targeting of αv integrin identifies a core molecular ... · itgavflox/flox;pdgfrb-cre hscs...
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Targeting of αv integrin identifies a core molecular pathway that regulates
fibrosis in several organs
Neil C Henderson*, Thomas D Arnold, Yoshio Katamura, Marilyn M Giacomini, Juan D
Rodriguez, Joseph H McCarty, Antonella Pellicoro, Elisabeth Raschperger, Christer
Betsholtz, Peter G Ruminski, David W Griggs, Michael J Prinsen, Jacquelyn J Maher, John
P Iredale, Adam Lacy-Hulbert, Ralf H Adams, Dean Sheppard*
*Address correspondence to: Neil Henderson (e-mail: Neil.Henderson@ed.ac.uk) or Dean
Sheppard (e-mail: Dean.Sheppard@ucsf.edu).
Nature Medicine: doi:10.1038/nm.3282
Nature Medicine: doi:10.1038/nm.3282
ALT
(IU/L
× 1
03 )
cb Control v Cre
Oil
20
864
j Controlv cre
CD31
(% a
rea)
50
201510
Oil CCl4
Control v Cre
NS
f Control v Cre
Oil
Neut
/ po
rtal t
ract
Controlv cre
Chr
onic
CC
l 4
Oil CCl4
NS
g
50
201510
NS
Oil
Acu
te C
Cl 4
Control v Cre
Oil
Chr
onic
CC
l 4
h
Oil CCl4
20
864
Controlv cre
F4/8
0 (%
are
a)
i
NSNS
NS
NS
Oil
Chr
onic
CC
l 4
Controlv cre
v SMAv / SMA
/ DAPI
a
10
d
Desm
in (%
are
a)
Cont v Cre
50
25201510
NS
PDG
FR
(% a
rea)
Cont v Cre
e
50
25201510
NS
CCl4
Supplementary Figure 2: Hepatic myofibroblasts express αv integrin in human fibrotic liver tissue, and αv integrin depletion on murine HSCs does not affect initial liver injury after CCl4 treatment, baseline HSC number, inflammatory infiltrates or the vasculature of itgavflox/flox;Pdgfrb-Cre livers.(a) Representative immunofluorescence micrographs of human fibrotic liver tissue stained for αv integrin (red), αSMA (green) and DAPI (blue) (n = 5 cases). Arrow (right panel) indicates αv integrin and αSMA double-positive cell. Scale bar, 10 μm. (b) Micrographs of hematoxylin / eosin stained liver sections from control or itgavflox/flox;Pdgfrb-Cre (αv Cre) mice 24 h after a single i.p. injection of olive oil or CCl
4 (n = 6 male mice per group). Scale bar, 200μm. (c) Serum alanine
aminotransferase (ALT) levels. (d,e) Digital image analysis of desmin and PDGFRβ staining in uninjured control and itgavflox/flox;Pdgfrb-Cre livers (n = 6 male mice per group). (f) Gr1 immunohistochemistry of liver tissue after control or chronic CCl
4 treatment of control and itgavflox/flox;Pdgfrb-Cre mice. (n = 8 male mice per group). Scale bar, 50μm.
(g) Neutrophil counting. (h) F4/80 immunohistochemistry of liver tissue after control or chronic CCl4 treatment of
control and itgavflox/flox;Pdgfrb-Cre mice. Scale bar, 200μm. (i) Digital image analysis quantification of F4/80 staining. (j) CD31 immunohistochemistry of liver tissue (left) after control or chronic CCl
4 treatment of control and
itgavflox/flox;Pdgfrb-Cre mice. Scale bar, 100μm. Digital image analysis quantification of CD31 staining (right). Data are mean ± s.e.m. (Student’s t test).
