), and DMAP (0.024 g, 0.2 mmol) were dissolved in DCM. Then, DCC (0.648 g, 3.141 mmol) was added, and 4 h later, HPLC indicated complete consumption of the starting materials. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (1.0−8.0% MeOH/ DCM) to provide 19 (1.37 g, 99% yield). 1H NMR (500 MHz, DMSO) δ 7.46 (m, 5H), 7.22 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 5.80 (td, J = 10.6, 6.6 Hz, 1H), 5.61 (ddd, J = 23.8, 15.4, 7.5 Hz, 2H), 4.68 (s, 2H), 4.12−3.95 (m, 3H), 3.69 (q, J = 15.4 Hz, 2H), 3.18 (dt, J = 13.8, 7.7 Hz, 1H), 2.45 (ddd, J = 13.4, 8.3, 5.3 Hz, 1H), 2.24 (t, J = 7.4 Hz, 2H), 2.19−2.00 (m, 3H), 1.52−1.41 (m, 3H), 1.35−1.05 (m, 9H), 0.90 (s, 9H), 0.08 (s, 6H). HRMS (ESI) (m/z) calcd for C38H54F2NO6Si+ ([M + H]+) 686.3683; found 686.3700. Ethyl 7-((R)-2-((R,E)-3-(2-(4-((5S,8S)-13-Amino-8-((4(hydroxymethyl)phenyl)carbamoyl)-13-imino-5-isobutyl-3,6-dioxo2-oxa-4,7,12-triazatridecyl)phenyl)acetoxy)-4,4-difluoro-4-phenylbut-1-en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (20). Compound 19 (0.32 g, 0.47 mmol) in THF (1.6 mL) was treated with TBAF (1.0 M in THF, 520 μL). After 30 min, TLC indicated complete consumption of the starting material. Saturated NH4Cl solution (40 mL) was added to quench the reaction, and the mixture was extracted with MTBE (30 mL × 3). The MTBE extracts were dried over MgSO4, and the solvent was removed under reduced pressure to give 8 (0.27 g, quant. yield) as a colorless oil, which was used directly in the following reaction. Compound 17a (0.30 g, 0.57 mmol) was dissolved in MeOH (10 mL); Pd/C (10%)(30 mg) was added, and with uniform stirring atmospheric pressure hydrogen was passed through the solution. Two hours later, TLC indicated complete consumption of starting material. The reaction mixture was filtered by Celite, after which methanol was removed under reduced pressure to provide compound 17b (223 mg; 99% yield) that was used directly in the next reaction. Compound 8 (0.27 g, theoretically 0.47 mmol) and PhNMe2 (0.13 mL, 1.05 mmol) in THF (2.4 mL) were treated with COCl2 (1.4 M in toluene, 0.38 mL, 0.53 mmol) and stirred for 30 min. Compound 17b (0.19 g, 0.48 mmol) and Et3N (0.30 mL, 2.1 mmol) were dissolved in DMF (2.4 mL), and the DMF solution was poured into the THF reaction mixture. After 3 h, the solvents were removed under high vacuum, and the residue was purified by column chromatography on silica gel (4−30%, MeOH/DCM) to yield 20 (400 mg, 84% yield). 1H NMR (500 MHz, DMSO) δ 10.07 (s, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.59−7.39 (m, 8H), 7.25 (t, J = 8.2 Hz, 4H), 7.13 (d, J = 7.6 Hz, 2H), 5.91−5.74 (m, 1H), 5.62 (ddd, J = 23.6, 15.3, 7.6 Hz, 2H), 5.12 (s, 1H), 4.98 (dt, J = 45.4, 22.8 Hz, 2H), 4.43 (s, 3H), 4.20− 3.93 (m, 4H), 3.70 (q, J = 15.3 Hz, 2H), 3.22−3.09 (m, 3H), 2.45 (d, J = 8.2 Hz, 1H), 2.25 (t, J = 7.2 Hz, 2H), 2.20−2.01 (m, 3H), 1.76 (m, 1H), 1.63 (d, J = 6.1 Hz, 2H), 1.47 (dd, J = 14.0, 7.1 Hz, 7H), 1.28 (s, 1H), 1.26−1.19 (m, 3H), 1.16 (t, J = 7.1 Hz, 3H), 1.10 (d, J = 7.0 Hz, 2H), 0.86 (t, J = 7.0 Hz, 6H). HRMS (ESI+) (m/z) calcd for C52H70F2N7O10+ ([M + H]+) 990.5147, found 990.5154. HPLC purity 98% (conditions 1). Ethyl 7-((R)-2-((R,E)-4,4-Difluoro-3-hydroxy-4-(4-iodophenyl)but1-en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (22). Compound 26 (2.7 g, 4.9 mmol), Et3N (0.79 mL, 4.93 mmol), and HCOOH (0.22 mL, 5.9 mmol) were dissolved in dichloromethane (20 mL). The mixture was cooled in a chilling bath (−5 °C internally), and Ru-TsDPEN-Cy (0.094 g, 0.15 mmol) was added. The reaction was stirred at −5 °C overnight and then quenched with saturated NaHCO3 solution. The mixture was extracted with ethyl acetate (50 mL × 3). The organic 7024

