Design, Synthesis, and Biological Evaluation of Novel Matrix

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Design, synthesis and biological evaluation of novel matrix metalloproteinase inhibitors as potent antihemorrhagic agents: from hit identification to an optimized lead. Josune Orbe, Juan A Sanchez-Arias, Obdulia Rabal, Jose A Rodriguez, Agustina Salicio, Ana Ugarte, Miriam Belzunce, Musheng Xu, Wei Wu, haizhong Tan, hongyu Ma, Jose A Paramo, and Julen Oyarzabal J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501940y • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Medicinal Chemistry

Design, synthesis and biological evaluation of novel matrix metalloproteinase inhibitors as potent antihemorrhagic agents: from hit identification to an optimized lead.

Authors: Josune Orbe,1 Juan A. Sánchez-Arias,2 Obdulia Rabal,2 José A. Rodríguez,1 Agustina Salicio,1 Ana Ugarte,2 Miriam Belzunce,1 Musheng Xu,3 Wei Wu,3 Haizhong Tan,3 Hongyu Ma,3 José A. Páramo,1,4,* and Julen Oyarzabal2,∗ 1

Atherosclerosis Research Laboratory,

2

Small Molecule Discovery Platform, Molecular

Therapeutics Program, Center for Applied Medical Research (CIMA), University of Navarra, Avda. Pio XII 55, E-31008 Pamplona, Spain; 3WuXi Apptec (Tianjin) Co. Ltd., No 111 HuangHai Road, 4th Avenue, TEDA, Tianjin 300456, PR China; 4Hematology Service, Clínica Universidad de Navarra. University of Navarra. Avda. Pio XII 36, E-31008 Pamplona, Spain.

∗

To whom correspondence should be addressed. For J.A.P.: +34 948 194700 (ext. 3015), e-mail, [email protected]. For J.O.: phone, +34 948 194700 (ext 2044); e-mail, [email protected].

ACS Paragon Plus Environment

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Abstract

Growing evidence suggests that matrix metalloproteinases (MMP) are involved in thrombus dissolution; then, considering that new therapeutic strategies are required for controlling hemorrhage, we hypothesized that MMP inhibition may reduce bleeding by delaying fibrinolysis. Thus, we designed and synthesized a novel series of MMP inhibitors to identify potential candidates for acute treatment of bleeding. Structure-based and knowledge-based strategies were utilized to design this novel chemical series, α-spiropiperidine hydroxamates, of potent and soluble (>75 µg/mL) pan-MMP inhibitors. The initial hit, 12, was progressed to an optimal lead 19d. Racemic 19d showed a remarkable in vitro phenotypic response and outstanding in vivo efficacy; in fact, the mouse bleeding time at 1 mg/kg was 0.85 min compared to 29.28 min using saline. In addition, 19d displayed an optimal ADME and safety profile (e.g., no thrombus formation). Its corresponding enantiomers were separated, leading to the preclinical candidate 5 (described in drug annotations series, ref18).


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INTRODUCTION

Bleeding represents a dangerous complication that can result in the need for blood transfusion and is associated with substantial morbidity and mortality. Hemorrhage is responsible for almost 50% of deaths occurring within the first 24 hours of traumatic injury and for up to 80% of intraoperative trauma mortality.1 Physiologically, coagulation occurs to prevent excesive blood loss after injury and fibrinolysis is required to remove the blood clots after wound repair. Pathological alterations of this delicate balance can lead to thrombosis or hemorrhage. Plasmin, the key protein in fibrinolysis, results from plasminogen activation and dysregulation of this system can lead to hyperfibrinolysis, which plays an important role in major bleeding events.2 Thus, antifibrinolytic agents are widely used in major surgeries and trauma episodes to reduce blood loss and transfusion and in the treatment of heavy menstrual bleeding.3 Two synthetic lysine analogs, ε-aminocaproic acid (EACA, 1) and tranexamic acid (TXA, 2), are the only antifibrinolytics commercially available to control bleeding (chemical structures described in Chart 1); in fact, they are the current standard of care (SoC). Recently, AZD6564 (3, Chart 1), a piperidyl-isoxazolone with improved potency over compound 2, was reported as a preclinical candidate for further development.4 Antifibrinolytics elicit their effects by competitively reducing the binding of plasminogen to fibrin, thus inhibiting the activation of plasminogen to plasmin, an enzyme that degrades fibrin clots, fibrinogen and other plasma proteins.5 Molecule 2 has been associated with adverse side effects and requires a high oral dose,6 and 1 is less effective.7 Moreover, there are some concerns with these compounds due to the potential risk of thrombotic complications.8,9 Although aprotinin, a polypeptidic, bovine-derived protease inhibitor, was an effective agent used to reduce bleeding during complex surgery, safety issues


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led to its temporary withdrawal from the market in 2008.10 However, very recently, the European Medicines Agency (EMA) has recommended that the suspension be lifted for a restricted range of indications.11 Thus, in this scenario, new therapeutic strategies are required for the prevention of major bleeding events.