Nature Medicine: doi:10.1038/nm.3282
Hep LSEC Kupffer HSC
-actin
v
Cre
Con
trol
v
Cre
Con
trol
v
Cre
Con
trol
v
Cre
Con
trol
PDGFR
v
b
- TMLC
- TMLC
+ TMLC
+ TMLC
TMLC
c d
SM
A (fo
ld c
hang
e)
Col
1A1
(fold
cha
nge)
e
Contro
l
Contro
l
+ TGF
+ TGF
NS
NS Controlv CreTMLC
0
100
200
300
0
200
400
600
- TMLC
- TMLC
+ TMLC
+ TMLC
TMLC
TGF
1 (p
g/m
l) NS
NS
Controlv Cre
0
1
2
3NS
NS
* **
Contro
l
Contro
l
+ TGF
+ TGF
0
1
2
3
TGF
1 (p
g/m
l)
a
Supplementary Figure 3: αv integrin and PDGFRβ expression in purified hepatic cell populations from itgavflox/flox;Pdgfrb-Cre mice, total TGF-β1 concentrations in control and itgavflox/flox;Pdgfrb-Cre HSCs, and addition of TGF-β1 to itgavflox/flox;Pdgfrb-Cre HSCs rescues pro-fibrotic gene expression. (a) Western blotting of αv integrin and PDGFRβ expression in purified hepatocytes (Hep), liver sinusoidal endothelial cells (LSECs), kupffer cells and HSCs from control and itgavflox/flox;Pdgfrb-Cre (αv Cre) male mice 5 days post isolation. (b) Total TGF-β1 concentrations measured by ELISA in cell lysates from control and itgavflox/flox;Pdgfrb-Cre HSCs cultured with and without TMLCs (c) Total TGF-β1 concentrations measured by ELISA in supernatants from control and itgavflox/flox;Pdgfrb-Cre HSCs cultured with and without TMLCs. (d) qPCR analysis of αSMA expression in control and itgavflox/flox;Pdgfrb-Cre (αv Cre) HSCs treated with control or recombinant TGF-β1 (1ng ml-1) for 24 h. (e) qPCR analysis of Col1A1 expression in control anditgavflox/flox;Pdgfrb-Cre (αv Cre) HSCs treated with control or recombinant TGF-β1 (1ng ml-1) for 24 h. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (Student’s t test).
Nature Medicine: doi:10.1038/nm.3282
0.0005
0
0.0010
0.0015TG
F1
/ 18S
HSC
Kupffe
rLS
EC
Hepato
cytes
c
b
dControl v Cre
TUN
EL
Contro
l
Fibron
ectin
Collag
en I
Collag
en IV
Lamini
n
Fibrino
gen
0
40
30
20
10
50
% a
dhes
ion
Controlv cre
% m
igra
tion
0
40
30
20
10
Control FCS
Controlv cre
e
TUNE
L-po
sitive
nuc
lei (
%)
0
0.4
0.3
0.2
0.1
Controlv Cre
Con v Cre
NS
NS
NS
NS
NS
NS
NS
NS
a
Supplementary Figure 4: TGF-β1 expression in hepatic cell populations isolated from CCl4-induced fibrotic liver, and assessment of adhesion, migration and anoikis in control and itgavflox/flox;Pdgfrb-Cre HSCs.(a) qPCR analysis of TGFb1 mRNA expression in purified HSCs, hepatocytes, liver sinusoidal endothelial cells (LSECs) and kupffer cells isolated from CCl4-induced fibrotic livers (n = 4 male mice per group). (b) Adhesion assay of control and itgavflox/flox;Pdgfrb-Cre HSCs plated on different matrices. (c) Migration assay of control and itgavflox/flox;Pdgfrb-Cre HSCs (d) TUNEL immunofluorescence staining of control and itgavflox/flox;Pdgfrb-Cre HSCs. Arrows indicate TUNEL positive nuclei (green), DAPI (blue). (e) Quantitation of TUNEL-positive nuclei. Data are mean ± s.e.m. (Student’s t test).
