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

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Design, synthesis and pharmacokinetics of a bonetargeting dual-action prodrug for treatment of osteoporosis Haibo Xie, Gang Chen, and Robert N. Young J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00951 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Journal of Medicinal Chemistry

Design, synthesis and pharmacokinetics of a bone-targeting dual-action prodrug for treatment of osteoporosis

Haibo Xie, Gang Chen, and Robert N. Young*

Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada

*Author to whom correspondence should be addressed Tel: +1(778) 782-3351 Fax: +1(778) 782-3765 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT A dual-action bone-targeting prodrug has been designed, synthesized and evaluated for in vitro and in vivo metabolic stability and in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double labelled 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-carboxymethylphenylmethyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). The optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the LeuArg-PABA element will liberate alendronic acid. Studies with a doubly radiolabeled 16 provide proof-ofconcept for 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 treatment of bone disorders such as osteoporosis.

INTRODUCTION Healthy bones turnover constantly 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 health 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 suppression of 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,


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cathepsin K (Cat K), to cleave and process the collagen matrix that is the other major structural component of bone. Inhibitors of Cat K are effective anti-resorptive agents and some Cat K inhibitors such as odanicatib2,3 are in late stages of development for 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 to release the acid necessary for resorption. Due to 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 While BPs such as alendronic acid (ALN) (1) are effective drugs, osteoporosis is often not treated until substantial bone loss has already occurred and while 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 formation9 and a stabilized analog, teriparatide, has been developed and is marketed for this purpose. However, 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, EP 1, 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 cells15 and a number of potent and very selective EP4 receptor agonists have been reported in recent years and 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 bone forming osteoblast cells and osteoid precursor cells (such as preosteoblasts) found in bone marrow.14 Pre-osteoblasts 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



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agonists exert side effects in vivo19 (including vasodilation and gastrointestinal disturbance) that render their direct use as bone anabolic drugs impractical.

O OH P OH OH O P OH OH

O CO 2H O OH P OH OH O P OH OH

H 2N

O

HO

1

OH

HO

CO 2R

O

O

OH

3

2

CO 2H

N H

O

CO 2Et

O N F

N F

F

F O

HO

HO

O

O S

O

4

O P OH OH O P OH OH

O

5a: R=H 5b: R= Et

H N

O

OH P OH OH P OH O OH

O

6

To avoid such side effects and to 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 therefore would exert both bone formation effects and inhibition of resorption, leading to an additive or synergistic effect (Figure 1).


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Local hydrolytic enzymes

BP

EP4a

LNK

Bone binding

BP

LNK

EP4a BP

Slow release EP4a LNK

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

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 the systemic circulation. Initially, a 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 (ca. 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 a 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 so 4 did not test the potential of the hoped-for synergism.



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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 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 4hydroxyphenylacetic acid via an ester bond to the carboxylic acid function and ALN to the phenol function via a carbamate group.21 The ester 5b was used for ease of synthesis and was shown to be rapidly hydrolyzed in blood to yield the active carboxylic acid 5a. Conjugate 6 was radiolabeled (with 3H on 5 and 14

C on the linker) and was shown to bind largely intact to bones (ratio of 3H/14C maintained) after IV

dosing to rats. Following the loss of the labels from bones over time indicated that the 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 The 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 anti-resorptive effects expected from such a dose of alendronate were not evident.23,24 It is possible that a component of the loss of 14C-label 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 about 200 days in the rat25 but more recent studies measuring loss of tetracycline from rat bone surface have indicated half-times of about 3 weeks in female rats.26 If the latter measure were more representative we would conclude that the conjugate 6 did not release the free ALN component at a sufficient rate to have truly tested the potential of the dual, anti-resorption/growth stimulation as originally proposed. Therefore, to better explore the potential of dual action conjugates, we have 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


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component would be unambiguously liberated by the cleavage of the peptide linker via action of Cat K. Cat K is the major peptidase released by osteoclasts at the bone surface.27 Cat K-cleavable conjugates including N-(2-hydroxypropyl)methacrylamide copolymer-based conjugates designed to release prostaglandin E1 (PGE1)28,29 have been previously described in the literature for treatment of osteoporosis and bone diseases. Cat K efficiently cleaves small peptide substrates such as Cbz-Gly-Pro-Arg-AMC or Cbz-Leu-Arg-AMC30 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.

CO 2Et F F

O

R N H

O

N

O


O O

O

H N O

N H

N H

R

Peptide

O

O

Bisphosphonate (ALN)

7

Esterase Release EP4 agonist by esterase

O

CO2H O F

O

F OH O

OH

N

R N H

5a

O

Bisphosphonate Release BP by CatK 1,6-Elimination -CO2

O

OH

R

N H

N H CatK

Peptide

O

EP4 agonist

O

O

O

O

H N


R N H

O

H N O

Peptide

H 2N OH

R


OH H 2N

Bisphosphonate

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



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We proposed to elaborate the benzyl function of the N-terminal 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 similar to that used in 6, and to replace the 7-amino-4-methylcoumarin (AMC) group with a para-aminophenylbenyzl alcohol (PABA) moiety which would be linked to ALN via a carbamate function to form the 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 aminofunctionalized drugs (for a review see Tranoy-Opalinski 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 to bind intact to bone, then subsequent hydrolysis of the ester would liberate 5a and cleavage of the peptide would liberate ALN (which would remain bond 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 (half-time of about 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 (ca 1.4 mg/kg) which is dosed orally once weekly. Notably however the oral bioavailability of alendronate very low, 1.7% in rats and 0.64% in women.32 Given that about 50% of absorbed alendronate is taken up into bones, the actual amount of alendronate reaching the bones is about 0.01 mg/kg/week. 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 assume a 5 mg/kg dose (the dose found to be effective for conjugate 6 and for the generic conjugate 7 equivalent to about 1 mg/kg of alendronate) and that about 5% of the dose were taken up into bones (as was the case for conjugate 6) and that the release half-time were one week, we can estimate the dose of active alendronate from a conjugate such as 7 would be about 0.025 mg/kg/week 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 labelled with tritium and the linking


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PABA element was labelled 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.