Chart 1. Known antifibrinolytics (1, 2 and 3), MMP inhibitors (4, 6, 7, 8, 9 and 10) and structure of a novel antihemorrhagic agent CM-352 (5)

EACA (1)

TXA (2) AZD6564 (3)

R

SC-78080/SD-3590 (6)

CM-352 (5) GM6001/Ilomastat (4)

RS-130830 (8)

SC-77774 (7)

(10) (9)


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The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution.12 MMPs can act either through direct fibrin/ogen targeting or by enhancing tissue plasminogen activator (tPA)-induced fibrinolysis.13 This provides a new pharmacological target for fibrinolysis control (Figure 1), as demonstrated by the markedly reduced fibrinolytic activity and bleeding time found in MMP-10 null mice, that can be reversed by administration of active recombinant human MMP-10.13 Moreover, MMP-3, another fibrinolytic MMP 82% homologous to MMP-10,12 has also been associated with intracranial hemorrhage.14 Thus, the inhibition of MMP-10, MMP-3 and/or other MMPs may provide a new opportunity for controlling bleeding. In spite of limited clinical success of hydroxamate-based MMP inhibitors in human cancer trials, mainly due to lack of efficacy and to reports of side-effects (i.e., musculoskeletal pain and inflammation) which occurred after 2-3 months of treatment15,16 and subsided within 1-3 weeks of discontinuation,16 there is still hope for MMP inhibition as a therapeutic approach.17 In fact, the doses of MMP inhibitors required for acute use in antihemorrhagic treatment are much lower than those for reducing metastasis;18,19 and, in addition, this indication only requires short-term MMP inhibition. Thus, these conditions lead to a new therapeutic opportunity for MMP inhibitors, as antihemorrhagic agents. In this regard, we have recently demonstrated this hypothesis with GM6001 (Ilomastat, 4), a synthetic broad-spectrum MMP inhibitor, and a novel, preclinical antihemorrhagic candidate, CM-352 (5, Chart 1).18 This novel compound (5) showed remarkable efficacy and safety; thus, 5 became a preclinical candidate for the acute treatment of hemorrhage. Herein we present a detailed account of the discovery of this series from hit identification to an optimized lead.

Figure 1. Simplified scheme for the proposed cooperation between fibrinolytic and MMP systems in thrombus dissolution.


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PAIs: plasminogen activator inhibitors.

Results and discussion Validation of a novel α-spiropiperidine hydroxamate chemical series as MMP inhibitors. MMP inhibitors (MMPi) have been explored as anticancer, anti-inflammatory and antiviral agents, and different structures have been designed to target the zinc catalytic center of these enzymes, with the majority of them containing a hydroxamic acid group acting as a chelating zinc binding group (ZBG).20 Particularly, α-sulfonyl-4-tetrahydropyranyl (THP) and α-sulfonylα-piperidinyl hydroxamates have been explored as potent MMP-2, -9 and -13 inhibitors, including the development candidate 6 (SC-78080/SD-2590) and the α-THP 7 (SC-77774) by Pfizer (Chart 1).21 These compounds spare MMP-1. The α-sulfone α-THP series possesses better ADME and PK properties than the closely related β-sulfone α-THP core of the Roche clinical candidate RS-130830 (8).22 Wyeth’s initial work on the α-sulfone α-piperidinyl series described compound 9 as a potent, selective and orally active MMP inhibitor in the clinically relevant advanced rabbit osteoarthritis model.23 Recent work on this chemical series by Wyeth has


6

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evolved towards selective TNF-α converting enzyme (TACE/ADAM17) inhibitors over MMPs by incorporating a butynyloxyphenyl P1’ moiety.24

Figure 2a Structural information for MMP-13 and MMP-3 isoforms, in complex with ligands, guided the design of novel chemical series 5’.

