We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFβ production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.
We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFβ production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.
Neuropilin- 1 (NRP1)
is a cell-surface coreceptor for a number
of different growths factors, including several different isoforms
of vascular endothelial growth factor (VEGF), transforming growth
factor-β1 (TGF-β1), PLGF, HGF (also known as scatter factor)
as well as Semaphorins 3A, 4F.[1] As such,
NRP1 plays key roles in both vascular and neuronal development.[2,3] It has also been shown that NRP1 has an important immunological
function.[4] NRP1 is expressed on several
types of immune cells, including T cells and dendritic cells, where
it is one of the components of the immunological synapse.[5] NRP1 is implicated in potentiating the function
and survival of regulatory T cells (Tregs).[6] This T cell fragility is linked to responses to PD1 checkpoint inhibitors.[7] NRP1 expression can be used to distinguish Treg
subsets arising in vivo, thus NRP1 is present on thymus derived Tregs
(natural Tregs),[8] whereas it is not present
on Foxp3+ positive inducible Tregs.[9,10] The
Ikaros family protein Helios has been suggested as an additional and
more general marker for thymic derived Tregs.[11]NRP1 is also important in the control of the M2 shift in tumor
associated macrophages/microglia in gliomas.[12] NRP1 interacts with TGFβR1 to activate SMAD2/3 and drive secretion
of TGF-β1, which results in expansion of Treg subsequent immune
suppression.[13−15] As the role of the immune system in cancer development
becomes better understood,[16] NRP1 is emerging
as an attractive anticancer target.[17] Novel
drug compounds which act as NRP1 antagonists could therefore exhibit
their anticancer effects in three different ways: blocking tumor angiogenesis
by blocking the NRP1/VEGF-A interaction,[18] preventing tumor cell migration by binding to NRP1,[19] and reducing Treg or macrophage mediated suppression of
the immune response.[20]A number of
peptide antagonists of neuropilin are known: ATWLPPR[21] is a low affinity linear peptide, whereas a
bicyclic disulfide bonded peptide, EG3287, is derived from the C-terminal
domain of VEGF-A[22] (Scheme ). N-Terminal modification (N-octanoyl) resulted in a high affinity antagonist EG00086 (KD = 76 nM).[23] EG00086
was also shown to inhibit VEGF-A mediated cell signaling, including
cell adhesion, through reduction in p130Cas tyrosine phosphorylation.
Its usefulness for in vivo studies was limited, however, by its low
plasma stability (t1/2 < 5 min).[23] In addition, NRP1 antibodies[18] and a mini-protein based on the kalata cyclotide have been
reported.[24]
Scheme 1
Previously Identified
Small Molecule and Peptidic Antagonists of
NRP1
Development of a potent, small
molecule NRP1 antagonist, with increased
in vivo stability, would therefore be attractive. Despite the interest
in this area, only a small number of molecules have been identified[25−27] These molecules are reported to have micromolar potencies, and some
antitumor effects have been claimed in vivo. The best characterized
of these is (S)-2-(3-(benzo[c][1,2,5]thiadiazole-4-sulfonamido)thiophene-2-carboxamido)-5-((diaminomethylene)amino)pentanoic
acid (EG00229), which has been previously identified as a specific
inhibitor of the NRP1/VEGF-A interaction.[27] Other compounds, such as the benzimidazole-based inhibitor exemplified
by N-((5-(1H-benzo[d]imidazol-2-yl)-2-methylphenyl)carbamothioyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide were identified through screening
approaches (Scheme ).[25] EG00229 was shown to inhibit the
binding of biotinylated VEGFA (bt-VEGF-A) to NRP1 with an IC50 of 8 μM. It was also demonstrated to have functional effects
on cell-migration and VEGF-R2 phosphorylation.[27] Further studies have shown EG00229 to reverse an immune
phenotype elicited by the immunomodulatory peptide tuftsin by blocking
canonical TGFβ signaling through SMAD3/AKT.[28] When delivered locally, the compound also inhibits glioma
proliferation in vivo, replicating genetic ablation studies.[12] In squamous cell carcinoma, the compound suppresses
epidermal stem cell function and tumor formation in vivo.[29] The binding mode of EG00229 has been confirmed
by NMR and crystallographic studies, providing a useful starting point
for the development of new NRP1 antagonists.[27] Herein, we utilize EG00229 as a starting point for the discovery
of potent and bioavailable inhibitors of the NRP1/VEGF-A interaction,
resulting in the identification of 1 (EG013777) as a
new lead.
Results and Discussion
Structure-Based Design of New NRP1 Antagonists
The
crystal structure of EG00229 bound to human NRP1-b1 was previously
solved to 2.9 Å resolution (PDB 3I97), and two different binding poses for
the ligand were identified (Figure A,1B).[27] The crystal structure
revealed a close fit of the arginine portion of the molecule into
the NRP1 binding pocket with near identical conformations observed
for this part of the molecule. As previously noted, the 3-aminothiophene-2-carboxylic
acid displays an H-bonded constrained conformation in the bound molecule,
indicating the presence of an alternate tautomeric form of this substructure.
In contrast, the positions of the benzothiadiazole group were markedly
different. Although the two binding poses of EG00229 were distinct,
the positions of the protein side chains were identical when overlapping
the two chains, except for E348. We hypothesized that through further
modification and elaboration of the EG00229 scaffold, more potent
NRP1 antagonists could be synthesized. Using chain B as an illustration
(Figure B), two key
amino acids were identified which could be targeted either to improve
existing H-bond interactions (S298) or to introduce new ones (E348).[27]
Figure 1
Two poses of EG00229
(PDB 3I97):
C atoms in magenta and brown. In (B), a H-bond is
formed with S298.
Chemistry
To target outer pocket
residues, such as
S298 and E348, a range of substituted dihydrobenzofurans were prepared.
These were designed to be able to make potential hydrogen bonding
or salt bridge contacts with the S298 and E348 residues. The first
part of the general synthetic route for the 5-substituted dihydrobenzofuran
series is shown (Scheme ). The synthesis began with sulfonamide formation between 5-bromo-2,3-dihydrobenzofuran-7-sulfonyl
chloride 2 and methyl 3-aminothiophene-2-carboxylate,
to give sulfonamide 3. Hydrolysis of the methyl ester
with LiOH gave acid 4, which was then coupled with the
Pbf-protected arginine methyl ester to give 5. Subsequent
hydrolysis of 5 gave the key intermediate 6, which was suitable for Suzuki–Miyaura couplings with a range
of arylboronic acids.
Scheme 2
Synthesis of Brominated Dihydrobenzofuran
Scaffold
Reagents and conditions: (a)
methyl 3-aminothiophene-2-carboxylate, pyridine, 0 °C to rt,
20 h; (b) LiOH, THF, MeOH, H2O, 45 °C, 20 h; (c) H-l-Arg(Pbf)-OMe, PyBrop, DIPEA, CH2Cl2,
rt, 18 h; (d) LiOH·H2O, THF, H2O, rt, 3
h.
Synthesis of Brominated Dihydrobenzofuran
Scaffold
Reagents and conditions: (a)
methyl 3-aminothiophene-2-carboxylate, pyridine, 0 °C to rt,
20 h; (b) LiOH, THF, MeOH, H2O, 45 °C, 20 h; (c) H-l-Arg(Pbf)-OMe, PyBrop, DIPEA, CH2Cl2,
rt, 18 h; (d) LiOH·H2O, THF, H2O, rt, 3
h.From this common brominated intermediate 6, a range
of azaheterocycles were prepared as shown in (Scheme ). First, intermediate 6 was
coupled to either 2- or 3-formylphenyl boronic acid to give 7a,b, and then reductive aminations were carried
out using the desired amine to furnish substituted analogues 8 (for definitions of R, see Tables –3). Removal
of the Pbf-protecting group in acidic conditions gave the final products 9a–e, 10a–e (Table ), 11a–e, 12a–e, and 13a (Table ).
Scheme 3
Synthesis of Aryl Substituted Dihydrobenzofurans
For definitions of R, see Tables –3. Reagents and conditions: (a) Pd(PPh4)Cl2, K2CO3, DME, H2O, 90°; (b)
amine, AcOH, then NaHCNBH3, DMF; (c) DCM, TFA 1:1.
Table 1
Equilibrium Binding Constants for
Substituted Azaheterocycles
Table 3
Equilibrium Binding Constants for
Methylamino Analogues
ID
R
SPR KD [μM]
13
3′-CH2NMe2
4.52 ± 1.36
15a
2′-CH2NH2
1.87 ± 0.32
15b
3′-CH2NH2
2.44 ± 0.71
1
4′-CH2NH2
1.32 ± 0.08
18a
2′,3′-(CH2NHCH2)
2.47 ± 0.25
18b
3′,4′-(CH2NHCH2)
2.17 ± 0.32
Table 2
Equilibrium
Binding Constants for
Substituted Methylpiperidines
Synthesis of Aryl Substituted Dihydrobenzofurans
For definitions of R, see Tables –3. Reagents and conditions: (a) Pd(PPh4)Cl2, K2CO3, DME, H2O, 90°; (b)
amine, AcOH, then NaHCNBH3, DMF; (c) DCM, TFA 1:1.The synthesis of primary
methylaminoaryl analogues was achieved
by the use of preformed boronic acids (Scheme ). Thus, 6 under Suzuki–Miyaura
conditions with the 2- or 3-methylaminoboronic acids gave the intermediates 14a–c, which with Pbf removal gave the
target molecules 15a, 15b, and 1. For large scale batches of 1, a slightly modified
synthetic route was employed with a Boc protected methylaminoboronic
acid (Supporting Information, Scheme S1). For the synthesis of cyclized isoindolyl analogues, 6 was transformed into the functionalized boronic acid 16 using bispinacolato diboron and Pd(dppf)Cl2, and this
was used directly for the Suzuki–Miyaura coupling using potassium
acetate as the base and Pd(PPh4) as the palladium catalyst (Scheme ). In this case,
cesium carbonate was preferred as the base. Final deprotection of
the Pbf group furnished the isoindolyl analogues 18a and 18b.