RESULTS AND DISCUSSION

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 15-hydroxyl function by esterification with 4hydroxymethylphenylacetic acid and the peptide component would be prepared by standard peptide synthesis and the C-terminus reacted 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). Due to the extreme polarity and poor non-aqueous 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 humans 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



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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. While 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 To evaluate these requirements model peptide-PABA-ALN conjugates Cbz-Pro-Arg-PABA-ALN (10), Cbz-Leu-Arg-PABA-ALN (11) and Cbz-Gly-Pro-Arg-PABA-ALN (12) were synthesized. To prepare 10, Cbz-arginine hydrochloride was first coupled with p-aminobenyl alcohol under standard amide formation conditions to provide the amide 13a (Scheme 1). The Cbz was removed by hydrogenation and the liberated amine was coupled with Cbz-proline to form the 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 p-nitrophenyl 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 the desired carbamate 10 in excellent yield. Cbz-Leu-Arg-PABA-ALN (11) and Cbz-Gly-Pro-Arg-PABAALN (12) were readily prepared in a similar manner.

Scheme 1. Synthesis of model peptide-bisphosphonate conjugate 10a.


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Cbz


O

H N

R

OH

O

H N

N H

(i)

H 2N

H 2N

NH

N Cbz O

H N

NH

NH

H 2N

O

O

NH 14 O

N

N (v)

N H

OH N H

13a: R = Cbz 13b: R = H O

(ii)

(iv)

N Cbz O

(iii)

O

H N

NH

NH

HCl

OH

N Cbz O

O

H N

N H

N H

NH H 2N

O

OH PO 3H 2 PO 3H 2

NH

NH

H 2N

NH

15

10 (Cbz-Pro-Arg-PABA-ALN) 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.

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 minutes incubation with human and rat Cat K respectively in keeping with observations in the literature34 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 hour incubation with human and rat Cat K respectively.

Table 1. Comparison of rates of cleavage of peptide substrates with rat and human cathepsin K. Cbz-Pro-Arg-PABA-ALN

Cbz-Leu-Arg-PABA-ALN

Cbz-Gly-Pro-Arg-PABA-ALN

(10) (% cleaved)a

(11) (% cleaved)a

(12) (% cleaved)a



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Time (h)

Hum Cat K

Rat Cat K

Hum Cat K

Rat Cat K

Hum Cat K

Rat Cat K

1

90

5

66

73

91

11

2

98

10

75

80

100

20

52

79 (3 h)

95

20

a

80

Compounds 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, 71% cleaved in 6min; with human Cat K, 63% in 6 min.

To demonstrate that these peptide conjugates could survive in the blood stream 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 ºC 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 at least 2 hours (with 80% or more remaining), long enough to allow efficient uptake into bones. Somewhat surprisingly, 12 was much less stable with only about 40% remaining in serum after 2 hours. 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 the conjugate 16 became our synthetic goal. Of course it was 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.

Table 2. Stability of model peptide conjugates in rat plasma and serum. Cbz-Pro-Arg-PABA-ALN

Cbz-Leu-Arg-PABA-ALN

Cbz-Gly-Pro-Arg-PABA-

(10) (% remaining )a

(11) (% remaining)a

ALN (12) (% remaining)a


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Time (h)

Plasma

Serum

Plasma

Serum

Plasma

Serum

0

96

93

82

99

95

91

1

95

89

78

97

51

82

2

90

78

78

95

38

72

4

90

77

64

86

17

68

24

65

69

59

70

16

64

24(control)

86

97

83

95

49

93

a

Compounds were incubated in rat plasma or serum at 37 °C and sampled at various times and compared

to inactivated control plasma or serum.

Synthesis of the 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 PABA-alcohol (Scheme 2). Thus Cbz-Leu-Arg-PABA (17a) was deblocked by hydrogenation (over Pd/C) to provide the free amino compound 17b. 4-Hydroxymethylphenylacetic acid was exhaustively silyl protected with tbutyldimethylsilylchloride 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-n-butylammonium fluoride gave the alcohol 8 which was reacted with an equivalent of phosgene to give the intermediate chlorocarbonate which was then reacted with 17b to form the 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 reaction to be carried out in anhydrous dimethylacetamide (alendronic acid is virtually insoluble in any solvent other than water either as the free



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acid or as a disodium salt). The product, tetra-n-butylammonium salt form of 16, was converted to the potassium salt and then purified by chromatography on a strong anion exchange column with elution with pH 8 potassium hydrogen phosphate buffer followed by removal of excess salts by C-18 chromatography and lyophilization to give the final conjugate 16.

Scheme 2. Synthesis of conjugate 16. Cbz

OH

O

H N

R

OH

O

H N

OH N H

(i)

R

(iii)

+

H 2N

N H

O

NH

NH Cl H 2N

H 2N

NH 2

NH

NH

H 2N

13a: R = Cbz 13b: R = H

(ii)

OH

O

H N

N H

NH

17a; R = Cbz 17b; R = H

(ii)

O CO 2Et N F

O F

CO 2Et N

(iv)

OH HO

OH

O

TBSO

F

5b HO O

O

F

(vii)

O

(v)

18 OR

19; R = TBS 8; R = H

(vi)

CO 2Et

CO 2Et O

F F

O

N H

O O

H N

O

O

F

N H

O

O

OH F

(viii)

O O

N O

O

NH

20

H 2N

O O

F O

N H

O O

O

NH

21

F

N

N H

O

NH

(ix)

N

N

CO 2Et H N

O

O N H

O

N H

H 2N

NH


N O

NH

16

a

N H

H N

O

O

H 2N

NH

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 ºC to rt; 2) K2CO3, MeOH/THF = 1/2; 68%


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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. Synthesis of 3H-labeled 16. In order 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 have used 5 labeled with tritium at the 15-position in the past but use of this labeled compound in the synthesis developed for 16 (Scheme 2) would require carrying the label through multiple steps and this was not optimal. It was 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 palladium-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 analog of 5b. Thus the analog 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 the difluorinated ester 23 that was converted to the Wittig reagent 24 by reaction with the anion derived from dimethyl methylphosphonate. 24 was then reacted with the aldehyde 25 followed by selective reduction of the resulting ketone 26 to give 22 following the same procedure as previously described for the synthesis of 513,36 (Scheme 3).