C)


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5’ a

(A) Complex of SC-78080 6 with MMP-13 (PDB accession code: 3KRY). (B) Complex of 10 with MMP-3 (PDB accession code: 1D8F). The molecular surface is drawn to demonstrate the narrowness of the S1’ pocket. (C) Markush formula of novel α-spiropiperidine hydroxamates 5’.

According to the X-ray structure of 6 with MMP-13,21b PDB accession code 3KRY, and the Xray structure of MMP-3 complexed with the 1-sulfonyl-4-benzyloxycarbonyl-piperazine 10, PDB accession code 1D8F,25 the piperidine N-substituent is directed towards the solvent region (Figures 2a and 2b). In fact, structural modifications in this region, the solvent accessible area, generally have a minor effect on potency and can provide a handle for modulating solubility and pharmacokinetic parameters. From this structural information, we focused our efforts on replacing the piperidine ring with a conformationally constrained ring system. Conformationally constrained ring systems are considered privileged structures.26 Among them, the azaspirocyclic system and, in particular spiropiperidines, have emerged as valuable fragments in drug discovery programs.27 Potentially, the reduced molecular flexibility of these fragments and their spatial conformation may have a favorable impact on the molecular properties as well as their off-target profile (i.e., hERG and P450s). Thus, we designed a new series of MMP inhibitors incorporating


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a spiropiperidine unit (5’, Figure 2C) while maintaining the hydroxamic acid moiety as a zinc binding group (ZBG). The presence of an ionizable basic nitrogen in our molecules has a positive impact on the water solubility of these compounds, an issue of the THP chemical series.28,29 In addition, proposed molecules bearing this chemotype assure an intellectual property position for this novel chemical series of α-spiropiperidine hydroxamates (5’).30

To validate our hypothesis, compounds 11-18, bearing different S1’ fragments selected according to structure-based and knowledge-based approaches, together with their synthetic accessibility, were synthesized and evaluated for their inhibition against MMP-3 and MMP-10 (Table 1). The simple 4-methoxyphenyl derivative 12 exhibited nanomolar potency, with IC50 values of 15000

96

84

≤400

9

12

≤400

178

189

≤400

F

12 O

13 O

OMe

14 O

OMe


10

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15

20000

25400

3700

4

4

≤400

20

32

≤400

114

184

900

O

OMe

16 O

OCF3

17 OCF3 O

18 OCF3 N H

a

Human whole blood

Following our screening funnel, compounds with inhibition activities in the low nM range (IC50 15000

H

314

713

900

H

46

54

≤400

Cpd

R1

19c

OCF2

Thromboelastography EC50 (nM)

O

19d

N H

19e

N O N

19f 19g

OMe N

19h OMe

19i MeO

19j O

19k

N H

H N O O

19l 19m

N OMe H


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O

19n

H

39

68

≤400

H

476

435

900

Cl

9600

8700

>15000

HN

19o

N O

19p a

OCF3

Human whole blood

Exploration of N-substituted aryloxyphenyl α-spiropiperidine hydroxamates. The next stage involved an exploration around the basic nitrogen of the spiropiperidine in our chemical series 5’. As anticipated and according to the proposed binding mode (Figure 2b), the impact on primary activity due to growing the vector (R’) from the nitrogen facing the solvent-accessible area was minimal. Thus, the corresponding secondary amine of the spiro ring in compound 13 was converted into a tertiary amine (e.g., R1 = Me, 20a), a fluorinated alkyl-containing tertiary amine (20b) and an amide (20c), and their corresponding IC50 values against MMPs, as well as their antifibrinolytic activities, were essentially unchanged (Table 3). These compounds were initially designed to explore a variety of pKa values and to evaluate their impact on ADME properties, mainly focused on solubility and hERG binding. Therefore, considering that the primary and functional activities are maintained, these molecules, covering a diversity in physicochemical properties, may provide alternative profiles from an ADME perspective. In fact, based on the estimated pKa’s (Table 3), compounds 13, 20a and 20b will be ionized to different extents at physiological pH, but molecule 20c, bearing a tertiary amide considered as "non-basic" and "non-acidic" under physiologic conditions, will not be protonated. However, because the synthesized molecules bearing a secondary amine did not show any hERG-related issues and


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optimal solubility was achieved as described below, no further exploration around this position has been performed.

Table 3. Exploration of N-substituted α-spiropiperidine hydroxamates

Cpd

R1

13

-H

MMP3 IC50 (nM) 9

20a CF3

20b

MMP10 IC50 (nM) 12

a

Thromboelastography EC50 (nM) ≤400

b

Estimated pKa

10

17

≤400

7.35 7.33

6

16

≤400

6.66

5

9

400

O

20c a

Human whole blood; bIn silico estimation performed using the algorithm implemented in

Pipeline Pilot v 8.5.