Scheme 4
Synthesis of Methylaminoaryl Substituted Analogues
To further
investigate the binding of 1 to NRP-1, X-ray crystallography
studies were carried out. The differences in binding modes between 1 and EG00229 were then analyzed. The structure of 1 bound to NRP1-b1 was determined in two conformations: a high (0.9
Å) and a low (2.8 Å) resolution structure (Figure ). The high-resolution crystal
structure provides us with the most detailed view of the ligand-binding
site to date. The refined model includes residues 273–427 of
NRP1-b1, 39 non-hydrogen atoms of 1 and 472 water molecules.
High resolution allowed us to observe multiple conformations of the
side chains; 24 side chains were refined with at least two alternative
rotamers. Comparison of NRP1-b1/compound 1 complexes
indicate that the ligand can bind in two different conformations.
In the low-resolution 2.8 Å structure (PDB 6FMF), the ligand’s
bulky aromatics extend out of the back of the binding pocket. In the
high resolution 0.9 Å structure (PDB 6FMC), they extend out of the top of the binding
pocket (Figure ).
The difference in ligand conformation originates from a rotation about
the carbon–carbon bond axis of the carboxyl group, which forms
hydrogen bonds to S346 and T349. There is an approximate 77°
rotation along this bond (Figure B and Supporting Information, Figure S1), resulting in more than 1 Å separation between the
two different conformations. By exiting out of the top of the binding
pocket, the ligand in the high-resolution structure forms additional
interactions with the N-terminal residues (in particular G271–M276)
of a symmetry mate located above the NRP1-b1 binding pocket. It is
likely these interactions improve the crystal contacts, increasing
crystal order, which is necessary to produce the higher resolution
data explaining the difference in resolution between the two conformations.
These contacts are however a crystallographic artifact, with the lower
resolution structure more likely to represent the true conformation
of 1 bound to NRP1-b1. The difference in ligand conformations
results in a significant change in the side chain rotamer of E348.
In the low resolution structure, E348 points away from the binding
pocket and forms a hydrogen bond with the aryl-NH2 of 1, which may help to explain the compound’s increase
in potency. In the high resolution structure, the aryl-NH2 of 1 does not interact with E348 changing the side
chain rotamer such that it now faces toward the center of the binding
pocket. The detection of the hydrogen-bond to E348 in the low resolution
structure confirmed our modeling predictions.
Figure 2
Two crystallized forms
of 1 with NRP1-b1. (A) Low resolution 2.8
Å structure PDB 6FMF (cyan) shows the H-bond from the methylamino of 1 to
E348. (B) High resolution 0.9 Å PDB 6FMC (green) showing alternate conformation
of 1. The conformation of E348 is also different. (C)
Overlay of low (cyan) and high (green) resolution structures showing
bond rotation around the Cα-COO bond.
Two poses of EG00229
(PDB 3I97):
C atoms in magenta and brown. In (B), a H-bond is
formed with S298.Two crystallized forms
of 1 with NRP1-b1. (A) Low resolution 2.8
Å structure PDB 6FMF (cyan) shows the H-bond from the methylamino of 1 to
E348. (B) High resolution 0.9 Å PDB 6FMC (green) showing alternate conformation
of 1. The conformation of E348 is also different. (C)
Overlay of low (cyan) and high (green) resolution structures showing
bond rotation around the Cα-COO bond.
Biological Evaluation
All the compounds were evaluated
using an SPR binding assay (Biacore) where recombinant NRP1-b1b2 protein
was immobilized on a dextran coated chip.[23] Selected compounds were then evaluated in competitive binding assay
systems using biotinylated VEGF. As part of an extensive structure–activity
investigation, the binding of 9a and 10a to the NRP1-b1 domain was assessed by SPR and promising activity
noted for the morpholine extended analogue 10a with binding
affinity of 3.76 ± 0.52 μM by SPR as opposed to 14.43 ±
3.76 μM for the unsubstituted compound 9a. This
encouraging result prompted us to conduct a more focused structure–activity
study around the 10a structure.The first group
of analogues examined heteroaryl substituents on the 2′ and
3′ positions. All of the synthesized compounds 9b–e and 10a–e showed binding to the NRP1-b1 domain, with some compounds demonstrating
nanomolar KD values. Substitution at the
3-position seemed generally favorable, with all of the 3-substituted
compounds, 10a–e, showing higher
binding affinities than the 2-substituted analogues.A further
range of analogues, 11a–e and 12a–e, were designed which
contained a functionalized piperidine linker to add length and flexibility
to further explore outer-pocket interactions. Binding affinities to
the NRP1-b1 domain were again assessed by SPR (Table ).The resulting compounds once again
showed binding to NRP1, although
the binding affinities were generally lower than had been observed
for the previous azaheterocyclic compounds. The highest binding affinity
for this series was obtained for the nonfunctionalized piperidine 12a (KD = 1.17 μM). The
generally lower binding affinities for 11a–e suggested that the addition of the piperidine linker was
not an effective strategy to introduce specific interactions with
any additional surface amino acid residues, and so this series was
not pursued further. With these results in hand, a compound set with
smaller methylamino substituents that could be accommodated at the
4-position was synthesized (Table ). Compounds 1 and 13a–e showed consistent activity although
this declined with the methylated analogue 13c. Compound 1 showed reasonable affinity (Figure A,B), which was encouragingly maintained
in both cell-based and cell-free competition assays with bt-VEGF (Table ). Isothermal calorimetry data for 1 fitted to
a one-site binding model and provided an orthogonal assay system (Figure B). Evaluation of 1 against NRP2, a closely related receptor to NRP1, showed
no detectable binding (Supporting Information, Figure S2), indicating very good selectivity. These results
prompted us to investigate the pharmacokinetic profiles of some selected
analogues.
Figure 3
Biophysical
binding data supports the interaction of compound 1 with
NRP1-b1 protein. (A) Dose response analysis of equilibrium
binding of 1 determined by SPR. (B) Isothermal calorimetry
of 1 with NRP1-b1 using 19 consecutive injections of 1 (200 μM stock solution) applied at 2 min intervals.
Table 4
Cell-Free and Cell-Based
Binding Data
for Compound 1
binding assay
value
ITC NRP1-b1 (Ka)
4.14 × 106 ± 3.84 × 105
cell-free NRP1-a1,b1 (IC50)
0.609 ± 0.066 μM
DU145/cells.Ad.NRP1, IC50 (HS)
1.6 μM (0.9)
Biophysical
binding data supports the interaction of compound 1 with
NRP1-b1 protein. (A) Dose response analysis of equilibrium
binding of 1 determined by SPR. (B) Isothermal calorimetry
of 1 with NRP1-b1 using 19 consecutive injections of 1 (200 μM stock solution) applied at 2 min intervals.
Pharmacokinetics
Both of the compounds from the dihydrobenzofuran
series exhibited improved PK profiles over the historical compound
EG00229, which has a relatively short half-life of 0.5 h.[12] Compound 10d had a longer half-life
(1.2 h) with an improved Vd of 1103 mL/kg
(Table ). Compound 1 also exhibited an encouraging half-life of 4.29 h, sufficient
to sustain once per day dosing. The methylated analogue 13c showed less favorable parameters with a notably higher clearance
and lower AUC than 1. With this data in hand demonstrating 1 to be a reasonably potent and stable inhibitor, we undertook
a thorough biological characterization of 1 examining its antiangiogenic,
antitumor, and immune effects.
Compound 1 Inhibits VEGF-A Stimulated Tyrosine
Phosphorylation of VEGF-R2/KDR
VEGF-A signaling through VEGF-R2/KDR
plays an important role in cell function in endothelial, tumor, and
other cell types.[30] We investigated the
effect of 1 on VEGF-R2/KDR tyrosine phosphorylation induced
by VEGF-A in HUVECs. VEGF-A (1 ng /mL) stimulated a significant
increase in VEGF-R2/KDR tyrosine phosphorylation at 10 min, which
was inhibited by 50% on treatment with 1 at 30 μM
(Figure ). Studies
with 1 had previously shown a 20% inhibition at 30 μM.[27] These results once again indicate the importance
of NRP1 for optimal VEGF function and signaling[1] and confirmed the higher potency of 1 compared
to EG00229 as indicated by its higher affinity for NRP1-b1 and higher
potency in a cell-free binding assay.
