PhD Final Defense

1. PART I. UNIFIED PHARMACOPHORIC
PROTEIN MODELS OF THE
BENZODIAZEPINE RECEPTOR SUBTYPES
PART II. SUBTYPE SELECTIVE LIGANDS
FOR 5 GABAA/Bz RECEPTORS
Terry S. Clayton
1

2. Outline
• Introduction
• Part I
• Chemistry
• Part 2
• Pharmacophore Modeling
• Part 3
• Homology Modeling
• Part 4
• Protein Ligand Docking
• Part 5 What is next?
• Conclusion
2

3. GABAA/ BzR/ Chloride Channel Complex
The major inhibitory neuro transmitter
system in the CNS, which modulates many
of the neurological functions in the CNS
Complicated molecular
composition:pentamaric protein ploymer
comprised , and subunits which formed
transmemberane ion channel. When GABAA
binds to the receptor, it opened the Cl- ion
channel resulting hyperpolarization in
neuronal transmission. 21 subunits, no x-
ray crystal structure
Benzodiazepine receptor ligands, a class of
the most popularly prescribed drugs, of
which most are used as anxiolytic and
anticonvulsant agents, offer no selectivity,
broad pharmacological effect
3

4. Cross-section of GABAA Receptor
Absolute subunit arrangement of the a1b2g2 GABAA receptor when viewed from the synaptic cleft. The GABA binding
sites are located at the b+a- subunit interfaces and the modulatory Bz BS (Bz) is located at the a+g- subunit interface.18,[
The GABAA binding sites are located at the  subunit interfaces and the modulatory benzodiazepine binding site is 4
located at the  subunit interface. The part of the schematically drawn subunits marked by the + indicates loop C of the
respective subunits .

5. • Figure I. GABA function in C. elegans. GABA released from neurons activates the inhibitory GABAA receptor; the
influx of chloride ions causes the relaxation of the body muscles. GABA is cleared from the cleft by the plasma
membrane transporter, SNF-11. (Adapted from Trends Neurosci 27, Schuske, K., Beg, A. A., and Jorgensen, E. M.
The GABA nervous system in C. elegans. 407-414,
5

6. Hippocampus
throughout
cortex
striatum
thalamus
Amygdala
Temporal Hippocampus
lobe
cerebellum
6

7. Action of benzodiazepines at GABAA
receptor subtypes
Subtype Associated Effect
Sedation, anterograde amnesia,
Some anticonvulsant action, ataxia,
addiction at higher dose
Anxiolytic, hypnotic (EEG), some
muscle relaxation
Some anxiolytic action,
anticonvulsant action at higher dose
Maybe some muscle relaxation
Diazepam-insensitive site
Cognition, temporal and spatial
memory
(Maybe memory component of
anxiety)
Diazepam-insensitive site
7

8. Synthesis of 8-substituted imidazobenzodiazepines
NH2 NHCOCH2Br
O
NH3 / CH3OH
O Br
Br O
reflux
5L NaHCO3 / CHCl3 5L* 83%
24 hours, r.t. 2 days*
500g
H O H O
N N
2eq Br2 / H2SO4 / HOAc
N 66% Br N
2L ~7 days
solid
100g
8

9. Synthesis of 8-substituted imidazobenzodiazepines
O N O
H
N 1, NaH, ClPO(C2H5)2 / THF
N O C2H5
2, NaH, CNCH2COOC2H5 / THF
Br N 45% Br N
3
4
O
N
N O C2H5
SnBu3
N reflux 12 hours
Pd(PPh3)4
tol
64%
5
DM-I-81
9

10. Synthesis of 8-substituted Imidazobenzodiazepine Dimers
H H O
N O H COOH N Br2, NaOAc
DMSO
+ N H
O H 3C H reflux AcOH, rt.
90 %
N
O CH3 80 %
O
6 7
N
H O CO2C2H5
N 1). LDA, THF N
ClPO(OEt)2 0 o C
Br N 2). LDA, THF
Br N
CH3 CNCH2CO2Et CH3
O O
8 45%
9
10

