Current Programs in Medicinal/Biological Chemistry
1. Nitric oxide mimetic drug discovery
• Alzheimer’s and neurodegenerative disorders
• Colon cancer chemoprevention
2. Selective estrogen receptor modulators
• chemical toxicology & cancer promotion/prevention
• postmenopausal antidepressants (inc. botanical)
NIH/NCI CA 102590
NIH/NCCAM AT002299
NIH/NIA AG027425
Institute for Study of Aging-Elan Pharmaceuticals
A Brief Presentation of Research from the Thatcher Group
Better Living Through Chemistry
1.Nitric oxide mimetic drug discovery
►Clinical drugs: nitroglycerin (GTN); isosorbide dinitrate (ISDN); organic nitrates
-angina (1874); CHF; cardioprotection (2005)
Biological activity mimics NO: bioactivation to NO in vivo?
RONO2 + 3e- + 3H+  NO
►NO biology: Nobel Prize 1998 Medicine: “NO sex; NO wonder; NO way”
NO signaling ubiquitous (diffusible free radical gas binds to Fe-heme)
Endogenous NO: from Arg +NO synthase (NOS)
eNOS: smooth muscle relaxation, anti-atherogenic
iNOS: immune response; antibacterial
nNOS: neurotransmission, learning & memory
NO is essential for normal physiological function in the CNS, including learning &
memory, & is compromised in disease states including neurodegenerative disorders
ONO2
H3CO
2
NCX 4016
NCX 4040
O
ASA
NCX 4215
O
Better Living Through Chemistry
naproxen
sulindac
F
AZD 3582
NCX 1102
ASA
NO-ASA
versions
1.Nitric oxide mimetic drug discovery
►Clinical drugs: nitroglycerin (GTN); isosorbideNO-NSAIDs
dinitrate (ISDN); organic nitrates
-angina (1874); CHF; cardioprotection (2005)
NO chimeras
EtO2C
S S
O2N
O
S
.HCl
N
O2NO
O2NO
anti-inflammatory
GT 094
GABAA potentiator
GT 1061
GT 1061 in Phase 1 clinical trials for Alzheimer’s (FDA approved IND)
NO is essential for normal physiological function in the CNS, including learning &
memory, & is compromised in disease states including neurodegenerative disorders
Better Living Through Chemistry
2.
Selective estrogen receptor modulators
-tissue specific estrogen agonist effect: bone and lipids
-tissue specific antiestrogen effect: mammary and uterine
-Anti-osteoporosis; hormone replacement therapy (HRT); chemopreventive;
-anticancer; antiinflammatory
-endometrial cancer; breast cancer; thrombosis + stroke
Risk: long-term use in healthy (menopausal) women
► SERMs are polyaromatic phenols: oxidative metabolism by
oxidase/peroxidase
Are oxidative metabolites responsible for toxicity or therapeutic
activity?
Can new SERMs be designed to improve efficacy and safety
profile?
Common Themes & Vision
1. Nitric oxide mimetic drug discovery
2. Selective estrogen receptor modulators
•
both families are reactive molecules; metabolism is
important for activity and potentially toxicity; these are
good drugs clinically proven over decades or a century
•
NO signaling and biological redox systems are intrinsic to
the actions of both families
Aims
► Molecules hitting multiple targets; diseases are multifactorial
► Moderate potency, multiple mechanisms, high safety
► Translational research: bench-to-bedside
Tools of the Trade
1. Synthetic organic chemistry: drug candidates; model compounds;
reactive intermediates; novel biological probes Examples
2. Physical - Mechanistic organic chemistry: reaction mechanisms; kinetics;
identification of reaction intermediates; computational methodsExamples
3. Cell biology and proteomics: Perturbation of cell growth and protein/gene
biomarkers; ROS and NOx production; LC-MS-MS Examples
4. Animal models- cancer*, cardio** and cerebrovascular**: murine tissue