Figure 4
Compound 1 inhibits VEGF-A
stimulated tyrosine phosphorylation
of VEGF-R2/KDR. HUVECs were grown to confluence and serum-starved
with medium containing 0.5% serum for 16 h. Cells were preincubated
for 30 min with medium containing 0.1% DMSO (Veh) 3, 10, and 30 μM
1 or medium alone followed by stimulation with 1 ng mL–1 VEGF-A or with no further treatment (control) for
10 min. Cell lysates were then prepared, blotted, and probed with
the indicated antibodies. The data shown are representative of three
independent experiments. Quantitation of pVEGF-R2/KDR phosphorylation
was performed by densitometry using ImageJ; see Materials and Methods.
Data are presented as pVEGF-R2/KDR phosphorylation relative units
(RU; means ± SEM) normalized to total VEGF-R2/KDR; p < 0.05 = * and p < 0.001 = ***.
Compound 1 inhibits VEGF-A
stimulated tyrosine phosphorylation
of VEGF-R2/KDR. HUVECs were grown to confluence and serum-starved
with medium containing 0.5% serum for 16 h. Cells were preincubated
for 30 min with medium containing 0.1% DMSO (Veh) 3, 10, and 30 μM
1 or medium alone followed by stimulation with 1 ng mL–1 VEGF-A or with no further treatment (control) for
10 min. Cell lysates were then prepared, blotted, and probed with
the indicated antibodies. The data shown are representative of three
independent experiments. Quantitation of pVEGF-R2/KDR phosphorylation
was performed by densitometry using ImageJ; see Materials and Methods.
Data are presented as pVEGF-R2/KDR phosphorylation relative units
(RU; means ± SEM) normalized to total VEGF-R2/KDR; p < 0.05 = * and p < 0.001 = ***.
Angiogenesis, Inhibition of VEGF-Induced
Migration in HUVEC
Cells
To investigate the importance of blocking NRP-1 in
HUVEC cells, we performed transwell assays of chemotaxis and in vitro
scratch assays of wound closure (chemokinesis). The transwell assay
examines cell chemotaxis, the directional cell migration toward the
chemo-attractant. To understand if 1 could inhibit VEGF-A-induced
migration of HUVEC cells, 2 × 105 HUVEC cells were
plated in serum-free medium (EBM) with the addition of either 0.1%
DMSO, 25 ng/mL VEGF-A, 1 (30 μM), or a combination
of VEGF-A and 1 on the bottom chamber. Cells were allowed
to migrate through the pores of the insets for 4 h. Data collected
was consistent with previous reports,[31] with VEGF-A being able to induce HUVEC cells migration by almost
3 times more compared to DMSO control (Figure A,B). Treatment of HUVEC cells with 1 alone did not influence the migratory ability of these cells
but the administration of 1 at 30 μM in the presence
of VEGF-A significantly reduces, by more than 60%, the ability of
cells to migrate toward VEGF-A stimulus (Figure B). These results suggest that 1 has a higher potency than the previously reported compound, EG00229,[27] that only displayed significant inhibition (≈34%
reduction) once used at 100 μM in combination with VEGF-A.
Figure 5
Compound 1 is able to significantly reduce HUVEC cell
migration in response to VEGFA. (A) 8× magnified images representing
HUVEC cells (stained in blue) that migrate through membrane pores
toward serum free medium supplemented with 0.1% DMSO as vehicle control
(Veh), VEGF 25 ng/mL, 1 (30 μM), and 1 (30uM) + VEGF 25 ng/mL. (B) Graphical representation. Data represent
the average number of migrated cells of five independent experiments
± SEM; ***P ≤ 0.001. (C) HUVEC cells
were starved overnight in 1% EBM before a precise scratch was generated
using the WoundMaker (Essen BioScience). Migration was assessed in
the presence or absence of medium containing 0.1%DMSO (Veh), VEGF
25 ng/mL, 1 (30 μM), and 1 (30 μM)
+ VEGF 25 ng/mL, using an IncuCyte ZOOM live-cell imaging platform.
The graph represents three independent experiments: means ± SEM.
Each treatment per experiment was performed in 12 replicates.
Compound 1 is able to significantly reduce HUVEC cell
migration in response to VEGFA. (A) 8× magnified images representing
HUVEC cells (stained in blue) that migrate through membrane pores
toward serum free medium supplemented with 0.1% DMSO as vehicle control
(Veh), VEGF 25 ng/mL, 1 (30 μM), and 1 (30uM) + VEGF 25 ng/mL. (B) Graphical representation. Data represent
the average number of migrated cells of five independent experiments
± SEM; ***P ≤ 0.001. (C) HUVEC cells
were starved overnight in 1% EBM before a precise scratch was generated
using the WoundMaker (Essen BioScience). Migration was assessed in
the presence or absence of medium containing 0.1%DMSO (Veh), VEGF
25 ng/mL, 1 (30 μM), and 1 (30 μM)
+ VEGF 25 ng/mL, using an IncuCyte ZOOM live-cell imaging platform.
The graph represents three independent experiments: means ± SEM.
Each treatment per experiment was performed in 12 replicates.
Wound Healing Scratch Assay
HUVEC cells were plated
and once confluent a scratch was made as described in the methods.
Cells were kept in culture for 5 days in 1% EGM with 0.1% DMSO, 25
ng/mL VEGF-A, 1 (30 μM), or a combination of VEGF-A
and 1. Data shows that 1 can delay the VEGF-induced
wound closure (Figure C).
Compound 1 in Combination with VEGF-A Reduces Network
Area, Length, and Branching Points
Next, we used an organotypic
endothelial–fibroblast coculture assay to recapitulate the
endothelial tube formation characteristic of VEGF-A stimulated angiogenesis.
The coculture assay of angiogenesis is a simple in vitro assay where
HUVEC cells are cultured with human embryonic fibroblasts (HDF). The
layer of fibroblasts secretes a complex extracellular matrix that
contains collagen I with fibronectin, tenascin-C, decorin, and versican,
mimicking the composition of tissue stroma. This matrix becomes remodelled
into a 3D environment, allowing HUVECs to reorganize into a network
of tubes.This assay is particularly suited to test factors
that promote or inhibit angiogenesis. Thus, we next analyzed endothelial
tubulogenesis in coculture HUVEC cells treated with either VEGF-A
or VEGF-A + 1 during 4 days (Figure ). Data collected shows that HUVEC cells
stained for the endothelial marker Von Willebrand factor (VWF), have
a ≈41% reduction in the number of VEGF-induced branch points
in tubular networks upon NRP1 inhibition with 1 (Figure B). This reduction
was also observed when overall network area (≈50%) and length
(≈40%) (Figure B) were assessed. Results suggest that NRP1 inhibition can significantly
influence the angiogenic properties of endothelial cells, thus being
an attractive target to test on highly metastatic cancers that express
NRP1.
Figure 6
Blocking NRP1 is important for angiogenesis in a coculture assay.
(A) Human dermal fibroblasts were grown in a 24-well format to confluence.
HUVECs were seeded, treated with medium supplemented with 0.1%DMSO
as vehicle control (Veh), VEGF 25 ng/mL, 1 (30 μM),
and 1 (30 μM) + VEGF 25 ng/mL and kept in culture
for 4 days. Endothelial cells were then visualized by immunofluorescence
for Anti-von Willebrand. (B) Quantitation of coculture assays in A.
The bar graph shows the mean ± SEM network branch points, area,
and length. Data corresponds to the average of three independent experiments;
*p < 0.05.
Blocking NRP1 is important for angiogenesis in a coculture assay.
(A) Human dermal fibroblasts were grown in a 24-well format to confluence.
HUVECs were seeded, treated with medium supplemented with 0.1%DMSO
as vehicle control (Veh), VEGF 25 ng/mL, 1 (30 μM),
and 1 (30 μM) + VEGF 25 ng/mL and kept in culture
for 4 days. Endothelial cells were then visualized by immunofluorescence
for Anti-von Willebrand. (B) Quantitation of coculture assays in A.
The bar graph shows the mean ± SEM network branch points, area,
and length. Data corresponds to the average of three independent experiments;
*p < 0.05.
Reduced VEGF-Induced Angiogenesis after Treatment with 1
To further analyze the effect of blocking NRP1
during angiogenesis, we next used an ex vivo mouse aortic rings assay.
The aortic ring assay provides a more complete picture of angiogenic
processes compared with traditional cell-based assays. In this model,
endothelial cells are able to proliferate and migrate, forming network
tubes and branching points without the need for cellular dissociation.[32] This allows us to assess different steps that
occur during the angiogenic process, which we aim to target. Rings
were embedded in collagen and kept in culture in medium containing
0.1% DMSO, 25 ng/mL VEGF-A, 1 (30 μM), or a combination
of VEGF-A and 1. As expected, after 7 days in culture,
VEGF-A increased vessel sprouting from WT aortic rings, but this response
was significantly suppressed (≈7-fold reduction) by the administration
of 30 μM of 1 (Figure A,B).
Figure 7
Compound 1 in combination
with VEGFA reduces angiogenesis.
