Download SUPPLEMENTAL INFORMATION

Structure-based identification of small molecule ACE2
activators as novel antihypertensives
José A. Hernández Prada1*, Anderson J. Ferreira1,5*, Michael J. Katovich3,
Vinayak Shenoy3, Yanfei Qi3, Robson A. S. Santos6, Ronald K. Castellano4,
Andrew J. Lampkins4, Vladimir Gubala4, David A. Ostrov2, Mohan K. Raizada1
McKnight Brain Institute1 and Departments of Physiology and Functional
Genomics1 and Pathology, Immunology and Laboratory Medicine 2, College of
Medicine; Department of Pharmacodynamics3, College of Pharmacy; and
Department of Chemistry4, College of Liberal Arts and Sciences, University of
Florida, Gainesville, Florida 32610, USA; Department of Morphology5 and
Department of Physiology and Biophysics6, Biological Sciences Institute, Federal
University of Minas Gerais, Belo Horizonte, MG 31270-901, Brazil.
Address for Correspondence:
Mohan K Raizada, Ph.D. Department of Physiology and Functional Genomics
PO Box 100274, e-mail: [email protected] Phone: (352) 392-9299
Fax: (352) 294-0191
*Jose A. Hernandez Prada and Anderson J. Ferreira contributed equally to this
work.
Running title: ACE2 activators as antihypertensive agents
MS# 108944
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SUPPLEMENTAL INFORMATION:
Compounds and synthesis:
Initial functional screening was performed using compounds obtained from the
NCI/DTP. Dry compounds were dissolved in 100% dimethyl sulfoxide (DMSO)
and used in an in vitro angiotensin-converting enzyme (ACE) 2 assay. A member
of the xanthone family, CAS# 86456-22-6, 1-[[2-(dimethylamino)ethyl]amino]-4(hydroxymethyl)-7-[[(4-methylphenyl)sulfonyl]oxy]- 9H-xanthen-9-one (hereafter
referred to as XNT) was dissolved in saline at low pH (2-2.5) for in vivo studies.
This compound was consistently prepared 24-48 hours before delivery in
animals. The XNT was prepared on a gram scale in six synthetic steps from 5methoxysalicylic acid and m-chloroiodobenzene through modifications of a
published procedure.1,2 Synthetic details and the first structural characterization
data (1H NMR, 13C NMR, and HRMS) for XNT and its precursors are as follows.
Synthesis of xanthenone3 (1)
General:
Reagents and solvents were purchased from commercial sources and used
without further purification unless otherwise specified. THF, ether, CH 2Cl2, and
DMF were degassed in 20 L drums and passed through two sequential
purification columns (activated alumina; molecular sieves for DMF) under a
positive argon atmosphere. Thin layer chromatography (TLC) was performed on
SiO2-60 F254 aluminum plates with visualization by UV light or staining. Flash
column chromatography was performed using Purasil SiO2-60, 230−400 mesh
MS# 108944
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from Whatman. 300 (75) MHz 1H (13C) NMR spectra were recorded on Varian
Mercury 300, Gemini 300, or VXR 300 spectrometer. Chemical shifts () are
given in parts per million (ppm) relative to TMS and referenced to residual
protonated solvent (CDCl3: H 7.27 ppm, C 77.00 ppm; DMSO-d6: H 2.50 ppm,
C 39.50 ppm). Abbreviations used are s (singlet), d (doublet), t (triplet), q
(quartet), quin (quintet), hp (heptet), b (broad), and m (multiplet). ESI spectra
were recorded on a Bruker APEX II FTICR spectrometer.
MS# 108944
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2-(3-Chlorophenoxy)-5-methoxybenzoic acid (4):
A suspension of 5-methoxysalicylic acid (2) (14.1 g, 83.9 mmol) and anhydrous
K2CO3 (20.9 g, 151 mmol) in dry DMF (200 mL) was heated to 120 °C for 10 min.