Scheme 3. Synthesis of p-iodo analog of 5ba.



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O OEt

Cl

O

(i)

O

F F

(ii)

OEt

OEt

O

I

F F

(iii)

O

I

Page 16 of 51

O

I

24

23

O CO2Et

O

O

(iv)

CO2Et

N

(v) N O

OH

25

O

O

CO2Et

(vi)

N F

26

CO2Et

(vii)

N F

F

O

a

CO2Et

N OTBS

OMe P OMe O

F

HO I

22

I

Reagents and conditions: (i) PhI, AlCl3, DCM, rt; 34%; (ii) DAST, Toluene, rt; 34%; (iii) MePO(OMe)2,

n-BuLi, THF, -78 ºC; 72%; (iv) TBAF, THF; (v) (COCl)2, DMSO, Et3N, DCM, -78 ºC; (vi) 24, NaH, ZnCl2, THF; 73% (vii) Ru-TsDPEN-Cy, HCOOH, Et3N, DCM, -5 ºC, 24 h; 74%. We then evaluated the reduction of 22 to provide 5b and optimized the reduction for efficient introduction of a tritium atom. To confirm the efficiency of the reduction, 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 ºC in 90% yield (Scheme 4A). 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, while 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 were used. To evaluate this possibility, model studies were performed where reductions were carried out on 22 in DMF using 5% Pd(OAc)2 and a mixture of


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NaBH4 (1.2 equivalent) and NaBD4 (1.2 equivalent). 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).

Scheme 4. Model studies on reduction p-iodo precursors to provide labeled 5ba.

F F OEt

A O

I

OEt O

H

23

27 HPLC yield 90%

O

O CO 2Et

B

F F

(i)

N F

CO 2Et N F

F

HO

22

(ii)

F

HO I

5b

H/D

H/D = 1.43

HPLC yield 95%

a

Reagents and conditions: (i) Pd(OAc)2 (10 mol%), NaBH4; (1.0 equiv), DMF, 70 ºC, 30 min; (ii)

Pd(OAc)2 (5 mol%), NaBH4 (1.2 equiv), NaBD4 (1.2 equiv), DMF, rt, 30 min.

However, to further maximize potential radiochemical yield in the preparation of [3H]-16, we envisaged a reaction where the iodo-precursor would be first reacted with less than one equivalent of sodium borotritiide and then the reaction would be driven to completion with 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 minutes and then followed by excess NaBH4 to complete the reduction. This



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Page 18 of 51

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-analog of the conjugate 16. Thus the iodo-EP4a (22) was converted in a sequence identical as used before for the proteo-conjugate to provide the 4-iodophenyl analog of conjugate 16 (compound 31) (Scheme 5). Model reduction of 31 under the same conditions Pd(OAc)2 (5%); NaBH4 (1 equivalent in DMF) gave clean and complete conversion to the proteo-product 16.

Scheme 5. Synthesis of tritium labeled conjugate 16a.

CO 2Et

CO2Et

CO2Et

I

I

F

O O

F

(iii)

(i)

N

O

OR

N

O

O

O O

F

(iv)

O

N H

O O

O

O

H N

N H

N H

O

OH N H

O

H 2N

NH


N O

NH

31

H 2N

NH

CO2Et

O H( 3H)

F F

(v)

O O

O

O N H

H N

O

O N H

O

N H


N O

[ 3H]-16

NH H 2N

a

O

NH

30

CO 2Et

F

H N

O

28; R = TBS 29; R = H

I

N H

N

O

22 (ii)

O

F

F OH

O

I

F

F

NH

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


Page 19 of 51

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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. 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. Several reductions were carried out at milligram scale to provide ample [3H]16 for use in stability and biodistribution studies.

Stability of free and bone bound conjugate 16 in rat plasma. With the availability of [3H]-16 it became possible to evaluate the stability of the conjugate in vitro and its uptake into bones in vivo. If significant hydrolysis took place at any place on the linker in blood or in liver prior to binding of the bisphosphonate to bone, it would lead to loss of tritium and reduced labeling of the bones. Other conjugates such as 6 have been shown to give about 5% of total label uptake in bone and to survive largely intact based on dual labeling studies. Conjugate 16 joins the EP4 agonist to the linker via an ester functionality in essentially identical manner as in 6 and the model conjugate Cbz-Leu-Arg-PABAALN (11) had been shown to be stable in plasma and serum so we were hopeful the final conjugate would be similarly stable or at least have a lifetime in the systemic circulation long enough to reach and attach to the bone. Using [3H]-16, the conjugate was shown to bind rapidly and essentially completely (> 95%) to bone powder (bovine) within 30 minutes when stirred in PBS buffer and in rat plasma. The conjugate was then incubated in rat plasma at 37 ºC, and after fixed times the incubate was stirred with bone powder, to adsorb the intact conjugate, and any cleaved bisphosphonate, and the supernatant solution was counted to quantify any unbound radioactivity into the supernatant. 16 was shown to liberate 27% of the label after 30 minutes, 44% after 4 hours and 53% over 24 hrs. When [3H]-16 was allowed to bind to bone powder for 30 minutes in buffer and then the bone powder filtered, resuspended, and then incubated in fresh rat serum, buffer or inactivated serum, only about < 3% of the tritium label was liberated after 24 hours indicating the high stability of the conjugate (to serum esterases or proteases) once bound to bone.



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It is known that uptake of bisphosphonates into bones is rapid in vivo25,37,38 and so although significant partial hydrolysis 16 was observed in plasma, the rate was somewhat similar to what had been found previously for conjugate 4 (ca. 15% over 4 hours)20 while somewhat higher than observed for conjugate 6 (ca 6% hydrolysis in 24 hrs).21 As both 4 and 6 were significantly taken up into rat bone in vivo (3.5% and 5.9% respectively), we decided to evaluate the uptake of [3H]-16 into bone in a probe experiment in rats.