(A) Representative pictures of mouse aortic rings stained with isolectin
B4 (red) and SMA (green). Rings were treated with medium containing
0.1%DMSO as vehicle control (Veh), VEGF 25 ng/mL, 1 (30
μM), and 1 (30 μM) + VEGF 25 ng/mL. (B) Sprouts
outgrowth area was quantified using imageJ. Data represent the average
of three independent experiments ± SEM, ***P ≤ 0.001.
Compound 1 in combination
with VEGFA reduces angiogenesis.
(A) Representative pictures of mouse aortic rings stained with isolectin
B4 (red) and SMA (green). Rings were treated with medium containing
0.1%DMSO as vehicle control (Veh), VEGF 25 ng/mL, 1 (30
μM), and 1 (30 μM) + VEGF 25 ng/mL. (B) Sprouts
outgrowth area was quantified using imageJ. Data represent the average
of three independent experiments ± SEM, ***P ≤ 0.001.Studies have described
that NRP1 up-regulation is associated with
the tumor invasive behavior and metastatic potential, for instance,
in melanoma and breast cancer.[17,33] Thus, our data reinforces
the importance of targeting NRP1 and suggest a possible attractive
therapeutic approach for cancers that are so far resistance to the
traditional angiogenic therapies.
Antitumor: Blocking NRP1
Reduces Melanoma Invasion in a 3D Spheroid
Assay
To further investigate the effects of NRP1 blockade
on cancer cells, we used a three-dimensional (3D) spheroid assay.
3D spheroids are a useful tool to replace the commonly used 2D cell
culture systems. By using this system, we aimed to recapitulate how
cells grow in vivo in three dimensions.NRP1 expression is associated
with melanoma progression and invasiveness. In addition, studies have
shown that these properties can be inhibited by the use of anti NRP1
antibodies or shRNA constructs.[34] Thus,
we hypothesize that NRP1 is a potential target for the treatment of
the metastatic melanoma. In our study, we have used A375P (malignant
melanoma) cells that express NRP1 (data not shown).A375P spheroids
were embedded in collagen and treated with medium
supplemented with 0.1% DMSO, 25 ng/mL VEGF-A, 1 (30 μM),
or a combination of VEGF-A and 1 (Figure A). Data collected shows that treatment with 1 in A375P cells significantly inhibited invasion induced
by VEGF-A, whereas 1 treatment on its on its own had
no significant effect on radial invasion compared to the DMSO control
(Figure B). These
results further establish an important role for blocking NRP1 in regulating
VEGF-A mediated signaling, which are essential for cell motility and
invasion in melanoma cells.
Figure 8
Compound 1 in combination with
VEGFA reduces A375P
spheroid outgrowth. (A) A375P cells were resuspended and used to generate
spheroids as described in Materials and Methods. Then 24 h after spheroid
production, spheroids were imbedded in a collagen gel and incubated
in medium containing 0.1% DMSO as a vehicle control (Veh), VEGF 25
ng/mL, 1 (30 μM), and 1 (30 μM)
+ VEGF 25 ng/mL for 7 days. (B) Spheroid outgrowth area was measured
by quantifying the area corresponding to the invasion rim minus the
area of the core for at least four different spheroids per condition.
Data from at least three independent experiments ± SEM are presented
as arbitrary units (a.u.) *p < 0.05.
Compound 1 in combination with
VEGFA reduces A375P
spheroid outgrowth. (A) A375P cells were resuspended and used to generate
spheroids as described in Materials and Methods. Then 24 h after spheroid
production, spheroids were imbedded in a collagen gel and incubated
in medium containing 0.1% DMSO as a vehicle control (Veh), VEGF 25
ng/mL, 1 (30 μM), and 1 (30 μM)
+ VEGF 25 ng/mL for 7 days. (B) Spheroid outgrowth area was measured
by quantifying the area corresponding to the invasion rim minus the
area of the core for at least four different spheroids per condition.
Data from at least three independent experiments ± SEM are presented
as arbitrary units (a.u.) *p < 0.05.
Blocking NRP1 on Regulatory T Cells (Treg)
with 1 Reduces Their Production of TGFβ in the
Presence of Glioma-Conditioned
Media (GCM)
Nrp1 is upregulated on the surface of Treg and
is vital to their maintenance. Nrp1+ Treg populations have
also been shown to induce allograft tolerance and limit potential
antitumorigenic responses in murine models. Depletion of Nrp1+ Treg leads to enhancement of antitumoral immune responses,
making them a favorable population of cells to target pharmacologically.[6,35] To determine whether 1 had the potential to block the
pro-tumorigenic polarization of Nrp1+ Treg, we isolated
and purified Nrp1+, FoxP3+, and CD25+ populations of Treg from mice (Figure A) and exposed them to glioma conditioned
media (GCM)[12] for 12 h after pretreating
the cells with 1. TGFβ is normally present in GCM[36,37] and contributes to the immunosuppressive tumor microenvironment
because interference with TGFβ expression has been shown to
strongly promote recognition of glioma cells by cytotoxic T cells
and NK cells.[37] Treatment of the Nrp1+ Tregs with GCM alone activated the Tregs, which resulted
in further increased TGFβ cytokine production, while pretreatment
of the cells with 1 inhibited the GCM-induced production
of TGFβ (Figure B).
Figure 9
Compound 1 blocks the production of TGFβ by
Nrp1+ Tregs in the presence of tumor cell-derived factors.
(A) Primary T cells were isolated from the spleens of mice and purified
for CD4 and CD304 (Nrp1) expression. The expression of these cell
surface receptors as well as FOXP3 expression were verified by flow
cytometry. (B) Nrp1+ Tregs were either exposed or not to
500 nM 1 for 2 h prior to incubation with glioma conditioned media
(GCM). Production of TGFβ by the cells was assessed via ELISA
after 12 h. The lanes correspond to media alone (Ctr), media with
GCM (GCM alone), media with 1377 (1377), and then Nrp1+ Treg cells exposed to GCM (+GCM), or GCM + 1377 (1377 + GCM).
Compound 1 blocks the production of TGFβ by
Nrp1+ Tregs in the presence of tumor cell-derived factors.
(A) Primary T cells were isolated from the spleens of mice and purified
for CD4 and CD304 (Nrp1) expression. The expression of these cell
surface receptors as well as FOXP3 expression were verified by flow
cytometry. (B) Nrp1+ Tregs were either exposed or not to
500 nM 1 for 2 h prior to incubation with glioma conditioned media
(GCM). Production of TGFβ by the cells was assessed via ELISA
after 12 h. The lanes correspond to media alone (Ctr), media with
GCM (GCM alone), media with 1377 (1377), and then Nrp1+ Treg cells exposed to GCM (+GCM), or GCM + 1377 (1377 + GCM).
Conclusion
A focused
set of novel NRP-1 antagonists were designed using structure-based
drug design to allow targeting of specific residues located close
to the binding pocket of arginine. X-ray crystallography was able
to confirm that these interactions were being formed and enabled the
design of further analogues.Compounds were tested in several
different assays to confirm binding
to NRP1 and inhibition of NRP1–VEGF complex formation. Of these
new inhibitors, compound 1 shows consistent biological
activity and good stability in vivo. It exhibits submicromolar potency
in inhibition of VEGF-A binding to NRP1 and good functional inhibition
of VEGF driven angiogenesis, cell migration, tumor invasiveness, and
notably Treg cell activation. The compound also demonstrates a sustained
IV PK profile, making it an exciting new proof-of-concept molecule
for the investigation of NRP-1 antagonists as anticancer therapies
(Table ).
Table 5
IV PK Data for Selected Compounds
Experimental
Section
Materials and Methods
Chemistry
All materials were obtained
from commercial
suppliers and used without further purification unless otherwise noted.
Anhydrous solvents were either obtained from Aldrich or Fisher Scientific
and used directly. All reactions involving air- or moisture-sensitive
reagents were performed under a nitrogen atmosphere. Routine analytical
thin layer chromatography was performed on precoated plates (Alugram,
SILG/UV254). Reaction analyses and purity were determined by reverse-phase
LC-MS using an analytical C18 column (Phenomenex Luna C18 (2) 50 mm
× 4.6 mm, 5 μm for 4.5 and 13 min methods), using a diode
array detector and an A:B gradient starting from 95% A:5% B at a flow
rate of 2.25 mL/min or 1.5 mL/min, where eluent A was 0.1% formic
acid/H2O and eluent B was 0.1% formic acid/MeOH or eluent
A was 10 mM NH4HCO3 (aq) and eluent B was MeOH.
Silica gel chromatography was performed with prepacked silica gel
Biotage SNAP (KP-Sil) cartridges. Ion exchange chromatography was
performed using Isolute Flash SCX-2 cartridges. Reverse-phase preparative
HPLC was carried out on a Waters ZQ instrument using mass-directed
purification on a preparative C18 column (Phenomenex Luna C18 (2),
100 mm × 21.2 mm, 5 μm). Depending upon the retention time
and the degree of separation of the desired compound from any impurities,
an A:B gradient was employed starting from high %A/low %B at a flow
rate of 20 mL/min. The following combinations of A and B were typically
used: A = H2O + 0.1% formic acid and B = MeOH + 0.1% formic
acid, or A = 10 mM NH4HCO3 (aq) and B = methanol. 1H and 13C spectra were measured with Bruker NMR
spectrometers as indicated. All observed protons are reported as parts
per million (ppm) and are aligned to the residual solvent peak, e.g.,
for DMSO-d6 at δH 2.50
and δC 39.5 and for CDCl3 at δH 7.26. Data are reported as follows: chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, br = broad, m = multiplet),
coupling constants (J) recorded in Hz, and a number
of protons. Low-resolution mass spectrometry data were determined
on Waters ZQ4000 single quadrupole, Micromass Ultima triple quadruple
mass spectrometers or Agilent 6100 single quadrupole/1200 series.