DOI: 10.1021/acs.jmedchem.6b00951 J. Med. Chem. 2017, 60, 7012−7028

Journal of Medicinal Chemistry

Article

phase was dried over MgSO4, filtered, and concentrated. The crude product was purified by chromatography on silica gel. (Gradient: 50 to 100% ethyl acetate/hexane) to provide 22 as a light yellow oil (2.0 g, 74% yield) 1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 5.71−5.63 (m, 2H), 4.54 (td, J = 9.7, 3.0 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 4.06−4.00 (m, 1H), 3.41 (ddd, J = 13.8, 8.8, 6.8 Hz, 1H), 2.75 (ddd, J = 13.9, 8.7, 5.4 Hz, 1H), 2.38−2.25 (m, 4H), 2.22−2.13 (m, 1H), 1.70−1.56 (m, 3H), 1.47−1.22 (m, 9H). HRMS (ESI+) (m/z) calcd for C23H31F2INO4+ ([M + H]+) 550.1260, found 550.1267. Ethyl 2,2-Difluoro-2-(4-iodophenyl)acetate (23). Ethyloxalyl chloride (8.2 g, 60 mmol) was added dropwise at room temperature to a solution of iodobenzene (11 g, 54 mmol) and aluminum chloride (8.0 g, 60 mmol) in dichloromethane (100 mL) over 15 min, and the mixture was left stirring at room temperature for a further 3 h. The reaction mixture was then poured into ice-cold 3 M hydrochloric acid. The mixture was stirred for a further 10 min and then extracted with dichloromethane. The organic phase was washed with 1 M hydrochloric acid and then saturated aqueous sodium chloride solution, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was chromatographed on silica gel to yield 5.6 g of ethyl 2-(4-iodophenyl)-2-oxoacetate (34% yield). 1H NMR (500 MHz, CDCl3) δ 7.89 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 4.44 (q, J = 7.0 Hz, 1H), 1.42 (t, J = 7.2 Hz, 2H). Ethyl 2-(4-iodophenyl)-2-oxoacetate (5.60 g, 18.4 mmol) in toluene (10 mL) was treated with DAST (3.26 g, 20.2 mmol) added dropwise over 15 min. The reaction mixture was stirred at room temperature overnight. Ice−water-NH3·H2O (5 mL) was added dropwise to quench the reaction. Then, the solution was extracted by MTBE (20 mL × 2). The MTBE solution was dried with MgSO4, and the solvent was removed under reduced pressure. The crude product was chromatographed on silica gel to provide ethyl 2-(4-iodophenyl)-2oxoacetate (3.1 g, 34% yield). 1H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 4.30 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H). 19F NMR (471 MHz, CDCl3) δ −104.37 (s). Dimethyl (3,3-Difluoro-3-(4-iodophenyl)-2-oxopropyl)phosphonate (24). Dimethyl methylphosphonate (1.30 g, 10.5 mmol) in THF (40 mL) was cooled to −78 °C; then, n-BuLi (1.6 M in hexane, 6.6 mL, 10.5 mmol) was added dropwise, and the mixture was stirred at −78 °C for 0.5 h. Compound 23 (3.10 g, 9.5 mmol) in THF (20 mL) was then added dropwise, and the reaction mixture was stirred at −78 °C for 4 h. After that, the reaction was quenched at −78 °C with CH3COOH (0.630 g, 10.5 mmol). The mixture was warmed to room temperature, and 60 mL of saturated NH4Cl solution and 30 mL of water were added. The mixture was extracted with EtOAc (30 mL × 3). The organic phase was dried over MgSO4, filtered, and concentrated. The crude product was purified on silica gel (gradient: 50 to 100% ethyl acetate/hexane) to provide 24 as a light yellow solid (2.76 g, 72% yield). 1H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 3.75 (d, J = 11.3 Hz, 3H), 3.36 (d, J = 22.0 Hz, 1H). Ethyl (R),(E)-7-(2-(4,4-Difluoro-4-(4-iodophenyl)-3-oxobut-1-en1-yl)-5-oxopyrrolidin-1-yl)heptanoate (26). Compound 24 (2.74 g, 6.77 mmol) in THF (60 mL) was treated with NaH (60% in mineral oil, 0.28 g, 7.1 mmol) at room temperature for 30 min. The mixture was cooled to 0 °C; then, ethyl (R)-7-(2-formyl-5-oxopyrrolidin-1yl)heptanoate (25)40 (1.83 g, 6.77 mmol) in THF (30 mL) and ZnCl2 (1.9 M in 2-methyltetrahydrofuran) were added, and the mixture was heated to 50 °C for 6 h. The reaction was quenched with saturated NH4Cl solution and extracted with EtOAc (50 mL × 3). The organic phase was dried over MgSO4, filtered, and concentrated. The crude product was purified by chromatography on silica gel (gradient: 50 to 100% ethyl acetate/hexane) to provide compound 26 as a light yellow oil (2.70 g, 73% yield). 1H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 6.96 (dd, J = 15.6, 7.7 Hz, 1H), 6.57 (d, J = 15.6 Hz, 1H), 4.24 (td, J = 7.9, 4.6 Hz, 1H), 4.12 (q, J = 7.1 Hz, 3H), 3.56 (ddd, J = 13.9, 8.9, 7.0 Hz, 1H), 2.76 (ddd, J = 13.9, 8.7, 5.3 Hz, 1H), 2.45−2.33 (m, 2H), 2.31−2.24 (m, 3H), 1.84−1.76 (m, 1H), 1.63−1.56 (m, 2H), 1.50−1.35 (m, 2H), 1.31−1.23 (m, 7H).