Uptake of the conjugate [3H]-16 into bones, bio-distribution and elimination in rats. To determine tolerability and bio-distribution of 16 and whether a sufficient amount was able to reach the bones intact, an experiment was planned where [3H]-16 would be administered intravenously at a dose of 5 mg/kg to rats and blood samples were drawn at intervals over 6 hrs. 16 was not sufficiently soluble at the concentrations desired for dosing (2 mg/mL) in PBS alone and therefore was further evaluated in formulations of PBS with 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 which yielded a homogeneous solution and this was used for subsequent in vivo experiments. Thus 3 rats were injected with [3H]-16 (5 mg/kg, 31 µCi per rat) and blood samples were taken at intervals over 6 hours. The dose was well tolerated and the experiment was terminated after 6 hours and tissue samples, including samples of long bones were taken and analyzed for radioactivity. Processed samples showed the conjugate was rapidly cleared from the bloodstream (Figure 4A) with most radioactivity found in the liver at 6 hours and about 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 about 5% was found in bone after 6 hours21,22 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 levelpotentially 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 in order to carry


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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.

A

B

Figure 4. Pharmacokinetics of elimination over 6 hours (Panel A) and tissue distribution (Panel B) of radiolabel 6 hours after a single IV dose of [3H]-16 in rats. Synthesis of 14C-labelled conjugate 16. Incorporation of 14C into the PABA-ALN carbamate linker was achieved by 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. Scheme 6. Synthesis of 14C-labeled conjugate 16a.



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CO 2Et

CO 2Et O

F F O

O O

N

H N

N H

O

OH

O

F O

NH H 2N

O O

F

(i)

N H

O

20

O O

N

N H

CO 2Et

O O

F

O

O O

N

N H

H N

14 C

O

O

N H

N H

O

O

14 C

O

O

N

N

N H

O

NH [14C]-21

F

H N

O

NH

(ii)

H 2N

NH


NH [14C]-16

a

Page 22 of 51

H 2N

NH

Reagents and conditions: (i) CDI [carbonyl-14C] (1.0 equiv), 1.6 mg in 50 µL THF, rt, 1 h; (ii) alendronic

acid bis-tetra-n-butylammonium salt (2.4 equiv), DMAc, rt; 52% radiochemical yield for 2 steps.

Bio-distribution, uptake and release of [3H,14C]-16 in rats. For these experiments, a mixture of [3H]-16 and [14C]-16 (respectively 8.9 and 1.8 µCi/rat) was diluted with unlabeled 16 and dosed by IV administration to 15 rats at a total dose of 5 mg/kg per rat. Groups of 3 were terminated after 6 hours, 1 day, 7 days, 14 days and 28 days and the bones and tissues (liver, spleen, kidney, heart, and brain) were collected and analyzed quantitatively for both 3H and 14C label. The radioactivity observed was corrected for whole body/organ weight and the results are depicted in Figure 5 as percentage of the original total administered dose. It was immediately apparent that the uptake of 14C into bones was at least two-fold higher than 3H and this difference remained constant over 28 days while 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 appeared to both plateau between two and four weeks post-dosing. Levels in other tissues decayed exponentially and did not plateau over 28 days when residual levels were very low (Table 3).


Page 23 of 51

Bone Radioactivity in Bone 8 3

H

14

6

% DOSE

C

4

2

0 .0

0

2

4

8

.0

0 .0

1

1

7

.0

.2

0

5

0

0

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Time tim (Days e ( post d a y )dose)

! 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

6-hour

1-day

7-day

14-day

28-day

2.10

2.12

1.30

0.80

0.74

C

5.52

4.63

3.51

2.52

2.44

H

8.95

2.33

0.48

0.30

0.08

C

7.03

2.50

0.61

0.27

0.10

H

0.35

0.22

0.076

0.039

0.037

C

0.34

0.23

0.086

0.050

0.039

H

0.21

0.11

0.053

0.026

0.017

0.11

0.047

0.020

0.013

0.0092

H

14

Liver

3

14

Spleen

3

14

Kidney

3

14

C



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Heart

3

H

0.025

0.021

0.014

LOD

LOD

C

0.017

0.009

LOD

LOD

LOD

H

0.022

0.025

LOD

LOD

LOD

0.014

0.012

LOD

LOD

LOD

14

Brain

3

14

a

Page 24 of 51

C

Levels are expressed as a percentage of the originally administered radioactivity and are the average of

data from three rats. Bone data was calculated for long bone samples assuming total bone represented 8% of body weight. LOD, limit of detection.

These data indicate that significant loss of the tritiated EP4 agonist component occurred prior to attachment to the bones while thereafter, the rate of release of the bound tritium was very similar to what had been observed in previous studies with 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 3 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, that 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 2 weeks may suggest that overlay of new bone has trapped some conjugate where it is no longer accessible.

CONCLUSIONS A 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-para-aminobenylalcohol (Leu-Arg-PABA) linker. We have also


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developed syntheses of radiolabelled analogs of 16 designed to interrogate the pharmacokinetic, tissue distribution uptake into bones and release of the EP4 agonist and the alendronate components in vivo. A tritium labeled analog ([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 (ca. 55% over 24 h). This stability was considered sufficient to warrant further in vivo evaluation and thus a second labeled conjugate ([14C]-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 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 about 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 4.4 days) is similar to that observed for other conjugate prodrugs such as 6 in earlier studies21,22 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 about 4 µg/kg/day. This is somewhat lower than the calculated daily release of 5a (ca 18 µg/kg/day)23 for the efficacious dose24 of conjugate 6. The alendronate component was released with a roughly 5 days half-time which is much faster than previously observed for conjugate 6. Based on the 5% bone uptake of 14

C this would allow calculation of release of active alendronate at a rate of about 5 µg/kg/day. Compound

16 was designed so that alendronate component would be released through protease cleavage the peptideNH-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



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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 contemplated. 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.