High-resolution mass spectroetry was determined using an Orbitrap.
All compounds tested (bioassays) were determined to be at least 95%
pure by LC-MS unless otherwise stated.
To a stirred solution
of 5-bromo-2,3-dihydro-benzofuran-7-sulfonyl chloride (1.25 equiv,
13.6 g, 47.6 mmol) in pyridine (anhydrous, 50 mL) under nitrogen (balloon),
at 20 °C, was added dropwise over 2 h a solution of methyl-3-aminothiophene-2-carboxylate
(1 equiv, 6 g, 38.1 mmol) in pyridine (anhydrous, 10 mL). The resulting
suspension was stirred at 20 °C for 18 h, and after this time
the reaction mixture was cooled (approx 0 °C) and water (50 mL)
added dropwise. Precipitation occurred and the mixture was further
diluted with water (100 mL) and the desired product collected by filtration,
washed with ice-cold water (2 × 50 mL), and dried in vacuo to
afford 3. Yield: 15.6 g, 37.4 mmol, 98%. Off-white solid.
LC-MS: tR = 4.42 min. MS: m/z 416/418 [M – 1]−. HRMS:
Calculated for C14H13BrNO5S2 417.9419. Measured mass: 417.9428. 1H NMR (600 MHz, DMSO-d6) δ 9.88 (s, 1H), 7.86 (d, J = 5.5 Hz, 1H), 7.71–7.67 (m, 1H), 7.63–7.59 (m, 1H),
7.22 (d, J = 5.5 Hz, 1H), 4.67 (t, J = 8.8 Hz, 2H), 3.82 (s, 3H), 3.27–3.20 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 163.24,
155.98, 141.86, 134.12, 133.88, 133.53, 128.23, 121.05, 120.39, 111.30,
110.77, 74.21, 52.37, 28.31.
The ester (3) (1 equiv,
15.6 g, 37.4 mmol) was stirred with lithium hydroxide monohydrate
(5 equiv, 7.9 g, 187 mmol) in a tetrahydrofuran/methanol/water mixture
(5:3:2; 40 mL) at 50 °C for 4 h. After this time, the organic
solvents were removed in vacuo, and the residue diluted with water
(50 mL) and then acidified to pH 1 with 6 M hydrochloric acid upon
which precipitation occurred. The precipitate was washed with water
(3 × 50 mL), collected by filtration, and dried in vacuo to give 4. Yield: 13 g, 32.2 mmol, 86%. Off-white solid. LC-MS: tR = 4.54 min. MS m/z 402/404 [M – 1]−. HRMS: Calculated
for C13H11BrNO5S2 403.9262,
Measured mass: 403.9246. 1H NMR (600 MHz, DMSO-d6) δ 10.11 (s, 1H), 7.79 (d, J = 5.5 Hz, 1H), 7.71–7.66 (m, 1H), 7.63–7.58 (m, 1H),
7.19 (d, J = 5.5 Hz, 1H), 4.67 (t, J = 8.8 Hz, 2H), 3.27–3.20 (m, 2H). 13C NMR (151
MHz, DMSO-d6) δ 164.75, 155.96,
141.69, 134.07, 133.51, 133.08, 128.24, 121.06, 119.85, 112.19, 110.77,
74.19, 28.32.
The acid (4) (1 equiv, 13 g, 32.2 mmol) and bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBrOP; 1.1 equiv, 16.4 g, 35.4 mmol) were suspended
in dichloromethane (100 mL) under nitrogen (balloon) and stirred at
20 °C for 10 min. N,N-Diisopropylethylamine
(7 equiv, 39.2 mL, 225.4 mmol) was added to the mixture and stirred
for a further 15 min. The protected amino acid H-l-arginine(Pbf)-OMe (hydrochloric acid salt; 1.1 equiv, 16.7
g, 35.4 mmol) was added as a single portion, and the reaction was
then stirred for 18 h at 20 °C. After this time, the solvents
were removed in vacuo and the resulting residue dissolved in EtOAc
(200 mL) washed with 1 M hydrochloric acid (3 × 80 mL). The organic
layer was washed with NaCl (aqueous satd solution, 80 mL), dried (Mg2SO4), and concentrated in vacuo. The residue was
purified by flash column chromatography (silica; EtOAc in iso-hexane
50/50 to 0/100). The desired fractions were collected and concentrated
in vacuo to give 5. Yield: 21 g, 25.5 mmol, 79%. Off-white
solid. LC-MS tR = 4.77 min. MS m/z −826/828 [M + H]+. HRMS: Calculated for C33H41BrN5O9S3 826.1250. Measured Mass: 826.1279. 1H NMR (600 MHz, DMSO-d6) δ
11.02 (s, 1H), 8.64 (d, J = 7.3 Hz, 1H), 7.73 (d, J = 5.3 Hz, 1H), 7.65 (s, 1H), 7.57 (s, 1H), 7.16 (d, J = 5.4 Hz, 1H), 4.67–4.54 (m, 2H), 4.41–4.30
(m, 1H), 3.65 (s, 3H), 3.19 (s, 2H), 3.10–3.02 (m, 2H), 2.93
(s, 2H), 2.47 (s, 3H), 2.42 (s, 3H), 1.97 (s, 3H), 1.84–1.66
(m, 2H), 1.50–1.43 (m, 1H), 1.40 (m, 6H). 13C NMR
(151 MHz, DMSO-d6) δ 172.08, 163.43,
157.48, 156.11, 155.98, 141.05, 137.27, 133.99, 133.31, 131.47, 130.56,
128.27, 124.35, 121.26, 120.02, 116.29, 113.47, 110.67, 86.33, 73.99,
52.20, 52.04, 42.48, 40.10, 30.42, 29.02, 27.54, 22.12, 13.97.
(S)-2-{[3-(5-Bromo-2,3-dihydro-benzofuran-7-sulfonylamino)-thiophene-2-carbonyl]-amino}-5-(2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonyl-guanidino)-pentanoic
acid methyl ester (1 eq, 2.45 g, 2.96 mmol) was stirred with 1 M lithium
hydroxide (5 equiv, 14.82 mL mg, 14.82 mmol) in tetrahydrofuran (29
mL) at 20 °C for 3 h. After this time, the organic solvents were
removed in vacuo and the (aqueous) residue diluted with water (30
mL) and then acidified to pH 1 with 6 M hydrochloric acid. Ethyl acetate
(200 mL) was added to the resulting suspension and, after thorough
mixing, the organic layer separated. The aqueous layer was further
extracted with ethyl acetate (150 mL), and the organic extracts were
combined, washed with brine (saturated aqueous solution; 3 ×
75 mL), dried over magnesium sulfate, filtered, and the solvent removed
in vacuo. The product (pale-yellow foam, 2.42 g, 100%) was used without
further purification. LC-MS: Rt 4.89 min.
MS m/z −812/814 [M + H]+. HRMS: Calculated for C32H39BrN5O9S3 812.1093. Measured mass: 812.1063. 1H NMR (700 MHz, DMSO-d6, plus
20 μL of D2O, 353.2 K) δ 7.52 (dd, J = 2.2, 0.8 Hz, 1H), 7.38 (dt, J = 2.2,
1.2 Hz, 1H), 7.29 (d, J = 5.4 Hz, 1H), 7.14 (d, J = 5.4 Hz, 1H), 4.54–4.45 (m, 2H), 4.26 (dd, J = 8.6, 4.8 Hz, 1H), 3.19–3.06 (m, 4H), 2.95 (s,
2H), 2.57 (s, 1H), 2.46–2.43 (m, 3H), 2.01 (s, 3H), 1.83–1.74
(m, 1H), 1.74–1.65 (m, 1H), 1.64–1.54 (m, 2H), 1.42
(s, 7H). 13C NMR (151 MHz, DMSO-d6, plus 20 μL D2O) δ 173.65, 163.18,
158.46, 158.25, 157.51, 156.18, 156.13, 155.49, 137.32, 131.53, 128.35,
124.42, 118.17, 116.36, 116.19, 114.22, 109.91, 86.38, 51.74, 42.44,
34.16, 28.38, 28.31, 28.18, 18.95, 17.62, 12.25.