m-Chloroiodobenzene (3) (20.0 g, 83.9 mmol), Cu powder (0.39 g, 6.2 mmol),
and freshly purified CuI (0.25 g, 1.2 mmol) were then added, and the mixture was
allowed to reflux under argon for 48 h. The cooled suspension was suction
filtered and the filtrate was concentrated to a gummy residue, which was taken
up in DI water and acidified with HCl. The resulting mixture was extracted three
times with EtOAc, and the combined organics were washed with water and brine,
dried over MgSO4, clarified with activated carbon, and concentrated under
reduced pressure. The resulting residue was recrystallized from hexanes/EtOAc
to give a mixture of 2 and 4. This mixture was recrystallized from 60% MeOH in
water to give 4 (10.9 g, 47%) as colorless needle-like crystals: 1H NMR (CDCl3) δ
3.85 (s, 3H), 6.84 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 6.93 (d, J = 2.4 Hz, 1H), 6.95
(d, J = 9.3 Hz, 1H), 7.07 (dd, J = 8.4 Hz, J = 0.9 Hz, 1H), 7.10 (dd, J = 9.0 Hz, J =
3.3 Hz, 1H), 7.23 (t, J = 8.4 Hz, 1H), 7.59 (d, J = 3.3 Hz, 1H). 13C NMR (CDCl3) δ
56.4, 115.5, 116.2, 116.7, 120.3, 122.5, 124.9, 126.6, 131.8, 134.4, 147.3, 156.8,
160.5, 166.6. HRMS (ESI) calculated for C14H11ClO4 + Na (M+) 301.0238,
experimentally determined 301.0253.
1-Chloro-7-methoxy-9H-xanthen-9-one (5):
To polyphosphoric acid (84%, 50 g) preheated to 120 °C was added 4 (8.5 g, 30
mmol). The solution was stirred for 6 h, after which it was poured into water (300
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mL). After dissolving the yellow gum, the resulting white suspension was
neutralized with 10% NaOH. The mixture was filtered and washed with water.
The solid was dried in vacuo to give a dull white mixture of 5 and the 3-chloro
isomer (6.8 g, 85%); the mixture was carried through to the next step without
further purification: 1H NMR (CDCl3) δ 3.91 (s, 3H), 3.92 (s, 3H), 7.32–7.45 (m,
6H), 7.51 (d, 1H), 7.55 (t, 1H), 7.68 (d, 1H), 7.69 (d, 1H), 8.26 (s, 1H), 8.29 (s,
1H). HRMS (ESI) calculated for C14H10ClO3 + Na (M+) 261.0312, experimentally
determined 261.0318.
1-[[2-(Dimethylamino)ethyl]amino]-7-methoxy-9H-xanthen-9-one (6):
In a sealed pressure tube, a solution of 5 (8.00 g, 30.8 mmol) in freshly distilled
pyridine (20 mL) was added N,N-dimethylethylenediamine (11.2 mL, 98.4 mmol)
and the solution was heated to 120–130 °C for 12 h. Upon cooling to rt, the
solvent was evaporated and the residue was diluted with CH 2Cl2 and extracted
twice with 1 M HCl. The combined aqueous extracts were made basic with 10%
NaOH and extracted an additional three times with CH2Cl2. The combined
organic layers were dried over MgSO4 and concentrated to a crude residue,
which was purified using column chromatography (15:1 CH2Cl2 / MeOH) to afford
6 (2.88 g, 30%) as a bright yellow solid: 1H NMR (CDCl3) δ 2.35 (s, 6H), 2.68 (t, J
= 6.6 Hz, 2H), 3.35 (dt, J = 6.3 Hz, J = 5.1 Hz, 2H), 3.90 (s, 3H), 6.39 (d, J = 8.4
Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 7.24 (ddd, J = 9.3 Hz, J = 3.3 Hz, J = 0.8 Hz,
1H), 7.32 (d, J = 9.0 Hz, 1H), 7.44 (t, J = 8.4 Hz, 1H), 7.65 (d, J = 3.0 Hz, 1H),
9.59 (bs, 1H).
MS# 108944
13C
NMR (CDCl3) δ 41.0, 45.5, 55.8, 58.0, 102.1, 103.1, 105.6,
5
106.6, 118.5, 122.3, 123.8, 135.7, 150.0, 151.6, 155.6, 158.1, 179.6. HRMS
(ESI) calculated for C18H21N2O3 + Na (M+) 313.1546, experimentally determined
313.1556.
1-[[2-(Dimethylamino)ethyl]amino]-7-hydroxy-9H-xanthen-9-one (7):
A solution of 6 (6.0 g, 19 mmol) in 60 mL of 56% HI was heated to reflux for 6h.
The mixture was cooled, and the HI salt of 6 was filtered and washed with ether.