Nature Medicine: doi:10.1038/nm.3282
Cou
nts
Fluoresence
Cou
nts
Cou
nts
a b
c
0 105102 103 104
0 105102 103 104
Control 5-PE
Fluoresence
Fluoresence
v Cre 5-PE
Control Iso-PE
v Cre Iso-PEControl 1-PE
v Cre 1-PE
Control Iso-PE
v Cre Iso-PE
Control 1-PE
v Cre 1-PE
Control Iso-PE
v Cre Iso-PE
0 105102 103 104
0
50
100
25
75%
reco
mbi
natio
n
Desm
inPD
GFR
PDG
FR
Desm
in
SM
A
Saline Bleomycin
d
Supplementary Figure 5: FACS analysis of α1, α5 and β1 integrin expression levels in control and itgavflox/flox;Pdgfrb-Cre HSCs, and recombination efficiency in lung pericytes and myofibroblasts in Ai14;Pdgfrb-Cre mice.FACS analysis of (a) α1, (b) α5 and (c) β1 integrin expression levels in control and itgavflox/flox;Pdgfrb-Cre HSCs cultured on tissue culture plastic for 5 days. (d) Analysis of recombination efficiency in lungs from saline treated (control) or bleomycin treated Ai14;Pdgfrb-Cre reporter mice (n = 4 male mice per group) stained for desmin, PDGFRβ or αSMA. Data are mean ± s.e.m.
Nature Medicine: doi:10.1038/nm.3282
Reporter / DAPI SMA / DAPIb
d
Lung
sK
idne
ys
Mergeda-actin-SMA
c-actin-SMA
Salin
eBl
eoSa
line
Bleo
Sham
UUO
Sham UU
OReporter / DAPI SMA / DAPI Merged
e v-actin
v CreControlSaline
g
v-actin
v CreControlBleomycin
fv
-actin
v CreControlOil
v-actin
v CreControl
CCl4
h i v-actin
v CreControlSham
v-actin
v CreControlUUO
j
Control v Cre
Control v Cre Control v Cre
CC
l 4O
il
UU
OS
ham
Ble
oS
alin
e
Supplementary Figure 6: Myofibroblast induction in Ai14-Td tomato positive cells sorted from lungs and kidneys of Ai14;Pdgfrb-Cre mice, and analysis of αv and β6 integrin expression in uninjured and fibrotic lung, liver and kidney in control and itgavflox/flox;Pdgfrb-Cre mice.(a) Western blotting of αSMA in freshly sorted Td tomato positive cells from saline or bleomycin treated (28 days post instillation) Ai14;Pdgfrb-Cre mice (n = 4 male mice per group). (b) Immunofluoresence staining of Td tomato positive cells sorted from the uninjured lungs of Ai14;Pdgfrb-Cre mice and plated on tissue culture plastic for 7 days. Endogenous report (red), αSMA (green), DAPI (blue). Scale bar, 100μm. (c) Western blotting of αSMA in freshly sorted Td tomato positive cells from sham operated or UUO (day 7) Ai14;Pdgfrb-Cre mice (n = 4 male mice per group). (d) Immunofluoresence staining of Td tomato positive cells sorted from the uninjured kidneys of Ai14;Pdgfrb-Cre mice and plated on tissue culture plastic for 7 days. (e) Western blotting of αv expression in whole lung homogenates from saline or bleomycin treated control and itgavflox/flox;Pdgfrb-Cre (αv Cre) mice. n = 5 male mice per group. (f) Representative sections of αvβ6 immunohistochemistry in lungs from saline or bleomycin treated control and itgavflox/flox;Pdgfrb-Cre (αv Cre) mice. Scale bar, 50 μm. (g) Western blotting of αv expression in whole liver homogenates from olive oil or CCl
4 treated control and itgavflox/flox;Pdgfrb-Cre (αv Cre) mice. n = 5 male mice per group. (h)
Representative sections of αvβ6 immunohistochemistry in livers from olive oil or CCl4 treated control and itgavflox/flox;Pdgfrb-Cre (αv Cre)
mice. Arrows indicate αvβ6 positive biliary epithelium. Scale bar, 50 μm. (i) Western blotting of αv expression in whole kidney homogenates from sham operated or UUO (day 14) control and itgavflox/flox;Pdgfrb-Cre (αv Cre) mice. n = 5 male mice per group. (j) Representative sections of αvβ6 immunohistochemistry in kidneys from sham operated or UUO (day 14) control and itgavflox/flox;Pdgfrb-Cre (αv Cre) mice. Scale bar, 50 μm.
