Design, synthesis and pre-clinical characterization of selective Factor

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Design, synthesis and pre-clinical characterization of selective Factor D inhibitors targeting the alternative complement pathway. Rajeshri Karki, James Powers, Nello Mainolfi, Karen Anderson, David B. Belanger, Donglei Liu, Nan Ji, Keith Jendza, Christine F. Gelin, Aengus Mac Sweeney, Catherine Solovay, Omar Delgado, Maura Crowley, Sha-Mei Liao, Upendra A Argikar, Stefanie Flohr, Laura R. LaBonte, Edwige Lorthiois, Anna Vulpetti, Ann Brown, Debby Long, Melissa Prentiss, Nathalie Gradoux, Andrea de Erkenez, Frederic Cumin, Christopher M. Adams, Bruce Jaffee, and Muneto Mogi J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00271 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Design, synthesis and pre-clinical characterization of selective Factor D inhibitors targeting the alternative complement pathway. Rajeshri G. Karki,*† James Powers,† Nello Mainolfi,†,║ Karen Anderson,†,¥ David B. Belanger,†,§ Donglei Liu,† Nan Ji,†,║ Keith Jendza,† Christine F. Gelin,†,§ Aengus Mac Sweeney,‡,¶ Catherine Solovay,† Omar Delgado,† Maura Crowley,† Sha-Mei Liao,† Upendra A. Argikar,† Stefanie Flohr,‡ Laura R. La Bonte,† Edwige L. Lorthiois,‡ Anna Vulpetti,‡ Ann Brown,† Debby Long, † Melissa Prentiss,† Nathalie Gradoux, ‡ Andrea de Erkenez†, Frederic Cumin,‡,† Christopher Adams,† Bruce Jaffee† and Muneto Mogi† †Novartis

Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA.

‡Novartis

Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.

KEYWORDS. Alternative complement pathway, factor D inhibitors, structure-based design, benzylamines.

ABSTRACT: Complement Factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular

degeneration

(AMD),

atypical

hemolytic

uremic

syndrome

(aHUS),


1

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membranoproliferative glomerulonephritis type II (MPGNII) and paroxysmal nocturnal hemoglobinuria (PNH). Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structurebased approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide (LPS)-induced AP activation model, as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.

INTRODUCTION The complement system is part of the innate immune system that has a central immunoregulatory function and also a pathogenic role during ischemic, inflammatory, and autoimmune diseases.1 Depending on the molecular recognition pattern, activation of the complement system can be initiated by three distinct routes: the classical, the lectin and the alternative pathways. Complement factor D (FD; also known as adipsin) is a member of the chymotrypsin family of serine proteases that is essential for alternative pathway (AP) activation (Figure 1).2, 3 FD cleaves factor B (FB) when it is complexed with C3b or C3 (H2O) to generate an active C3 convertase (C3bBb) that cleaves C3 into C3a and C3b. C3b is deposited onto acceptor surfaces (opsonization). Further recruitment of a C3b molecule to the membrane-bound C3 convertase generates a C5 convertase (C3bBbC3b), leading to C5a release and membrane attack complex (MAC; C5b to C9) formation on targeted surfaces with subsequent membrane disruption and cell lysis.2


2


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Figure 1. Schematic representation of the alternative complement pathway (AP) from initiation to cell lysis. Dysregulation of AP activity by genetic mutations, neutralizing antibodies to complement regulatory proteins, or stabilizing antibodies to complement convertase predisposes individuals to diverse disorders including paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS) and C3 glomerulonephritis (C3G).4-9 We have a long standing interest identifying oral FD inhibitors, due in part to the array of diverse diseases involving AP activation.10-13 In addition to our efforts, others have also made significant progress in developing FD inhibitors culminating in the clinical investigation of ACH4471.

14, 15

We have previously reported the discovery of the benzylamine-based FD inhibitors (1

and 2, Figure 2) targeting an active conformation of FD.13 In this report we describe the structurebased optimization of 1 that led to the discovery of 12, an orally bioavailable and selective FD


3

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inhibitor which demonstrated systemic suppression of AP activation in a lipopolysaccharide (LPS)-induced AP activation model, as well as local ocular suppression in, intravitreal injectioninduced AP activation model in mice expressing human FD. H N H

O

H N O

H 2N

H 2N 1

2

Figure 2. Structures of benzylamine-based FD inhibitors13 RESULTS AND DISCUSSION In the native state, FD exists in an inactive conformation with a non-competent arrangement of the catalytic triad: His57, Ser195 and Asp102 (chymotrypsinogen numbering has been used throughout this paper). FD is autoinhibited by its self-inhibitory loop (residues 214 to 219) with Arg218 forming a salt bridge with Asp189, thus trapping it at the bottom of the S1 pocket (Figure 3). When an arginine sidechain from the substrate (or potentially inhibitors containing a basic amine functionality) occupies the S1 pocket, the salt bridge between Asp189-Arg218 is broken, thereby releasing Arg218 from the S1 pocket. The self-inhibitory loop undergoes a conformational change eventually leading to a productive arrangement of the catalytic triad3,

12, 13

and a

catalytically active FD.


4


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Figure 3. FD conformational change upon substrate or benzylamine binding. Crystal structure of inactive FD (5FBE.pdb10, white) is shown relative to active FD (6FUT.pdb13, green). The substrate binding sites are labeled S1-S2’. Salt bridge between Arg218-Asp189 is shown in black dotted lines. His57, Asp102 and Ser195 form the catalytic triad. Inhibitors bound with FD in the crystal structures are not shown for clarity. We sought to exploit this conformational flexibility by designing modifications of the benzylamine compounds with the goal of engaging FD in a unique conformation not seen in other serine proteases, thereby enabling selectivity. From the previously disclosed X-ray structure of 1 with FD13 (also represented in Figure 4a), it was clear that the tetrahydronaphthalene moiety was in close proximity to the key Arg218 residue. So we decided to replace the tetrahydronaphthalene moiety in 1 with substituents capable of hydrogen bonding with the sidechain of Arg218. For preliminary modeling from protein-ligand co-crystal structures, an interactive 3D-editor tool in FOCUS16 (Molsoft, L.L.C.) was employed. The 3D-editor tool provides a simple interface to


5

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explore modification of an initial ligand within a protein−ligand complex, followed by constrained local minimizations of ligand molecules in the protein pocket. Starting from the co-crystal structure of 1 with FD, compound 3 (Table 1) was designed in absence of the succinic acid with aid of the 3D-editor tool, to interact with the Arg218 sidechain (Figure 4a). Acid 3 was synthesized (vide infra) and assessed for FD inhibition via a time-resolved fluorescence energy transfer (TRFRET) in vitro assay. Compound 3 inhibited FD with a TR-FRET IC50=120 nM, an almost 270fold improvement in potency relative to 1. In addition, 3 exhibited functional inhibition of AP activation in vitro in a membrane attack complex (MAC) deposition assay using 50% human whole blood.10 The significant improvement in potency with the first synthesized compound was very encouraging, hence we decided to determine the binding mode of the compound. Surprisingly, docking analysis of 3 using Glide17 (Schrödinger Inc.) generated an alternate binding mode than was originally designed, with the phenyl acetic acid H-bonding with residues of the catalytic triad instead of Arg218. X-ray crystallography confirmed that the binding mode predicted by Glide was in fact accurate (Figure 4b). The 1.64 Å resolution X-ray structure afforded two copies of FD in the asymmetric unit. In both the monomer copies, the benzylamine group of 3 makes direct or water mediated H-bonding with Asp189 in addition to H-bonding interactions with Ser190 and water mediated interactions with Thr214 and Ile227 in the S1 pocket. The carboxylic acid group of 3 is nestled in the catalytic site making H-bonding interactions with His57, Gly193 and Ser195. Upon comparison of the FD bound co-crystal structure of 3 with that of 1, we observed differences in the conformation of the Arg218 sidechain. Also, the carboxylic acid group of 3 overlapped perfectly with the succinic acid from the co-crystal structure of 1 (Figure 4). Interactions of carboxylic acid and phosphonic acid at the oxyanion hole have been reported earlier with FD13 and other S1 serine proteases,18-20 albeit with different chemotypes.


6


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Although the binding mode of 3 was different from the initial design rationale using FOCUS, the serendipitous discovery gave us new insights into improving potency. However, the high level of structural homology across serine proteases as it relates to this particular binding mode raised questions of selectivity. Hence we profiled compound 3 in a panel of more than 50 proteases. Factor XIa (FXIa; IC50=2.8 µM; 23-fold) and tryptase-β2 (IC50=2.3 µM; 19-fold) were identified as the major off-targets. Activity against the rest of the proteases assessed was 100 to 1000-fold weaker relative to FD biochemical potency. Apart from potency and selectivity we also characterized the solubility, in vitro ADME parameters, and the in vivo mouse pharmacokinetic profile of compound 3.

Figure 4. Binding mode of 3 complexed with FD (a) 3D-editor generated orientation of 3 (green) shown relative to X-ray of 1 (cyan, 6FUT.pdb13). Alternate positions of succinic acid cocrystallized with 1 are shown in two shades of orange. (b) Crystal structure 3 (yellow) illustrating a productive arrangement of the catalytic triad residues His57, Asp102 and Ser195. The ligand binding pocket is shown in grey and H-bonding interactions are shown as back dotted lines. Sidechain of Arg218 has shifted upon binding of 3.


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Table 1. In vitro profiling data of FD inhibitors O

O N H

N

R1

NH2

N H

X O

O

OH

3 R1=COOH 4 R1=COOCH3 5 R1=2H-tetrazole 6 R1=SO2NHCOCH3

OH

7 NH2

NH2

Y

8 X=NH, Y=CH2 9 X=CH2, Y=O 10 X=O, Y=CH2

Compound FD TR-FRET IC50 (µM)a 1 32

Human 50% WB MAC IC50 (µM)b NDd

Log Papp cm/s (permeability rank)c -3.9 (high)

3

0.120

4.80

2500-fold). Further homologating the hydroxyl group as in 13 led to an even greater enhancement in selectivity but at the expense of a modest decline in FD potency.


