Synchrotrons, Mannitol, Prazosin, etc.
Synchrotron radiation is accessible, decisive
Hydrogen bonding affects molecular conformation
Work performed with
V.V. Chernyshev, A.V. Yatsenko, O.B. Ryabova, V.A. Makarov,
C.E. Botez, R. Suryanarayanan, C. Nunes.
See also poster #14 on R-albuterol, S. Cuffini
Physical adsorption
of Kr on graphite
X-ray diffraction of Kr
on exfoliated graphite.
Crystallography with one
Bragg peak - Kr(1,0)
Experiments at SSRL in 1979.
(Moncton, Birgeneau, Horn, Brown, PWS)
20x better angular resolution
Substrate coherence length is ~2000Å
Completely new picture!
There is a disordered phase between
the commensurate and incommensurate
solids. Interesting new physics.
Heroic age of synchrotron radiation.
Parasitic on high energy physics.
Huge investment of effort to get one
or two weeks of access per year.
National Synchrotron Light Source at
Brookhaven National Laboratory
Produces electromagnetic radiation from IR to -rays.
Easy to obtain access.
~75 experimental stations
~2500 users per year
Typical of many facilities worldwide: APS, ESRF, SLS,
SRS Daresbury, SSRL, …, which are eager for users.
Synchrotron access is for:
Academic:
Industrial:
Who publish in the open literature*
Hold data for proprietary reasons**
The people who operate these facilities need to
have them widely used!
* Access by writing competitive proposals or arranging
collaboration.
** You have to pay for the prorated cost of operating
the facility - ~$250/hour = $2000/pattern at NSLS.
Compare lab vs. synchrotron data sets.
This drug has two polymorphs that can’t be quantified except by Rietveld.
Form 1 -- Lab
Form 2 -- Lab
Form 1 -- Synchrotron
Form 2 -- Synchrotron
THEORY OF POWDER DIFFRACTION
(series of elementary recipes)
CONTENTS
OF UNIT CELL
UNIT CELL
RECIPROCAL LATTICE,
SPACE GROUP . . . . . . . . .
. . . .
. . . .
.
.
.
.
.
. . . .
. . . .
. . . . . . . . .
INTENSITIES
2
2i ( hkl )( xyz ) j
I hkl ~
f je

POWDER PEAK POSITIONS
2
3
4
5
6
7
8
9
2 (degrees)
10
11
12
13
14
15
INSTRUMENT
AFFECTS PEAK
SHAPES
2
3
4
5
6
7
8
9
2 (degrees)
10
11
PHYSICAL SAMPLE
AFFECTS LINESHAPES
OTHER ARTIFACTS:
PREFERRED ORIENTATION, ETC.
12
13
14
15
POWDER DIFFRACTION PATTERN
USE OF POWDER DIFFRACITON TO SOLVE A CRYSTAL STRUCTURE
Chemical
knowledge
of contents
Data
2
3
4
5
6
7
8
9
2 (degrees)
10
11
12
13
14
15
1. Start with the best data you can get (but no better).
2. Get a list of accurate diffraction peak positions.
3. Figure out a lattice that explains the peaks.
4. Guess the space group (systematic absences, # molecules).
5. Search for the best place to put the molecule(s), best
conformation of the molecule.
6. Refine, refine, refine, refine, refine, …
At any stage, you can be forced to jump back to any stage.
Don’t think that people only use powders for organic molecules, or
that direct space modelling is the only useful technique
Lausenite:
Fe2(SO4)3·5H2O
J. Majzlan, …, PWS
Direct Methods (EXPO)
Souzalite
(Fe,Mg)3(al,Fe)4(PO4)4(OH)6·2H2O
A. Le Bail, …, PWS
Real Space (ESPOIR)
#1, 2 This is a data-driven enterprise.
Students may think that we spend all our time talking about algorithms,
software, etc., but the results are no better than the data!
Powder diffraction station at X3B1
beamline, National Synchrotron
Light Source, Brookhaven National
Laboratory, U. S. A.
From
storage
ring
GE (111)
analyzer crystal
Scintillation
detector
Monochromatic
X-ray beam
Si(111) double
monochromator
Ion chamber
sample
Analyzer crystal geometry measures angles – eliminates significant
aberrations of familiar Bragg-Brentano diffractometer.
Capillary sample geometry is very helpful. Eliminates preferred
orientation, peak shifts that bother flat plate
#3. Indexing
Given some values of d spacings, find a lattice that fits them, i.e.,
find {A,B,C,D,E,F } such that every d can be expressed as
1/d 2 = Ah 2 + Bk 2 + Cl 2 + Dkl + Ehl + Fhk
for some integers h, k, l.
Familiar programs, in the public domain:
TREOR, ITO, DICVOL, have their quirks, but basically they always
work, given sufficiently good data. (Often possible with good lab
diffractometers, nearly always with synchrotron data.)
TOPAS (Alan Coehlo, Bruker AXS) has indexing tools that are
qualitatively more powerful.
Prazosin
Designer drug – selective antagonist for α1-adrenoceptors
(blood pressure).
