Dr' Andrew Stachulski

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Carbohydrate Metabolites Their Importance in
Drug Discovery
Dr. Andrew Stachulski Mar 23rd 2009
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The molecule that changed my life?
Morphine 6-ß-D-glucuronide (M6G)
A human metabolite of morphine at least as potent
an analgesic as morphine itself and better
tolerated. In contrast, the 3-glucuronide is not
pharmacologically active.
R. J. Osborne, S. P. Joel, D. Trew and M. Slevin,
Lancet, 1988, i, 828 F. Scheinmann, K. W.
Lumbard, R. T. Brown and S. P. Mayalarp,
International Patent WO 93/3051, 1993 A. V.
Stachulski, F. Scheinmann, J. R. Ferguson, J. L.
Law, K. W. Lumbard, P. Hopkins, N. Patel, S.
Clarke, A. Gloyne and S. P. Joel, Bioorg. Med.
Chem. Lett., 2003, 13, 1207.
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Why are carbohydrate metabolites important?

• Recent guidelines issued by the FDA-Metabolites
  in Safety testing (MIST)
• All metabolites accounting for gt10 of drug dose
  must in general be screened
• for toxicity/ safety
• Among these, glucuronides- especially acyl
  glucuronides- were specifically
• mentioned
• Through a series of Ph D studentships-
  collaborating with AstraZeneca,
• GlaxoSmith Kline and Pfizer- we have developed
  effective synthetic routes for
• a wide range of glucuronide metabolites
• These have been used for analytical standards but
  will increasingly form the basis
• for toxicological studies
• Current work is focused on better understanding
  of structure-activity properties
• of the metabolites and studying protein
  interactions, especially through proteomics
• Some case studies follow

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Glucuronide Structures
Glucuronides are the commonest phase 2
metabolites and occur in a wide variety of
structures O, N, S and C-glucuronides are all
known. O-glucuronides are most common and are
subdivided into aryl, alkyl and acyl types. Some
typical glucuronides of common drugs
Glucuronides are generally regarded as purely
detoxifying metabolites -certainly not
always true!
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Steroidal Glucuronides Fulvestrant
Glucuronides of endogenous steroids are very
common, e. g. of estradiol, androsterone,
progesterone
Fulvestrant (ICI182,780) is an antiestrogen and
used in breast cancer therapy. Both the 3-and
17-O-glucuronides were detected as human
metabolites.
Samples of both were required as standards and
for toxicological evaluation
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Synthesis
The 3-glucuronide was relatively straightforward

• The 17-formate was prepared by ester exchange
• (HCO2Et,Tos-OH, distil)
• Large amount of BF3.OEt2 needed (1.5 eq.)
• strong complexation by sulfoxide

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17-Glucuronide
Synthesis of the 17-(alkyl) glucuronide was much
more difficult
Inverse addition imidate added dropwise at
-15C to a mixture of steroid and catalyst
Cf. direct addition only 7 of desired
conjugate obtained. The inverse addition has
some generality for alcohols.
J. R. Ferguson, J. R. Harding, K. W. Lumbard, F.
Scheinmann and A. V. Stachulski, Tetrahedron
Lett., 2000, 41, 389 J. Chem. Soc., Perkin
Trans.1, 2001, 3037.
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Acyl Glucuronides in context

• Reactive metabolites are one of the major
  concerns in drug development
• Acyl glucuronides (AGs) are relatively reactive
  with proteins are they toxic? No causal link yet
  proved
• To answer the question definitively, we need
  efficient synthesis of AGs as the
  naturally-biosynthesised 1ß-anomers

A. V. Stachulski, J. R. Harding, J. C. Lindon, J.
L. Maggs, B. K. Park and I. D. Wilson, J. Med.
Chem., 2006, 49, 6931 (Review).
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Case Study Diclofenac Acyl Glucuronide

• The well-known antiinflammatory agent diclofenac
  has a complex metabolic profile
• Its acyl glucuronide is short-lived and suspected
  to cause adverse reactions
• We developed a convenient synthesis of this and
  many other AGs by selective acylation-superseding
  an older Mitsunobu method