Exploration of acyl linker aryl moieties. Therapeutic scenarios where the stoppage of bleeding is required (surgery, trauma and first aid, etc.) involve an acute treatment; therefore, we also designed molecules that have very short half-lives, i.e., immediate elimination after achieving their goal, allowing for subsequent wound healing. Thus, taking into account the structural description of the S1’ pocket (Figure 2b), we explored ester-linked analogs (21), and those corresponding to compounds 13 and 19d (21a and 21b) were synthesized (Table 4). Compound 21a exhibited potency and functional activity similar to compound 13.


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Some of synthesized compounds (13, 16, 19d, 19p and 20a) were screened against other MMP isoforms (Supplementary Information). In general, no selectivity was observed against any isoform at 10 µM, with inhibitory activities greater than 90% for most of the screened isoforms (MMP-1, -2, -8, -9, -12, -13, -14, and -20), except for MMP-1 and MMP-7 vs compound 19p, whose inhibition were 9% and 26%. Non-planarity induced by chlorine atom at meta position led to this selective MMP inhibitor, 19p, inactive against MMP-1, -3, -7 and -10; its R1 substructure did not accommodate within S1’ pocket. Unfortunately, this molecule did not interact with the MMPs of interest (MMP-3 and MM-10) and, consequently, its functional antifibrinolytic response was really poor (EC50 > 15µM).

Table 4. Exploration of ester-linked analogs

21 Cpd

MMP3 IC50 (nM)

R1

21a

OMe

MMP10 IC50 (nM)

a

Thromboelastography EC50 (nM)

15

18

≤400

132

103

7500

O

21b a

N H

Human whole blood


17


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In vitro ADME data (Table 5) guided us during the lead optimization process to prioritize which compounds were to progress to the in vivo assay. Our compounds showed low permeability (PAMPA Pe < 1 nm/s), which is a desirable feature; in fact, to be efficacious as antifibrinolytics, the compounds must be maintained in the bloodstream, minimizing tissue distribution and avoiding undesirable side effects. Additionally, all of these compounds showed low inhibition of the five major cytochrome P450 isoforms (1A2, 2C9, 2C19, 2D6, 3A4); in fact, the percentage inhibition values, at 10 µM, were less than 50% for all tested compounds. The metabolic stability of this set of compounds was evaluated in human, mouse and rat liver microsomes after 20 min of incubation at a concentration of 1 µM. All compounds, except for compound 21a with the metabolically labile ester functionality, showed good microsomal stability in human and mouse species (>70% remaining). Replacement of the –OCF3 group with the methylaminocarbonyl group tended to increase the stability, especially in the case of the pair 17 and 19n (68.9% vs. 97.6%; 71.1% vs. 90.2%; and, 68.7% vs. 91.4%, for human, mouse and rat, respectively). In addition, there was minimal interaction with the hERG channel according to the binding assay; in fact, all assayed molecules showed hERG IC50 values >30 µM, and some of them, compounds 16, 19d, 19e and 19n, even had IC50 values ≥100 µM.

Table 5. ADME profile.a Cpd

PAMPA 1A2 2C9 2C19 2D6 3A4 HLM MLM RLM hERG b b b b b c c c Pe (%) (%) (%) (%) (%) (%) (%) (%) IC50 (µM) (nm/s) 13 0.009 6.1 0 19.4 6.3 0 87.7 83.8 52.3 37 16 0.007 4.9 7.4 8.4 10.6 0 92.1 87.2 95.6 100 17 N/D 13.6 8.8 30.5 19.9 0 68.9 71.1 68.7 50.3 19d 0.010 0 0 0 5.7 0 91.1 97.5 99.8 >100 19e N/D 0.4 0 18.9 28.8 8.3 90.2 90.6 92.6 >100 19n N/D 25.6 12.3 40.2 37.2 11.6 97.6 90.2 91.4 >100 21a 0.002 3.8 0 11.1 5.1 0 1.8 0 3.3 63.4 a N/D: not determined; b% inhibition at 10 µM; c% compound remaining after a 20-min incubation in human, mouse or rat liver microsomes (HLM, MLM and RLM, respectively).


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In vivo efficacy in murine bleeding models. The most active compounds in the functional antifibrinolytic assay (EC50

Design, Synthesis, and Biological Evaluation of Novel Matrix

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