Four other polymorphs claimed in US Patents 4092315,
4739055, 4816455, and JP Patent 03206088. Department
of Medicinal Chemistry, State Scientific Center of
Antibiotics, Moscow, could not reproduce any of them.
Patent literature : Literature
Military intelligence : Intelligence
50000
Powder diffraction pattern of prazosin HCl
 = 1.15019Å
compared to peak positions from PDF 42-1864
40000
30000
20000
10000
0
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Four of the lines in the Powder Diffraction File for that compound
are impurities, not seen in our pattern.
Throw them away and use TOPAS to index the lab data.
0.1
2theta (measured - indexed)
0.08
0.06
0.04
0.02
0
0
-0.02
-0.04
-0.06
5
xx
10
x x
15
20
25
30
2theta
Errors 0.021° ± 0.024°
Triclinic
TOPAS FOM = 15.99
a = 8.717Å
b = 7.572Å
c = 16.381Å
α = 90.01°
β = 72.43°
γ = 108.95°
Vol = 969Å3
-0.08
(There is a lot of not-quite-good data in the data bases.
Is not-quite-good distinguishable from bad?)
Given sufficiently good data
-> pattern can be indexed easily
Data quality = sample  instrument
If a pattern from a good instrument*
cannot be indexed, there is something
wrong with the sample
*Test your instrument by trying to index
known phases of comparable complexity.
Acetaminophen and Ibuprofen are good
organic test cases to get started.
#5. Make a model of the molecule, put it into the lattice.
Move the model around seeking best agreement between calculated
and observed diffraction patterns.
Lots of options: software DASH, PSSP, FOX,
TOPAS, PowderSolve, …
_
In this case, assumed P1, coauthor searched
nine parameters with software developed
with H. Schenk.
Cl
2
#6. Refine, refine, …
If your presumed rough solution is close enough, you can roll down hill
to the correct solution, using refinement programs such as GSAS,
TOPAS, FULLPROF, …
~100 refined variables
THE GLOBAL TOPOLOGY MAY
LOOK MORE LIKE THIS
Any fit looks
good on this scale
d = 1.47Å
χ2 = 2.31, Rwp=5.92%. No restraints except for tethering all H atoms.
Same steps for prazosin free base – only 6 search coordinates
Monoclinic, Cc, χ2 = 2.78, Rwp=5.92%
Planarity of the aromatic rings gives a measure of the degree
of accuracy of the finished atomic geometry.
Prazosin refinements. Geometry of piperazine ring
Hydrochloride
N17
C18
Bonds
Hydrochloride
Free Base
N17-C18
1.489Å
1.521Å
C18-C19
1.509Å
1.491Å
C19-N20
1.513Å
1.507Å
N20-C21
1.471Å
1.489Å
C21-C22
1.538Å
1.610Å
C22-N17
1.472Å
1.543Å
N17
123.1
117.9
C18
102.3
107.7
C19
114.2
114.6
N20
115.8
111.0
C21
106.2
111.0
C22
105.1
102.8
Angles
Free base
Hydrochloride
3 N-H…Cl
Free base
1 N-H…N 3.02Å
1 N-H…O 3.02Å
Prazosin conclusions:
•That wasn’t so hard
•Of relevance to quantitative modeling of
structure-activity relationships
Hydrochloride
Free base
Hydrochloride methanol solvate (single xtl)
Prazosin2 tetrachloro-copper(II) (s x)
The rest of the talk
1. Enalapril Maleate. Y.H. Kiang, Merck
2. Proxy for a real business problem.
3. Delta D-Mannitol
4. Mannitol hemihydrate
Enalapril Maleate is a potent angiotensin converting enzyme
(ACE) inhibitor with two known polymorphs, Form I and Form
II. The single crystal structure of Form I has been known for
almost twenty years. On the other hand, the crystal structure
of Form II has never been reported before because of the
difficulty to obtain single crystals of this polymorph, which is
made by water slurry of Form I.
The crystal structure of Form II is of interest for several
reasons:
1. Form II is the more stable of the two polymorphs.
2. The two forms are structurally similar based on X-ray, IR,
and solid-state NMR.
3. The conformation of ACE inhibitors is important to their
biological activity.
Lab(Sealed Tube) and Synchrotron XRD patterns of Enalapril Maleate
Form I
Cu Ka1
Form II
4
9
14
19
2q
24
29
34
39
Form I
=1.15Å,
Form II
5
9
13
17
2q
21
25
29
33
Enalapril Maleate
form II
a=17.838
b=6.640
c=11.649
b=106.29
form I
Monoclinic P21
Orthorhombic P212121
a=33.987
b=6.642
c=11.210
23 parameters: 11 enalapril torsions
(+2 maleate) + 6 orientation + 6 position
At the time of the original work, we couldn’t solve from simulated annealing.
We could have benefited from the systematic geometric insights presented
by Claire Gervais
Form I
green:carbon
yellow:nitrogen
red:oxygen
Form II
Y.-H. Kiang of Merck found the solution by hand, using Cerius.