11

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Figure 6. X-ray of 12 (yellow) complexed with FD (white). The ligand binding pocket is shown in grey and H-bonding interactions are shown as back dotted lines.


12


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Table 2. In vitro profiling data of benzylamine FD inhibitors.

R1

O OH O

R2 NH2

Compou nd

R1

R2

FD TRFRET IC50 (µM)a

Human 50% WB MAC IC50 (µM)b

FXIa IC50 (µM)d

Tryptase β2 IC50 (µM)d

Log Papp PAMPA cm/s (permeabil ity rank)c

9

H

H

0.008

0.270

0.60

0.35

-4.5 (high)

11

H

CH3

0.025

1.3

5.20

21

-4.5 (high)

12

H

CH2OH

0.012

0.26

7.7

6.5

-5.1 (medium)

13

H

(CH2)2O H

0.030

0.70

>100

55

-5.2 (medium)

14

Br

CH2OH

0.003

0.160

4.0

3.3

-5.0 (medium)

15

CH2OH

CH2OH

0.005

0.07

8.09

3.7

-5.4 (low)

16

CH2OCH3

CH2OH

0.010

0.02

4.8

3.6

-5.3 (low)

17

C(CH3)2O H

CH2OH

0.008

0.013

0.65

3.3

-5.5 (low)

18

NHiPr

CH2OH

0.015

0.07

2.1

8.7

-5.5 (low

aHalf-maximal

inhibition of recombinant human complement FD as determined in a TR-FRET assay. bHalf-maximal inhibition of soluble MAC complex formation, as determined in an ELISA assay. cPermeability measurement by PAMPA. dScreening assay with >50 protease targets. Data represent mean values of duplicate measurements. We next turned our attention toward improving interactions in the S1β sub-site with the aim of enhancing functional potency. Compounds 14-18 were synthesized with diverse substituents to


13

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occupy different regions in S1β sub-site. While modifications in the S1β sub-site did in fact lead to modest improvements in biochemical and functional potency in most instances (e.g. 15-18), it also resulted a reduction in permeability in the PAMPA assay with introduction of polar substituents (Table 2). With this data in hand compounds 12, 13 and 14, were selected for in vivo pharmacokinetic assessment (1 mg/kg IV and PO at 10 mg/kg; C57BL6 mice Table 3). All three compounds exhibited low clearance upon IV administration, but only 12 afforded robust oral bioavailability (83%). In light of the encouraging mouse PK profile compound 12 was selected for in vivo pharmacology assessment (vide infra).

Table 3. Pharmacokinetic Parameters of 12, 13 and 14 in C57BL6 mice dosed at 1 mg/kg IV and 10 mg/kg PO. Compound

oral Cmax AUC(0-7)

CL

(nM)

(mL/min/kg) (L/kg)

(nM*Hours)

Vdss

Elimination t1/2 (IV) %F (Hours)

IV/PO 12

31000

16100/125000 2.6

0.3

1.6

83

13

10700

4970/19500

8.7

0.3

1.3

39

14

26000

48700/11600

0.7

0.1

2.5

25

CHEMISTRY Compounds 3-7 can be generally accessed from three primary building blocks: benzylamines of type A, 3-halobenzoic acids of type B, and the appropriate amino phenyl acetate derivatives of type C via two separate routes. Route A, as exemplified by the synthesis of 4 begins with a Suzuki cross-coupling between A and B followed by a peptide coupling with aniline C.


14


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Alternatively, Route B can be used to access compounds such as 5 by first coupling building blocks B and C to afford the requisite amides which are then subjected to Suzuki cross-coupling with A (Scheme 1). Scheme 1. R HN

NH2

O

O

OH

OH B OH

X

A

R'

O

A A= CH or N

B

C

Route A

O

Boc HN

Boc HN

OH B OH

19

HO

3-bromobenzoic acid PdCl2(dppf).CH2Cl2 adduct 2M aq. K3PO4 CH3CN, 90 °C

1. methyl 2-(2aminophenyl)acetate O hydrochloride, HATU, TEA DMF, rt

N H O

2. 2M HCl in ether

20

O

4 NH2

Route B

O 3-(Aminomethyl)benzeneboronic acid hydrochloride PdCl2(dppf).CH2Cl2 adduct

O

NH2 O O

21

HATU, DIPEA N

DMF, rt, Br

N H

22

O O

2M aq. K3PO4 CH3CN, 110 °C

N H

N

OH O

5 NH2

The core of compounds 9-18, which are devoid of the amide linkage, were typically prepared from methyl 2-(2-hydroxyphenyl)acetate and the corresponding 3-bromobenzyl bromide to furnish cores such as 25. Alternatively, for the opposite regioisomer (e.g. 26), by starting from methyl 2(2-(chloromethyl)phenyl)acetate and coupling with the corresponding 3-bromophenol as outlined in Scheme 2. With the cores in place, final compounds can then be accessed via Suzuki coupling with the appropriate boronic acid. In the case of 9 and 10, (3-(aminomethyl)phenyl) boronic acid hydrochloride was utilized avoiding the need for protection of the basic amine. Scheme 2.


15

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X Cl

HO O

or

O

23

O

24

O

X

3-Bromobenzyl bromide or 3-Bromophenol

(3-(aminomethyl)phenyl) boronic acid hydrochloride, O PdCl2(dppf).CH2Cl2 adduct

Y O

Br

K2CO3, DMF

25 X=C, Y=O 26 X=O, Y=C

2M aq. K3PO4, CH3CN, 90 °C, followed by LiOH, H2O, THF, NH2

Y O OH

9 X=C, Y=O 10 X=O, Y=C

More elaborate building blocks of type A needed to access compounds 11-18 were readily accessed from the corresponding bromide, which are either commercially available or known in the literature, by employing Miyaura-type conditions as depicted in Scheme 3. Similarly more complex building blocks of type B required for compounds 14-18, could be accessed from commercial sources or from standard transformations as delineated in the experimental section. Scheme 3.

R Boc

N H

Br

Bis(pinacolato)diboron K3PO4, PdCl2(dppf)·CH2Cl2, MeCN/H2O, 90 oC

R Boc

N H

O B

O

IN VIVO PHARMACOLOGY As stated above, the FD inhibitory potency of 12, combined with favorable protease selectivity and pharmacokinetic profile, led to its selection for evaluation in in vivo pharmacology studies. To enable in vivo efficacy assessment, it was necessary to generate mice expressing human FD in place of the murine enzyme (human FD knock-in mice) as previously described10 due to compounds in this chemical series lacking species cross-reactivity. A pharmacodynamic model was developed with these mice by administration of lipopolysaccharide (LPS), a component of outer membrane of gram-negative bacteria.

Intraperitoneal administration of LPS induces

activation of the AP, as detected by increased levels of AP breakdown products, in particular Ba which is a direct product of FD-mediated cleavage of C3bB.10 When 12 was administered in this


16


model by oral gavage at 3 and 10 mg/kg, AP modulation was observed in a dose responsive manner as measured by plasma Ba levels (Figure 7). Encouragingly, the AP pathway was fully inhibited for up to 10 hours at the 10 mg/kg dose.

3

Relative Plasma Ba Levels  SEM

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

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10 mg/kg 3 mg/kg LPS

2

PBS

1

0 0

4

8

12

16

20

24

Time after compound 12 p.o. (hours)

Figure 7. Inhibitor 12 tested at 3 and 10 mg/kg in the human FD knock-in mouse pharmacodynamic model. Given the evidence of the role of AP in AMD25 we also investigated the ability of 12 to achieve exposure in posterior tissues of the eye, specifically the retina and the posterior eye cup (PEC) which consists of the primary target tissue for AMD the RPE/choroid as well as the posterior sclera. Due to the small size of the mouse eye, which hampers precise tissue dissection, the ocular tissue distribution profile of 12 was evaluated in Brown Norway rats. Upon oral administration of 12 (10 mg/kg) to Brown Norway rats absolute plasma exposure was lower relative to the mouse (Cmax = ~4x lower; AUC = ~54x lower), but encouragingly exposure in the posterior eye cup was


17

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above the IC50 of 12 in the 50% whole blood MAC deposition functional assay (260 nM) for over 24h (Figure 8).

Plasma

PEC

Retina

10000 Mean Concentration (nM)

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


1000 100 10 1 0

5

10 15 Time (hr)

20

25

Rat ocular PK AUC Plasma/PEC/retina nM*hours (DN)

2300/1160 /8

Concentration in Plasma/PEC/retina @24 hours [nM] 34 /290 /BQL

Figure 8. Concentration versus time profile for 12 in retina, PEC, and plasma and PK parameters after a single dose (PO 10 mg/kg) in Brown Norway rats. DN = dose normalized.

In light of the encouraging ocular PK profile of 12 we examined the ability to suppress AP activation in ocular tissues. To examine ocular efficacy we chose a model where AP is activated locally via ocular injury, specifically an intravitreal injection (IVT). This model has previously been described26 and shown to increase AP locally in ocular tissues. Oral administration of 12


18


showed a dose dependent decrease in the ocular Ba levels (Figure 9) with up to 86% inhibition at 10 mg/kg dose. Relative Ocular Ba Levels  SEM

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 20 of 49

2

68%

1

86%

0

veh

veh

10mpk

3mpk

Compound 12 IVT

Figure 9. Ocular efficacy of 12 after a single oral dose in a model where AP is activated locally via intravitreal injection; efficacy assessments were made 4h post intravitreal injection.26

The encouraging in vivo pharmacology profile wherein 12 demonstrates suppression of AP activation both in plasma and in ocular tissues warranted advanced in vitro and in vivo profiling of 12. Compound 12 was evaluated in an internal panel of over 70 enzymes, receptors and ion channels and it did not exhibit any activity against these off-targets at concentrations ≤ 10 µM. 12 also did not inhibit CYP3A4, CYP2C9, CYP2D6, or hERG (Q-patch assay) at concentrations ≤ 30 µM in vitro. The PK profile of 12 was also evaluated in beagle dogs at a dose of 1 mg/kg IV and 10 mg/kg PO. Consistent with the observed rodent PK profiles, 12 exhibited low clearance in dogs (3.8 mL/min/kg) and excellent oral bioavailability (~70%) (Table 4).