EXPERIMENTAL General Chemistry Methods. 1

H 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.38 NMR spectra for all compounds containing a bisphosphonate group were run in DMSO-d6 with 20 µL (35% by weight) DCl in D2O (Sigma Aldrich). LC-MS were recorded with an ESI ion source on an Agilent 6200 Time-of-Flight spectrometer coupled with Agilent 1200 series HPLC. Analytical thin-layer chromatography (TLC) was performed on aluminum plates pre-coated 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 HPsilica cartridges from BioTage or SiliCycle Inc. Derivatized silica was obtained from SiliCycle Inc. Tetrahydrofuran (THF) was distilled from Na and benzophenone under nitrogen. Dichloromethane


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(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. Purity of all final products was 95% or higher as determined by HPLC analysis. Non-radioactive compounds were analyzed using an Agilent 1100 HPLC and PDA detector at 254 nm with conditions 1: Agilent Zorbax SB-C8 column (3.0 x 150 mm, 5 µm) with gradient of acetonitrile: 0.1% formic acid from 5:95 to 95:5 over 5 minutes flow rate 2 mL/min or conditions 2: Advanced Materials Technology Halo C18 column (4.6 x 50 mm, 5 µm) with gradient: methanol:0.1% formic acid from 5:95 to 95:5 over 5 minutes, flow rate 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 minutes flow rate 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) but-1-en-1-yl)-5-oxopyrrolidin-1yl)heptanoate ([3H]-5a). Compound 22 (3.6 mg, 6.56 µmol) in 100 µL DMF was treated with Pd(OAc)2 (0.33 µmol in 66 µL DMF). After 15 min, NaB3H4 (Specific activity: 15.6 Ci/mmol) (12.13 mCi in 100 µL DMF) was added, and the mixture was stirred for 1 hour. Then NaBH4 (6.0 µmol in 131 µL 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 wash 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), stirred for 30 min. The MeOH was removed under reduced pressure and the residue was dissolved in water and directly loaded on a C18 cartridge. After washing with water (6 column volumes), MeOH was used to elute the hydrolysis product, [3H]-5a (3.8 mCi. Radiochemical yield 31%).



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(4-((((4-((S)-2-((S)-1-((Benzyloxy)carbonyl)pyrrolidine-2-carboxamido)-5-guanidinopentanamido)benzyl)oxy)carbonyl)amino)-1-hydroxybutane-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 and taken up in ACN/H2O (1/1) and loaded on an anion-exchange column (Strong anion exchange, Silicycle R66430B, Lot 53470). First, it was washed with ACN/H2O =1/1 (5.0 column volume); 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, 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-guanidinopentanamido)benzyl)oxy)carbonyl)amino)-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) (11). Compound 17a (54 mg, 0.10 mmol), CDI (25 mg, 0.15 mmol) was dissolved in DMF (0.5 mL ) and the mixture was stirred at room temperature. 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 hours. The reaction mixture was reduced to dryness in vacuo and taken up in ACN/H2O (1/1) and loaded on an anion-exchange column (strong anion exchange, Silicycle R66430B, Lot 53470). First, it was washed with ACN/H2O =1/1 (5.0 column volume); 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


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obtained as 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-2-carboxamido)-5-guanidinopentanamido)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 methanol, palladium on carbon (10%) (0.030g) was added to the solution. With uniform stirring atmospheric pressure hydrogen was passed through the solution. 2 h later, TLC indicated completed consumption of start material. The reaction mixture was filtered through Celite®, and solvent was removed to give crude pyrrolidine-2-carboxylic 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 DMF. 4.0 h later, the solvent was removed under high vacuum. The residue was separated by column chromatography on silica gel (gradient: methanol/dichloromethane 5% to 34%), then solvent was removed in vacuo to provide benzyl ((S)-2-(((S)5-guanidino-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1carbonyl)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), CDI (46 mg, 0.28 mmol) was dissolved in 0.8 mL DMF. The mixture was stirred at room temperature. 2 h later HPLC indicated completed consumption of start material. A solution of alendronic acid bis-tetra-n-butyl ammonium salt in DMF (0.5 mL)(230mg, 0.160mmol) was added and the mixture was stirred at room temperature for 4.0 h. The reaction mixture was reduced to dryness in vacuo and taken up in ACN/H2O (1/1) and loaded on an anion-exchange column (Strong Anion Exchange, Silicycle R66430B, Lot 53470). First, it was washed with ACN/H2O =1/1 (5.0



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column volume); 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 white power (60 mg). 1H NMR (500 MHz, DMSO) δ 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)-1-oxopentan-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) was dissolved in 50 mL DMF. 4 h later, the solvent was removed by high vacuum. The residue was separated by c`olumn chromatography (120 g silica gel. gradient: methanol/dichloromethane 5% to 34%) 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). 13

C 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-2yl)carbamoyl)pyrrolidine-1-carboxylate (14). Compound 13a (0.83 g, 2 mmol) was dissolved in 20 mL 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


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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, 2mmol) and EDCI (0.46 g, 2.4 mmol) were dissolved in 10 mL DMF and 4 h later, the DMF was removed under high vacuum. The residue was separated by column chromatography (50 g silica gel; gradient: methanol/dichloromethane 5% to 34%), 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)-1,1difluoro-1-phenylbut-3-en-2-yl)oxy)-2-oxoethyl)benzyl)oxy)carbonyl)amino)-4-methylpentanamido)5-guanidinopentanamido)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 THF, then compound 20 (10 mg, 0.011 mmol) was added. The mixture was stirred at room temperature for 1h, after which HPLC analysis indicated complete consumption of 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 ACN/H2O =1/1 (5 column volumes), then product was eluted with



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KH2PO4-K2HPO4 buffer (0.08M, pH = 8.0, in ACN/H2O = 2/3). The salts were then removed by C18 column and lyophilisation of selected fractions gave 16 as 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), 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 DMF was treated with Pd(OAc)2 (0.15 µmol in 30 µL DMF) at room temperature. After 15 min NaB3H4 (5.29 mCi, 1.53 µmol, specific activity: 10.9 Ci/mmol) in 50 µL 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 wash with water (6 column volume), product was eluted by acetonitrile and water mixture (ACN/H2O =1/1), 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, specific activity: 56 mCi/mmol) was dissolved in 50 µL THF, then compound 20 (11 mg, 10 µmol) was added. The mixture was stirred at room temperature for 1 h. Alendronic acid bis-tetra-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), then diluted with 300 µL 50% acetonitrile-water before being loaded on anion-exchange


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column (Strong anion exchange, Silicycle R66430B, Lot 53470). The column was washed with ACN/H2O =1/1 (5.0 column volume). Then, product was eluted by KH2PO4-K2HPO4 solution (0.08M pH = 8.0, ACN/H2O = 2/3). The salts were then removed by C18 column. The product was dissolved in 4.0 mL water i-PrOH mixture (ratio 1/1), and counted 0.26 mCi. Radiochemical yield 52%.