General Procedure
for Suzuki–Miyaura Couplings
(S)-2-{[3-(5-Bromo-2,3-dihydro-benzofuran-7-sulfonylamino)-thiophene-2-carbonyl]-amino}-5-(2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonyl-guanidino)-pentanoic
acid (approx 1 g, 1.23 mmol, 1.0 equiv), boronic acid (1.85 mmol,
1.5 equiv), and tetrakis(triphenylphosphine)palladium(0) (71 mg, 0.06
mmol, 0.05 equiv) were suspended in degassed 1,2-dimethoxyethane (3
mL). Potassium phosphate (4.92 mmol, tribasic, 2 M aqueous solution
2.46 mL, 4 equiv), also degassed, was further added and the reaction
mixture heated using microwave conditions (100 W, 90 °C, ramp
time = 10 min). After this time, the solvent was removed in vacuo
and the resulting residue was partitioned between ethyl acetate (200
mL) and hydrochloric acid (1 M aqueous solution; 150 mL). The phases
were separated and the aqueous phase further extracted with ethyl
acetate (200 mL). The organic extracts were combined, washed with
brine (saturated, aqueous solution; 2 × 100 mL), dried over magnesium
sulfate, filtered, and the solvent removed in vacuo. The crude product
(typically a yellow solid; approx 1.5 g) was purified by flash column
chromatography on silica gel (eluent: dichloromethane increasing to
dichloromethane/methanol; 75:25) to afford the desired product.
A solution
of the aldehyde (1 equiv) in tetrahydrofuran/methanol (1:1, 1.5 mL)
was added to the amine (commercially available; 1.1 equiv) followed
by acetic acid (1–2 drops ∼ pH 6). The reaction was
stirred at 20 °C for 2 h before sodium cyanoborohydride (2 equiv)
in methanol (0.1 mL) was added in one portion. The reaction was stirred
for a further 16 h at 20 °C. The reaction was filtered through
a preconditioned SCX-2 (1 g) cartridge and the product eluted with
2 M ammonia in methanol. Solvent evaporation gave the product as a
yellow oil, which was dissolved in dichloromethane/trifluoroacetic
acid (1:1, 8 mL) and stirred at 20 °C for 1 h. The solvent was
removed in vacuo and the crude residue dissolved in dimethyl sulfoxide
and purified by (mass-directed) preparative LC-MS using a preparative
C-18 column (Phenomenex Luna C18 (2), 100 mm × 21.2 mm, 5 μM)
and a linear AB gradient of 5–95% for B over 12 min at a flow
rate of 20 mL/min, where, (i) eluent A was 0.1% formic acid/water
and eluent B was 0.1% formic acid/methanol or, (ii) eluent A was 10
mM ammonium bicarbonate (pH 9) and eluent B was 100% methanol. The
purified compounds were isolated via solvent evaporation and carried
through to Pbf removal.
General Procedure for Pbf Removal
The residue was dissolved
in dichloromethane/trifluoroacetic acid (1:1, 5 mL) and stirred at
room temperature for 1 h. The solvent was removed in vacuo and the
crude residue dissolved in dimethyl sulfoxide and purified by (mass-directed)
preparative LC-MS using a preparative C-18 column (Phenomenex Luna
C18 (2), 100 mm × 21.2 mm, 5 μM) and a linear AB gradient
of 5–95% for B over 12 min at a flow rate of 20 mL/min, where
eluent A was 0.1% formic acid/water and eluent B was 0.1% formic acid/acetonitrile.
The purified peptidomimetics were isolated via solvent evaporation.
(S)-2-{[3-(5-Bromo-2,3-dihydro-benzofuran-7-sulfonylamino)-thiophene-2-carbonyl]-amino}-5-(2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonyl-guanidino)-pentanoic
acid methyl ester (5 g, 6.00 mmol), bispinacolato diboron (1.85 g,
7.3 mmol), Pd(dppf)2Cl2 (443 mg, 0.6 mmol),
and KOAc (1.78 g, 18.1 mmol) were combined and suspended in dioxane
(30 mL). The suspension was degassed with nitrogen for 30 min before
heating at 100 °C for 16 h. The reaction was allowed to cool
to room temperature and partitioned between EtOAc (200 mL) and water
(200 mL). The aqueous phase was extracted with EtOAc (2 × 100
mL), and the combined organic extracts were washed with water (300
mL) then brine (300 mL) and dried over Na2SO4. Concentration in vacuo provided the crude product as a brown residue
(6.6 g). The crude product was purified by flash column chromatography
on silica gel (eluent: neat ethyl acetate, increasing to ethyl acetate/MeOH
95:5), affording the desired product as a brown foam (3.73 g, 71%).
Used directly in the Suzuki reactions. LC-MS (pH 2): Rt 3.27 min. MS m/z
873 [M + H]+.
Using the standard Suzuki
procedure but with CsCO3 (3 equiv) as the base and boronic
acid 16 following the Suzuki procedure used for 18a above and t-butyl-5-bromoisoindoline-2-carboxylate
as the coupling partners (scale 2 × 0.11 mmol and 1 × 0.23
mmol – total scale = 0.46 mmol of boronic acid). Product 17b white solid, 71 mg, 0.074 mmol, 16%. LC-MS (pH 2): Rt 3.40 min. MS m/z −965 [M + 1]+. Rt 3.46
min. MS m/z −951 [carboxylic
acid + 1]+. Hydolysis of the methyl ester using LiOH followed
by standard Pbf removal. To a stirred solution of the methyl ester
(71 mg, 0.073 mmol) in THF (2.5 mL) was added 1 M lithium hydroxide
(aq, 0.37 mL, 0.36 mmol) and the reaction mixture stirred at room
temperature for 1.5 h. The solvent was removed in vacuo and the residue
dissolved in DCM/TFA (1:1, 1.8 mL) and stirred at room temperature
for 18 h and then 40 °C for 24 h. The solvent was removed in
vacuo and the crude residue dissolved in DMSO and purified by (mass-directed)
preparative LC-MS using a preparative C-18 column (Phenomenex Luna
C18 (2), 100 mm × 21.2 mm, 5 μM) and a linear AB gradient
of 5–95% for B over 12 min at a flow rate of 20 mL/min, where
eluent A was 0.1% formic acid/water and eluent B was 0.1% formic acid/MeOH.
The combined HPLC fractions were concentrated in vacuo to provide
the title compound as a white solid, 29 mg, 0.05 mmol, 66%. LC-MS
(pH 2): Rt 4.03 min. MS m/z −599 [M + H]+. HRMS (ES +ve):
Calculated for C25H26N5O6S2 597.1590. Measured mass: 597.1576. 1H NMR
(600 MHz, DMSO-d6) δ 12.88 (s, 1H),
11.08 (s, 1H), 8.71–8.35 (m, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.75–7.70 (m, 2H), 7.69–7.65 (m, 2H),
7.60 (dd, J = 7.9, 1.7 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 5.5 Hz, 1H),
4.72–4.64 (m, 2H), 4.60–4.51 (m, 4H), 4.39–4.29
(m, 1H), 3.32–3.19 (m, 2H), 3.15–3.07 (m, 2H), 1.91–1.82
(m, 1H), 1.81–1.66 (m, 1H), 1.59–1.46 (m, 2H). 13C NMR (151 MHz, DMSO) δ 173.15, 171.49, 163.48, 156.79,
156.58, 156.28, 141.36, 138.90, 136.25, 130.31, 126.67, 124.74, 123.58,
120.20, 119.88, 113.17, 73.79, 52.05, 40.32, 36.53, 28.37, 27.35,
22.55, 1.20.
Biology
United Kingdom: All in vivo
study protocols,
husbandry, and anesthesia were performed by followed guidelines of
United Kingdom Home Office Scientific Procedures Act (1986). USA:
All animal work was approved by the Stony Brook University IACUC.
Protein
Expression, Purification, and Crystallization
Recombinant
human NRP1-b1 domain was expressed in Escherichia coli strain Rosetta Gami 2 (DE3) pLysS
cells. Gene sequence corresponding to NRP1-b1 residues 273–427
was subcloned into pET15b vector, resulting in a protein product that
contains a TEV protease- cleavable His6 tag on the N-terminus.
The protein was purified to a high level of homogeneity using a combination
of Ni-NTA (GE Healthcare) affinity chromatography with size exclusion
chromatography (Superdex S75, GE Healthcare) and ion exchange chromatography
using an SP FF Sepharose column (GE Healthcare). Purified protein
was dialyzed into 20 mM Tris-HCl pH 7.9 and 50 mM NaCl prior to crystallization.Crystallization of both the high and low resolution crystals was
performed using the vapor diffusion methods using a 1:1 ratio of protein
to mother liquor. Crystallization of the low resolution structure
was carried out at 20 °C, with NRP1-b1 concentration of 10 mg/mL
supplemented with 1 mM of 1 and with the precipitant
solution containing 16% w/v PEG3350 and 200 mM ammonium chloride.
Crystals of the high resolution structure were grown at 4 °C
with protein solution at 10 mg/mL supplemented with 2.3 mM of 1. Crystallization condition contained 12% w/v PEG3350 and
200 mM ammonium chloride. Prior to data collection, both crystals
were cryoprotected in mother liquor containing an additional 22% PEG300.
