Design, Synthesis, and Preclinical Characterization of Selective Factor D Inhibitors Targeting the Alternative Complement Pathway

Article abstract of DOI:10.1021/acs.jmedchem.9b00271

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, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria. 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 structure-based 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-induced AP activation model as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.

Full text of DOI:10.1021/acs.jmedchem.9b00271

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Article
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.
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*
†
†
†,║
†,¥
†,§
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),
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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 structure-
based 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.
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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 (H O) to generate an active C3 convertase (C3bBb) that
2
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
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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 ACH-
4471. 1
4, 15
We have previously reported the discovery of the benzylamine-based FD inhibitors (1
13
and 2, Figure 2) targeting an active conformation of FD. In this report we describe the structure-
based optimization of 1 that led to the discovery of 12, an orally bioavailable and selective FD
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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 injection-
induced AP activation model in mice expressing human FD.
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O
H
H
N
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O
H N
2
H N
2
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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
catalytically active FD.
, 12, 13
and a
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Figure 3. FD conformational change upon substrate or benzylamine binding. Crystal structure of
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inactive FD (5FBE.pdb , white) is shown relative to active FD (6FUT.pdb , 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
1
3
with FD (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
1
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FOCUS (Molsoft, L.L.C.) was employed. The 3D-editor tool provides a simple interface to
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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
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(vide infra) and assessed for FD inhibition via a time-resolved fluorescence energy transfer (TR-
FRET) in vitro assay. Compound 3 inhibited FD with a TR-FRET IC =120 nM, an almost 270-
50
fold 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
1
0
blood. 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,
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docking analysis of 3 using Glide (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
1
3
carboxylic acid and phosphonic acid at the oxyanion hole have been reported earlier with FD and
other S1 serine proteases,18-20 albeit with different chemotypes.
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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; IC =2.8 µM; 23-fold) and tryptase-β2 (IC =2.3 µM; 19-fold) were identified
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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)
1
3
shown relative to X-ray of 1 (cyan, 6FUT.pdb ). Alternate positions of succinic acid co-
crystallized 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
X
N
N
H
Y
H
N
O
O
1
1
1
1
1
1
1
1
1
1
2
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3
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3
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3
4
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5
5
5
5
5
5
6
0
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0
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R1
OH
OH
8 X=NH, Y=CH2
3
4
5
R =COOH
1
7
R =COOCH
1
3
9
X=CH , Y=O
R =2H-tetrazole
1
2
NH2
NH2
NH2
10 X=O, Y=CH2
6 R =SO NHCOCH
3
1
2
Compound FD TR-FRET Human 50% WB Log Papp cm/s
a
b
c
IC (µM)
MAC IC (µM)
(permeability rank)
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3
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9
1
32
NDd
4.80
NDd
NDd
NDd
0.02
2.4
-3.9 (high)
0.120
13.6
64
<-5.3 (low)
-4.3 (high)
<-5.3 (low)
<-5.5 (low)
-5.2 (medium)
-5.0 (medium)
-4.5 (high)
8.2
0.03
0.270
0.008
2.5
0.270
>100
0
-4.8 (medium)
a
Half-maximal inhibition of recombinant human complement FD as determined in a TR-FRET
b
assay. Half-maximal inhibition of soluble MAC complex formation, as determined in an ELISA
assay. Permeability measurement by PAMPA. ND=Not determined. Data represent mean values
of duplicate measurements
c
d
Compound 3 had good solubility (0.22 mM at pH 6.8) but poor permeability as assessed by a
PAMPA assay (Table 1). When administered to C57BL6 mice at 1mg/kg (IV) and 10mg/kg (PO),
3
exhibited moderate clearance (22 mL/min/kg), and 10% oral bioavailability. The low oral
bioavailability was presumed to be due to permeability limitations consistent with relatively low
Papp value in the PAMPA assay.
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With this profile in hand, the goal for medicinal chemistry optimization became improving
permeability, protease selectivity and potency. To this end we routinely used in vitro permeability
(PAMPA assay) to guide our optimization efforts and prioritize compounds for pharmacokinetic
studies.
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Initial efforts to improve permeability focused on carboxylic acid of 3. Methyl ester analog 4
resulted in a 113-fold (IC =13.6 µM) reduction in activity compared to 3. Replacing the
50
carboxylic acid with bioisosteres also negatively impacted biochemical potency (5 and 6).