The salt was then dissolved in H2O (300 mL) and made alkaline with NaOH. The
solution was clarified by filtration through a bed of Celite and then the filtrate was
carefully neutralized with dilute acetic acid. The yellow crystals that resulted were
filtered, washed with H2O, and dried to yield 4.2 g (73 %) of 7. 1H NMR (DMSOd6) δ 2.18 (s, 6H), 2.50 (t, J = 5.7 Hz, 2H), 3.22 (t, J = 5.1 Hz, 2H), 6.39 (d, J =
8.4 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 7.17 (m, 1H), 7.39 (m, 3H), 9.45 (s, 1H),
9.85 (bs, 1H).
13C
NMR (DMSO-d6) δ 40.2, 45.1, 57.3, 101.6, 103.5, 105.6,
108.5, 118.4, 122.0, 123.5, 136.1, 148.2, 151.0, 153.6, 157.4, 178.4. HRMS
(ESI) calculated for C17H19N2O3 + Na (M+) 299.1390, experimentally determined
299.1393.
1-[[2-(dimethylamino)ethyl]amino]-7-[[(4-methylphenyl)sulfonyl]oxy]-
9H-
xanthen-9-one (8):
To a stirring solution of 7 (4.0 g, 13 mmol) was added p-TsCl (3.0 g, 16 mmol)
and the mixture was allowed to stir at 50–60 °C, under argon, for 4 h. Upon
cooling, the solvent was evaporated and the residue was dissolved in EtOAc / DI
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H2O and poured into a separatory funnel. The organic layer was separated and
the aqueous layer was extracted a second time with EtOAc. The organic layers
were combined, washed with brine (containing a few mLs aq. HCl to scavenge
pyridine), dried over MgSO4 and concentrated to a crude residue, which was
purified using column chromatography (15:1 CH2Cl2 / MeOH) to afford 8 (5.3 g,
88%) as an orange/yellow solid: 1H NMR (CDCl3) δ 2.32 (s, 6H), 2.41 (s, 3H),
2.63 (t, J = 6.3 Hz, 2H), 3.30 (q, J = 6.3 Hz, 2H), 6.36 (d, J = 8.4 Hz, 1H), 6.47 (d,
J = 8.4 Hz, 1H), 7.24–7.41 (m, 5H), 7.69 (m, 3H), 9.41 (bs, 1H).
13C
NMR
(CDCl3) δ 21.6, 40.9, 45.5, 57.8, 102.0, 103.9, 106.3, 118.7, 119.1, 122.6, 128.4,
128.5, 129.9, 131.9, 136.3, 145.1, 145.6, 151.5, 153.5, 157.8, 178.4. HRMS
(ESI) calculated for C24H25N2O5S + Na (M+) 453.1478, experimentally determined
453.1482.
1-[[2-(Dimethylamino)ethyl]amino]-4-(hydroxymethyl)-7-[[(4methylphenyl)sulfonyl]oxy]-9H-xanthen-9-one (1):
In a sealed pressure tube, a solution of 8 (4.6 g, 10 mmol), 37% aqueous
formaldehyde (400 mL), and 5 M HOAc (10 mL) was allowed to stir at 70–80 °C
for 72 h. Upon cooling, the solution was made basic by addition of aqueous
Na2CO3, poured into a separatory funnel and extracted with CHCl3. The organics
were combined, dried over MgSO4 and concentrated to a crude residue. EtOAc
and CH2Cl2 were added and the mixture was sonicated at ambient temperature
for 15 min prior to removal of the solid material by suction filtration. The filtrate
was concentrated and purified via column chromatography (15:1 CH2Cl2 / MeOH)
MS# 108944
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to afford 1 (1.37 g, 29%) as an orange/yellow solid: 1H NMR (CDCl3) δ 2.35 (s,
6H), 2.45 (s, 3H), 2.65 (t, J = 6.0 Hz, 2H), 3.07 (bs, 1H), 3.30 (q, J = 5.1 Hz, 2H),
4.76 (s, 2H), 6.34 (m, 1H), 7.30 (m, 4H), 7.46 (d, J = 8.7 Hz, 1H), 7.71 (m, 3H),
9.40 (bs, 1H).