Ethyl 7-((R)-2-((R,E)-3-(2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)-2-oxoethoxy)-4,4-difluoro-4-(4-iodophenyl)but-1-en-1-yl)5-oxopyrrolidin-1-yl)heptanoate (28). Compound 18 (0.12 g, 0.44 mmol), compound 22 (0.20 g, 0.36 mmol), and DMAP (4 mg, 0.036 mmol) were dissolved in DCM (2 mL). DCC (0.11 g, 0.55 mmol) was added, and 4 h later, HPLC indicated complete consumption of 22. The reaction mixture was then filtered, and the filtrate was concentrated. The residue was separated by column chromatography on silica gel (1.0−8.0% MeOH/DCM) to yield 28 (0.190 g, 64% yield). 1H NMR (500 MHz, DMSO) δ 7.80 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 7.9 Hz, 2H), 5.79 (dd, J = 17.1, 10.8 Hz, 1H), 5.63 (ddd, J = 23.7, 15.3, 7.3 Hz, 2H), 4.69 (s, 2H), 4.09−3.99 (m, 3H), 3.73−3.64 (m, 2H), 3.22−3.15 (m, 1H), 2.47−2.43 (m, 1H), 2.24 (t, J = 7.3 Hz, 2H), 2.21−2.03 (m, 3H), 1.48 (dd, J = 14.6, 7.3 Hz, 3H), 1.32−1.07 (m, 9H), 0.90 (s, 9H), 0.08 (s, 6H). HRMS (ESI+) (m/z) calcd for C38H53F2INO6Si+ ([M + H]+) 812.2649, found 812.2639. Ethyl 7-((R)-2-((R,E)-3-(2-(4-((5S,8S)-13-Amino-8-((4(hydroxymethyl)phenyl)carbamoyl)-13-imino-5-isobutyl-3,6-dioxo2-oxa-4,7,12-triazatridecyl)phenyl)acetoxy)-4,4-difluoro-4-(4iodophenyl)but-1-en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (30). Compound 28 (0.18 g, 0.22 mmol) in THF (0.50 mL) was treated with TBAF (1.0 M in THF, 240 μL). After 30 min, TLC indicated complete consumption of starting material. Saturated NH4Cl solution (40 mL) was used to quench the reaction, and the mixture was extracted with MTBE (30 mL × 3). Finally, the MTBE solution was dried over MgSO4 and concentrated under reduced pressure to provide compound 29 (0.16 g) as a colorless oil. This material was used directly in the next reaction. Compound 29 (0.16 g, theoretically 0.22 mmol) and PhNMe2 (0.06 mL, 0.488 mmol) in THF (2.0 mL) were treated with COCl2 (0.17 mL, 0.24 mmol) (1.4 M in toluene) and stirred for 30 min. Compound 17b (0.087 g, 0.48 mmol) and Et3N (0.140 mL, 0.98 mmol) were dissolved in DMF (2.0 mL) and added to the THF reaction mixture. After stirring for 3 h, the solvents were removed under reduced pressure, and the residue was separated by column chromatography on silica gel (4−30%, MeOH/DCM) to yield 30 (0.180 g, 73% yield). 