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The acceptable pharmacokinetic profile in two species combined with the ability of 12 to suppress AP activation both systemically and in ocular tissue led to its progression into more advanced preclinical evaluation, which will be reported in due course. Table 4. Pharmacokinetic parameters of 12 in beagle dogs dosed at 1 mg/kg IV and 10 mg/kg PO represented as mean values (n=3). PK Parameters

Mean

± SD

CL (mL/min/kg)

3.8

1

Vdss (L/kg)

0.8

0.2

T ½ (Hours)

3.8

0.4

AUC0-last (nM*hours) 10 mg/kg PO

85000

16100

Cmax (nM) 10 mg/kg PO

16500

2000

Tmax (hours)

0.8

0.3

%F 70% * N/A denotes not applicable.

N/A

CONCLUSION A rationally conceived approach exploiting the FD conformational dynamics to optimize the benzylamine FD inhibitors13 led to the discovery of lead molecule 3. The novel serendipitous binding mode of 3 while offering acceptable potency presented challenges related to protease selectivity. Structure-based optimization for selectivity in combination with optimization of ADME properties culminated in the discovery of 12, an orally bioavailable, selective FD inhibitor. Compound 12 has demonstrated suppression of AP activation both in plasma and ocular tissues in mice. Compound 12 has been selected for advanced preclinical evaluation.


20


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EXPERIMENTAL SECTION Experimental procedures and compound characterization for novel compounds General Chemistry Information Unless otherwise specified, all solvents and reagents were obtained from commercial suppliers and used without further drying or purification. NMR spectra were recorded on a Bruker Avance II 400 MHz spectrometer. All chemical shifts are reported in parts per million (δ) relative to tetramethylsilane. The following abbreviations are used to denote signal patterns: s = singlet, d = doublet, t = triplet, m = multiplet, and br = broad. Flash chromatography was conducted using grade 60 230−400 mesh silica gel from Fisher Chemical (S825-1) or by utilizing the CombiFlash Companion from Teledyne Isco, Inc. and RediSep Rf disposable normal phase silica gel columns (4−300 g). Thin layer chromatography was performed using 2.5 × 7.5 cm glass-backed TLC Silica Gel 60 F254 plates from EMD Chemicals, Inc. (15341-1) and visualized by UV light. HPLC purifications were performed on a Gilson preparative HPLC system controlled by Unipoint software using X-Bridge Phenyl, C8, C18, or RP18 30 × 50 mm columns with 5 μm particle size. Low resolution mass spectra were recorded using an Agilent 1100 series LC-MS spectrometer. The purity of all exemplified compounds was ≥95%, as determined by both 1H NMR and HPLC-UV at a wavelength of 214 nm, unless otherwise stated. Unless otherwise stated, chiral starting materials were commercially available with e.e. ≥98%. Experimentals 2-(2-(3'-(Aminomethyl)-[1,1'-biphenyl]-3-carboxamido)phenyl)acetic

acid

(3):

Triethylamine (0.276 mL, 1.984 mmol) was added to a mixture of 3-bromobenzoic acid (199 mg, 0.992 mmol) and HATU (415 mg, 1.091 mmol) in DMF at 23 °C. In a separate vial, methyl 2-(2aminophenyl)acetate hydrochloride (CAS # 49851-36-7) (200 mg, 0.992 mmol) was stirred with


21

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TEA (0.276 mL, 1.980 mmol) in DMF (0.5 mL). After 5 min, the solution of methyl 2-(2aminophenyl)acetate hydrochloride (200 mg, 0.992 mmol) was added to the 3-bromobenzoic acid (199 mg, 0.992 mmol) mixture and the resulting mixture was stirred at rt for 30 min. The reaction mixture was diluted with EtOAc and washed with water. The aqueous layer was extracted with EtOAc. The combined organics were washed with 5% aq LiCl, dried (Na2SO4) and concentrated. The resulting residue was purified by silica gel chromatography (EtOAc-heptanes 0-60%) to provide methyl 2-(2-(3-bromobenzamido)phenyl)acetate (194 mg, 94%). MS (ESI+) m/z 348.0, 350.0 (M+H). A mixture of methyl 2-(2-(3-bromobenzamido)phenyl)acetate (90 mg, 0.258 mmol), prepared as described above, (3-(aminomethyl)phenyl)boronic acid hydrochloride (CAS # 146285-80-5) (63.0 mg, 0.336 mmol), PdCl2(dppf).CH2Cl2 adduct (CAS # 95464-05-4) (10.55 mg, 0.013 mmol), and 2M aq. K3PO4 (0.388 mL, 0.775 mmol) in 9:1 MeCN/H2O (2.8 mL) was heated in a microwave reactor at 110 °C for 60 min. The organic layer was filtered and the filtrate was directly purified by reverse phase HPLC (Stationary phase: Gemini® NX 5µ C18 110A 100x30 mm. Mobile phase: acetonitrile gradient, water with 0.1% (28% ammonium hydroxide) / acetonitrile). Fractions containing the desired product were pooled, the pooled fractions were frozen and lyophilized to provide the title compound (0.063 g, yield 68%). 1H NMR (400 MHz, DMSO-d6, one drop of TFA added) δ ppm 10.13 (s, 1 H) 8.28 (s, 1 H) 8.23 (br s, 3 H) 7.98 (d, J=7.83 Hz, 1 H) 7.87 - 7.94 (m, 2 H) 7.81 (d, J=7.83 Hz, 1 H) 7.66 (t, J=7.77 Hz, 1 H) 7.54 - 7.61 (m, 1 H) 7.46 - 7.53 (m, 2 H) 7.30 - 7.39 (m, 2 H) 7.21 - 7.28 (m, 1 H) 4.15 (q, J=5.81 Hz, 2 H) 3.70 (s, 2 H) HRMS calcd. for C22H20N2O3 (M+H)+ 361.1552, found 361.1550. Methyl 2-(2-(3'-(aminomethyl)-[1,1'-biphenyl]-3-ylcarboxamido)phenyl)acetate (4): Triethylamine

(0.255

mL,

1.833

mmol)

was

added

to

a

mixture

of

3'-(((tert-

butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3-carboxylic acid (200 mg, 0.611 mmol), 20, and


22


Page 24 of 49

HATU (256 mg, 0.672 mmol) in DMF at 23 °C. After 15 min, methyl 2-(2-aminophenyl)acetate hydrochloride (CAS # 35613-44-6) (130 mg, 0.611 mmol) was added and the resulting mixture was stirred at room temperature overnight. The resulting mixture was partitioned between with EtOAc and H2O. The layers were separated and the aqueous layer was extracted with 4:1 EtOAc/heptane. The combined organics were washed with 5% aqueous LiCl solution, the washed organics were dried with Na2SO4, the dried solution was filtered, and the filtered solution was concentrated. The resulting crude was purified by silica gel chromatography (10-70% EtOAc/heptanes)

to

provide

methyl

2-(2-(3'-(((tert-butoxycarbonyl)amino)methyl)-[1,1'-

biphenyl]-3-ylcarboxamido)phenyl)acetate (63 mg, 22% ). MS (ESI-) m/z 473.4 (M-H). Methyl 2(2-(3'-(((tert-butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3-ylcarboxamido)phenyl)acetate (50 mg, 0.105 mmol) was added to a solution of HCl (2 M in Et2O, 1.05 mL, 2.11 mmol). The resulting mixture was stirred at room temperature overnight, then concentrated. The residue was purified by reverse phase HPLC (Waters SunFireTM Prep C18 OBDTM 5µm, 30x100 mm. Mobile phase: MeCN gradient, water with 0.1% TFA / acetonitrile). Fractions containing the desired product were pooled, the pooled fractions were frozen and lyophilized to provide the title compound as a TFA salt (11 mg, yield 23%). 1H NMR (TFA salt, 400 MHz, methanol-d4) δ ppm 8.26 (s, 1 H), 7.98 (d, J=7.7 Hz, 1 H), 7.88 - 7.94 (m, 1 H), 7.77 - 7.84 (m, 2 H), 7.65 (t, J=7.8 Hz, 1 H), 7.59 (t, J=7.8 Hz, 1 H), 7.47 - 7.55 (m, 2 H), 7.34 - 7.42 (m, 2 H), 7.25 - 7.32 (m, 1 H), 4.22 (s, 2 H), 3.78 (s, 2 H), 3.63 (s, 3 H). HRMS calcd. for C23H22N2O3 (M+H)+ 375.1700, found 375.1696. N-(2-((2H-tetrazol-5-yl)methyl)phenyl)-3'-(aminomethyl)-[1,1'-biphenyl]-3carboxamide (5): To a solution of 5-[(2-nitrophenyl)methyl]-1H-1,2,3,4-tetrazole (CAS# 177595-29-8, 280 mg, 1.27 mmol) in THF (6.8 mL) was added TEA (0.43 mL, 3.1 mmol). The mixture was cooled to 0 oC and SEMCl (0.3 mL, 1.7 mmol) was added. After ~ 1.5 h the