(5S,8S)-13-Amino-8-((4-(hydroxymethyl)phenyl)carbamoyl)-5-isobutyl-3,6-dioxo-1-phenyl-2-oxa4,7,12-triazatridecan-13-iminium chloride (17a). Compound 13a (0.625 g, 1.58 mmol) was dissolved in 20 mL 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 DMF and 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 silica gel; gradient: methanol/dichloromethane 5% to 34%) 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).



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4-(Hydroxymethyl)phenylacetic acid (3.33 g, 20 mmol), imidazole (3.40 g, 50 mmol) was 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 was added drop-wise. After 2 h TLC indicated complete consumption of starting material. The cold bath was removed and 200 mL 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 solid and 2 h later, the mixture was filtered and the solvents were removed under reduced pressure. The residue was dissolved in 150 mL water and the pH was adjusted to 2.0 with HCl. The solution was extracted with MTBE (50 mL x 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 colourless 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)-4,4difluoro-4-phenylbut-1-en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (19). Compound 18 (0.65 g, 2.33 mmol), compound 5b (0.85g, 2.0 mmol) and DMAP (0.024g, 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 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.


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Ethyl 7-((R)-2-((R,E)-3-(2-(4-((5S,8S)-13-amino-8-((4-(hydroxymethyl)phenyl)carbamoyl)-13-imino5-isobutyl-3,6-dioxo-2-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 starting material. Saturated NH4Cl solution (40mL) was added to quench the reaction, and the mixture was extracted with MTBE (30 mL x 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 colourless 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. 2 h 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)



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Ethyl 7-((R)-2-((R,E)-4,4-difluoro-3-hydroxy-4-(4-iodophenyl)but-1-en-1-yl)-5-oxopyrrolidin-1yl)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 ºC internally), and Ru-TsDPEN-Cy (0.094 g, 0.15 mmol) was added. The reaction was stirred at -5 ºC overnight, and then quenched with saturated NaHCO3 solution. The mixture was extracted with ethyl acetate (50 mL x 3). The organic phase was dried over MgSO4, filtered and concentrated. The crude product was purified by chromatography on silica gel. (Gradient: ethyl acetate/hexane 50% to 100%) to provide 22 as 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 drop-wise 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 minutes 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 sulphate, and concentrated under reduced pressure. The crude product was chromatographed on silica gel to yield 5.6 g of ethyl 2-(4-iodophenyl)-2oxoacetate (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).


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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 drop-wise over 15 min. The reaction mixture was stirred at room temperature overnight. Ice-water-NH3.H2O (5 mL) was added drop-wise to quench the reaction. Then the solution was extract by MTBE (20 mL x 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-(4iodophenyl)-2-oxoacetate (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 ºC and then n-BuLi (1.6M in hexane) (6.6 mL, 10.5 mmol) was added dropwise and the mixture was stirred at -78 ºC for 0.5 h. Compound 23 (3.10 g, 9.5 mmol) in THF (20mL) was then added drop-wise and the reaction mixture was stirred at -78 ºC for 4 h. After that the reaction was quenched at -78 ºC with CH3COOH (0.630 g, 10.5 mmol). The mixture was warmed to room temperature and 60 mL saturated NH4Cl solution, 30 mL water were added. The mixture was extracted with EtOAc (30 mL x 3). The organic phase was dried over MgSO4, filtered and concentrated. The crude product was purified on silica gel (gradient: ethyl acetate/hexane 50% to 100%), to provide 24 as 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-en-1-yl)-5-oxopyrrolidin-1yl)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 ºC, then ethyl (R)-7-(2-formyl-5oxopyrrolidin-1-yl)heptanoate (25)40 (1.83 g, 6.77 mmol) in THF (30 mL) and ZnCl2 (1.9 M in 2-



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methyltetrahydrofuran) were added, and the mixture was heated to 50 ºC for 6 h. The reaction was quenched with saturated NH4Cl solution, and extracted with EtOAc (50 mL x 3). The organic phase was dried over MgSO4, filtered, and concentrated. The crude product was purified by chromatography on silica gel (Gradient: ethyl acetate/hexane 50% to 100%) to provide compound 26 as 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)-4,4difluoro-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-imino5-isobutyl-3,6-dioxo-2-oxa-4,7,12-triazatridecyl)phenyl)acetoxy)-4,4-difluoro-4-(4-iodophenyl)but-1en-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (30).


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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 x 3). Finally, the MTBE solution was dried over MgSO4 and concentrated under reduced pressure to provide compound 29 (0.16 g) as a colourless 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 30 min. Compound 17b (0.087 g, 0.48 mmol) and Et3N (0.140 mL, 0.98 mmol) was dissolved in DMF (2.0 mL) and added to the THF reaction mixture. After stirring 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)-1,1difluoro-1-(4-iodophenyl)but-3-en-2-yl)oxy)-2-oxoethyl)benzyl)oxy)carbonyl)amino)-4methylpentanamido)-5-guanidinopentanamido)benzyl)oxy)carbonyl)amino)-1-hydroxybutane-1,1diyl)-bis(phosphonic acid) (31). CDI (2.0 mg, 0.012 mmol) was dissolved in 50 µL 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



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consumption of starting material. Alendronic acid bis-tetra-n-butylammonium 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 50% acetonitrile-water before loading onto an anion-exchange column (strong anion exchange, Silicycle R66430B, Lot 53470). The column was first washed with ACN/H2O =1/1 (5.0 column volume). Then, product was eluted by KH2PO4K2HPO4 buffer (0.08 M, pH = 8.0, in ACN/H2O = 2/3). The salts were then removed by C18 column. After lyophilisation of selected fractions, 31 was obtained as 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 = 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, 11 and 12 in rat plasma and serum. Stock solutions of 10, 11 and 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 ºC for 2 h). The samples were incubated in a 37 ºC 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, 4.6x50 mm, on ACNwater-TFA gradient, UV 254 nm) to assess the remaining concentration of 10, 11 and 12.

