Work performed withV.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

Synchrotrons, Mannitol, Prazosin, etc.

Synchrotron radiation is accessible, decisiveHydrogen bonding affects molecular conformation

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 yearTypical of many facilities worldwide: APS, ESRF, SLS, SRS Daresbury, SSRL, …, which are eager for users.

Synchrotron access is for:

Academic: Who publish in the open literature*

Industrial: 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.

Form 1 -- Lab

Form 2 -- Lab

Form 1 -- S ynchrotron

Form 2 -- S ynchrotron

Compare lab vs. synchrotron data sets.This drug has two polymorphs that can’t be quantified except by

Rietveld.

THEORY OF POWDER DIFFRACTION

UNIT CELL

RECIPROCAL LATTICE,SPACE GROUP

POWDER PEAK POSITIONS

POWDER DIFFRACTION PATTERN

CONTENTSOF UNIT CELL

PHYSICAL SAMPLEAFFECTS LINESHAPES

OTHER ARTIFACTS:PREFERRED ORIENTATION, ETC.

2)()(2~ jxyzhkli

jhkl efI

INTENSITIES

2 3 4 5 6 7 8 9 10 11 12 13 14 152 (degrees)

2 3 4 5 6 7 8 9 10 11 12 13 14 152 (degrees)

. . . . . . . . .

. . . . . . . .

. . . . .

. . . . . . . .

. . . . . . . . .

(series of elementary recipes)

INSTRUMENTAFFECTS PEAKSHAPES

USE OF POWDER DIFFRACITON TO SOLVE A CRYSTAL STRUCTURE

2 3 4 5 6 7 8 9 10 11 12 13 14 152 (degrees)

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.

Data Chemical knowledge of contents

Don’t think that people only use powders for organic molecules, or that direct space modelling is the only useful technique

Lausenite: SouzaliteFe2(SO4)3·5H2O (Fe,Mg)3(al,Fe)4(PO4)4(OH)6·2H2OJ. Majzlan, …, PWS A. Le Bail, …, PWSDirect Methods (EXPO) Real Space (ESPOIR)

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

#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.

Ion chambersample

GE (111) analyzer crystal

Scintillation detector

MonochromaticX-ray beam

Si(111) double monochromator

From storagering

#3. IndexingGiven 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 : LiteratureMilitary intelligence : Intelligence

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

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P ow der d iffraction pattern of prazosin H C l = 1.15019Åcom pared to peak positions from P D F 42-1864

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.

xxxx

TriclinicTOPAS FOM = 15.99a = 8.717Åb = 7.572Åc = 16.381Åα = 90.01°β = 72.43°γ = 108.95°Vol = 969Å3

(There is a lot of not-quite-good data in the data bases. Is not-quite-good distinguishable from bad?)

Errors 0.021° ± 0.024°

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

#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, …

2

~100 refined variables

THE GLOBAL TOPOLOGY MAY LOOK MORE LIKE

THIS

χ2 = 2.31, Rwp=5.92%. No restraints except for tethering all H atoms.

Any fit looks good on this scale

d = 1.47Å

Monoclinic, Cc, χ2 = 2.78, Rwp=5.92%

Same steps for prazosin free base – only 6 search coordinates

Planarity of the aromatic rings gives a measure of the degree of accuracy of the finished atomic geometry.

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

Angles

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

Prazosin refinements. Geometry of piperazine ring

N17C18

Hydrochloride

Free base

Hydrochloride3 N-H…Cl

Free base1 N-H…N 3.02Å1 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.

5 9 13 17 21 25 29 33

Form I

Form II

Lab(Sealed Tube) and Synchrotron XRD patterns of Enalapril Maleate

2

=1.15Å,

4 9 14 19 24 29 34 39

2

Cu K1

Form I

Form II

a=17.838

b=6.640c=11.64

9form I

a=33.987 b=6.642 c=11.210

form II

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

23 parameters: 11 enalapril torsions (+2 maleate) + 6 orientation + 6 position

Enalapril Maleate

Monoclinic P21

Orthorhombic P212121

Form I Form IIgreen:carbonyellow:nitrogenred:oxygen

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 500/7.5Gross tablet 607 mgAcetaminophen 500 mg – known lattice & structureOxycodone (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.

Endocet (in tact tab le t)500 m g Acetam inophen7.5 m g. oxycodone H C l

2 4 6 8 10 12 14 16 18 20

2theta (degrees)

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-ra

y In

ten

sity

(co

un

ts p

er

10

^6 m

on

itor)

Acetam inophen

data: endocet.631

M easured

Profile fitx 50 of acetaminophen

Powder patterns of oxycodone hydrochloride from ICDD Powder Diffraction File. Strucutures and lattices are not known.

0 4 8 12 16 202 (degrees) a t = 0 .70Å

0

50

100

0

50

100

Pe

ak

Inte

nsi

ty

PD F 38-1799

PD F 06-0014

Endocet (in tact tab le t)500 m g Acetam inophen7.5 m g. oxycodone H C l

3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0

2theta (degrees)

0

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X-r

ay

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ty

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^6 m

on

itor)

0

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Oxy

cod

on

e H

Cl p

ea

ksfr

om

PD

F

A cetam inophen

data: endocet.631M easured P rofile fit

?

?

Back to structures: Mannitol H H OH OH H H | | | | | | H - C - C - C - C - C - C - H | | | | | | OH OH H H OH 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 21 21

8.942 x 18.798 x 4.893Å205.6 Å3 / moleculeMiddle melting

P 21 21 21

8.672 x 16.875 x 5.560Å203.4 Å3 / moleculeHighest melting

P 21

5.089 x 18.250 x 4.917Åβ = 118.304°201.0 Å3 / moleculeLowest melting

Present work

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

Alpha D-mannitol, H bonds(beta is very similar)

4-cycle

Zig-zag chain

One zig-zag chain

Delta D-mannitol, H bonds

alpha

beta

delta

The molecules in alpha, beta, delta D-mannitol are essentially identical

Start with the best data possible?

Play the hand you’re dealt!

Mannitol hemihydrate

Int

en

sit y

(c ou nt

s)

2 (degrees)

(a)

(e)

(d)

(c)

(b)

-10

-8

-6

-4

-2

0

20 40 60 80 100 120 140 160 180 200

Temperataure (°C)

Hea

t fl

ow

(W

/g)

94

95

96

97

98

99

100

Wei

gh

t (%

)

120 140 160 180

Temperature ( C)

TGA

DSC

Lab x-ray (Minnesota)

Lab x-rays identify the sampleTGA -> hemihydrate

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Tw o Th e ta (d e g )

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x5

H y d ra teD e ltaB e taIce

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.

A

B

Search two independent mannitol molecules, one O atom.Several different starts.

Hydrogen bonding pattern in hemihydrate

Normal conformation

Conformation with one leg

lifted

water

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 dataFundamental understanding of the available toolsMotivation

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.