23

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mixture was partially purified by directly absorbing onto a plug of silica gel and eluting with 050% EtOAc/hepatane, the eluent was concentrated and the resulting residue was dissolved in THF (4.8 mL), charged with PtO2 (11 mg, 0.05 mmol) and placed under a H2 gas atmosphere. After ~ 2h the mixture was filtered through a plug of Celite® and concentrated to which a solution of 20 (125 mg, 0.38 mmol) in DMF (1.9 mL) was added, followed by HATU and TEA (133 µL, 0.96 mmol) and the mixture was stirred for ~14h at which time it was passed through a plug of silica (0-50% EtOAc/heptanes) and concentrated, the resulting oil was then diluted with DCM (1 mL) and treated with TFA (300 µL, 3.8 mmol) and stirred for 3h. The mixture was concentrated to dryness and purified directly by RP-HPLC (Stationary phase: Gemini® NX 5µ C18 110A 100x30 mm. Mobile phase: MeCN gradient, water with 0.1% (28% ammonium hydroxide) / acetonitrile)) to afford the title compound in 85% purity by 1H HNMR (40 mg, yield 18%,): 1H NMR (TFA salt, 400 MHz, DMSO-d6) δ 16.15 (s, 1H), 10.15 (s, 1H), 8.21 (t, J = 1.9 Hz, 3H), 7.95 – 7.87 (m,3H), 7.82 (dt, J = 7.9, 1.3 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.61 – 7.53 (m, 1H), 7.49 (dt, J = 7.7, 1.4 Hz,1H), 7.46 – 7.41 (m, 1H), 7.37 (ddd, J = 7.9, 6.3, 2.5 Hz, 1H), 7.33 – 7.25 (m, 2H), 4.37 (s, 2H), 4.16 (d,J = 5.7 Hz, 2H). HRMS calcd. for C22H21N6O (M+H)+ 385.1776 found 385.1766 N-(2-((N-acetylsulfamoyl)methyl)phenyl)-3'-(aminomethyl)-[1,1'-biphenyl]-3carboxamide (6): Was prepared in a similar fashion as 5, by starting with N-((2aminobenzyl)sulfonyl)acetamide which can be prepared as follows. To a solution of (2nitrophenyl)methanesulfonamide (CAS# 51145-00-7; 230 mg, 1.064 mmol) in DCM (10 mL) was added bismuth(III) trifluoromethanesulfonate (40 mg, 0.061 mmol) and acetyl chloride (600 µL, 8.44 mmol) and the mixture was stirred at rt for 40 minutes at which time it was filtered and the eluent was concentrated to dryness and the resulting crude N-((2-


24


Page 26 of 49

aminobenzyl)sulfonyl)acetamide, which could be used without further purification in the subsequent step. Spectroscopic data for the title compound, N-(2-((Nacetylsulfamoyl)methyl)phenyl)-3'-(aminomethyl)-[1,1'-biphenyl]-3-carboxamide (6): 1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.54 (t, J = 1.8 Hz, 1H), 8.22 (s, 3H), 8.04 (dt, J = 7.8, 1.5 Hz, 1H), 8.00 – 7.90 (m, 3H), 7.86 (dt, J = 7.6, 1.5 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.55 (t, J = 7.6Hz, 1H), 7.47 (dt, J = 7.7, 1.3 Hz, 1H), 7.35 (ddd, J = 15.0, 7.4, 1.6 Hz, 2H), 7.17 (td, J = 7.5, 1.3 Hz,1H), 4.42 (s, 2H), 4.15 (s, 2H), 1.56 (s, 3H). HRMS calcd. for C23H24N3O4S (M+H)+ 438.1487 found 438.1469. 2-(2-(6-(3-(Aminomethyl)phenyl)picolinamido)phenyl)acetic acid (7): The title compound was prepared from methyl 2-(2-(6-chloropicolinamido)phenyl)acetate (22) and 3aminomethylphenyl boronic acid HCl (CAS # 146285-80-5) using a method similar to that described for 3. 1H NMR (400MHz, DMSO-d6) δ ppm 12.22 (br. s, 1H), 9.32 (br. s, 1H), 9.13 (br. s, 2H), 8.32 (d, J=7.5 Hz, 1H), 8.20 - 8.06 (m, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.54 - 7.41 (m, 2H), 7.29 - 7.17 (m, 3H), 7.09 (t, J=6.9 Hz, 1H), 4.10 (s, 2H), 3.41 (s, 2H). HRMS calcd. for C21H19N3O3 (M+H)+ 362.1505, found 362.1502. 2-(2-(((3'-(Aminomethyl)-[1,1'-biphenyl]-3-yl)amino)methyl)phenyl)acetic acid (8): A degassed

mixture

of

3-bromoaniline

(600

mg,

3.49

mmol),

3-((tert-

butoxycarbonylamino)methyl)phenylboronic acid (1051 mg, 4.19 mmol), Pd(PPh3)4 (202 mg, 0.174 mmol), and 2M aq. K3PO4 (1481 mg, 6.98 mmol) in dioxane (15 mL) and water (3 mL) was heated at 100 °C for 1.5 h. The reaction mixture was diluted with EtOAc and washed with water. The aqueous layer was washed with EtOAc. The combined organics were washed with brine, dried (Na2SO4) and concentrated. The residue was purified by silica gel chromatography (EtOAcheptane 0-100%) to provide tert-butyl ((3'-amino-[1,1'-biphenyl]-3-yl)methyl)carbamate (552 mg,


25

Page 27 of 49 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


yield 53 %). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.30 - 7.51 (m, 4 H) 7.18 (d, J=7.20 Hz, 1 H) 7.09 (t, J=7.77 Hz, 1 H) 6.81 (t, J=1.83 Hz, 1 H) 6.74 (d, J=7.58 Hz, 1 H) 6.55 (dt, J=6.92, 1.15 Hz, 1 H) 5.14 (s, 2 H) 4.17 (d, J=6.06 Hz, 2 H) 1.40 (s, 9 H). Sodium triacetoxyborohydride (111 mg, 0.523 mmol) was added to a solution of tert-butyl ((3'-amino-[1,1'-biphenyl]-3yl)methyl)carbamate (104 mg, 0.349 mmol) and methyl 2-(2-formylphenyl)acetate (CAS # 6396983-5) (74.5 mg, 0.418 mmol) in DCE (4 mL) at rt. The reaction mixture was stirred at rt for 2.5 h. The reaction mixture was partitioned between EtOAc and sat. aq. NH4Cl. The aqueous layer was extracted with EtOAc. The combined organics were washed with brine, dried (Na2SO4) and concentrated. The residue was purified by silica gel chromatography (EtOAc-heptane 0-50%) to provide

methyl

2-(2-(((3'-(((tert-butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3-

yl)amino)methyl)phenyl)acetate (132 mg, yield 82%). MS (ESI+) m/z 461.4 (M+H). A solution of 2-(2-(((3'-(((tert-butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3yl)amino)methyl)phenyl)acetate (110 mg, 0.239 mmol) in DCM (1 mL) and TFA (1 mL) was stirred at room temperature for 15 hour. DCM (10 mL) and sat. aqueous NaHCO3 solution (15 mL) was added. Then solid NaHCO3 was added until pH ~ 8. The two layers were separated and the organic layer was concentrated and the resulting residue was dissolved in THF (1 mL) and aq. LiOH (2M solution, 0.598 mL, 1.195 mmol) was added. The mixture was stirred at room temperature for 2 hours. The mixture was evaporated and the residue was diluted in 2 mL water. HCl was added until pH=4 and the aqueous layer was extracted with EtOAc. Then NH4OH was added until pH=10 and the aqueous layer was extracted with EtOAc. The organic layer was dried (sodium sulfate), filtered evaporated and purified by reverse phase HPLC (Stationary Phase: Agilent Eclipse XDB-C18; particle size 1.8 m; column size 4.6 x 50 mm; Mobile phase: eluent/gradient 20-100% CH3CN/H2O (CH3CN and H2O containing 0.1% of TFA)) to give the


26


Page 28 of 49

title compound, 2-(2-(((3'-(Aminomethyl)-[1,1'-biphenyl]-3-yl)amino)methyl)phenyl)acetic acid, (60 mg, yield 73%). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.80 (s, 1 H) 7.52 - 7.61 (m, 1 H) 7.30 - 7.41 (m, 2 H) 7.03 - 7.26 (m, 5 H) 6.85 - 6.91 (m, 1 H) 6.81 (t, J=1.96 Hz, 1 H) 6.77 (ddd, J=8.02, 2.34, 0.88 Hz, 1 H) 4.52 (s, 2 H) 4.09 (s, 2 H) 3.59 (s, 2 H). HRMS calcd. for C22H22N2O2 (M+H)+ 347.1760, found 347.1756. 2-(2-((3'-(Aminomethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic

acid

(9):

A

microwave vial was charged with methyl 2-(2-((3-bromobenzyl)oxy)phenyl)acetate (25) (109 mg, 0.325 mmol), PdCl2(dppf).CH2Cl2 (13.28 mg, 0.016 mmol) and (3-(aminomethyl)phenyl)boronic acid hydrochloride (CAS # 146285-80-5) (76 mg, 0.406 mmol). Acetonitrile (1.3 mL), 2M aqueous K3PO4 (0.488 mL, 0.976 mmol) and water (0.130 mL) were added, the vial was flushed with nitrogen, sealed and heated in the microwave for 60 min at 110°C. THF (4 mL) and 2M aqueous LiOH (0.813 mL, 1.626 mmol) were added and the mixture was heated at 50oC for 4 hours. Additional 2M aqueous LiOH (0.813 mL, 1.626 mmol) was added and the reaction was heated at 50oC overnight. The mixture was acidified to pH 4-5 with 2N HCl, extracted with ethyl acetate, washed with water and brine, dried with sodium sulfate, filtered and concentrated. The crude was purified by preparative HPLC (Stationary phase: Gemini® NX 5µ C18 110A 100x30 mm. Mobile phase: MeCN gradient, water with 0.1% (28% ammonium hydroxide) / acetonitrile) to provide the title compound (30 mg, yield 25%). 1H NMR (400MHz, ACETONITRILE- d3) δ ppm 7.89 - 7.84 (m, 1H), 7.84 - 7.80 (m, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.57 (d, J=7.2 Hz, 1H), 7.40 (dt, J=1.8, 7.6 Hz, 2H), 7.27 - 7.19 (m, 2H), 7.16 (d, J=7.5 Hz, 2H), 6.88 (dt, J=0.9, 7.4 Hz, 1H), 6.83 (d, J=7.8 Hz, 1H), 5.00 (s, 2H), 3.92 (s, 2H), 3.57 (s, 2H). HRMS calcd. for C22H21NO3 (M+H)+ 348.1600, found 348.1589.