J. Med. Chem., 2004, 47, 2816 Org Lett., 2005,
7, 2591
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Aqueous stability data in summary
Half-lives in buffer, pH 7.4, 37C for AGs of
representative drugs
Acyl migration (half lives) Arylacetic acid
typelt arylpropionatelt a,a-disubstituted
type. Half lives 0.5 to 80h Benzoic acid type
follow a Hammett correlation ArCX2CO2H
steric/electronic effects both important
Cf. T. Ebner, G. Heinzel, A. Prox, K. Beschk and
H. Wachsmuth, Drug Metab. Dispos., 1999, 27,
1143 M. Shipkova, V. W. Armstrong, M. Oellerich
and E. Wieland, Ther. Drug Monit.,2003, 25, 1.
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Aqueous Stability of Diclofenac Acyl Glucuronide
S. Clarke, B. K. Park, J. R. Kenny, J. L. Maggs,
D. Sinnott, X. Meng and A. V. Stachulski, J. Med.
Chem., 2004, 47, 2816.
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Case study Overcoming intramolecular cyclisation
A series of neutral endopeptidase inhibitors was
found to metabolise mainly as their acyl
glucuronides. In one case, however, the AG
exhibited a remarkably short half life
The highly acidic NH group (pK ca. 9.5) lead to
facile cyclisation of the AG, even at
physiological pH
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Careful choice of protecting groups led to an
effective synthesis of the AG

• Again Mitsunobu coupling possible- but as ever
  gives ßa mixture.
• DABCO (rather than NMM) is essential for a good
  rate here.
• Evaluation of protein reactivity of the AG (and
  imide) now possible.

X. Meng, J. L. Maggs, D. C. Pryde, S. Planken,R.
M. Jenkins, T. M. Peakman, K. Beaumont, C. Kohl,
B. K. Park and A. V. Stachulski, J. Med.
Chem., 2007, 50, 6165.
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Reactivity with nucleophiles

• The imide could be made independently, from the
  parent drug
• Reaction of both AG and imide with model
  nucleophiles studied
• Reaction of imide with HSA also
  examined-proteomics 8 Lys residues modified
• Conclusion The reactivity of the AG is, in this
  case, the same as the imide

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Some current work
We have studied a series of phenylacetic acid AGs
with varying substitution at the a-carbon. The
following were prepared in good yield by
selective acylation
R1 R2 H R1 Me, R2 H- both
diastereoisomers R1 R2 Me

• Half-lives of 1ß-isomers measured by NMR
• Molecular modelling study- predict rates from
  transition state analysis
• Important model study for NSAIDs like ibuprofen
  (2R), (2S) mono-Me differ
• in acyl migration rates
• Ideally need many more data points-but first
  results show good correlation between
• NMR rates and DFT calculations

Org. Biomol. Chem., accepted for publication
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Calculated half-lives by DFT
Limited number of data points- but excellent
correlation Especially the difference between
(R) and (S) is well predicted
Drs. N. Berry, P. Jayapal
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Studying protein reactivity

• As noted earlier- NSAIDs such as Ibuprofen are
  widely-used drugs
• A series of arylacetic acid AGs including that of
  Ibuprofen was synthesised
• Their reactivity with highly purified human serum
  albumin (HSA) has been studied and compared with
  some model compounds

Acylating ability of the AG directly compared to
an N-hydroxysuccinimido ester -a standard peptide
coupling intermediate Then the series of NSAIDs
with varying a-substitution
R1 R2 H Ibufenac (withdrawn from market) R1
H, R2 Me both (R) and (S) Ibuprofen R1
R2 Me Bibufenac
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In conclusion, for AGs

• A number of acyl glucuronides of important drugs
  and model carboxylic acids
• have been made by an efficient three-step
  procedure
• Selective acylation of a glucuronate monoester is
  the key step
• By suitable choice of ester, a wide variety of
  functional groups are compatible
• Patterns of structure-reactivity are being built
  up in vitro and in vivo
• As proteomics assumes greater importance, we can
  supply the necessary AGs

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Introducing N-Glucuronides
These are still a relatively little-studied
class. Both neutral and charged (zwitterionic)
N-glucuronides are known. Some structures
Tamoxifen is of interest here the N-glucuronide
may be partly responsible for its activity.
T. Kaku, K. Ogura, T. Nishiyama, T. Ohnuma, K.
Muro and A. Hiratsuka, Biochem. Pharm., 2004, 67,
2093.
Effective new synthesis developedLisa Iddon,
ongoing Ph. D. studies
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Acknowledgements
University of Liverpool Chemistry Jennifer
Perrie Paul Meath Eiizabeth Bowkett Amy
Jones Xiaoli Meng Mazhar Iqbal Lisa Iddon
Neil Berry Prabha Jayapal Pharmacology
Prof. Kevin Park Roz Jenkins James Maggs
Astra Zeneca Alderley Park John Harding Ian
Wilson Ryan Bragg Pfizer Kevin Beaumont Chris
Kohl Analytical Services International Prof.
David Holt Prof. Atholl Johnston
University of Oxford Prof. Ben Davis Rune Monrad
Funding Astra Zeneca GSK Pfizer ASI The EPSRC
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