Compound 7 was synthesized with the goal of restricting exposure of polar functional groups by
promoting intra-molecular H-bonding of the pyridine nitrogen with the amide N-H. While 7
showed a significant improvement in potency (TR-FRET IC =0.03 µM and MAC IC =0.02 µM),
5
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it showed only a marginal improvement in permeability (Table 1). We next turned our attention
toward removing the amide linker of 3 altogether, as it was not directly interacting with FD and
posed a potential liability as it pertains to permeability. Compound 8, lacking the carbonyl group
showed a significant improvement in permeability while maintaining modest biochemical potency.
Compound 9 with an ether linker provided the breakthrough in potency and permeability (Table
1
, TR-FRET IC =0.008 µM; MAC IC =0.27 µM, Papp = -4.5 log cm/s). Interestingly, the
50 50
position of the oxygen in the ether linker (i.e 9 vs 10) had a significant impact on biochemical and
functional potency for reasons that are not clear at this time.
To study the impact of the improved PAMPA permeability, 9 was assessed in a mouse
pharmacokinetic study (1mg/kg IV and PO at 10 mg/kg; C57BL6 mice), where 9 showed low
clearance (~14mL/min/kg) and excellent oral bioavailability (100%). With regards to protease
selectivity, 9 exhibited <100-fold selectivity on only 3 of 50 proteases evaluated (FXIa IC =0.630
5
0
µM, 80-fold; tryptase-β2 IC =0.360 µM, 45-fold; and urokinase IC =0.77 µM, 96-fold). While
5
0
50
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encouraged by the off-target profile we recognized an opportunity to further enhance the selectivity
while maintaining permeability.
In an attempt to address the selectivity question, we compared the FD bound 3 X-ray structure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
with a reported FXIa structure (Figure 5). Upon close examination of the loop spanning residues
Ser214-Gly218 in FXIa, we observed Gly216 of FXIa making a van der Waals contact (3.5Å) with
the benzylic carbon atom of 3, which may account for the weaker activity against FXIa. The
corresponding residue in FD is at a distance of 6.3Å because of the conformational difference of
the self-inhibitory loop spanning residues Thr214-Val219 (Figure 5). Upon analysis of this loop
2
2
23
in published crystal structures of other key off-targets tryptase-β2 and urokinase , we found the
loops of these proteases to be more similar to FXIa than FD, presenting an opportunity to confer
selectivity by focusing on the Gly216 region.
21
Figure 5. Crystal structure of 3 bound with FD (yellow and white carbons) and FXIa (green
carbons). Only selected residues in the ligand-binding pocket are shown. FD residues are labeled
in black and FXIa in green.
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9
1
1
1
1
1
1
1
1
1
1
2
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2
3
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4
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5
5
5
5
5
5
6
We designed and analyzed substitutions on the benzylic carbon atom of 9 and predicted that
even a methyl substituent would make enough of a steric clash to further attenuate affinity with
off-target proteases while maintaining FD binding affinity. We also rationalized that the extra
space offered by FD may enable placement of a moiety capable of H-bonding with Thr214.
Furthermore, we were cognizant of the potential metabolic liability associated with an
unsubstituted benzylamine.24 Thus compounds 11-14 (Table 2) were synthesized with the aim of
improving potency, selectivity and metabolic profile. In practice, we found off-target protease
potency was in fact reduced, with compounds becoming >200-fold weaker relative to FD
biochemical potency. Interestingly, despite the extra space offered by FD, the FD potency of
compounds 11-14 was sensitive to stereochemistry of the substituent with only one isomer
showing favorable potency. Furthermore, consistent with the design rationale, the hydroxyl
methyl group in 12 when crystallized with FD, occupied the small pocket created in the active
conformation of FD making H-bonding interactions with Thr214 and Ile227 (Figure 6). However,
despite this additional interaction the biochemical and functional potency of 12 was similar to 9,
but encouragingly selectivity was substantially enhanced (FXIa IC =7.7 µM, 640-fold; tryptase-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
β2 IC =6.5 µM, 540-fold; and urokinase IC >30 µM, >2500-fold). Further homologating the
50
50
hydroxyl group as in 13 led to an even greater enhancement in selectivity but at the expense of a
modest decline in FD potency.
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5
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6
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. In vitro profiling data of benzylamine FD inhibitors.