27

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2-(2-(((3'-(Aminomethyl)-[1,1'-biphenyl]-3-yl)oxy)methyl)phenyl)acetic acid

(10):

The title compound was prepared in a similar fashion as (9), by starting with the coupling of 3bromophenol (CAS # 591-20-8) and methyl 2-(2-(chloromethyl)phenyl)acetate (CAS # 95360-331). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.71 (s, 1 H) 7.54 (d, J=7.83 Hz, 1 H) 7.13 7.43 (m, 9 H) 7.02 (ddd, J=8.21, 2.53, 0.88 Hz, 1 H) 5.33 (s, 2 H) 3.96 (s, 2 H) 3.60 (s, 2 H). HRMS calcd. for C22H21NO3 (M+H)+ 348.1600, found 348.1593. (R)-2-(2-((3'-(1-Aminoethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic acid (11): Methyl 2-(2-((3-bromobenzyl)oxy)phenyl)acetate (25) (124 mg, 0.370 mmol), (R)-tert-butyl (1(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethyl)carbamate, (CAS# 887254-66-2, 135 mg, 0.388 mmol), and PdCl2(dppf).CH2Cl2 (15.11 mg, 0.018 mmol) were added to a microwave vial. Acetonitrile (1.5 mL), 2M aqueous K3PO4 (0.555 mL, 1.110 mmol) and water (0.300 mL) were added, the vial head space was flushed with nitrogen and the suspension was heated in a microwave for 60 min at 110 oC. At this point, 2 mL of 2M aq. LiOH solution were added and the reaction was heated at 60oC for 2 hours. The mixture was acidified with 2N HCl, extracted with ethyl acetate, dried over sodium sulfate, filtered, and concentrated. The crude product was dissolved in 1:1 TFA/DCM (2 mL) and stirred at room temperature for two hours. The reaction was concentrated and purified by preparative HPLC (Stationary phase: Gemini® NX 5µ C18 110A 100x30 mm. Mobile phase: MeCN gradient, water with 0.1% (28% ammonium hydroxide) / acetonitrile) to provide the title compound (16.5 mg, yield 12%). 1H NMR (400MHz, DMSO-d6) δ ppm 8.36 (s, 1H), 8.12 - 8.09 (m, 1H), 7.69 - 7.62 (m, 2H), 7.47 - 7.35 (m, 3H), 7.33 - 7.26 (m, 1H), 7.13 - 7.06 (m, 2H), 6.93 (d, J=8.5 Hz, 1H), 6.81 (t, J=1.0 Hz, 1H), 5.21 - 5.17 (m, 2H), 4.32 - 4.24 (m, 1H), 3.45 - 3.35 (m, 2H), 1.52 (d, J=6.7 Hz, 3H). HRMS calcd. for C23H23NO3 (M+H)+ 362.1756, found 362.1756.


28


Page 30 of 49

(S)-2-(2-((3'-(1-Amino-2-hydroxyethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic acid (12): To a solution of methyl 2-(2-((3-bromobenzyl)oxy)phenyl)acetate (25) (21 g, 62.7 mmol) in DMF (100 mL) was added 4,4,4’,4’,5,5,5’,5’-octamethyl-2,2’-bi(1,3,2-dioxaborolane) (CAS # 73183-34-3) (22.27 g, 88 mmol), potassium acetate (18.45 g, 188 mmol) and PdCl2(dppf).CH2Cl2 adduct (CAS # 95464-05-4) (1.834 g, 2.506 mmol). The reaction was heated at 110°C for 90 minutes. The organic phase was washed with water, brine, and then dried over Na2SO4 before filtration and evaporation. The crude material was purified by flash column chromatography on silica gel (heptane/EtOAc with 10% MeOH = 100:0 to 70:30) to give methyl 2-(2-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acetate (22 g, 72%). MS (ESI+) m/z 383.1 (M+H).

A mixture of methyl 2-(2-((3-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)benzyl)oxy)phenyl)acetate (8.82 g, 24.3mmol) and (S)-tert-butyl (1-(3bromophenyl)-2-hydroxyethyl)carbamate (CAS# 910308-92-8; 8.14 g, 24.3 mmol) in CH3CN (50 mL)/water (25 mL) with solid K3PO4 (15.5 g, 73 mmol) and PdCl2(dppf).CH2Cl2 adduct (CAS # 95464-05-4) (0.45 g, 0.62 mmol) was heated to 45°C for ca. 15 hours and then diluted with EtOAc and water. The layers were separated and the aqueous phase was extracted with EtOAc. The combined organic phase was washed with water and brine and then dried over Na2SO4. The crude material was purified by flash column chromatography on silica gel (heptane/EtOAc 4:1 to 2:1) to give

(S)-methyl

2-(2-((3'-(1-((tert-butoxycarbonyl)amino)-2-hydroxyethyl)-[1,1'-biphenyl]-3-

yl)methoxy)phenyl)acetate (9.25 g, yield 77%). 1H NMR (400MHz, DMSO- d6) δ ppm 7.70 (s, 1H), 7.56 - 7.67 (m, 2 H), 7.44 - 7.56 (m, 2 H), 7.37 - 7.44 (m, 2 H), 7.18 - 7.33 (m, 4 H) 7.09 (d, J=8.2 Hz, 1H), 6.91 (t, J=7.4 Hz, 1H), 5.18 (s, 2H), 4.79 (t, J=5.6 Hz, 1H), 4.57 HCl in 1,4dioxane (27.5

mL,

110 mmol) was

then added to (S)-methyl 2-(2-((3'-(1-((tert-

butoxycarbonyl)amino)-2-hydroxyethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetate

(9.0 g,


29

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18.3 mmol) and the mixture was stirred 2 hours. The mixture was then directly concentrated to dryness, and to the resulting solid was added THF (30 mL), MeOH (30 mL), water (40 mL) followed by LiOH (1.75 g, 73.2 mmol). The mixture was stirred for 15h and then diluted with Et2O (350 mL) and the resulting white precipitate was collected by filtration. The resulting solid was then added to a stirring solution of 1N aqueous HCl (40 mL) and the mixture was then further diluted with EtOAc. The resulting layers were separated and the aqueous layer was extracted 2 additional times with EtOAc. The organic layers were combined washed with brine, dried over MgSO4, filtered and concentrated. The resulting solid was then neutralized by suspending in MeOH (15 mL) and water (15 mL) and then charging with 7M NH3 in MeOH (2.091 mL, 14.64 mmol) to afford a solution with a pH of ~7. After stirring for 1 h a precipitate formed which was collected by filtration. To the resulting solid was added MeOH and the mixture was heated at reflux for 2 h and then allowed to cool to room temperature to afford a white precipitate that was collected by filtration to furnish the title compound, (S)-2-(2-((3'-(1-amino-2-hydroxyethyl)-[1,1'biphenyl]-3-yl)methoxy)phenyl)acetic acid (3.7 g, 54%). 1H NMR (400MHz, DMSO-d6) 8.24 (s, 1H), 8.01 (s, 1H), 7.67 (t, J=9.3 Hz, 2H), 7.48 - 7.38 (m, 3H), 7.30 (d, J=7.6 Hz, 1H), 7.13 - 7.07 (m, 2H), 6.94 (d, J=8.2 Hz, 1H), 6.81 (t, J=7.0 Hz, 1H), 5.20 (s, 2H), 4.17 (dd, J=4.6, 6.6 Hz, 1H), 3.77 - 3.65 (m, 2H), 3.46 (d, J=15.3 Hz, 1H), 3.36 (d, J=15.4 Hz, 1H). HRMS calcd. for C23H23NO4 (M+H)+ 378.1706, found 378.1721. (R)-2-(2-((3'-(1-amino-3-hydroxypropyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic

acid

(13): The title compound was prepared in a similar fashion as 12, employing (R)-3-amino-3-(3bromophenyl)propan-1-ol hydrochloride (CAS # 1213637-86-5).