11. Improved Imidazo Process
>70%
12

12. Synthesis of 8-substituted Imidazobenzodiazepine Dimers
N N N
CO2C2H5 CO2C2H5 CO2C2H5
TMS H N
N N TBAF, THF/H2O, rt
Pd(OAc)2(PPh3)2
N
Et3N/CH3CN, reflux N 88 % N
Br CH3
CH3 80 % TMS CH3 H O
O O
O RY80
N O O
N N
2 N NaOH N OH CDI, DMF
N O O N
C2H5OH, 70 o C HOCH2CH2CH2OH, DBU
N
90 % CH3 60 % N N
H O CH3 H3C
H O O H
XLi093
N O O
N
N O O
Pd/C, H2 N
N XLI-356
C2H5OH, CH2Cl2
90% N
O
O
13

13. Binding data
14

14. Add dimers and binding data
15

15. Synthesis of PWZ-029
N
H THF/DMF, NaH
H O COOEt
N O N ClP(O)(OC2H5)2, 0 oC; N
CH3NHCH2COOH
O THF/DMF, NaH
Cl DMSO, 150oC Cl N Cl N
o
O 84% CNCH2COOC2H5, 0 C
O O
1 2 45% 3
LiBH4-CH3OH,
67% THF/ethyl ether
reflux
N N
CH2OCH3 CH2OH
N N
DMSO, KOH, CH3I, rt
Cl N 95% Cl N
O O
5 PWZ-029 4
16

16. Oocyte and Selectivity of PWZ-029
Modulation of EC3 in oocytes currents
By PWZ-029
N
N O
Cl N
O
Affinity of PWZ-029 for axb3g2 (x = 1-6) benzodiazepine receptor isoforms
Alpha 1 Alpha 2 Alpha 3 Alpha 4 Alpha 5 Alpha 6
Merck >300 >300 >300 ND 38.5 >300
Moltech 920 ND ND ND 30 ND
UNC-Roth 17
362.4 180.330 328.2 ND 6.185 ND

17. Fear conditioned contextual memory
10 mg/kg PWZ-029 in mouse
18

18. Visual/Audio Cue Data for Xli356
Scopolamine (1 mg/kg) reduces freezing (ie impairs memory) typically caused by pairing the context (the cage) with a
shock. The drug Xli356 when given at 10mg/kg attenuates the impairment of memory returning the freezing to the level
that one typically sees the mouse freeze at (ie veh). In audio cued, memory is triggered by sound not the context. Xli356
is not able to reverse this type of memory. 19

19. Full Panel Receptor Binding Results
from Case Western Reserve
20

20. 21

21. Synthesis of Chiral Benzodiazepines
O
O
NH2 N O
O H DCC H
NH
O + N O
HO
CH2Cl2 O
O
(81%)
1. HCl(g), 20 min
2. MeOH/H2O(pH 8.5)
r.t. 24 hrs
(83%)
O
N H O
O 1. NaH, THF/DMF H O N
N ClPO(OEt)2, -40°C N Br2 con. H2SO4
glacial AcOH
N
Br N Br N
2. NaH, DMF 81%
CNCH2CO2Et, -40°C
(40%)
Pd(OAc)2(PPh3)2
84% TEA, CH3CN reflux
TMS
O O
N N
O
O TBAF, THF N
N
85% N
N
TMS TMS
22
Sunjie, v.; Kajfez, F.; Stromar, I.; Blazevic, N.; Kolbah, D. J. Heterocycl. Chem. 1973, 10, 591.

22. Chiral HPLC
• Regis Whelk 0,1 SS
• 45% CH2Cl2, 42% Hex,
3% IPA, 10% EtOAc
23

23. Chromatograms of SH-053-S-CH3 (8) and SH-053-R-CH3
(15) by chiral HPLC
24

24. Chromatograms of SH-I-S66 and SH-I-R66 by
chiral HPLC.
25

25. Benzodiazepine Conformation
Scheme 1. Dynamic Chirality of 1a-c and Stereochemical Cooperativity in 2b ( G Values Were Determined by 1H NMR
Spectroscopy (Coalescense))
Carlier, P., Zhao, H., Deguzman, J., Lam, P., Enantioselective Synthesis of “Quaternary” 26
1,4-Benzodiazepin-2-one Scaffolds via Memory of Chirality, J. Am. Chem. Soc. 2003, 125, 11482-11483.