R1
O
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
R2
NH2
Compou R1
nd
R2
FD TR-
Human
FXIa
IC50
(µM)
Tryptase Log Papp
β2 IC PAMPA
FRET
IC
50% WB
MAC IC
50
50
d
d
(µM)
cm/s
5
0
b
(
µM)a
(µM)
(permeabil
ity rank)
c
9
1
1
H
H
H
H
0.008
0.025
0.012
0.270
1.3
0.60
5.20
7.7
0.35
21
-4.5 (high)
-4.5 (high)
-5.1
1
2
CH3
CH OH
0.26
6.5
2
(medium)
1
3
H
(CH ) O 0.030
0.70
>100
4.0
55
-5.2
2 2
H
(medium)
14
Br
CH OH
0.003
0.160
3.3
-5.0
2
(medium)
15
16
17
CH OH
CH OH
0.005
0.010
0.008
0.07
0.02
0.013
8.09
4.8
3.7
3.6
3.3
-5.4 (low)
-5.3 (low)
-5.5 (low)
2
2
CH OCH
CH OH
2
3
2
C(CH ) O CH OH
0.65
3
2
2
H
1
8
NHiPr
CH OH
0.015
0.07
2.1
8.7
-5.5 (low
2
a
Half-maximal inhibition of recombinant human complement FD as determined in a TR-FRET
b
assay. Half-maximal inhibition of soluble MAC complex formation, as determined in an ELISA
assay. Permeability measurement by PAMPA. Screening assay with >50 protease targets. Data
represent mean values of duplicate measurements.
c
d
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
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9
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).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Pharmacokinetic Parameters of 12, 13 and 14 in C57BL6 mice dosed at 1 mg/kg IV and
1
0 mg/kg PO.
Compound oral Cmax AUC(0-7)
CL
Vdss
Elimination t (IV) %F
1/2
(nM)
(nM*Hours) (mL/min/kg) (L/kg) (Hours)
IV/PO
1
1
1
2
3
4
31000
10700
26000
16100/125000 2.6
4970/19500 8.7
48700/11600 0.7
0.3
0.3
0.1
1.6
1.3
2.5
83
39
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.
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9
1
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1.
R
O
NH2
HN
O
OH
R'
OH
A
O
B
A= CH or N
OH
X
A
B
C
Route A
O
1
. methyl 2-(2-
N
Boc
HN
Boc
HN
HO
H
aminophenyl)acetate
hydrochloride,
HATU, TEA DMF, rt
O
O
3-bromobenzoic acid
PdCl (dppf).CH Cl adduct
OH
2
2
2
O
B
OH
2M aq. K3PO4
2. 2M HCl in ether
CH CN, 90 °C
19
3
20
4
NH2
Route B
O
N
O
3-(Aminomethyl)benzeneboronic
acid hydrochloride
H
NH2
N
OH
O
HATU, DIPEA
DMF, rt,
N
H
PdCl2(dppf).CH2Cl2 adduct
O
N
O
O
O
2M aq. K3PO4
CH3CN, 110 °C
Br
5
21
22
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.
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9
X
Y
X
(3-(aminomethyl)phenyl)
Cl
3-Bromobenzyl bromide
Y
O
boronic acid hydrochloride,
PdCl (dppf).CH Cl adduct
HO
or
O
2
2
2
OH
O
or
O
3-Bromophenol
Br
O
2M aq. K PO , CH CN, 90 °C,
O
O
K2CO3, DMF
3
4
3
2
3
24
25 X=C, Y=O
6 X=O, Y=C
followed by LiOH, H O, THF,
2
9 X=C, Y=O
0 X=O, Y=C
2
NH2
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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.
Bis(pinacolato)diboron
K3PO4, PdCl2(dppf)·CH2Cl2,
R
R
O
B
o
MeCN/H2O, 90 C
Boc
Br
Boc
N
N
O
H
H
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
1
0
in place of the murine enzyme (human FD knock-in mice) as previously described 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
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9
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
10 mg/kg
3
mg/kg
LPS
PBS
2
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.
2
5
Given the evidence of the role of AP in AMD 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
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above the IC of 12 in the 50% whole blood MAC deposition functional assay (260 nM) for over
5
0
2
4h (Figure 8).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Plasma
PEC
Retina
1
0000
1
000
1
00
1
0
1
0
5
10
15
20
25
Time (hr)
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
2
6
been described and shown to increase AP locally in ocular tissues. Oral administration of 12
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
showed a dose dependent decrease in the ocular Ba levels (Figure 9) with up to 86% inhibition at
1
0 mg/kg dose.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
68%
86%
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 ≤
3
0 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.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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)
T ½ (Hours)
AUC0-last
0.8
3.8
0.2
0.4
(nM*hours)
85000 16100
1
0 mg/kg PO
Cmax (nM)
0 mg/kg PO
1
6500 2000
1
Tmax (hours)
0.8
0.3
%F
70%
N/A
*
N/A denotes not applicable.
CONCLUSION
A rationally conceived approach exploiting the FD conformational dynamics to optimize the
1
3
benzylamine FD inhibitors 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.