Data Collection and Processing
Low resolution (2.8
Å) data was collected at the Institute for Structural and Molecular
Biology X-ray crystallography facilities equipped with Rigaku Micromax
007 generator and a Rigaku Saturn 944+ CCD detector. The 600 images
were collected with a 0.5° oscillation which were processed using
d*TREK.[38]High resolution (0.9 Å)
data was obtained at Soleil, Paris. Diffraction data was collected
at the PROXIMA 1 beamline with a DECTRIS PILATUS 6 M detector. The
1800 images of 0.1° oscillations were collected and processed
using XDS software.[39]Molecular replacement
was performed using Phaser[40] with the NRP1-b1
domain structure (PDB 3I97) as a model. Refinement
of the lower resolution structure was performed using Phenix.[41] The high resolution structure was initially
refined with Phenix before completing the refinement using ShelXL.[42] Model building of both structures was performed
using COOT.[43] Data quality and refinement
statistics are shown in Table S1 (Supporting
Information).
SPR Experimental
Surface plasmon
resonance experiments
were performed using a Biacore 4000 instrument at 25 °C. Sensor
chips, buffer stock solutions, and immobilization reagents were from
GE Healthcare. Recombinant human NRP1-b1 was as above. Other reagents
were obtained from Sigma. Immobilization: PBS (containing 0.05% surfactant
P20) was used as the running buffer. The four flow cells were treated
in the same way to optimize throughput. In summary, using a CM5 chip
spots 1 and 2 were activated with the coupling reagents EDC and NHS
for 10 min. NRP1-b1 at a concentration of 20 μg/mL in 10 mM
sodium acetate pH 5 was injected onto the surface for 10 and 5 min
in spots 1 and 2, respectively, to generate surfaces with high and
low density. The immobilization levels ranged from 2302 to 1823 RU
on spot 1 and from 948 to 1112 RU in spot 2. The unmodified spot 3
was used as a reference. Kinetics and affinity measurements: PBS containing
0.05% surfactant P20 and 3% DMSO was used as the running buffer and
sample dilution buffer. Dose–responses were obtained using
a 2-fold sample dilution from 16 μM to 31 nM, using an injection
time of 60 s. Surface regeneration between injections was not necessary,
but a wash step with 1 M NaCl was included after injection of the
highest concentration sample for each compound. Data processing: Binding
curves were corrected for variations in DMSO concentration and normalized
by molecular weight. Binding results to high and low-density surfaces
were processed independently and the average ± SD is presented. KDs reported are derived from steady-state binding
responses and therefore correspond to the equilibrium binding affinity
of the compounds.
Isothermal Titration Calorimetry
Isothermal titration
calorimetry (ITC) experiments were carried out in a reaction buffer
(20 mM Tris pH 7.9, 50 mM NaCl) using a MicroCal iTC200 system (Malvern)
at 20 °C. Prior to the experiments, the Nrp1-b1 sample was dialyzed
in the reaction buffer and all solutions, including the buffer that
was used for heat dilution measurements, were degassed and filtered
just before loading into the calorimeter. Then 20 μM Nrp1-b1
in the reaction cell was titrated with the 200 μM stock solutions
of 1. Nineteen consecutive compound injections of 2 μL
at 0.4 s/μL were applied at 100 s intervals while stirring the
reaction solution at 1000 rpm constant speed. For heat dilution of
the protein, 0.2 mL of reaction buffer in the reaction cell was titrated
by 40 μL of 200 μM compound, and this value was subtracted
from the measured heats of binding. Protein concentrations of the
samples used in these experiments were estimated by UV absorbance
measurements at 280 nm. Data were evaluated using Origin version 7.0
software (OriginLab) with the ITC plug-in provided by the instrument
manufacturer.
Pharmacokinetics
To test compound
drug-like properties,
selected compounds with low IC50 were further tested for
pharmacokinetics (PK) profile. First, 6–8 week-old BABL/c female
mice were used, then 2 mg/kg of compounds was formulated in 7.5% DMSO
and 92.5% solution and intravenously dosed from the tail vein as a
bolus. Blood samples were collected by cardiac puncture at 5, 15,
30, 60, 180, and 240 min post dosing. Plasma samples were prepared
by centrifugation at 7000 rpm for 5 min, and supernatants were collected,
immediately snap-frozen on dry ice, and stored at −20C. Samples
were analyzed by liquid chromatography–tandem mass spectrometry
using electrospray ionization and data was analyzed by WinNonlin software.
btVEGF-A Cell-Free Binding Assay
The 96-well plates
were precoated with NP1 protein at 3 μg/mL overnight at 4 °C.
On the following day, the plates were treated with blocking buffer
(PBS containing 1% BSA) and washed three times with wash buffer (PBS
containing 0.1% Tween-20). The various concentrations of compounds
diluted in PBS containing 1% DMSO were added, followed by addition
of 0.25 nM of bt-VEGF-A165. After 2 h of incubation at
room temperature, the plates were washed three times with wash buffer.
The bound bt-VEGF-A165 to NP-1 was detected by streptavidin–horseradish
peroxidase conjugates and the enzyme substrate, and measured using
a Tecan Genios plate reader at A450 nm
with a reference wavelength at A595 nm.
Nonspecific binding was determined in the absence of NP-1 coated wells
of the plates.
Immunoblotting
Cells were lysed
with RIPA buffer (Sigma)
supplemented with protease inhibitor (Roche) and phosphatase inhibitors
II and III (Sigma) and analyzed by SDS-PAGE with 4–12% Bis·Tris
gels (Nupage, Invitrogen), followed by electrotransfer onto Invitrolon
PVDF membranes (Invitrogen). Membranes were blocked with nonfat dry
milk (5% w/v) and Tween-20 (0.1% v/v) in tris-buffered saline (TBS-T),
for 1 h at room temperature before being probed with the primary antibody
by overnight incubation at 4 °C, followed by incubation
for 1 h at room temperature with a horseradish-peroxidase-linked secondary
antibody (Cell Signaling) and detection with the aid of Clarity Western
ECL Substrate (BioRad), by the manufacturer’s protocol. Immunoblots
were quantified by scanning the films and then performing densitometry
by use of ImageJ (US National Institutes of Health; http://rsb.info.nih.gov/ij/). Differences between three concentrations of 1 were
evaluated by the two-way analysis of variance (ANOVA) with Sidak’s
multiple comparisons test. Values represent means ± SEM determined
from the results of three independent experiments. A value of p < 0.05 was considered statistically significant.
Cell Culture
Human umbilical vein endothelial cells
(HUVECs) were obtained from TCS Cell Works (Buckingham, UK) and were
cultured on tissue culture-coated flaks in endothelial basal medium
(EBM; Cambrex BioScience Ltd., Nottingham, UK) supplemented with gentamycin–ampicillin,
epidermal growth factor, and bovine brain extract (Singlequots; Cambrex)
and 10% fetal bovine serum (FBS) (complete EBM). For experimental
purposes, fully confluent HUVECs at passages 1–3 were preincubated
overnight with 1% FBS in EBM prior to addition of factors and other
treatments. DU145/cells.Ad.NRP1 cells: DU145 prostate cancer cells
were from ATCC and infected with an adenovirus expressing WT NRP1
as previously described[27] (cells were grown
in 10% FCS in DMEM). A375P melanoma cells were from ATCC. Cells were
grown in 10% FCS in DMEM.Human dermal fibroblasts (HDF) were
obtained from TCS cells Works Buckingham, UK) and were cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco).
For experimental purposes, fully confluent HDF at passages 1–3
were preincubated overnight with serum-free DMEM prior to other procedures.
GL261 cells were purchased from ATCC and cultured as described.[44,45]
Transwell Migration
Transwell cell culture inserts
made of transparent, low pore density polyethylene terephthalate (PET)
with 8 μm pore size (Falcon; BD Biosciences, Oxford, UK) were
inserted into a 24-well plate. Serum-free media supplemented with
or without 25 ng/mL VEGF-A, 0.1% DMSO, or 1 (30 μM)
were placed in the bottom chamber, and HUVECs in suspension (1.5 ×
105 cell/well in serum-free EBM) were added to the top
chamber of a 24-well plate and incubated at 37 °C for 4 h. HUVECs
that had not migrated or had only adhered to the upper side of the
membrane were removed before membranes were fixed and stained with
a Reastain Quik-Diff kit (IBG Immucor Ltd., West Sussex, UK) using
the manufacturer’s protocols. Plates were allowed to dry overnight
and HUVECs that had migrated to the lower side of the membrane were
counted in three random fields per well.
Coculture
In vitro
angiogenesis was determined by using
a coculture assay. Briefly, HDF cells were grown to confluence in
24-well plates in 10% DMEM, 1% penicillin/streptomycin. Medium was
replaced, and 10000 HUVECs were plated on top of the fibroblast layer
cultured in complete endothelial growth medium supplemented with 1%
FBS. HUVECs, or medium with 0.1% DMSO, VEGF 25 ng/mL, 1 (30 μM), and 1 (30 μM) + VEGF 25 ng/mL
and fibroblasts were propagated in coculture for 4 days at 37 °C
and 5% CO2. Cells in coculture were fixed in absolute ethanol
for 1 h at room temperature, washed twice in PBS, and blocked using
PBS 5% milk. HUVECs were identified by incubating with anti-von Willebrand
factor antibody (Dako, 1:1000) in PBS 5% milk overnight at 4 °C.