1H NMR (500 MHz, DMSO) δ 10.07 (s, 1H), 8.14 (t, J = 10.8 Hz, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.63 (dd, J = 11.2, 5.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.30− 7.19 (m, 6H), 7.13 (d, J = 7.9 Hz, 2H), 5.80 (td, J = 10.8, 6.6 Hz, 1H), 5.64 (ddd, J = 23.8, 15.3, 7.5 Hz, 2H), 5.11 (t, J = 5.6 Hz, 1H), 5.01 (dd, J = 38.9, 12.7 Hz, 2H), 4.44 (t, J = 8.0 Hz, 3H), 4.15−3.99 (m, 4H), 3.69 (q, J = 15.6 Hz, 2H), 3.26−3.03 (m, 3H), 2.47−2.39 (m, 1H), 2.24 (q, J = 7.4 Hz, 2H), 2.21−2.03 (m, 3H), 1.76 (m, 1H), 1.63 (dd, J = 12.1, 6.2 Hz, 2H), 1.58−1.35 (m, 7H), 1.29 (dd, J = 13.7, 5.8 Hz, 1H), 1.25−1.18 (m, 3H), 1.16 (t, J = 7.1 Hz, 3H), 1.10 (dd, J = 14.9, 7.7 Hz, 2H), 0.94−0.77 (m, 6H). HRMS (ESI+) (m/z) calcd for C52H69F2IN7O10+ ([M + H]+) 1116.4113, found 1116.4093. HPLC purity 95% (conditions 1). (4-((((4-((S)-2-((S)-2-((((4-(2-(((R,E)-4-((R)-1-(7-Ethoxy-7-oxoheptyl)-5-oxopyrrolidin-2-yl)-1,1-difluoro-1-(4-iodophenyl)but-3-en-2yl)oxy)-2-oxoethyl)benzyl)oxy)carbonyl)amino)-4-methylpentanamido)-5-guanidinopentanamido)benzyl)oxy)carbonyl)amino)-1hydroxybutane-1,1-diyl)-bis(phosphonic acid) (31). CDI (2.0 mg, 0.012 mmol) was dissolved in 50 μL of THF; then, compound 30 (11 mg, 0.010 mmol) was added. The mixture was stirred at room temperature for 1 h, after which HPLC indicated complete consumption of starting material. Alendronic acid bis-tetra-nbutylammonium salt stock solution in DMAc (40 μL, 0.024 mmol) was added. The mixture was stirred at room temperature for 1.0 h. The reaction was quenched with AcOH (3.0 μL, 0.052 mmol) and then diluted with 300 μL of 50% acetonitrile/water before loading onto an anion-exchange column (strong anion exchange, Silicycle R66430B, lot 53470). The column was first washed with 1:1 ACN/H2O (5.0 column volume). Then, the product was eluted by KH2PO4−K2HPO4 buffer (0.08 M, pH 8.0, in 2:3 ACN/H2O). The salts were then removed by C18 column. After lyophilization of selected fractions, 31 was obtained as a white power (6 mg, 44% yield). 1H NMR (600 MHz, DMSO) δ 9.02 (s, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 1.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.24 (d, J 7025