26. Developed Vehicle Options*
1. 10% cyclodextrin in DI water
2. 85% DI water: 14% propylene glycol: 1%
Tween 80
3. a) dissolve in propylene glycol, dilute
50% in DI water
4. 60% propylene glycol: 20% ethanol: 20%
water*
•Mortar and Pestle and sonication in all vehicles
•when traditional saline is not working
28

27. Chemistry Summary
• Scale up to 200 grams and faster
work up using 10 Liter rotovap
• Improved yield 30% with imidazo
process improvements
• Easier work up of dimers
• R/S chiral benzo and natural products
intermediates resolution using chiral
HPLC
• Developed some recipes for vehicles
for our collaborators 29

28. Pharmacophore Modeling
30

29. Classes of Ligands Employed for the Study of
Pharmacophore/ Receptor Models of BzR
Subtypes
N
CO2Et CO2tBu
N H
N N
N N
N
N N H
CH3 N
O N
Ro15-4513 BCCT H
pyridodiindole
CH3
O N
N CH3
N N N
H3 C CH3
Cl N N
O
N
N(CH3)2
diazepam zolpidem CF3 CL218,872
H3 C N
N iPr
N N N
N N O
N N
O Cl N
N
N CH3
H Cl O
CGS-8216 triazolam FG 8205 31

30. Pharmacophore Modeling
LDi
The pyrazolo [3,4-c]quinolin-3-one ligand CGS-9896 (dotted line), diazepam (thick line), and planar diindoles (thin line)
fitted to a schematic representation of the inclusive pharmacophore model for the BzR. The descriptors H1 and H2
designate hydrogen bond donor sites on the receptor protein while A2 represents a hydrogen bond acceptor site necessary 32
for potent inverse agonist activity in vivo. L1, L2, L3 and LDi are four lipophilic regions in the binding

31. Interactive Ligand Library
• Single largest BzR database at present.
• Features include:
– Selectivity Filtering Ki value (nm)
– Log P
– Volume
– MW
– Query based on mathematical expressions
• (Ex. = min (A1:A5)/A1)
– Substructure search
– Similarity search and R-group analysis
– Import/ export
• Today over 600 compounds in an interactive table
33

32. Molecular Databases
ChemDraw for Excel ChemDBsoft
34

33. Hardware for Modeling
Current SYBYL Version: SYBYL X 1.3
• OS: Linux RedHat 4
• 2 Intel(R) Pentium(R) 4 CPU 3.20GHz,
3200.763, 1024 KB
• Video Card NVIDIA Corporation
35

34. Synthesis of 5 selective BzR bivalent ligands (XLi093)
N
CO2C2H5 N O
N CO2C2H5 N
TBAF, THF/H2O, rt N OH
2N NaOH N
N 88% C2H5OH, 70 oC
TMS O CH3 N
CH3 90% N
H O CH3
RY79 RY80 H O
O O
N N
CDI, DMF O O
N N
HOCH2CH2CH2OH, DBU
60 % N N
CH3 XLi093 H3C
H O O H
O
N
5g
RY24
OtBu
N (nM)
N >1000 >1000 858 1550 15 >2000
CH3
H O
(nM)
RY24 26.9 26.3 18.7 N/A 0.4 5.1 (10g)
RY79 121 142 198 159 5.0 114 (10g)
RY80 28.4 21.4 25.8 53 0.49 28.8 (10g)
36

35. X-ray structure Xli093
Jeffrey R. Deschamps, Ph.D.
Research Chemist
Naval Research Laboratory
37

36. Xli093
38

37. PWZ-029 docked in 2
40

38. Receptor binding data of methyl ester analogs related to
RY 80 and PWZ-029 in nM.
32.74 13.22 24.1 ND 3.548 ND
Binding affinity at αxβ3γ2 GABA A/BzR subtypes
(Values are reported in nM).
2.531 5.786 5.691 ND 0.095 ND
Binding affinity at αxβ3γ2 GABA A/BzR subtypes
(Values are reported in nM).
41