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EXPERIMENTAL SECTION
Experimental procedures and compound characterization for novel compounds
General Chemistry Information
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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
1
spectrometer. The purity of all exemplified compounds was ≥95%, as determined by both H
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-(2-
aminophenyl)acetate hydrochloride (CAS # 49851-36-7) (200 mg, 0.992 mmol) was stirred with
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TEA (0.276 mL, 1.980 mmol) in DMF (0.5 mL). After 5 min, the solution of methyl 2-(2-
aminophenyl)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 (Na SO ) and concentrated.
1
1
1
1
1
1
1
1
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0
2
4
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,
3
50.0 (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), PdCl (dppf).CH Cl adduct (CAS # 95464-05-4) (10.55
2
2
2
mg, 0.013 mmol), and 2M aq. K PO (0.388 mL, 0.775 mmol) in 9:1 MeCN/H O (2.8 mL) was
3
4
2
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
1
00x30 mm. Mobile phase: acetonitrile gradient, water with 0.1% (28% ammonium hydroxide) /
acetonitrile). Fractions containing the desired product were pooled, the pooled fractions were
1
frozen and lyophilized to provide the title compound (0.063 g, yield 68%). H NMR (400 MHz,
DMSO-d , 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,
6
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 C H N O (M+H) 361.1552, found 361.1550.
2
2
20
2
3
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
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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
0
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EtOAc and H O. The layers were separated and the aqueous layer was extracted with 4:1
2
EtOAc/heptane. The combined organics were washed with 5% aqueous LiCl solution, the washed
organics were dried with Na SO , the dried solution was filtered, and the filtered solution was
2
4
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 Et O, 1.05 mL, 2.11 mmol). The resulting
2
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
1
TFA salt (11 mg, yield 23%). H NMR (TFA salt, 400 MHz, methanol-d ) δ ppm 8.26 (s, 1 H),
4
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 C H N O (M+H) 375.1700, found 375.1696.
23 22
2
3
N-(2-((2H-tetrazol-5-yl)methyl)phenyl)-3'-(aminomethyl)-[1,1'-biphenyl]-3-
carboxamide (5): To a solution of 5-[(2-nitrophenyl)methyl]-1H-1,2,3,4-tetrazole (CAS#
1
77595-29-8, 280 mg, 1.27 mmol) in THF (6.8 mL) was added TEA (0.43 mL, 3.1 mmol). The
o
mixture was cooled to 0 C and SEMCl (0.3 mL, 1.7 mmol) was added. After ~ 1.5 h the
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mixture was partially purified by directly absorbing onto a plug of silica gel and eluting with 0-
5
0% EtOAc/hepatane, the eluent was concentrated and the resulting residue was dissolved in
THF (4.8 mL), charged with PtO (11 mg, 0.05 mmol) and placed under a H gas atmosphere.
2
2
1
1
1
1
1
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0
1
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0
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
1
hydroxide) / acetonitrile)) to afford the title compound in 85% purity by H HNMR (40 mg, yield
1
1
8%,): H NMR (TFA salt, 400 MHz, DMSO-d ) δ 16.15 (s, 1H), 10.15 (s, 1H), 8.21 (t, J = 1.9
6
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
3
.33 – 7.25 (m, 2H), 4.37 (s, 2H), 4.16 (d,J = 5.7 Hz, 2H). HRMS calcd. for C H N O (M+H)
22 21 6
85.1776 found 385.1766
N-(2-((N-acetylsulfamoyl)methyl)phenyl)-3'-(aminomethyl)-[1,1'-biphenyl]-3-
carboxamide (6): Was prepared in a similar fashion as 5, by starting with N-((2-
aminobenzyl)sulfonyl)acetamide which can be prepared as follows. To a solution of (2-
nitrophenyl)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-
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aminobenzyl)sulfonyl)acetamide, which could be used without further purification in the
subsequent step. Spectroscopic data for the title compound, N-(2-((N-
1
acetylsulfamoyl)methyl)phenyl)-3'-(aminomethyl)-[1,1'-biphenyl]-3-carboxamide (6): H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
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(
400 MHz, DMSO-d ) 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
6
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
1
4
.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,
+
.3 Hz,1H), 4.42 (s, 2H), 4.15 (s, 2H), 1.56 (s, 3H). HRMS calcd. for C H N O S (M+H)
2
3
24
3
4
38.1487 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 3-