DOI: 10.1021/acs.jmedchem.6b00951 J. Med. Chem. 2017, 60, 7012−7028

Journal of Medicinal Chemistry

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= 7.9 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 7.8 Hz, 2H), 5.75 (m, 1H), 5.69−5.50 (m, 2H), 5.11−4.86 (m, 4H), 4.42−4.36 (m, 1H), 4.03 (m, 4H), 3.65 (q, J = 15.3 Hz, 2H), 3.18−3.08 (m, 3H), 2.93 (t, J = 6.8 Hz, 2H), 2.46−2.41 (m, 1H), 2.22 (t, J = 7.4 Hz, 2H), 2.18−2.11 (m, 2H), 2.06 (dd, J = 12.6, 7.7 Hz, 1H), 1.86−1.78 (m, 2H), 1.75 (s, 1H), 1.71−1.58 (m, 4H), 1.57−1.40 (m, 7H), 1.25 (s, 1H), 1.18 (dd, J = 14.7, 7.4 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H), 1.07 (dd, J = 15.2, 7.2 Hz, 2H), 0.83 (m, 6H). HRMS (ESI+) (m/z) calcd for C57H80F2IN8O18P2+ ([M + H]+) 1391.4073; found 1391.4058. HPLC purity 96% (conditions 1). Stability of 10−12 in Rat Plasma and Serum. Stock solutions of 10−12 (10 mM in water) were used to prepare 100 μL solutions of each compound (100 μM) in fresh and inactivated rat plasma (inactivated by heating at 56 °C for 2 h). The samples were incubated in a 37 °C water bath over 24 h. Aliquots were analyzed at time points 0, 1, 2, 4, 24 h. At each time point, methanol (1.0 M HCl, 100 μL) was added; the resulting mixture was centrifuged, and the supernatant solution was analyzed by HPLC (Phenomenex Monolith C18, 4.6 × 50 mm, on ACN-water-TFA gradient, UV 254 nm) to assess the remaining concentrations of 10−12. General Procedure for Cathepsin K Assays. Human procathepsin K was obtained from Sigma-Aldrich (Catalog number SPR6001, recombinant, expressed in E. coli, ≥95% (SDS-PAGE)). Rat cathepsin K (recombinant, expressed in E. coli) was obtained from Merck Research Laboratories as a gift. Stock solutions of 10−12 (1.0 mM in water) were prepared by carefully weighing the compounds and dissolving each compound in a calculated amount of Milli-Q water. Enzyme activity was assayed using the cathepsin K standard substrate Cbz-Leu-Arg-AMC. Assay buffer: 50 mM MES, pH 5.5, 2 mM EDTA, 4 mM DTT, 0.15% chondroitin sulfate. Final concentration: human or rat Cathepsin K 0.2 μM; standard substrate Cbz-Leu-Arg-AMC, 100 μM; conjugates 10−12, 100 μM. Procathepsin K (human) was activated in 50 mM NaOAc-HOAc, 200 mM NaCl buffer, pH 4.1, at room temperature for 40 min. Reactions were incubated at 37 °C and analyzed after 1, 2, and 20 h. Twenty microliter aliquots were analyzed by HPLC (Agilent Zorbax C18 4.6 × 150 mm, 5 μm column) eluting with MeOH-water-NH3·H2O gradient, UV 240 nm. Conjugate hydrolysis rates were calculated according to the appearance of 4-aminobenyl alcohol and its DTT-trapped adduct. Dosing Rats for Blood Levels and Elimination from Blood. Three female Sprague−Dawley rats (Charles River) were approximately 23 weeks old when received. They were acclimatized to the facility and operators. The rats were dosed by intravenous tail vein injection at a total dose of 5 mg/kg of 16 and 31.0 μCi/rat radioactivity dose of [3H]-16. For formulation, conjugate 16 was suspended in 10% Poloxamer 188 solution, adjusted to pH 8.0 with NaHCO3, and then diluted with an equal volume of PBS. The dosing was done with a fixed volume of 500 μL. Blood samples (0.5 mL per time point per rat) were taken after 0.5, 1, 2, 4, and 6 h and placed in heparinized microcentrifuge tubes. Blood samples (500 μL) were centrifuged at ∼5000g until plasma had separated. Then, 100 μL of the supernatant was analyzed in a Harvey OX-300 biological oxidizer using the 4 min program. The collected scintillation mixture was counted on a Beckman Coulter LS-6500 scintillation counter. The counting time was 2 min. Rat Organ Distribution, Uptake, and Release of Conjugate 16 in Rat Bone. Female Sprague−Dawley rats (Charles River) were approximately 13 weeks old when received. They were acclimatized to the facility and operators and were randomly divided to five groups of three. Dose: [3H]-16: 8.90 μCi/rat, [14C]-16 1.77 μCi/rat, and total dose of 16 5.0 mg/kg. For formulation, conjugate 16 was suspended in 10% Poloxamer 188 solution, adjusted to pH 8.0 with NaHCO3, and then diluted with an equal volume of PBS. The dosing was done with a fixed volume of 500 μL. The bolus injection and blood sampling were done via the tail vein. Dosed animals showed no unusual behavior or reaction. After 6 h and 1, 7, 14, and 28 days, three animals were weighed and sacrificed by CO2 asphyxiation, and then blood, fat, muscle, brain, heart, kidney, spleen, liver, and femurs were harvested and cleaned free of any loose tissue. The amount of radioactivity was determined by incineration in a R. J. Harvey OX300 Biological