Y.-H. Kiang, Ashfia Huq, Peter W. Stephens, Wei Xu, Journal of Pharmaceutical
Sciences 92, 1844-53 (2003)
Real business problem:
_____ has a patented polymorph of _____ , and
suspects that _____ is selling material that infringes.
It is desired to examine the commercial tablets and
determine the polymorph of the API for potential
litigation.
Proxy:
Examine commercial tablet of Endocet 500/7.5
Gross tablet 607 mg
Acetaminophen 500 mg – known lattice & structure
Oxycodone (as HCl) 7.5 mg – pattern in PDF but lattice
unknown,*
* In general, I’d like to get better info into the
PDF database. Please get in touch if you can help.
X-ray Intensity (counts per 10^6 monitor)
80000
Endocet (intact tablet)
500 mg Acetaminophen
7.5 mg. oxycodone HCl
60000
data: endocet.631
Measured
x 50
Profile fit of acetaminophen
40000
20000
0
Acetaminophen
2
4
6
8
10
12
2theta (degrees)
14
16
18
20
Powder patterns of oxycodone hydrochloride from
ICDD Powder Diffraction File.
Strucutures and lattices are not known.
100
PDF 06-0014
Peak Intensity
50
0
100
50
PDF 38-1799
0
0
4
8
12
2q (degrees) at  = 0.70Å
16
20
X-ray Intensity
(counts per 10^6 monitor)
Endocet (intact tablet)
500 mg Acetaminophen
7.5 mg. oxycodone HCl
data: endocet.631
Profile fit
Measured
4000
?
3000
?
2000
1000
0
Oxycodone HCl peaks
from PDF
Acetaminophen
100
0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
2theta (degrees)
12.0
13.0
14.0
15.0
16.0
Back to structures: Mannitol
H
|
H - C |
OH
H
|
C |
OH
OH
|
C |
H
OH
|
C |
H
H
|
C |
OH
H
|
C - H
|
OH
D-Mannitol (naturally produced in various plants)
Long-standing confusion about how many forms of D-mannitol exist –
finally laid to rest by Burger, Henck, & co. (2000). (Their II, I, and
III are more commonly known as α, β, δ)
α and β were solved from single crystals in 1968.
δ (lowest melting) identified in 1968, but no single crystals grown
(until ~2002, Henck & Benet-Buchholz, unpublished).
We solved the structure of δ from a powder sample (with 20% β)
Mannitol is widely used as an excipient in freeze-drying; metastable
hydrate discovered by Lian Yu. TGA shows it is hemihydrate.
Structure solved from powder sample with 26% δ, 2% β, ~10% ice.
P 21 2 1 21
8.942 x 18.798 x 4.893Å
205.6 Å3 / molecule
Middle melting
P 21 21 21
8.672 x 16.875 x 5.560Å
203.4 Å3 / molecule
Highest melting
Present
work
P 21
5.089 x 18.250 x 4.917Å
β = 118.304°
201.0 Å3 / molecule
Lowest melting
All the same steps. At extraction, we did a Le Bail refinement of the δ
lattice along with Rietveld refinement of (known) β structure.
λ = 0.70224Å
4-cycle
Zig-zag chain
Alpha D-mannitol, H bonds
(beta is very similar)
Delta D-mannitol, H bonds
One zig-zag chain
alpha
beta
The molecules in alpha, beta, delta D-mannitol are essentially identical
delta
Mannitol hemihydrate
Start with the best data possible?
Play the hand you’re dealt!
Lab x-ray (Minnesota)
TGA
100
(a)
98
97
96
95
94
DSC
0
(b)
(c)
(d)
(e)
2q (degrees)
Heat flow (W/g)
Int
en
sit
y
(c
ou
nt
s)
Weight (%)
99
-2
-4
-6
-8
Lab x-rays identify the sample
TGA -> hemihydrate
120
140
160
180
Temperature ( C)
-10
20
40
60
80
100 120 140 160 180 200
Temperataure (°C)
25000
Normalized X-ray counts
20000
x5
data
fit
15000
10000
5000
Differnce
0
Hydrate
Delta
Beta
Ice
2000
0
-2000
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
Two Theta (deg)
Index the peaks that are not any other identified phase to a triclinic lattice:
9.896 x 10.542 x 4.786 Å, 102.59°, 86.09°, 116.08°, 2 mannitol in P1.
Search two independent mannitol molecules, one O atom.
Several different starts.
B
A
Hydrogen bonding pattern in hemihydrate
Conformation with
one leg lifted
water
Normal
conformation
Only mannitol hemihydrate has an OH twisted up into the
plane of the C-C-C-C-C-C backbone.
What is the energy cost relative to the conformation of all
other observed mannitol crystal structures?
Is there no way to pack “table” mannitols and a water of
solvation without straining the molecule?
Structure determination from powders requires:
Good data
Fundamental understanding of the available tools
Motivation
Choose good problems.
If you are stuck with a crystallographic problem, try a
synchrotron.* You pay taxes – claim your share. Find
one where there is already a strong program of
structure determination.
* Also single crystal.