Nature Medicine: doi:10.1038/nm.3282
a
Br
HN
NH O
OHN
NHN
OH
CO2H
OH
CWHM 12 CWHM 96
S-enantiomer
b
Br
HN
NH O
OHN
NHN
OH
CO2H
OH
R-enantiomer
c v1
0
1.0
2.0
0.5
1.5
10-3 10-1 101 103 105
Concentration (nM)
Abso
rban
ce (6
50 n
M) CWHM-12
CWHM-96
0
0.25
0.50
0.75
1.00
1.25
Abso
rban
ce (4
05 n
M)
Concentration (nM)10-2 100 102 104
v3d
e v5
100 101 102 103 104 1050
0.25
0.50
0.75
1.00
1.25
Abso
rban
ce (4
05 n
M)
Concentration (nM)
f v6
0
0.5
1.0
1.5
10-2100 10210-4 104 106
Abso
rban
ce (4
05 n
M)
v8
Abso
rban
ce (6
50 n
M)
Concentration (nM)10-4 10-2 100 102
g
Concentration (nM)
0
1
2
3
4
Integrin CWHM12 Mean IC50 (nM) + SD
CWHM96 Mean IC50 (nM) + SD
Fold difference
αvβ1 1.8 + 0.9 288 + 70 160 X
αvβ3 0.8 + 0.6 784 + 81 980 X
αvβ5 61 + 17 8762 + 4988 144 X
αvβ6 1.5 + 2.5 459 + 106 306 X
αvβ8 0.2 + 0.1 366 + 325 1,830 X
αIIbβ3 > 5000 > 5000 Both inactive
α2β1 > 5000 > 5000 Both inactive
α10β1 > 5000 > 5000 Both inactive
h
Abso
rban
ce (6
50 n
M)
Concentration (nM)
IIb3
10-2 100 102 1040
50
100
150
200
21 ki j
0
1.0
2.0
0.5
1.5
100 102 10410-2
Concentration (nM)
Abso
rban
ce (6
50 n
M)
101
Abso
rban
ce (6
50 n
M)
0
1.0
2.0
0.5
1.5
Concentration (nM)100 102 10410-2
Supplementary Figure 7: Inhibition of αv integrins by the novel small molecule CWHM 12.(a) Chemical structure of CWHM 12. (b) Chemical structure of CWHM 96 (control R-enantiomer of CWHM 12). (c–g) Ligand-binding functions of αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8 were measured as described in Materials and Methods.Representative CWHM 12 and CWHM 96 inhibition curves are shown for each assay. (h–j) Ligand-binding functions of αIIbβ3, α2β1 and α10β1 were measured as described in Materials and Methods. Representative CWHM 12 and CWHM 96 inhibition curves are shown for each assay. (k) Mean IC50 values and standard deviation from at least two identical experiments are summarized.
Nature Medicine: doi:10.1038/nm.3282
a PDGFR / DAPI
p-Smad3 / DAPI
PDGFR / p-Smad3/ DAPI
Con
trol
CW
HM
12b **
0
20
40
60
Cont C12
Arb
itrar
y un
its100
400
300
200
0Hydr
oxyp
rolin
e (u
g/g)f
Con
c
6 weeks CCl4
Control CWHM 96
PS
R
d
Siriu
s re
d (%
are
a)e
0
6
4
2
8
Con C96 C96
500CWHM96treatment
NS NS
Supplementary Figure 8: Phospho-Smad3 signaling is reduced in the livers of CWHM 12 treated mice following chronic CCl4 administration, and treatment with CWHM 96 (control R-enantiomer of CWHM 12) does not inhibit liver fibrosis.(a) Immunofluoresence micrographs of liver sections from control and CWHM 12 treated mice following chronic CCl
4
treatment (n =14 male mice per group). Scale bar, 25 μm. (b) Digital image analysis quantification of phospho-Smad3 staining. (c) Dosing regime in the CWHM 96 (control R-enantiomer of CWHM 12) liver fibrosis model. Mice were given CCl
4 I.P. twice weekly for 3 weeks, then Alzet osmotic minipumps containing either CWHM 96 or vehicle
(50% DMSO) were inserted, followed by a further 3 weeks of CCl4 I.P. twice weekly. (d) Picrosirius red staining of
liver tissue from vehicle control and CWHM 96 treated mice (n =10 male mice per group) after chronic CCl4 treatment.