General procedure for cathepsin K assays.


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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, 11 and 12 (1.0 mM in water) were prepared by carefully weighing the compounds and dissolving each compound in calculated amount of Milli-Q water. Enzyme activity was assayed using the cathepsin K standard substrate Cbz-Leu-ArgAMC. 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, 11 and 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 ºC and analyzed after 1, 2, 20 h. 20 µL aliquots were analyzed by HPLC (Agilent Zorbax C18 4.6x150 mm, 5 µm column) eluting with MeOH-water-NH3.H2O gradient, UV 240 nm. Conjugates 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 about 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 16 and radioactivity dose of [3H]-16: 31.0 µCi/rat. For formulation the conjugate 16 was suspended in 10% Poloxamer 188 solution, adjusted to pH 8.0 with NaHCO3, then diluted with 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 micro-centrifuge tubes. Blood samples (500 µL) were centrifuged at ~5000 x G until plasma had separated. 100 µL of the supernatant was analyzed in a Harvey OX-300 biological oxidizer using the four-minute program. The collected scintillation mixture was counted on a Beckman Coulter LS-6500 scintillation counter. The counting time was two minutes.

Rat organ distribution, uptake and release of conjugate 16 in rat bone.



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Female Sprague Dawley rats (Charles River) were about 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 the conjugate 16 was suspended in 10% Poloxamer 188 solution, adjusted to pH 8.0 with NaHCO3, then diluted with 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, 1, 7, 14, 28 days three animals were weighed, 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 skeleton was calculated as 8% of the body weight, the weight of the other organs was measured by balance.

ASSOCIATED CONTENT The supporting information is available free of charge on the ACS Publications website at DOI: xx.xxxx/acs.jmedchem.xxxx NMR spectra of key compounds (PDF) SMILES molecular formula strings (CSV)

AUTHOR INFORMATION Corresponding Author Correspondences should be directed to Robert Young: phone (778) 782-3351. Email : [email protected] ORCID ID Robert N. Young: 0000-0002-8235-6080


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Haibo Xie: 0000-0002-1350-9557

Current Addresses RY/GC: Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6 HX: School of Pharmacy, University of Wisconsin-Madison, 7218 Rennebohm Hall, 777 Highland Avenue, Madison, USA, WI 53705

Author contributions HX developed the design of the syntheses and synthesized all the analogs reported in the manuscript and also carried out the enzymatic assays and analyzed the biological sample, prepared schemes, wrote the experimental section and edited the manuscript. GC supported the radiochemical synthesis and contributed to formulation development and analysis of biological samples and edited the manuscript. RY conceptualized the project, designed the conjugates and general approaches for their synthesis and 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.

ACKNOWLEDGEMENTS The authors acknowledge the financial support of the Canadian Institutes for Health Research, Institute for Musculoskeletal Health and Arthritis (Grant # 122069) for this project. RNY 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-4-methylcoumarin; BP, bisphosphonate; Cat K, cathepsin K; CDI, carbonyldiimidazole; DAST, diethylaminosulfur trifluoride; DMAc, dimethylacetamide; DTT, dithiothreitol; EDCI, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; EP4, prostaglandin E2 receptor subtype 4; EP4a, EP4 receptor agonist; HOBt, 1hydroxybenzotriazole hydrate; IV, intravenous; LOD, limit of detection; OVX, ovariectomized; PABA, para-aminobenzylalcohol; Ru-TsDPEN-Cy, ((1S2S)-2-amino-12- diphenylethyl)(tosyl)-amido](pcymene)(pyridine)ruthenium(II)-tetrafluoroborate.

REFERENCES

(1)

Blume, S. W.; Curtis, J. R. Medical costs of osteoporosis in the elderly medicare population. Osteoporos. Int. 2011, 22, 1835-1844.

(2)

Gauthier, J. Y.; Chauret, N.; Cromlish, W.; Desmarais, S.; Duong, L. T.; Falgueyret, J. P.; Kimmel, D. B.; Lamontagne, S.; Leger, S.; LeRiche, T.; Li, C. S.; Masse, F.; McKay, D. J.; Nicoll-Griffith, D. A.; Oballa, R. M.; Palmer, J. T.; Percival, M. D.; Riendeau, D.; Robichaud, J.; Rodan, G. A.; Rodan, S. B.; Seto, C.; Therien, M.; Truong, V. L.; Venuti, M. C.; Wesolowski, G.; Young, R. N.; Zamboni, R.; Black, W. C. The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K. Bioorg. Med. Chem. Lett. 2008, 18, 923928.

(3)

Langdahl, B. Effect of odanacatib on bone density and estimated bone strength in postmenopausal women: a CT-based sub-study of the phase 3 long-term odanacatib fracture trial (LOFT). American Society for Bone and Mineral Research (ASBMR) 2015 Annual Meeting Oct. 9-12, 2015; Seattle, Abstract # 1056.


Page 44 of 51

Page 45 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(4)


Uludag, H. Bisphosphonates as a foundation of drug delivery to bone. Curr. Pharm. Des. 2002, 8, 1929-1944.

(5)

Hochdorffer, K.; Abu Ajaj, K.; Schafer-Obodozie, C.; Kratz, F. Development of novel bisphosphonate prodrugs of doxorubicin for targeting bone metastases that are cleaved pH dependently or by cathepsin B: synthesis, cleavage properties, and binding properties to hydroxyapatite as well as bone matrix. J. Med. Chem. 2012, 55, 7502-7515.

(6)

Luhmann, T.; Germershaus, O.; Groll, J.; Meinel, L. Bone targeting for the treatment of osteoporosis. J. Controlled Release 2012, 161, 198-213.

(7)

Erez, R.; Ebner, S.; Attali, B.; Shabat, D. Chemotherapeutic bone-targeted bisphosphonate prodrugs with hydrolytic mode of activation. Bioorg. Med. Chem. Lett. 2008, 18, 816-820.

(8)

Neer, R. M.; Arnaud, C. D.; Zanchetta, J. R.; Prince, R.; Gaich, G. A.; Reginster, J. Y.; Hodsman, A. B.; Eriksen, E. F.; Ish-Shalom, S.; Genant, H. K.; Wang, O.; Mitlak, B. H. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N. Engl. J. Med. 2001, 344, 1434-1441.