1H

NMR (400 MHz,

METHANOL-d4)  ppm 8.12 (s, 1 H) 8.03 (s, 1 H) 7.66 (dt, J=7.95, 1.28 Hz, 1 H) 7.60 (d, J=7.58 Hz, 1 H) 7.43 (q, J=7.74 Hz, 2 H) 7.35 - 7.39 (m, 1 H) 7.32 (d, J=7.58 Hz, 1 H) 7.17 (qd, J=7.44,


30


Page 32 of 49

1.65 Hz, 2 H) 6.96 (d, J=7.70 Hz, 1 H) 6.87 (td, J=7.40, 0.98 Hz, 1 H) 5.13 - 5.24 (m, 2 H) 4.41 (t, J=7.21 Hz, 1 H) 3.59 - 3.69 (m, 2 H) 3.45 - 3.58 (m, 2 H) 2.21 - 2.32 (m, 1 H) 2.09 - 2.19 (m, 1 H). HRMS calcd. for C24H25NO4 (M+H)+ 392.1862, found 392.1852. (S)-tert-Butyl

2-(2-((5-bromo-3'-(1-((tert-butoxycarbonyl)amino)-2-hydroxyethyl)-[1,1'-

biphenyl]-3-yl)methoxy)phenyl)acetate

(14): The synthesis of the title compound was

accomplished via the union of (S)-tert-butyl (2-hydroxy-1-(3-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)ethyl)carbamate

and

tert-butyl

2-(2-((3,5-

dibromobenzyl)oxy)phenyl)acetate as described below. (S)-tert-butyl

(2-hydroxy-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl)ethyl)carbamate was prepared as follows: to a solution of

(S)-tert-butyl (1-(3-

bromophenyl)-2-hydroxyethyl)carbamate (CAS# 910308-92-8; 5.8 g, 18.34 mmol) and 4,4,4’,4’,5,5,5’,5’-octamethyl-2,2’-bi(1,3,2-dioxaborolane) (CAS # 73183-34-3; 9.32 g, 36.7 mmol) in DMF (55.0 mL) was added KOAc (5.4 g, 55.0 mmol); this mixture was degassed for 10 minutes with N2(gas), and then PdCl2(dppf).CH2Cl2 adduct (0.750 g, 0.917 mmol) was added. The reaction was sealed and heated at 110 °C in an oil bath for 16 h. The reaction was cooled to room temperature, filtered over a plug of Celite® and the filtrate was partitioned between EtOAc/H2O and the layers separated; the organic phase was washed with brine, combined, dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (heptanes/EtOAc = 100:0 to 50:50) to afford the desired boronic ester (5.0 g, 75%).

1H

NMR

(400 MHz, DMSO-d6) δ ppm 7.61 (s, 1 H) 7.53 (d, J=7.20 Hz, 1 H) 7.37 - 7.42 (m, 1 H) 7.29 7.34 (m, 1 H) 7.27 (d, J=8.46 Hz, 1 H) 4.74 (t, J=5.81 Hz, 1 H) 4.49 (d, J=5.68 Hz, 1 H) 3.41 3.57 (m, 2 H) 1.36 (br. s, 9 H), 1.30 (s, 12 H).


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tert-butyl 2-(2-((3,5-dibromobenzyl)oxy)phenyl)acetate was prepared as follows: tertbutyl 2-(2-hydroxyphenyl)acetate (CAS# 258331-10-1; 0.84 g, 4.0 mmol) was dissolved in DMF (20.2 mL) and K2CO3 (0.641 g, 4.64 mmol) was added followed by 1,3-dibromo-5(bromomethyl)benzene (CAS # 56908-88-4; 1.46 g, 4.44 mmol). After stirring overnight at room temperature the reaction was diluted with ethyl acetate and water. The organic phase was washed with water, dried with MgSO4, filtered and concentrated. The reaction was purified by flash chromatography (0-30% EtOAc:Heptane) to provide the requisite di-bromo intermediate (1.78 g, yield 97%). 1H NMR (400 MHz, DMSO-d6)  ppm 7.79 (t, J=1.77 Hz, 1 H) 7.66 (d, J=1.77 Hz, 2 H) 7.16 - 7.29 (m, 2 H) 6.98 (d, J=7.58 Hz, 1 H) 6.92 (td, J=7.42, 0.95 Hz, 1 H) 5.13 (s, 2 H) 3.55 (s, 2 H) 1.34 (s, 9 H). To a solution of (S)-tert-butyl (2-hydroxy-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl)ethyl)carbamate

(0.98

g,

2.3

mmol)

and

tert-Butyl

2-(2-((3,5-

dibromobenzyl)oxy)phenyl)acetate (1.0 g, 2.19 mmol) in acetonitrile (20 mL) and water (2 mL) was added 2M aq. K3PO4 (3.3 mL, 6.6 mmol) and PdCl2(dppf).CH2Cl2 adduct (0.090 g, 0.110 mmol). The mixture was stirred at 100 oC for 1h, at which time the mixture was cooled to rt, diluted with EtOAc and water and the layers were separated. The aq. Layer was extracted two times with EtOAc and the combined organic layers were dried over MgSO4, filtered and concentrated. The resulting residue was purified by flash chromatography (0-100% EtOAc:Heptane)

to

provide

(S)-2-(2-((5-bromo-3'-(1-((tert-butoxycarbonyl)amino)-2-

hydroxyethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic acid (568 mg, 42%). MS (ESI+) m/z 612.1, 614.1 (M+H). Finally the title compound was prepared as follows, (S)-2-(2-((5-bromo-3'(1-((tert-butoxycarbonyl)amino)-2-hydroxyethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)acetic acid (100 mg, 0.16 mmol) in DCM (1.5 mL) was treated with TFA (1.5 mL) and stirred for 45


32


Page 34 of 49

minutes at which time the mixture was concentrated to dryness and purified by preparative HPLC (Stationary phase: Gemini® NX 5µ C18 110A 100x30 mm. Mobile phase: acetonitrile gradient, water with 0.1% (28% ammonium hydroxide) / acetonitrile)t to provide 14 (46 mg, yield 92%). 1H

NMR (400 MHz, METHANOL-d4)  ppm 8.17 (s, 1 H) 8.06 (s, 1 H) 7.74 - 7.80 (m, 1 H) 7.69

(d, J=7.96 Hz, 1 H) 7.55 (s, 1 H) 7.48 (t, J=7.71 Hz, 1 H) 7.36 (d, J=7.71 Hz, 1 H) 7.12 - 7.23 (m, 2 H) 6.95 (d, J=7.71 Hz, 1 H) 6.82 - 6.92 (m, 1 H) 5.11 - 5.25 (m, 2 H) 4.36 (dd, J=8.72, 4.55 Hz, 1 H) 3.95 (dd, J=11.68, 8.78 Hz, 1 H) 3.86 (dd, J=11.68, 4.61 Hz, 1 H) 3.50 - 3.68 (m, 2 H). HRMS calcd. for C23H22BrNO4 (M+H)+ 456.0810 and 458.0790, found 456.0804 and 458.0809. (S)-2-(2-((3'-(1-Amino-2-hydroxyethyl)-5-(hydroxymethyl)-[1,1'-biphenyl]-3yl)methoxy)phenyl)acetic acid (15): The title compound was prepared as described for 14. 1H NMR (400 MHz, METHANOL-d4) δ ppm 8.17 (s, 1 H) 7.99 (s, 1 H) 7.73 (d, J=8.07 Hz, 1 H) 7.61 (s, 1 H) 7.46 (t, J=7.70 Hz, 1 H) 7.29 - 7.38 (m, 2 H) 7.12 - 7.21 (m, 2 H) 6.96 (d, J=7.70 Hz, 1 H) 6.87 (td, J=7.37, 0.92 Hz, 1 H) 5.14 - 5.26 (m, 2 H) 4.70 (s, 2 H) 4.35 (dd, J=8.86, 4.58 Hz, 1 H) 3.95 (dd, J=11.74, 8.93 Hz, 1 H) 3.86 (dd, J=11.68, 4.58 Hz, 1 H) 3.51 - 3.68 (m, 2 H). HRMS calcd. for C24H25NO5 (M+H)+ 408.1811, found 408.1802. This synthesis required employment of tert-butyl 2-(2-((3-bromo-5-(hydroxymethyl)benzyl)oxy)phenyl)acetate which is prepared as follows: To a mixture of (5-bromo-1,3-phenylene)dimethanol (CAS# 51760-22-6 0.208 g, 0.960 mmol), tert-butyl 2-(2-hydroxyphenyl)acetate (CAS# 258331-10-1; 0.20 g, 0.96 mmol), and PPh3 (0.25 g, 0.96 mmol) at 0 oC was added DIAD (0.187 ml, 0.960 mmol). The mixture was allowed to warm to rt and stirred overnight, an then was quenched with water, extracted with EtOAc, dried over MgSO4, filtered and concentrated.

The residue was partially by purified by flash

chromatography (40g, 0-60% EtOAc:Heptanes) and the semi-purified material could be used


33

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directly in the next step without the need for further purification MS (ESI+) m/z 350.9, 352.9 (M(t-Bu)). (S)-2-(2-((3'-(1-Amino-2-hydroxyethyl)-5-(methoxymethyl)-[1,1'-biphenyl]-3yl)methoxy)phenyl)acetic acid (16): The title compound was prepared in a similar manner as 15. 1H

NMR (400 MHz, METHANOL-d4)  ppm 7.91 (s, 1 H) 7.82 (s, 1 H) 7.73 (dt, J=8.08, 1.26 Hz,

1 H) 7.59 (s, 1 H) 7.52 (t, J=7.77 Hz, 1 H) 7.36 - 7.45 (m, 2 H) 7.16 - 7.25 (m, 2 H) 6.97 - 7.06 (m, 1 H) 6.90 (td, J=7.45, 1.01 Hz, 1 H) 5.20 (s, 2 H) 4.55 (s, 2 H) 4.41 (dd, J=7.83, 4.93 Hz, 1 H) 3.85 - 3.98 (m, 2 H) 3.59 - 3.73 (m, 2 H) 3.42 (s, 3 H). HRMS calcd. for C25H27NO5 (M+H)+ 422.1967, found 422.1954. (S)-2-(2-((3'-(1-Amino-2-hydroxyethyl)-5-(2-hydroxypropan-2-yl)-[1,1'-biphenyl]-3yl)methoxy)phenyl)acetic acid (17): The title compound was prepared in a similar manner as 15. 1H

NMR (400 MHz, METHANOL-d4)  ppm 8.17 (s, 1 H) 7.95 (s, 1 H) 7.67 - 7.78 (m, 2 H) 7.42

- 7.52 (m, 2 H) 7.32 (d, J=7.71 Hz, 1 H) 7.11 - 7.22 (m, 2 H) 6.97 (d, J=7.83 Hz, 1 H) 6.83 - 6.91 (m, 1 H) 5.13 - 5.26 (m, 2 H) 4.35 (dd, J=8.97, 4.55 Hz, 1 H) 3.96 (dd, J=11.62, 8.97 Hz, 1 H) 3.86 (dd, J=11.68, 4.61 Hz, 1 H) 3.50 - 3.69 (m, 2 H) 1.60 (s, 6 H). HRMS calcd. for C26H29NO5 (M+H)+ 436.2124, found 436.2121. This synthesis required employment of tert-butyl 2-(2-((3bromo-5-(2-hydroxypropan-2-yl)benzyl)oxy)phenyl)acetate which was prepared by the following procedure.