Antibody was removed and cells washed with PBS. The secondary antibody,
goat anti-rabbit IgG, Alexa Fluor 488 conjugate (Life Technologies,
1:1000), was added on cells and left for 1 h in the dark. Solution
was removed and plate was scanned using IncuCyte.Photomicrographs
of von Willebrand factor-stained cocultures were analyzed using ImageJ
software. The Network area, length of all tubular structures, and
the number of branching points were measured in four representative
microscopic fields per well.
Scratch Assay
The experiment was performed using the
ESSEN IncuCyte system. Graduated 96-well plates from ESSEN were used
to seed HUVECs. When cells reached 95% confluence, a wound was made
on every well using the Wound Maker 96 instrument (ESSEN instruments).
Wells were supplemented with 1% EGM containing 0.1% DMSO, 25 ng/mL
VEGF-A, and/or 1 (30 μM). Cell migration toward
the wounds was monitored every 2 h and analyzed by the IncuCyte software.
Ex Vivo
Aortic Ring Sprouting Assay
This protocol was
adapted from previous studies.[32] All animal
and tissue procedures were carried out in accordance with United Kingdom
Home Office regulations and guidance. Briefly, female wild-type C57Bl/6
mice were killed in accordance with United Kingdom Home Office regulations.
Thoracic aorta was harvested from aortic arch. Aorta was placed into
a sterile Petri dish containing Opti-MEM Glutamax (Life technologies)
and 1% penicillin/streptomycin. Under the dissection micrioscope,
aortas were cleaned by sharp dissection and the vessel sliced into
0.5 mm rings with a scalpel. Rings were serum starved overnight at
37 °C in 5 mL of OptiMEM Glutamax supplemented with 1% penicillin/streptomycin.
On ice, 1.37 mL of purified type 1 rat-tail collagen (Millipore, Watford,
United Kingdom) was mixed with 0.5 mL of 10× DMEM (Gibco) and
3.13 mL of dH2O before adding 2 μL/ml of 5 M NaOH.
A 55 μL amount of this embedding matrix was pipetted per well
into a 96-well plate and aortic ring submerged within. Plates were
left for 15 min at room temperature before incubation for 60 min at
37 °C. A 150 μL amount of OptiMEM Glutamax containing 2.5%
FBS and 1% penicillin/streptomycin was added per well with medium
containing 0.1% DMSO, 25 ng/mL VEGF-A, and/or 1 (30 μM).
Aortic rings were incubated at 37 °C for 7 days with a medium
change on day 3 and 5. Wells were washed with 150 μL of PBS
containing 2 mM CaCl2 and 2 mM MgCl2 and fixed
in 4% formalin for 30 min. The collagen was permeabilized with three
15 min washes with PBS buffer containing 2 mM MgCl2, 2
mM CaCl2, and 0.25% Triton X-100. Rings were blocked in
30 μL of 1% BSA in PBLEC (PBS containing 100 μM MnCl2, 1% Tween-20, 2 mM CaCl2, 2 mM MgCl2) for 30 min at 37 °C. Then 100 μL of a mix containing
IsolectinB4 (1:100, Vector Laboratories) and anti smooth muscle, SMA
(1:100, Sigma) was added per well, followed by overnight incubation
at 4 °C. Wells were washed three times with 100 μL of PBS
containing 0.1% Triton X-100 and then with 100 μL of sterile
water. Aortic rings were imaged and the area of sprout growth was
quantified using ImageJ software.
Spheroid Assay
Spheroids were generated using the metho-cellulose
technique as described previously. Briefly, cells were trypsinized
and counted. A mix containing 10 mL of metho-cellulose-containing
medium (30% metho-cellulose, 70% culture medium) was prepared and
kept on ice. Approximately 5000 cells were added to the mix. Spheroids
were produced by pipetting 100 μL of the cell suspension into
a well of a 96-well round-bottomed nontissue-culture plate and incubating
for 24 h (37 °C, 5% CO2). Spheroids were collected
and embedded in a mix containing 700 μL of collagen type I (3.1
mg/mL), 200 μL of 5× DMEM, 100 μL of H2O, and supplemented with 0.1% DMSO, 25 ng/mL VEGF-A, and/or 1 30 μM. Spheroids were allowed to invade for 7 days,
followed by fixation in 4% formaldehyde. Spheroid invasion was determined
by measuring the circular area of the spheroid core and the rim of
invasion using ImageJ. The rim of invasion was determined as the circular
distance from the edge of the core to the edge of contiguous invading
cells.
Isolation and Treatment of Nrp1+ Regulatory T Cells
Primary murine splenocytes were isolated from 6-week-old C57/Bl6
mice. Briefly, mice were heavily anesthetized with avertin (0.02 mg/g
ip) and perfused with 1× PBS. Spleens were dissected, chemically
digested in papain for 15 min at 37 °C, minced, and dissociated
in PBS supplemented with 1% FBS and 1 mM EDTA. Cell suspensions were
filtered through 40 μm filters, washed thoroughly, and a Dynabeads
Untouched Mouse CD4 Cells kit (Invitrogen) was used to purify CD4+
cells following the manufacturer’s protocol. The cells were
then purified further for Nrp1 expression using positive selection
for Nrp1 using anti-Nrp1 antibody (Biolegend) and then using magnetic
Dynabeads (Invitrogen) to pull out this fraction. Cells were plated
at a density of 200000 cells per well. Plates were precoated with
anti-CD3ε (clone 145-2C11, BD Biosciences) overnight at 4 °C.
Cells were cultured in 250 μL of RPMI supplemented with 10%FBS,
1× penicillin/streptomycin, and anti-CD28 antibody (clone 37.51,
BD Biosciences) for costimulation. Cells were allowed to expand for
72 h, at which point they were treated with either DMSO control (untreated)
or 500 nM 1 for 2 h. Media was then supplemented with
50% glioma-conditioned media (GCM) isolated from confluent plates
of GL261 cells (ATCC)[44,45] that were serum starved for 24
h. Cultures were allowed to sit for 12 h, after which cells were isolated
for flow cytometric analysis of CD4 (eBioscience), CD25 (Biolegend),
Nrp1 (Biolegend), and FOXP3 (BD Biosciences) expression using a BD
LSR Fortessa flow cytometer (BD Biosciences). Medium was isolated
and TGFβ release by cells was quantified using a TGFβ
Ready Set Go ELISA kit (eBioscience) following the manufacturer’s
protocol.
Authors: Qi Pan; Yvan Chanthery; Wei-Ching Liang; Scott Stawicki; Judy Mak; Nisha Rathore; Raymond K Tong; Joe Kowalski; Sharon Fong Yee; Glenn Pacheco; Sarajane Ross; Zhiyong Cheng; Jennifer Le Couter; Greg Plowman; Franklin Peale; Alexander W Koch; Yan Wu; Anil Bagri; Marc Tessier-Lavigne; Ryan J Watts Journal: Cancer Cell Date: 2007-01 Impact factor: 31.743
Authors: Haiyan Jia; Rehan Aqil; Lili Cheng; Chris Chapman; Shaheda Shaikh; Ashley Jarvis; A W Edith Chan; Basil Hartzoulakis; Ian M Evans; Antonina Frolov; John Martin; Paul Frankel; Snezana Djordevic; Ian C Zachary; David L Selwood Journal: Chembiochem Date: 2014-04-25 Impact factor: 3.164
Authors: Mahesh Yadav; Cedric Louvet; Dan Davini; James M Gardner; Marc Martinez-Llordella; Samantha Bailey-Bucktrout; Bryan A Anthony; Francis M Sverdrup; Richard Head; Daniel J Kuster; Peter Ruminski; David Weiss; David Von Schack; Jeffrey A Bluestone Journal: J Exp Med Date: 2012-09-10 Impact factor: 14.307
Authors: Greg M Delgoffe; Seng-Ryong Woo; Meghan E Turnis; David M Gravano; Cliff Guy; Abigail E Overacre; Matthew L Bettini; Peter Vogel; David Finkelstein; Jody Bonnevier; Creg J Workman; Dario A A Vignali Journal: Nature Date: 2013-08-04 Impact factor: 49.962
Authors: Jabar A Faraj; Ali Jihad Hemid Al-Athari; Sharaf El Din Mohie; Iman Kareem Kadhim; Noor Muhsen Jawad; Weaam J Abbas; Abduladheem Turki Jalil Journal: Med Oncol Date: 2022-09-29 Impact factor: 3.738
Authors: Bartlomiej Fedorczyk; Piotr F J Lipiński; Anna K Puszko; Dagmara Tymecka; Beata Wilenska; Wioleta Dudka; Gerard Y Perret; Rafal Wieczorek; Aleksandra Misicka Journal: Molecules Date: 2019-05-06 Impact factor: 4.411
Authors: Daniel Conole; Yi-Tai Chou; Anastasia Patsiarika; Valery Nwabo; Eleni Dimitriou; Christelle Soudy; Filipa Mota; Snezana Djordjevic; David L Selwood Journal: Drug Dev Res Date: 2020-01-20 Impact factor: 4.360
Authors: Christopher A Chuckran; Chang Liu; Tullia C Bruno; Creg J Workman; Dario Aa Vignali Journal: J Immunother Cancer Date: 2020-07 Impact factor: 13.751
Scheme 1
Figure 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9