13C
NMR (CDCl3) δ 21.7, 40.8, 45.5, 57.7, 59.8, 103.6, 106.0,
113.5, 118.8, 119.1, 122.5, 128.4, 129.9, 131.9, 136.7, 137.6, 145.2, 145.6,
151.2, 153.1, 155.0, 178.3. HRMS (ESI) calculated for C25H27N2O6S + Na (M+)
483.1584, experimentally determined 483.1570.
Measurement of ACE2 activity:
Human recombinant ACE and ACE2 were obtained from R&D systems,
Minneapolis, MN, along with their respective fluorogenic substrates (ACE,
catalog ID: 929-ZN-10; ACE2, 933-ZN-10; ACE substrate, fluorogenic peptide V,
Mca RPPGFSAFK(Dnp)-OH, catalog ID: ES005; ACE2 substrate, fluorogenic
peptide VI, Mca-YVADAPK(Dnp)-OH, ES007). Enzymatic activity was measured
with a Spectra Max Gemini EM Fluorescence Reader (Molecular Devices) as
previously described.4 Compounds were tested against 50 μM substrate. All
assays were performed at least in triplicate in a reaction mixture containing 10
nM enzyme, 1 M NaCl, 75 mM Tris-HCl, 1% DMSO and 0.5 μM ZnCl2, at pH 7.4.
Samples were read every 15-30 seconds for at least 30 minutes immediately
after the addition of fluorogenic peptide substrates at 37 °C. DMSO did not affect
the activity of ACE or ACE2 under these conditions. Enzyme activity was
corrected for background.
MS# 108944
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Histological analysis:
At the end of the chronic study, hearts and kidneys were fixed in 10% buffered
formalin, embedded in paraffin, and sectioned to a thickness of 5 μm. Sirius red
staining was carried out to assess the extent of collagen deposition. Cardiac and
renal interstitial fibrosis at 100X magnification was measured by percent area
analysis. Perivascular fibrosis was measured at 250X magnification and data
was normalized to vessel lumen. An Olympus BX 41 microscope was used for
imaging and quantification of collagen density data was carried out with ImageJ
software from the NIH.5
Statistical analysis:
Data are expressed as mean ± SEM. Unpaired Student’s t-test and 1-way
ANOVA were performed for statistical analysis. For cardiac function, response to
Angiotensin II, bradykinin (BK), and losartan experiments, statistical significance
was estimated using 2-way ANOVA followed by the Bonferroni test. Differences
were considered significant at a p<0.05 or p<0.001, as indicated. Tests were
performed with the PRISM software package from GraphPad, San Diego.
References
(1) Archer S, Zayed AH, Rej R, Rugino TA. Analogues of hycanthone and
lucanthone as antitumor agents. J Med Chem. 1983;26:1240-1246.
MS# 108944
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(2) Archer S, Pica-Mattoccia L, Cioli D, Seyed-Mozaffari A, Zayed AH.
Preparation and antischistosomal and antitumor activity of hycanthone and
some of its congeners. Evidence for the mode of action of hycanthone. J
Med Chem.1988;31:254-260.
(3)
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings. Adv Drug Deliv Rev. 2001;46:3-26.
(4)
Huentelman MJ, Zubcevic J, Katovich MJ, Raizada MK. Cloning and
characterization of a secreted form of angiotensin-converting enzyme 2.
Regul Pept. 2004;122:61-67.
(5)
Abramoff MD, Magelhaes PJ, Ram SJ. Image Processing with ImageJ.
Biophotonics International. 2004;11:36-42.
MS# 108944
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Supplemental Table S1 – Top ten scoring compounds for the three different
docking sites.