aminomethylphenyl 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
mixture of 3-bromoaniline (600 mg, 3.49 mmol), 3-((tert-
degassed
butoxycarbonylamino)methyl)phenylboronic acid (1051 mg, 4.19 mmol), Pd(PPh ) (202 mg,
3
4
0
.174 mmol), and 2M aq. K PO (1481 mg, 6.98 mmol) in dioxane (15 mL) and water (3 mL) was
3 4
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
(
Na SO ) and concentrated. The residue was purified by silica gel chromatography (EtOAc-
2 4
heptane 0-100%) to provide tert-butyl ((3'-amino-[1,1'-biphenyl]-3-yl)methyl)carbamate (552 mg,
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yield 53 %). H NMR (400 MHz, DMSO-d ) δ ppm 7.30 - 7.51 (m, 4 H) 7.18 (d, J=7.20 Hz, 1 H)
6
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]-3-
yl)methyl)carbamate (104 mg, 0.349 mmol) and methyl 2-(2-formylphenyl)acetate (CAS # 63969-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
1
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0
8
3-5) (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. NH Cl. The aqueous layer was
4
extracted with EtOAc. The combined organics were washed with brine, dried (Na SO4) and
2
concentrated. The residue was purified by silica gel chromatography (EtOAc-heptane 0-50%) to
provide
yl)amino)methyl)phenyl)acetate (132 mg, yield 82%). MS (ESI+) m/z 461.4 (M+H). A solution of
-(2-(((3'-(((tert-butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3-
methyl
2-(2-(((3'-(((tert-butoxycarbonyl)amino)methyl)-[1,1'-biphenyl]-3-
2
yl)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 NaHCO solution (15 mL)
3
was added. Then solid NaHCO was added until pH ~ 8. The two layers were separated and the
3
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 NH OH was
4
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% CH CN/H O (CH CN and H O containing 0.1% of TFA)) to give the
3
2
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title compound, 2-(2-(((3'-(Aminomethyl)-[1,1'-biphenyl]-3-yl)amino)methyl)phenyl)acetic acid,
1
(
60 mg, yield 73%). H 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
C H N O (M+H)+ 347.1760, found 347.1756.
0
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(
2
2
22
2
2
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,
.325 mmol), PdCl (dppf).CH Cl (13.28 mg, 0.016 mmol) and (3-(aminomethyl)phenyl)boronic
0
2
2
2
acid hydrochloride (CAS # 146285-80-5) (76 mg, 0.406 mmol). Acetonitrile (1.3 mL), 2M
aqueous K PO (0.488 mL, 0.976 mmol) and water (0.130 mL) were added, the vial was flushed
3
4
with nitrogen, sealed and heated in the microwave for 60 min at 110°C. THF (4 mL) and 2M
o
aqueous LiOH (0.813 mL, 1.626 mmol) were added and the mixture was heated at 50 C for 4
hours. Additional 2M aqueous LiOH (0.813 mL, 1.626 mmol) was added and the reaction was
o
heated at 50 C 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)
1
to provide the title compound (30 mg, yield 25%). H NMR (400MHz, ACETONITRILE- d ) δ
3
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 C H NO
22 21
3
+
(
M+H) 348.1600, found 348.1589.
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Page 29 of 49
Journal of Medicinal Chemistry
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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 3-
bromophenol (CAS # 591-20-8) and methyl 2-(2-(chloromethyl)phenyl)acetate (CAS # 95360-33-
1
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). 1H NMR (400 MHz, METHANOL-d ) δ ppm 7.71 (s, 1 H) 7.54 (d, J=7.83 Hz, 1 H) 7.13 -
4
.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 C H NO (M+H)+ 348.1600, found 348.1593.
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(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 PdCl (dppf).CH Cl (15.11 mg, 0.018 mmol) were added to a
2
2
2
microwave vial. Acetonitrile (1.5 mL), 2M aqueous K
3
PO (0.555 mL, 1.110 mmol) and water
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(
0.300 mL) were added, the vial head space was flushed with nitrogen and the suspension was
o
heated in a microwave for 60 min at 110 C. At this point, 2 mL of 2M aq. LiOH solution were
o
added and the reaction was heated at 60 C 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
5
µ C18 110A 100x30 mm. Mobile phase: MeCN gradient, water with 0.1% (28% ammonium
1
hydroxide) / acetonitrile) to provide the title compound (16.5 mg, yield 12%). H NMR (400MHz,
DMSO-d ) 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
6
-
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,
H), 4.32 - 4.24 (m, 1H), 3.45 - 3.35 (m, 2H), 1.52 (d, J=6.7 Hz, 3H). HRMS calcd. for C H NO
2
2
3
23
3
+
(
M+H) 362.1756, found 362.1756.
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