Scale bar, 200μm. (e) Digital image analysis quantification of collagen (picrosirius red) staining. (f) Hydroxyproline analysis. Data are mean ± s.e.m. **P < 0.01 (Student’s t test).
Nature Medicine: doi:10.1038/nm.3282
Supplementary Methods
Synthesis of CWHM 12.
Instrumentation and General Methods. Chemical ionization mass spectra were recorded at 70-ev
ionizing voltage on a Hewlett-Packard 5973 CI quadrupole mass spectrometer connected to a
Hewlett-Packard 6890 gas chromatograph fitted with an Agilent Tech 12 m × 0.2 mm × 0.33 m
DB-1 (cross-linked methyl silicone) column.
Preparation of (3S)-N-[3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]
benzoyl]glycyl-3-(3-bromo-5-t-butylphenyl)--alanine (CWHM 12).
Step 1: Preparation of 3-hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)aminobenzoic acid.
3-Hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)aminobenzoic acid was synthesized according to literature procedures1.
Step 2: Preparation of 2-(3-hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)amino)benzamido) acetic acid.
Coupling of 3-hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)aminobenzoic acid with glycine ethyl ester:
CO2H
OH
HNN
NHHO
OH
HNN
NHHO
NH
O
OH
O
Nature Medicine: doi:10.1038/nm.3282
Glycine ethyl ester hydrochloride (5.02 g, 35.95 mmol) was added to a suspension of 3-
hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)aminobenzoic acid (9.013 g, 35.87
mmol) in a 1:1 mixture of DMF (50 ml) and DCM (50 ml), and the mixture was stirred at room
temperature under nitrogen atmosphere. Neat N,N′-diisopropylcarbodiimide (6.75 ml, 43.60
mmol) was added to above reaction mixture, and the mixture was stirred at room temperature
overnight to give a colorless suspension. The crude reaction mixture was used as such for the
hydrolysis of the above ester.
The above crude reaction mixture was cooled to 10 °C (ice bath), and a 2.5 N NaOH
solution (90 ml) was added slowly with stirring, the solution temperature was kept below 20 °C,
to give a pale yellow solution/suspension. The reaction mixture was stirred at room temperature
for 1.5 h. The reaction mixture was acidified with 5-N HCl with stirring to pH 5 to give a
colorless precipitate, and the mixture was stirred at room temperature for another 15 min and
filtered to give a colorless solid. The solid was washed with water (1 × 25 ml) and then with
acetonitrile (1 × 25 ml). The solid was dried in vacuo to give a colorless powder (9.686 g, yield
88%).
1H NMR (400 MHz, D2O): 3.37 (dd, J = 12.7 and 3.1 Hz, 2H), 3.50 (dd, J = 12.7 and
2.8 Hz, 2H), 4.17 (s, 2H), 4.37 (m, 1H), 6.97 (t, J = 2.01 Hz, 1H), 7.17–7.26 (m, 2H). 1H NMR
spectrum of the sample was consistent with the suggested structure of the product.
Step 3: Preparation of (S)-ethyl 3-amino-3-(3-bromo-5-t-butylphenyl)propionate hydrochloride.
(i) Preparation of 3-bromo-5-t-butylbenzaldehyde.