pathology; immunohistochemistry; ex vivo function Examples
5. Animal models- behavior: antidepressant; cognition enhancement;
anxiolytic; spatial working and reference memory; transgenics**
Examples
*
**
extradepartmental collaborations at UIC
extramural collaborations
Br
Br
1
H3CO
+
SHBr
KOH
EtOH-AcOEt
H3CO
S
N
OH
H2O2
Br
S
NaH, DMF
S
O
N
N
O
PPh3, TMSCl
1) HCl
O
O
O
Br
H3CO
Br
Br
S
5M NaOCH3, AcOEt
CuI, DMF-MeOH, 120oC
N
EtO2C
2
SH
CO2H
S
ONO2
1. EtOH, PTSA, Toluene, reflux
2. Allyl bromide, K2CO3, Acetone
3. AgNO3, I2, CH3CN
O2NO
ONO2 GT 794
+
EtO2C GT 947
S
i, ii, iii
ONO2
i. HNO3, H2SO4, DCM
S S
ONO2
ONO2
ii. Na2S2O3, CH3OH
ONO2
ONO2
iii. H2O2, H2SO4
GT 015
ONO2
SH 1. LiAlH , THF
4
SH
2. HNO3, (CH3CO)2O
S S
CO2H
OH
N
CuI,
proline,
NaOH
CH3SO2N
a, DMSO
120oC
S
CO2Et
CO2Et
S
O
O
3
HO
S
H3CO
O
1
O
2) BBr3
O
Br
OH
OH
O
Br
DCM-TFAH3CO
N
SH
S
Br
Br
Return
Br
H3CO
O
bromoacetamide
DCM-EtOHH3CO
PPA
O
ONO2
GT 4294
O
O
OMe
HO
S
SO2Me
HO
S
(H2C)5N(H2C)2OPh
2
(H2C)5N(H2C)2OPh
Tyr
P450
HO
O
OH
S
O
Return
(H2C)5N(H2C)2OPh
O
OH
OH
S
Raloxifene
O
MAJOR
2.5
GSH
nucleoside
GSH
nucleoside
conjugates
adducts
Ph3P
Mo-TPB
0.0
0
25
50
75
time, min
HPLC Chromatogram of raloxifene (0.05 mM), rat liver microsomes (1.0 nmol
P450/mL), GSH (0.5 mM), and a NADPH generating system in 50 mM phosphate
buffer ( 37 °C , 30 min). For control incubations, either GSH or NADP+ was omitted.
G (kcal/mol)
30
25
20
TS1
15
TS3
TS2
10
R1
TS1
R2
TS2
TS3
R3
R
R1
R3
2
G (kcal/mol)
25
20
15
TSb
TSa
10
5
0
Ra
TSa
Pa
R2
TSb
Rb
R
S
O
GTN
Legend
5.0
O
OH
HO
MINOR
O
conjugates
adducts
Response (corrected)
Tyr
P450
P450
S
HO
(H2C)5N(H2C)2OPh
O
P
Pb/R2
Rb
MW PBS
wash
3
MALDI-TOF + anti-GRP78 + rec. hGRP78 & PDIA4
Biotin PBS Biotin
eluate wash eluate
250
band 1
75
Return
78 kDa glucose-regulated protein (GRP 78; Bip)
72 kDa, protein disulfide isomerase A4 precursor
band 2
50
57 kDa, protein disulfide isomerase, precursor
57 kDa, protein disulfide isomerase, A3 precursor
LC-MS-MS
25
20
15
Cell cycle and
proliferation
coomassie
blue staining
rel. increse in cell
number as %
75
50
25
0
1.25 1.35 1.45 1.55 1.65 1.75 1.85 1.95 2.05
log ([GT094], uM)
Fig 9. Concentration response of cell number with GT094 in Caco-2 cell (48hr)
assayed by sulforhodamine B dye staining: EC50 = 40 μM.
rel % of cells in G2/M
0
36 ± 0.7
48.8 ± 2.5
14.9 ± 3.1
6
12
17.0 ± 6.4 28.5 ± 6.9
55.0 ± 4.6 38.1 ± 2.5
28.1 ± 1.8 33.5 ± 9.4
18
39.1 ± 2.3
36.5 ± 17.3
24.6 ± 15.1
24
26.4 ± 7.0
61.0 ± 5.6
12.7 ± 1.5
25
20
Figure 8. Cell cycle FACS analysis
of propidium bromide treated Caco2 cells incubated with GT 094 (100
uM, 48 h).
15
10
5
0
-5
0
6
12
t, hours
18
24
17 kDa, mGST 1
anti-mGST
10
100
Time, h
% G1
%S
% G2-M
band 3
Western blot
Analysis by
HRP-Streptavidin
Proteomic identification of
modified proteins in rat liver
4
Colon Cancer Chemoprevention
Return
1. GT094 significantly reduces ACFs compared
to no drug and ASA treatment.
no. of ACFs per colon
2. GT 094 significantly reduces colon cell
proliferation (p27 elevated) and tumor weight
and multiplicity in a 30 week AOM study
60
50
40
30
GT-094
iNOS
130 kDa
iNOS Quantity A.U
Aberrant Crypt Foci (ACF) correlate with
progression to tumors in animal models
AOM Treated Colon
GT-094
100
75
*
P=0.013
50
25
20
0
10
AOM Exposure Time (30 weeks)
0
4
09
T
G
N
A
ol
ntr
AS N/MD
o
c
ISM
+
A
AS
8 week drug treatment
* P< 0.01 vs Postsurgery
70
*
*
*
*
10
*
5
Percent correct
*
60
1
1
0.
5
0.
1
rg
Po
er
y
st
su
rg
er
y
50
es
u
Return
Pr
5
80
Donepezil
GT1061
(mg/kg)
(mg/kg)
-