(9) Jadav, S.; Käkelä, M., Bourgery, M.; Rimpilä, K.; Liljenbäck, H.; Siitonen, R.; Mäkilä, J.; Laitala-Leinonen, T.; Poijärvi-Virta, P.; Lönnberg, H.; Roivainen, A.; Virta, P. In vivo bone-targeting of bis(phosphonate)-conjugated double helical RNA monitored by positron emission tomography. Mol. Pharm. 2016, 13, 2588-2595.

(10) Jolette, J.; Wilker, C. E.; Smith, S. Y.; Doyle, N.; Hardisty, J. F.; Metcalfe, A. J.; Marriott, T. B.; Fox, J.; Wells, D. S. Defining a noncarcinogenic dose of recombinant human parathyroid



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

hormone 1-84 in a 2-year study in Fischer 344 rats. Toxicol. Pathol. 2006, 34, 929-940.

(11) Jee, W. S.; Ueno, K.; Kimmel, D. B.; Woodbury, D. M.; Price, P.; Woodbury, L. A. The role of bone cells in increasing metaphyseal hard tissue in rapidly growing rats treated with prostaglandin E2. Bone 1987, 8, 171-178.

(12) Jee, W. S.; Ke, H. Z.; Li, X. J. Long-term anabolic effects of prostaglandin-E2 on tibial diaphyseal bone in male rats. Bone Miner. 1991, 15, 33-55.

(13) Billot, X.; Colucci, J.; Han, Y.; Wilson, M.-C.; Young, R. N. EP4 Receptor Agonist, Compositions and Methods Thereof. US Patent 7238710, July 3, 2007.

(14) Machwate, M.; Harada, S.; Leu, C. T.; Seedor, G.; Labelle, M.; Gallant, M.; Hutchins, S.; Lachance, N.; Sawyer, N.; Slipetz, D.; Metters, K. M.; Rodan, S. B.; Young, R.; Rodan, G. A. Prostaglandin receptor EP(4) mediates the bone anabolic effects of PGE(2). Mol. Pharmacol. 2001, 60, 36-41.

(15) Fortier, I.; Gallant, M. A.; Hackett, J. A.; Patry, C.; de Brum-Fernandes, A. J. Immunolocalization of the prostaglandin E2 receptor subtypes in human bone tissue: differences in foetal, adult normal, 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.;


Page 46 of 51

Page 47 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

(18) 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 agonist-bisphosphonate conjugate drug (C1) promotes bone



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-(2hydroxypropyl)methacrylamide copolymer-alendronate conjugates. Mol. Pharm. 2008, 5, 548-558.

(30) Lecaille, F.; Chowdhury, S.; Purisima, E.; Bromme, D.; Lalmanach, G. The S2 subsites of


Page 48 of 51

Page 49 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 selfimmolative linkers for tumour-activated prodrug therapy. Anticancer Agents Med. Chem. 2008, 8, 618-637. (32) Karamustafa, F; Çelebi, N. Bisphosphonates and Alendronate. FABAD J. Pharm. Sci. 2006, 31, 31-42, 2006.

(33) Kalu, D. N. The ovariectomized rat model of postmenopausal bone loss. Bone Miner. 1991, 15, 175-191.

(34) Tada, S.; Tsutsumi, K.; Ishihara, H.; Suzuki, K.; Gohda, K.; Teno, N. Species differences between human and rat in the substrate specificity of cathepsin K. J. Biochem. 2008, 144, 499-506.

(35) Nagasaki, T.; Sakai, K.; Segawa, M.; Katsuyama, Y.; Haga, N.; Koike, M.; Kawada, K., ; Takechi, S.; Yen, W. L.; Mari, M.; Cao, Y.; Xie, Z.; Baba, M.; Reggiori, F.; Klionsky, D. J. A new practical tritium labelling procedure using sodium borotritide and tetrakis(triphenylphosphine)palladium(0). J. Labelled Compd. Radiopharm. 2001, 44, 9931004.

(36) Arns, S.; Moreau, A.; Young, R. N. Asymmetric [3H]‐labeling using ruthenium catalyzed transfer hydrogenation. J. Labelled Compd. Radiopharm. 2010, 53, 205-207.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 51

(37) Tower, R. J.; Campbell, G. M.; Muller, M.; Will, O.; Gluer, C. C.; Tiwari, S. Binding kinetics of a fluorescently labeled bisphosphonate as a tool for dynamic monitoring of bone mineral deposition in vivo. J. Bone Miner. Res. 2014, 29, 1993-2003.

(38) Weiss, H. M.; Pfaar, U.; Schweitzer, A.; Wiegand, H.; Skerjanec, A.; Schran, H. Biodistribution and plasma protein binding of zoledronic acid. Drug Metab. Dispos. 2008, 36, 2043-2049.

(39) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512-7515.

(40) Billot, X.; Chateauneuf, A.; Chauret, N.; Denis, D.; Greig, G.; Mathieu, M.-C.; Metters, K. M.; Slipetz, D. M.; Young, R. N. Discovery of a potent and selective agonist of the prostaglandin EP4 receptor. Bioorg. Med. Chem. Lett. 2003, 13, 1129-1132.

TABLE OF CONTENTS GRAPHIC CO2Et

O O

F F

O

R N H

O

O O

O

H N

O N H

R

N H


CatK

N

Bisphosphonate (ALN) (Binds to bone)

Peptide linker

O Esterase

CO2H O

R

F O

F OH

OH O

N

EP4 agonist (bone anabolic)

O

N H

O

H N O

H 2N OH R


Peptide Bisphosphonate (bone resorption inhibitor)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


CO 2Et

O O

F F

O

O

O

O

N

N H

R

CatK

O

N H

Bisphosphonate (ALN) (Binds to bone)

Peptide linker

O

F

O

F N

N H

O

O

H N

Esterase

CO 2H

O

R


OH

OH EP4 agonist (bone anabolic)

O

R N H

O

H N O

H 2N OH

R


Peptide Bisphosphonate (bone resorption inhibitor)


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

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