Oxidizer. The percent of the compound retained in the skeleton and the other organs at each time point was calculated by measuring activity per gram (μCi/g) in the collected samples and extrapolating to total activity retained in the skeleton and the other organs; the weight of the skeleton was calculated as 8% of the body weight, and the weight of the other organs was measured by a balance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00951. NMR spectra of key compounds (PDF) SMILES molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1(778) 782-3351. Fax: +1(778) 782-3765. E-mail: [email protected]. ORCID

Haibo Xie: 0000-0002-1350-9557 Robert N. Young: 0000-0002-8235-6080 Present Address †

H.X.: School of Pharmacy, University of Wisconsin−Madison, 7218 Rennebohm Hall, 777 Highland Avenue, Madison, Wisconsin 53705, United States Author Contributions

H.X. developed the design of the syntheses, synthesized all of the analogues reported in the manuscript, carried out the enzymatic assays, analyzed the biological samples, prepared schemes, wrote the Experimental Section, and edited the manuscript. G.C. supported the radiochemical synthesis and contributed to formulation development and analysis of biological samples and edited the manuscript. R.N.Y. conceptualized the project, designed the conjugates and general approaches for their synthesis, analyzed and interpreted data, and wrote and edited the manuscript. All authors have read and approved the final manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support of the Canadian Institutes for Health Research, Institute for Musculoskeletal Health and Arthritis (Grant # 122069) for this project. R.N.Y. also acknowledges the Canada Foundation for Innovation (Grant #16739) and the British Columbia Government Leading Edge Endowment Fund for financial support. The authors also acknowledge the technical staff of Animal Care Services in the Animal Resources Facility, Simon Fraser University, for animal experiments.

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ABBREVIATIONS USED ACN, acetonitrile; ALN, alendronate; AMC, 7-amino-4methylcoumarin; BP, bisphosphonate; Cat K, cathepsin K; CDI, carbonyldiimidazole; DAST, diethylaminosulfur trifluoride; DMAc, dimethylacetamide; DTT, dithiothreitol; EDCI, Nethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride; EP4, prostaglandin E2 receptor subtype 4; EP4a, EP4 receptor agonist; HOBt, 1-hydroxybenzotriazole hydrate; IV, intravenous; LOD, limit of detection; OVX, ovariectomized; 7026