1,3-Dibromo-5-t-butylbenzene (4.95 g, 16.95 mmol) was dissolved in anhydrous ether
(25.0 ml) in a dried flask under nitrogen. The reaction mixture was cooled to 78 °C and stirred
Nature Medicine: doi:10.1038/nm.3282
under nitrogen atmosphere. A 1.6-M solution of n-BuLi in hexanes (10.60 ml, 16.95 mmol) was
added dropwise to the above solution and the reaction mixture was stirred at 78 °C for 30 min
after complete addition of n-BuLi. After 30 min of stirring at 78 °C, the reaction mixture was
warmed to 30 °C. DMF (1.60 ml, 20.66 mmol) was added to above reaction mixture dropwise,
keeping the reaction mixture below 20 °C. After addition of DMF was complete, the reaction
mixture was warmed slowly to 0 °C (30 min) and then stirring at room temperature overnight
under nitrogen to give a yellow-orange solution. The reaction mixture was poured into 40 ml of
chilled 10% aqueous HCl and the reaction mixture was stirred for 15 min. The ether layer was
separated, washed with water (2 × 25 ml), dried over anhydrous MgSO4, filtered and evaporated
in vacuo to give the product as a pale yellow viscous liquid. The crude product was dissolved in
dichloromethane (25 ml) and passed through a small pad of silica gel (~100 mg). Evaporation of
the solvent in vacuo gave the product as a very pale yellow viscous liquid (4.096 g). GC-MS
analysis (CI mode/methane) shows the desired product’s mass: m/z 240 (79BrM+) and m/z 242
(81BrM+); Calcd for C11H13BrO: 241.12.
(ii) Preparation of 3-amino-3-(3-bromo-5-t-butylphenyl)propionic acid.
A suspension of 3-bromo-5-t-butylbenzaldehyde (4.17 g, 17.30 mmol), malonic acid
(2.15 g, 20.72 mmol) and ammonium acetate (2.66 g, 34.59 mmol) in isopropanol (35 ml) was
heated at reflux under nitrogen for 3 h to afford a thick colorless solid. The solid was filtered hot,
washed with hot isopropanol (2 × 25 ml) and dried in vacuo to give the desired racemic product
as a colorless solid (2.68 g).
(iii) Preparation of ethyl 3-amino-3-(3-bromo-5-t-butylphenyl)propionate hydrochloride.
Nature Medicine: doi:10.1038/nm.3282
Absolute ethanol saturated with anhydrous HCl gas (75 ml) was added to 3-amino-3-(3-
bromo-5-t-butylphenyl)propionic acid (4.78 g, 15.92 mmol), and the reaction mixture was heated
at reflux for 1.5 h to give a pale yellow solution. The solvent was removed in vacuo to give a
colorless solid. The solid was slurried with diethyl ether and heptane (2 25 ml). After the
solvent layer was decanted off, the residue was dried in vacuo to give the racemic -amino ester
hydrochloride salt as a cream solid (5.00 g).
(iv) Preparation of (S)-ethyl 3-amino-3-(3-bromo-5-t-butylphenyl)propionate hydrochloride. Enzymatic resolution of the racemic mixture:
A suspension of ethyl 3-amino-3-(3-bromo-5-t-butylpheny)propionate hydrochloride
(4.54 g, 12.45 mmol) in water (10 ml) was basified with 2.5-N NaOH (pH ~12) by dropwise
addition to give a creamy oily residue. The pH of the aqueous layer was adjusted to pH 8.20 by
the addition of 50 mM KH2PO4 solution. Amano lipase PS (5.24 g) was added to above reaction
mixture and the reaction mixture was stirred at room temperature overnight. The reaction
mixture was filtered after 22 h, and the solid was washed with acetone to give a colorless solid of
the resolved (S) enantiomer (1.72 g).
Absolute ethanol saturated with anhydrous HCl gas (35 ml) was added to (S)-3-amino-3-
(3-bromo-5-t-butylphenyl)propionicacid (1.83 g, 6.10 mmol), and the reaction mixture was
heated at reflux for 2 h to give a colorless solution. The solvent was removed in vacuo to give a
cream-yellow foamy solid. The solid was slurried with heptane. After the solvent was decanted
off, the residue was dried in vacuo to give the desired (S)--amino ester hydrochloride salt as a
pale yellow foamy solid (2.26 g).