39. Table 3. Receptor binding data of methyl ester analogs related to
RY 80 and PWZ-029 in nM.
15.31 87.8 60.49 ND 1.039 ND
Binding affinity at αxβ3γ2 GABA A/BzR subtypes
(Values are reported in nM).
945.9 326.8 245.9 ND 4.07 ND
Binding affinity at αxβ3γ2 GABA A/BzR subtypes
(Values are reported in nM).
42

40. PWZ-029 and Roche Cmpd 7
N
CF2H
N
Br N
N
N
Affinity for x (x = 1-6) benzodiazepine receptor isoforms
Alpha 1 Alpha 2 Alpha 3 Alpha 4 Alpha 5 Alpha 6
Roche 174.3 185.4 79.6 ND 4.6 ND
N
N O
Cl N
O Affinity of PWZ-029 for x (x = 1-6) benzodiazepine receptor isoforms
Alpha 1 Alpha 2 Alpha 3 Alpha 4 Alpha 5 Alpha 6
Merck >300 >300 >300 ND 38.5 >300
Moltech 920 ND ND ND 30 ND
UNC-Roth 43
362.4 180.330 328.2 ND 6.185 ND

41. Strategies for improving
selectivity
• By reducing the electrostatic potential of ester
terminal group some potency is lost but
selectivity is improved for 5 over 1
• Utilize bulkier groups extending into L2 pocket at
the 8 position
• Certain R isomers may bind to 5 selectivity
44

42. Pharmacophore
•Descriptors
•Included Volume
LDi
45

43. &
Alpha 1 is represented by the red volume. Alpha 2 is represented by the yellow, where orange are regions of overlap.
46

44. Alpha 2 & Alpha3
Alpha 2 is in red. Alpha 3 is represented in yellow. Regions of overlap are represented by orange.
47

45. Oocyte Data on SH-053-2'F-R-CH3
Compound
SH-053-2’F-R-CH3 759.1 948.2 768.8 >5000 95.17 >5000 48

46. R and S Conformation
S Conformation R Conformation
49

47. &
Overlay of the a5b3g2 receptor (yellow) subtype with the a1b3g2 receptor
(magenta) subtype. Orange surfaces indicate overlapping regions. 50

48. Subtype (solid) overlayed with the previous model (line).
51

49. Alpha 2 & Alpha 5
The alpha 5 included volume is illustrated in red. The alpha 2 is in yellow. Regions of overlap appear orange.
52

50. Alpha 5 & Alpha 6
Alpha 5 is represented in red. Alpha 6 is in yellow. Regions of overlap are orange.
53

51. New Conclusions Modeling
1. Discovery of an additional lipophilic pocket
(L4)
2. L2 pocket has been consistency manipulated to
enhance a5 selectivity on the oxo scaffold
3. Elimination of the ester moiety from the 1,4-
benzodiazpine template offers discrimination
between x and x receptor
compositions
a. Supported by Roche Cmpd 7 binding data
4. Evidence of conserved site entrance supported
by of dimer protrusion
54

52. GABAA Receptor Homology
Modeling
] Pritchett, D., Sontheimer, H., Shivers, B., Ymer, S., Kettenmann, H., Schofield, P., and
Seeburg, P., Importance of a novel GABAA receptor subunit for benzodiazepine
pharmacology, Nature, 1989, 338, 582-585.
J Neurochem 62(2):815-818, 1994
55

53. Figure 2. Longitudinal (A) and cross-sectional (B) schematic
representations of a ligand-gated ion channel. The numbers 1-4 refer
to the M1-M4 segments. The M2 segment contributes to the majority of
the pore lining within the membrane lipid bilayer.
56

54. Figure 1. Proposed topology of a GABAA receptor subunit.
The extracellular domain begins with the N-terminus and
M1-M4 represent the four transmembrane domains.
57