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osteoporotic and pagetic bone. Prostaglandins, Leukotrienes Essent. Fatty Acids 2004, 70, 431−439. (16) Xie, C.; Liang, B.; Xue, M.; Lin, A. S.; Loiselle, A.; Schwarz, E. M.; Guldberg, R. E.; O’Keefe, R. J.; Zhang, X. Rescue of impaired fracture healing in COX-2−/− mice via activation of prostaglandin E2 receptor subtype 4. Am. J. Pathol. 2009, 175, 772−785. (17) Yoshida, K.; Oida, H.; Kobayashi, T.; Maruyama, T.; Tanaka, M.; Katayama, T.; Yamaguchi, K.; Segi, E.; Tsuboyama, T.; Matsushita, M.; Ito, K.; Ito, Y.; Sugimoto, Y.; Ushikubi, F.; Ohuchida, S.; Kondo, K.; Nakamura, T.; Narumiya, S. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 4580−4585. (18) Pagkalos, J.; Leonidou, A.; As-Sultany, M.; Heliotis, M.; Mantalaris, A.; Tsiridis, E. Prostaglandin E(2) receptors as potential bone anabolic targets - selective EP4 receptor agonists. Curr. Mol. Pharmacol. 2012, 5, 174−181. (19) Konya, V.; Marsche, G.; Schuligoi, R.; Heinemann, A. E-type prostanoid receptor 4 (EP4) in disease and therapy. Pharmacol. Ther. 2013, 138, 485−502. (20) Gil, L.; Han, Y.; Opas, E. E.; Rodan, G. A.; Ruel, R.; Seedor, J. G.; Tyler, P. C.; Young, R. N. Prostaglandin E2-bisphosphonate conjugates: potential agents for treatment of osteoporosis. Bioorg. Med. Chem. 1999, 7, 901−919. (21) Arns, S.; Gibe, R.; Moreau, A.; Monzur Morshed, M.; Young, R. N. Design and synthesis of novel bone-targeting dual-action pro-drugs for the treatment and reversal of osteoporosis. Bioorg. Med. Chem. 2012, 20, 2131−2140. (22) Chen, G.; Arns, S.; Young, R. N. Determination of the rat in vivo pharmacokinetic profile of a bone-targeting dual-action pro-drug for treatment of osteoporosis. Bioconjugate Chem. 2015, 26, 1095− 1103. (23) Liu, C. C.; Hu, S.; Chen, G.; Georgiou, J.; Arns, S.; Kumar, N. S.; Young, R. N.; Grynpas, M. D. Novel EP4 receptor agonistbisphosphonate conjugate drug (C1) promotes bone formation and improves vertebral mechanical properties in the ovariectomized rat model of postmenopausal bone loss. J. Bone Miner. Res. 2015, 30, 670− 680. (24) Hu, S.; Liu, C. C.; Chen, G.; Willett, T.; Young, R. N.; Grynpas, M. D. In vivo effects of two novel ALN-EP4a conjugate drugs on bone in the ovariectomized rat model for reversing postmenopausal bone loss. Osteoporosis Int. 2016, 27, 797−808. (25) Lin, J. H.; Russell, G.; Gertz, B. Pharmacokinetics of alendronate: an overview. Int. J. Clin. Pract. Suppl. 1999, 101, 18−26. (26) Wolfe, M. S.; Klein, L. Sex differences in absolute rates of bone resorption in young rats: appendicular versus axial bones. Calcif. Tissue Int. 1996, 59, 51−57. (27) Troen, B. R. The role of cathepsin K in normal bone resorption. Drug News Perspect. 2004, 17, 19−28. (28) Pan, H.; Kopeckova, P.; Wang, D.; Yang, J.; Miller, S.; Kopecek, J. Water-soluble HPMA copolymer–prostaglandin E1 conjugates containing a cathepsin K sensitive spacer. J. Drug Targeting 2006, 14, 425−435. (29) Pan, H.; Sima, M.; Kopeckova, P.; Wu, K.; Gao, S.; Liu, J.; Wang, D.; Miller, S. C.; Kopecek, J. Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates. Mol. Pharmaceutics 2008, 5, 548−558. (30) Lecaille, F.; Chowdhury, S.; Purisima, E.; Bromme, D.; Lalmanach, G. The S2 subsites of cathepsins K and L and their contribution to collagen degradation. Protein Sci. 2007, 16, 662−670. (31) Tranoy-Opalinski, I.; Fernandes, A.; Thomas, M.; Gesson, J. P.; Papot, S. Design of self-immolative linkers for tumour-activated prodrug therapy. Anti-Cancer Agents Med. Chem. 2008, 8, 618−637. (32) Karamustafa, F.; Ç elebi, N. Bisphosphonates and Alendronate. FABAD J. Pharm. Sci. 2006, 31, 31−42. (33) Kalu, D. N. The ovariectomized rat model of postmenopausal bone loss. Bone Miner. 1991, 15, 175−191.

PABA, para-aminobenzylalcohol; Ru-TsDPEN-Cy, ((1S2S)-2amino-12-diphenylethyl)(tosyl)-amido](p-cymene)(pyridine)ruthenium(II) tetrafluoroborate

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