Step 4: Preparation of (3S)-N-[3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino] benzoyl]glycyl-3-(3-bromo-5-t-butylphenyl)--alanine (CWHM 12).
Nature Medicine: doi:10.1038/nm.3282
A mixture of 2-(3-hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-
yl)amino)benzamido)acetic acid (from step 2) (1.92 g, 6.23 mmol), and (S)-ethyl 3-amino-3-(3-
bromo-5-t-butylphenyl)propionate hydrochloride (from step 3) (2.25 g, 6.17 mmol) was
dissolved in DMF (20 ml) and dichloromethane (10 ml) to give a cream suspension. 1-
hydroxybenzatriazole hydrate was added to above reaction mixture and the reaction mixture was
stirred in an ice bath under nitrogen atmosphere for 10 min. N,N′-diisopropylcarbodiimide(DIC)
was added, and the reaction mixture was stirred at room temperature overnight under nitrogen
atmosphere. The solvent was evaporated in vacuo to give a pale yellow viscous gummy residue.
The residue was dissolved in acetonitrile (50 ml) and filtered to remove precipitated urea.
Evaporation of the filtrate in vacuo afforded a pale yellow to cream foamy residue of the crude
(3S)-N-[3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)imino]benzoyl]glycyl-3-(3-
bromo-5-t-butylphenyl)--alanine ethyl ester. To a suspension of the crude ester in a 1:1 mixture
of water/acetonitrile (14 ml), lithium hydroxide monohydrate (2.07 g, 49.38 mmol) at room
temperature and the reaction mixture was stirred at room temperature for 1.5 h. The reaction
mixture was acidified with TFA and evaporated in vacuo to give a cream foamy solid. The solid
was purified by reverse-phase preparative HPLC (10–90% water/acetonitrile containing 0.05%
TFA) to give the desired product, after lyophilization, as a colorless powder (2.244 g). LC/MS
shows the desired product’s mass: m/z 590 (79BrM+H) and 592 (81BrM+H); Calcd for
C26H32BrN5O6: 590.47. 1H NMR (400 MHz, DMSO-d6): 1.27 (s, 9H, (CH3)3C-), 2.70 (d, J =
7.31 Hz, 2H, -CH2-COOH), 3.16 (dd, J = 12.22, 3.10 Hz, 2H), 3.33 (brd, J = 12.44 Hz, 2H), 3.87
(d, J = 5.81 Hz, 2H), 4.08 (appt/m,J = 2.92 Hz, 1H), 5.18 (q, J = 7.62 Hz, 1H, -NH-CH-CH2-
COOH), 6.75 (t, J = 2.03 Hz, 1H), 7.13 (dt, J = 10.70 and 1.50 Hz, 2H), 7.34 (dt, J = 9.54 and
1.50 Hz, 2H), 7.39 (appt, J = 1.72 Hz, 1H), 8.09 (brs, 2H), 8.51 (d, J = 8.30 Hz, 1H), 8.61 (brt, J
= 5.88 Hz,1H), 9.57 (s,1H), 10.00 (brs, 1H), 12.35 (brs, 1H, -COOH). 1H NMR spectrum of the
sample was consistent with the suggested structure of the product.
Synthesis of CWHM 96. CWHM 96 was synthesized using the same method as described for
CWHM 12, except that (R)-ethyl-3-amino-3-(3-bromo-5-t-butylphenyl)propionate hydrochloride
Nature Medicine: doi:10.1038/nm.3282
is substituted for (S)-ethyl-3-amino-3-(3-bromo-5-t-butylphenyl)propionate hydrochloride in step
4. (R)-ethyl-3-amino-3-(3-bromo-5-t-butylphenyl)propionate hydrochloride is isolated from the
mother liquor in the enzymatic lipase reaction that produces (S)-3-amino-3-(3-bromo-5-t-
butylphenyl)propionate in the CWHM 12 method.
Reference:
1. Org. Process Res. Dev. 8, 571-575 (2004).
Nature Medicine: doi:10.1038/nm.3282
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