To

a

solution

of

tert-butyl

2-(2-((3-bromo-5-

(hydroxymethyl)benzyl)oxy)phenyl)acetate, prepared as described in 14 (28.5 g, 70.0 mmol) in DCM (350 mL) under nitrogen at room temperature, MnO2 (122 g, 1399 mmol) was added and this was heated at 40 °C. After overnight the reaction was filtered through Celite®, concentrated and purified directly by flash chromatography (100% DCM) to provide tert-butyl 2-(2-((3-bromo5-formylbenzyl)oxy)phenyl)acetate (10.3 g, yield 36%). 1H NMR (400 MHz, DMSO-d6)  ppm


34


Page 36 of 49

9.99 (s, 1 H) 8.05 (s, 1 H) 7.97 (s, 2 H) 7.16 - 7.30 (m, 2 H) 7.03 (d, J=8.08 Hz, 1 H) 6.93 (t, J=7.33 Hz, 1 H) 5.23 (s, 2 H) 3.57 (s, 2 H) 1.32 (s, 9 H). To a suspension of tert-butyl 2-(2-((3-bromo-5formylbenzyl)oxy)phenyl)acetate (0.30 g, 0.74 mmol) in MeOH (7.40 mL) under nitrogen, a solution of KOH (0.125 g, 2.22 mmol) in MeOH (3 mL) was added. This was cooled to 0 °C and a solution of iodine (0.244 g, 0.962 mmol) in MeOH (3 mL) was added. After overnight the reaction was quenched with saturated aqueous sodium thiosulfate, extracted with EtOAc, washed with water, dried with MgSO4, filtered and concentrated to give methyl 3-bromo-5-((2-(2-(tertbutoxy)-2-oxoethyl)phenoxy)methyl)benzoate (0.28 g, yield 87%), without the need for further purification. 1H NMR (400 MHz, DMSO-d6)  ppm 7.97 - 8.06 (m, 2 H) 7.94 (t, J=1.71 Hz, 1 H) 7.15 - 7.31 (m, 2 H) 7.02 (d, J=7.58 Hz, 1 H) 6.92 (td, J=7.42, 0.95 Hz, 1 H) 5.21 (s, 2 H) 3.87 (s, 3 H) 3.55 (s, 2 H) 1.32 (s, 9 H). To a solution of methyl 3-bromo-5-((2-(2-(tert-butoxy)-2oxoethyl)phenoxy)methyl)benzoate (0.28 g, 0.643 mmol) in THF (6.43 mL) at 0 °C under nitrogen, MeMgBr (3.0M in Ether, 0.643 mL, 1.93 mmol) was added and the reaction was warmed to room temperature. After 30 minutes additional MeMgBr (0.107 mL) was added. After 10 more minutes the reaction was quenched with saturated aqueous NH4Cl, extracted 2x with EtOAc, washed with water, dried with MgSO4, filtered and concentrated. The reaction was purified by flash chromatography (0-60% EtOAc:Heptanes) to provide the tert-butyl 2-(2-((3-bromo-5-(2hydroxypropan-2-yl)benzyl)oxy)phenyl)acetate (0.26 g, yield 93%). 1H NMR (400 MHz, DMSOd6)  ppm 7.60 (t, J=1.71 Hz, 1 H) 7.50 (s, 1 H) 7.43 - 7.49 (m, 1 H) 7.15 - 7.29 (m, 2 H) 7.01 (d, J=7.70 Hz, 1 H) 6.85 - 6.94 (m, 1 H) 5.18 (s, 1 H) 5.10 (s, 2 H) 3.54 (s, 2 H) 1.42 (s, 6 H) 1.32 (s, 9 H). (S)-2-(2-((3'-(1-Amino-2-hydroxyethyl)-5-(isopropylamino)-[1,1'-biphenyl]-3yl)methoxy)phenyl)acetic acid (18): The title compound was prepared in a similar fashion as 14,


35

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using tert-butyl 2-(2-((3-chloro-5-(isopropylamino)benzyl)oxy)phenyl)acetate.

1H

NMR (400

MHz, METHANOL-d4) δ ppm 7.80 (s, 1 H) 7.72 - 7.77 (m, 1 H) 7.64 (s, 1 H) 7.57 (t, J=7.71 Hz, 1 H) 7.45 - 7.50 (m, 1 H) 7.37 (s, 1 H) 7.29 (s, 1 H) 7.20 - 7.26 (m, 2 H) 7.00 (d, J=7.83 Hz, 1 H) 6.94 (td, J=7.45, 1.01 Hz, 1 H) 5.25 (s, 2 H) 4.45 (dd, J=8.02, 4.36 Hz, 1 H) 3.77 - 4.04 (m, 3 H) 3.71 (s, 2 H) 1.32 (d, J=6.44 Hz, 6 H). HRMS calcd. for C26H30N2O4 (M+H)+ 435.2239, found 435.2270. tert-butyl 2-(2-((3-chloro-5-(isopropylamino)benzyl)oxy)phenyl)acetate was prepared as follows. A pressure vessel was charged with a solution of tert-butyl 2-(2-((3-bromo-5chlorobenzyl)oxy)phenyl)acetate [prepared in a similar manner as tert-butyl 2-(2-((3,5dibromobenzyl)oxy)phenyl)acetate described for the synthesis of 14] (400 mg, 0.972 mmol) in MeCN (2 mL) was added Cs2CO3 (950 ng, 2.91 mmol) followed by BrettPhos Pd G1, Methyl tButyl Ether Adduct (39 mg, 0.05mmol) and isopropylamine (0.16 mL, 1.94 mmol). The vessel was sealed and heated at 110 oC for 1h. The mixture was then cooled to rt and diluted with EtOAc and water. The layers were separated and the organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by flash chromatography (0-60%

EtOAc:Heptane)

to

provide

tert-butyl

2-(2-((3-chloro-5-

(isopropylamino)benzyl)oxy)phenyl)acetate (150 mg, 40%). MS (ESI+) m/z 390.1 (M+H). 3'-(((tert-Butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3-carboxylic acid (20): A degassed mixture of 3-bromobenzoic acid (CAS # 585-76-2) (1 g, 4.97 mmol), 3-((tertbutoxycarbonylamino)methyl)phenylboronic acid (19, CAS # 832114-05-3) (1.25 g, 4.97 mmol), PdCl2(dppf).CH2Cl2 adduct (CAS # 95464-05-4) (0.20 g, 0.249 mmol), and 2M aq. K3PO4 (7.46 ml, 14.92 mmol) in CH3CN (25 mL) was heated at 90 °C for 2 h. The reaction mixture was diluted with EtOAc and washed with water. The aqueous layer was extracted with EtOAc. The combined


36


Page 38 of 49

organics were washed with brine, dried (Na2SO4) and concentrated. The residue was purified by FCC (EtOAc-heptane 0-100%) to provide the title compound (1.3 g, 80%). MS (ESI-) m/z 326.2 (M-H). Methyl 2-(2-(6-chloropicolinamido)phenyl)acetate (22): HATU (2.26 g, 5.95 mmol) was added in one portion at room temperature to a DMF solution (10 mL) of methyl 2-(2aminophenyl)acetate hydrochloride (CAS # 49851-36-7) (1g, 4.96 mmol), 6-chloropyridine-2carboxylic acid (CAS # 4684-94-0) (0.938 g, 5.95 mmol) and DIPEA (2.165 mL, 12.40 mmol). The resulting solution was allowed to stir at room temperature (2.5 hr). The reaction was diluted with ethyl acetate, washed with water (twice) and a saturated aq. NaCl solution. The organic phase was dried over sodium sulfate, filtered and concentrated. The residue was purified via FCC (10:1 Heptane/ethyl acetate to 1:1 Heptane/ethyl acetate) to give the title compound (1.07 g, yield 71%). MS (ESI+) m/z 305.3 (M+H). Methyl 2-(2-((3-bromobenzyl)oxy)phenyl)acetate (25): To a DMF (100 mL) suspension of methyl 2-(2-hydroxyphenyl)acetate (CAS # 22446-37-3) (10 g, 60.2 mmol) and K2CO3 (9.56 g, 69.2 mmol) was added 3-bromobenzyl bromide (CAS # 823-78-9) (16.54 g, 66.2 mmol). The mixture was stirred at room temperature for 18 hours. The mixture was diluted with EtOAc and water. The organic phase was washed four times with water, once with brine, and then dried over Na2SO4 before filtration and evaporation. The resulting title compound could be used without the need for further purification compound (21 g, yield 94%). MS (ESI+) m/z 334.9, 336.9 (M+H).