Rank #
Site 1
1
2
3
4
5
6
7
8
9
10
Site 2
1
*2
*3
4
5
6
7
8
*9
10
Site 3
1
*2
3
4
*5
*6
*7
*8
*9
*10
Catalog
Number
xlogP
NSC269897
NSC72361
NSC354677
NSC72756
NSC21221
NSC354317
NSC43058
NSC43083
NSC21044
NSC354297
NA**
3.41
NA
NA
1.84
NA
3.03
5.06
3.38
0.75
0
1
2
0
0
2
2
0
0
0
2
5
8
5
2
5
5
2
1
8
0
0
0
0
0
0
0
0
0
0
224
295
483
228
159
382
241
274
184
320
3
3
6
4
2
0
3
4
2
2
-19.26
-17.35
-16.03
-15.68
-14.00
-13.38
-12.15
-11.52
-11.09
-10.95
NSC121146
NSC243619
NSC324063
NSC90568
NSC371456
NSC42370
NSC631816
NSC103522
NSC624460
NSC371140
-0.7
-4.35
-1.42
1.46
2.49
2.62
1.12
5.33
2.55
-0.06
5
0
8
7
2
2
5
5
5
2
11
10
14
7
8
10
7
6
6
9
-2
-4
-1
0
0
-1
0
0
0
0
400
302
467
258
384
337
312
442
305
305
9
6
9
3
6
4
3
4
3
6
-32.43
-30.57
-28.99
-26.01
-25.94
-25.62
-25.56
-25.47
-25.30
-25.26
NSC83458
NSC138120
NSC658245
NSC152085
NSC138115
NSC82526
NSC694478
NSC704636
NSC657774
NSC407491
1.26
8.12
-1.12
2.55
7.77
0.54
2.41
2.98
8.01
2.24
6
1
5
8
1
6
5
2
0
1
10
3
11
5
3
10
9
10
6
6
2
0
2
2
0
2
0
-1
2
0
493
506
468
298
417
465
394
598
471
243
4
3
8
3
3
4
7
6
5
3
-27.71
-26.40
-26.31
-25.86
-25.63
-25.42
-25.35
-25.31
-24.91
-24.77
HHnet
molecular rotatable
donors acceptors charge
weight
bonds
Score
All compounds were requested from the NCI/DTP for in vitro testing. Active
compounds are highlighted. The top ten scoring compounds of each site share
some general characteristics. Site 1 clearly selected for smaller uncharged
compounds with relatively few hydrogen bond donors and acceptors. Site 2
favored neutral or negatively charged compounds and Site 3 seems to have
MS# 108944
11
favored positively charged compounds. Several compounds in sites 2 and 3 meet
Lipinski criteria less conservatively. The shared characteristics of these
compounds likely reflect the properties of the sites selected for virtual screening
and it appears site 1 is better fit for the ligation of a drug-like molecule. * Not
available from the NCI. **Information not available.
MS# 108944
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Supplemental Figure S1
a
225
mmHg
200
SHR
*
175
XNT
vehicle
150
WKY
125
b
0
3
5
10
15
20
4
d 4000
Dose (ng/Kg)
0
 MAP (mmHg)
1
2
Weeks
25
-30
30
*
-60
WKY
mmHg/sec
100
-1
e
10
15
20
25
30
-30
*
-60
SHR
mmHg/sec
 MAP (mmHg)
1000
SHR
Dose (ng/Kg)
5
MS# 108944
*
2000
0
0
-90
3000
0
-90
c
+ dP/dt
10
20
30
40
Time (minutes)
- dP/dt
4000
3000
2000
*
1000
SHR
0
0
10
20
30
40
Time (minutes)
13
Supplemental Figure S2
MS# 108944
14
Figure Legends
Supplemental Figure S1: Functional effects of chronic infusion of XNT. Nine
rats in each group were fitted with osmotic minipumps and infused with vehicle
(black bullets) or XNT at 60 µg/day (white bullets). Indirect blood pressure (BP)
was monitored as described in Methods. (a) Effect of chronic XNT infusion in BP
of SHR and WKY rats. The decrease of BP started at the first week of infusion
and it achieved the maximal decrease by the third week in SHR (p<0.05 n=9). (b,
c) Effect of BK on BP in WKY (b) and SHR (c). After 28 days of XNT infusion, as
described previously, rats were injected with the indicated doses of BK and BP
was monitored as described in Methods. The BK effect was more pronounced in
hypertensive rats. XNT treatment potentiated the BK hypotensive effect in both
strains. (d, e) Cardiac function in isolated hearts from XNT-treated SHR. Chronic
infusion of XNT resulted in an increase (n=8) in (d) + dP/dt and (e) - dP/dt in the
SHR. *p<0.05 and *** p<0.001 compared with vehicle-infused rats (n=6).
Supplemental Figure S2: Effects of XNT on cardiac and renal fibrosis:
representative images. Heart and kidney were sectioned and stained with Sirius
Red and examined. A significant increase in myocardial (b) and perivascular (e)
fibrosis was seen in the SHR compared with the WKY rat (a,d). XNT treatment
reduced this fibrosis (c,f). Increased fibrosis in the SHR kidney (h) compared to
the WKY kidney (g) was also diminished by XNT treatment (i).
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