55. GABA Ion Channel
58

56. Labeling
59

57. Cross-section of GABAA Receptor
Absolute subunit arrangement of the a1b2g2 GABAA receptor when viewed from the synaptic cleft. The GABA binding
sites are located at the b+a- subunit interfaces and the modulatory Bz BS (Bz) is located at the a+g- subunit interface.18,[i]
[i 60

58. GABA ligand docking
61

59. Protein Binding Site
Extracellular
BzR Entrance
N-terminus
Loop B
E
D
F
Loop A 62
Intracellular Loop C

60. Orthogonal views of the Location of Residues with respect to
pharmacophore in the BzR binding pocket
63

61. Orthogonal views of the Location of Residues with respect to
pharmacophore in the a1b3g2 BzR binding pocket
64

62. Affinity Labeling & Site Directed
Mutagenesis
65

63. Molecular Modeling
66

64. Pharmacophore Modeling
The pyrazolo [3,4-c]quinolin-3-one ligand CGS-9896 (dotted line), diazepam (thick line), and planar diindoles (thin line)
fitted to a schematic representation of the inclusive pharmacophore model for the BzR. The descriptors H1 and H2
designate hydrogen bond donor sites on the receptor protein while A2 represents a hydrogen bond acceptor site necessary 67
for potent inverse agonist activity in vivo. L1, L2, L3 and LDi are four lipophilic regions in the binding

65. Homology Modeling
Conclusions
• H1 is Tyrosine 210
• H2 is istidine 102
• A2 is Threonine 142
• Current Model assumes agonist bound
open channel conformation
• Further refinements necessary
68

66. AutoDock 4.2
• Prepare PDB files of Protein and Ligand
• Gasteiger (partial) charges are added
• non-polar hydrogens were merged
• aromatic carbons were identified
• rotatable bonds detected, and TORSDOF
set
69

67. Protein
• Identify Key residues
• Remove water molecules
• Add gasteiger charges
• Create a flexible residue file
• Choose an algorithm (unique)
70

68. Define binding site
71

69. Alpha 1 betacarboline dock
72

70. Rendering
73

71. Homology &Docking
Conclusions
• Agreement with site directed
mutagenesis
• Agreement with Photoaffinity labeling
• Entrance Theory supported
• Dimer extending to extracellular space
• Next steps
• Continued refinement
• Theoretical site directed mutagenesis
• Site directed mutagenesis 74

72. Overall Conclusions of Research
• Synthesized a number of benzodiazepine
ligands and dimers and made
improvements to synthetic procedures
(pendant and oxo series)
• Constructed a powerful Bz database and
further refined the Included volume
pharmacophores
• Correlated the unified pharmacophore to a
homology model
75

73. Thank You
Committee Members
Dr. Chen
Dr. Peng
Dr. Schwabacher Funding
Dr. Cook o NIH
Dr. Arnold o NIMH
Dr. Silvaggi o UWM
And to our collaborators:
Dr. Cromer RMIT University
Dr. Ramerstorfer Medical University Vienna
Professor Miroslav Savic University of Belgrade
Professor Werner Sieghart Medical University Vienna
Dr. Angela Duke Harvard Medical School
Professor James Rowlett Harvard Medical School
Dr. Cook’s Research Group University Wisconsin Milwaukee
76

74. Chlonazepam
O
H
N
-
O N
+
N
O
Cl
clonazepam
77

75. Previous Collaborators 5
• Subramaniam Sanker Case Western Reserve
– Binding affinity at GABA subtypes
• Mike Weed John Hopkins University
– Delayed matching to position in rhesus monkeys
• Klaus Miczek Tufts University
– Modulating alcohol heightened aggression
• Tim Delorey Molecular Research Institute
– Audio and Contextual Memory
• Jim Rowlett Harvard University
– Alcohol self-administration (primates)
• Harry June University of Maryland
– Alcohol self-administration (rats)
• Mark Galizio University of N.C.-Wilmington
– Anxiety and cognitive enhancement in rats
• Roman Furtmueller Brain Research Institute, Vienna Austria
– Electrophysiology traces of compounds in oocytes
• Galen Wenger University of Arkansas College of Medicine
– Titrated matching-to-position in rats and pigeons
78