Biological and in vivo experiments The Membrane attack complex (MAC) formation assay in 50% human whole blood (WB), the generation of the human FD knock-in mouse, and the LPS challenge model have previously been


37

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described in reference.10 Protocols, handling and care of animals were in accordance with the policy of the NIBR Cambridge Animal Care and Use Committee. Human complement FD TR-FRET assay Recombinant human FD (expressed in E. coli and purified using standard methods) labeled with biotin (10 nM), europium-labeled streptavidin (2 nM) and 2-((1E,3E,5E)-5-(1-(6-((((3S,5S)-1((1-carbamoyl-1H-indol-3-yl)carbamoyl)-5-((3-chloro-2-fluorobenzyl)carbamoyl)-3fluoropyrrolidin-3-yl)methyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indol-1-ium (Supporting Information Example 2.1.1) (10 nM) was incubated with test compound at various concentrations up to 2 hours at room temperature in 50 mM HEPES buffer, pH 7.4, containing 2.5 mM MgCl2, 0.01% (w/v) BSA and 0.05 % (w/v) CHAPS. The time-gated decrease in fluorescence intensity related to the competition between labeled and unlabeled FD ligands was recorded at both 620 nm and 665 nm, 70 µs after excitation at 337 nm using a microplate spectrofluorimeter. IC50 values were calculated from percentage of inhibition of FD-2-((1E,3E,5E)-5-(1-(6-((((3S,5S)-1-((1carbamoyl-1H-indol-3-yl)carbamoyl)-5-((3-chloro-2-fluorobenzyl)carbamoyl)-3fluoropyrrolidin-3-yl)methyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indol-1-ium (Supporting Information Example 2.1.1) displacement as a function of test compound concentration. Local ocular injury model of complement activation The local ocular injury model of complement activation has been previously described.26 Briefly compound 12 was administered via oral gavage in human FD knock-in mice at time 0. Two hours later, mice were anesthetized and 1µL sterile PBS was injected intravitreally per mouse


38


Page 40 of 49

eye. Eye tissues were collected 4 hours post intravitreal injection. Eye lysates were prepared and analyzed for complement Ba levels by Western blot. Plasma pharmacokinetic studies in mice. The pharmacokinetics of compounds 3, 9 and 12-14 were determined in C57BL/6 mice. The compounds were dosed intravenously (IV, via injection into jugular vein, 1 mg/kg, n=2 animals/compound); orally (PO, via oral gavage, 10 mg/kg, n=3 animals/compound). The IV solution formulations were prepared at 1mg/mL as described in the parenthesis for each compound – 3 (30% polyethylene glycol 300, 50% of 20% Cremophor EL, phosphate buffered saline), 9 (2 eq 1N HCl, 20%polyethylene glycol, 50% of 20% Cremophor EL, phosphate buffered saline), 12 (2 equivalents of 1 N HCl, 10% propylene glycol, 25% of a 20% solutol solution), 13 (10% polyethylene glycol 300, 25% of a 20% cremophor EL solution, phosphate buffered saline) and 14 (10% propylene glycol, 50% of a 20% solutol solution, phosphate buffered saline). The PO formulations were prepared as suspensions at 5mg/mL as described in the parenthesis for each compound – 3 (30% PEG300, 50% of 20% cremophor EL, phosphate buffered saline), 9 (0.1% Tween 80 and 0.5% methyl cellulose), 12 (0.5% Tween 80 and 0.5% methyl cellulose), 13 (0.5% Tween 80 and 0.5% methyl cellulose) and 14 (0.5% pluronic F68 and 0.5% hydroxypropyl cellulose). Approximately 50 L of whole blood was collected from the tails at 5 min (IV dose only), 15 min (PO dose only), 0.5, 1, 2, 4, and 7 hours post-dose and was transferred to EDTA tubes. Blood was centrifuged at 3,000 rpm and the resultant plasma was transferred to a capped PCR 96-well plate, and frozen at –20 °C until subsequent analysis by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The relevant pharmacokinetic parameters were estimated using non-compartmental methods using WinNonlin


39

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(Enterprise, Version 5.2) purchased from Pharsight Corporation (St. Louis, MO) or Watson LIMS (Thermo, Waltham, MA). Plasma pharmacokinetic studies in rats The pharmacokinetics of compound 12 was determined in Wistar Han rats. The compound was dosed intravenously (IV, via injection into the jugular vein catheter; 1 mg/kg, n=3 animals) and orally (PO, via oral gavage; 10 mg/kg, n=2 animals). The IV solution formulation was prepared in phosphate buffered saline containing 2% 1N HCl, polyethylene glycol 300, 50% of a 20% solutol solution. The PO formulation was a suspension in 20% captisol. Approximately 200 L of whole blood was collected from the tails at 5 min (IV dose only), 15 min, 0.5, 1, 2, 4, 7, and 24 hours post-dose and was transferred to EDTA tubes. Blood was centrifuged at 3,000 rpm and the resultant plasma was transferred to a capped PCR 96-well plate, and frozen at –20 °C until subsequent analysis by HPLC-MS/MS. The relevant pharmacokinetic parameters were estimated using non-compartmental methods using WinNonlin (Enterprise, Version 5.2) purchased from Pharsight Corporation (St. Louis, MO) or Watson LIMS (Thermo, Waltham, MA). Ocular pharmacokinetic studies in rats Three month old Brown Norway rats were administered, 12 via oral gavage as a suspension in 0.5% hydroxypropyl methyl cellulose and 0.1% Tween 80. Ocular tissues from both eyes and plasma were collected from 2 rats per timepoint at 0.25, 0.5, 1, 3, 6, and 24 hours after administration. The ocular tissues collected were the retina and the posterior eye cup (RPE/choroid and posterior sclera). The tissues were diluted with phosphate buffered saline containing 10% acetonitrile and homogenized, centrifuged prior to analyses. The concentrations of the test article were measured in plasma and supernatants of ocular homogenates by HPLCMS/MS in 4 individual retinas, 4 individual posterior eye cups, and 2 individual plasma samples


40


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at each timepoint. The relevant pharmacokinetic parameters were estimated using noncompartmental methods using WinNonlin (Enterprise, Version 5.2) purchased from Pharsight Corporation (St. Louis, MO) or Watson LIMS (Thermo, Waltham, MA). Plasma pharmacokinetic studies in dogs The pharmacokinetics of compound 12 was determined in beagle dogs. The compound was dosed intravenously (1 mg/kg, n=3 animals) and orally (10 mg per kg, n=3 animals/compound) by gavage. The IV formulation was a solution consisting of 2% 1N HCl, 20% polyethylene glycol 300, 50% of 20% solutol in phosphate buffered saline, whereas the PO formulation was a solution consisting of 20% polyethylene glycol 300, 10% solutol HS15 and 70% citrate buffer pH 3. Blood was collected at 5 min (IV dose only), 15 min, 0.5, 1, 2, 4, 7, and 24 hours post-dose. The subsequent blood or plasma samples were analyzed by HPLC-MS/MS under electrospray ionization in positive mode. Similarly, the relevant pharmacokinetic parameters were estimated using non-compartmental methods using WinNonlin (Enterprise, Version 5.2) purchased from Pharsight Corporation (St. Louis, MO) or Watson LIMS (Thermo, Waltham, MA). Other relevant calculations were performed in Microsoft Excel. Molecular modeling Docking poses were generated in a high resolution crystal structure of FD bound with 1 (PDB entry 6FUT13) using Glide (version 5.8)17 from Schrödinger. The crystal structure was first prepared using the standard protein preparation wizard within the Maestro interface27. Grid files were generated for docking after deleting the crystallographic water molecules and the succinic acid, a reagent used for crystallization. 3D-geometries of compounds were generated using ligPrep.27 Compounds were docked in the pre-generated grid files in standard precision mode. Three docking poses were saved for each compound and the docking results were analyzed visually.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website at DOI: Crystallographic structure determination of FD in complex with compounds 3 and 12; preparation of reagents for TR-FRET assay. Molecular formula strings and some data. Accession Codes Atomic coordinates and structure factors for the crystal structures of FD with compounds 3 and 12 are deposited in the protein data bank with accession codes 6QMT.pdb and 6QMR.pdb, respectively. Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Author *RGK Tel: +1 617-871-3913. E-mail: [email protected] Present Addresses §Kaleido

Biosciences, 65 Hayden Ave, Lexington, MA, 02421.

║Kymera

Therapeutics, 300 Technology Square, Cambridge, MA, 02139, USA.

§Janssen ¶

Research & Development, 3210 Merryfield Row, San Diego, CA 92121

Drug Discovery Biology, Idorsia Pharmaceuticals Ltd., Allschwil, Switzerland

¥Biogen,

225 Binney Street, Cambridge MA, 02142, USA.


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ACKNOWLEDGMENT The authors would like to thank Frederic Villard, Wendy Lee, Jakal Amin, Timothy Drew, Adam Amaral, Julia Kamholz, Peter Wipfli, Roland Feifel, Viral Kansara and Jaimie Spear for their excellent technical assistance, Jürgen Maibaum, Richard Sedrani, José Duca, and Jörg Eder for guidance and support. ABBREVIATIONS FD Complement Factor D; FXIa Factor XIa; AP alternative complement pathway; AMD agerelated macular degeneration; PNH paroxysmal nocturnal hemoglobinuria; MAC Membrane attack complex; FCC flash column chromatography; SEMCl 2-(Trimethylsilyl)ethoxymethyl chloride. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors declare no competing financial interest and all work described was funded by Novartis Institutes for BioMedical Research Inc. References 1. Thurman, J. M.; Holers, V. M. The central role of the alternative complement pathway in human disease. J. Immunol. 2006, 176, 1305-1310. 2. Lesavre, P. H.; Mullereberhard, H. J. Mechanism of action of factor-D of alternative complement pathway. J. Exp. Med. 1978, 148, 1498-1509.


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Table of Contents Graphic

H N H

O SBDD for potency and protease selectivity Med. Chem. optimization

O OH O

H 2N

HO 1

(S)

NH2

FD IC50=37 µM

12 FD IC50=0.012 µM Selective, orally bioavailable and in vivo efficacious


49

Design, synthesis and pre-clinical characterization of selective Factor

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