The synthesis of the glucuronide adduct of Trocade(TM)

Article abstract of DOI:10.1016/S0040-4039(00)01556-2

The synthetic preparation of the glucuronide adduct of Trocade(TM), a selective inhibitor of the MMP collagenase, is described. This was achieved by preparation of the protected O-glucuronsyl hydroxylamine (9) and subsequent coupling to the available carboxylic acid (7), followed by deprotection. The synthetic material was identical to authentic isolated metabolite, which confirmed that glucuronidation of Trocade(TM) occurs on the oxygen of the hydroxamic acid. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01556-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8829–8834
The synthesis of the glucuronide adduct of Trocade™
Mark B. Mitchell* and Ian W. A. Whitcombe
Chemical Synthesis Department, Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City,
Herts AL7 3AY, UK
Received 5 September 2000; accepted 14 September 2000
Abstract
The synthetic preparation of the glucuronide adduct of Trocade™, a selective inhibitor of the MMP
collagenase, is described. This was achieved by preparation of the protected O-glucuronsyl hydroxylamine
(9) and subsequent coupling to the available carboxylic acid (7), followed by deprotection. The synthetic
material was identical to authentic isolated metabolite, which confirmed that glucuronidation of
Trocade™ occurs on the oxygen of the hydroxamic acid. © 2000 Published by Elsevier Science Ltd.
Hydroxamic acids represent a class of compound which are currently of great interest with
respect to inhibition of matrix metalloproteases (MMPs).1 Trocade™ (1) is a selective inhibitor
of the MMP collagenase, and is currently undergoing Phase III clinical trials for the treatment
of rheumatoid arthritis (Fig. 1).
Figure 1.
Studies on the metabolism of Trocade™ in marmosets and by using human liver slice
microsomes indicated glucuronidation to be the predominant metabolic fate of the drug
substance. Glucuronidation is a well-known drug metabolising reaction and is generally
regarded as a ‘detoxification’ process, with the drug–glucuronic acid adducts usually being
cleared renally.2
* Corresponding author.
0040-4039/00/$ - see front matter © 2000 Published by Elsevier Science Ltd.
PII: S0040-4039(00)01556-2

8830
Two structures were proposed for the glucuronide adduct (Fig. 2), one oxygen linked (2) and
the other linked through nitrogen (3). These structures were based upon the following evidence,
obtained from a sample of the metabolite isolated from marmoset urine:
· the m/z of the adduct was consistent with a glucuronide
1
· the H NMR spectrum contained the elements of Trocade™ and glucuronic acid
· the molecule did not mutarotate, indicating the aglycone was coupled at the anomeric
position
· the chemical shift (l=4.7) and coupling constant (J=7.9 Hz) of the anomeric proton in the
1H NMR spectrum were consistent with a b-anomer
· treatment of the glucuronide with b-glucuronidase returned Trocade™. This supported
coupling to the sugar through the hydroxamic acid linkage.
Figure 2.
Only a few mg of isolated metabolite were available at this time therefore further studies were
not possible in order to distinguish between structures (2) and (3). A chemical synthesis was
required to confirm unambiguously the structure of the glucuronide and also to provide an
analytical reference sample of the metabolite.
Our initial efforts focused upon the Lewis acid mediated coupling of Trocade™ to the fully
benzyl protected glucuronic acid trichloroacetimidate (4) (Scheme 1). This process is well
precedented3 for the synthesis of alkyl glucuronides, but has not been reported for hydroxamic
acids. Additionally, the sugar (5) prepared as an anomeric mixture reacts with trichloroaceto-
nitrile to furnish the trichloroacetimidate (4) exclusively as the a-anomer.4 This in turn reacts
with nucleophiles to afford predominantly b-glucuronide as required.
Scheme 1. Reagents: (i) trichloroacetonitrile; (ii) Trocade™/BF3·OEt2 (cat.)/CH2Cl2

8831
Despite several attempts, no coupling was detected when Trocade™ was used as the
nucleophilic component; the hydrolysed sugar (5) was always obtained following work-up of the
reaction mixture. Activation of (5) as a reactive sugar triflate was also unsuccessful, whilst the
attempted coupling of (5) with (1) under Mitsunobu conditions degraded the aglycone. These
results suggested the hydroxamic acid was not sufficiently nucleophilic to react with the
activated glucuronic acid derivative. The literature also indicated that sugar uronates are
generally poor glycosyl donors.5 Thus we changed tack, and looked at the alternative disconnec-
tion depicted in Fig. 3.
Figure 3.
It was clear that an N-protected hydroxylamine would be required for the synthesis of (6)
since free hydroxylamine is known to react with sugars to afford oximes. The attempted
coupling of (5) with N-hydroxyphthalimide (HONFt) under Mitsunobu conditions returned
largely unreacted sugar. However, compound (8) was successfully prepared as depicted in
Scheme 2, based upon a method previously reported by Roy.6
Scheme 2. Reagents: (i) NaOMe/MeOH; (ii) acetic anhydride/pyridine (40% over two steps); (iii) HBr/AcOH (82%);
(iv) HOFt/TBAHS/NaHCO3/CH2Cl2 (66%); (v) hydrazine hydrate (1 equiv.)/MeOH 20 min (quant.)
The starting material ‘glucurone’ is readily available,7 and the intermediates are nicely
crystalline. The coupled product was obtained exclusively as the b-anomer, and was deprotected
to 9 by brief exposure to an equimolar quantity of hydrazine hydrate; the ester groups were
unaffected under these conditions.8 Carboxylic acid (7), a key intermediate in the synthesis of
Trocade™ was converted to the acid chloride, and successfully coupled to (9), albeit in low
1
yield. The product (11) appeared to be a single stereoisomer by H NMR spectroscopy.

8832
Scheme 3. Reagents: (i) (COCl)2/DMF (cat.)/toluene (quant.); (ii) (9)/triethylamine/CH2Cl2 (24%); (iii) NaOMe
(cat.)/MeOH; (iv) Duolite C255(H+) resin (97%, two steps); (v) 1 M NaOH (aq.); (vi) Duolite C255(H+) resin followed
by lyophilization (97%, two steps)
Scheme 4. Reagents: (i) b-glucuronidase/deuterated acetate buffer 40°C
The acetyl groups were rapidly cleaved by treatment with sodium methoxide/methanol and
the methyl ester subsequently saponified (Scheme 3). The product (12) had the desired m/z but
1
the H NMR was not identical when compared with the authentic metabolite. The material also
had a tendency to undergo slow intramolecular cyclization in solution, as judged by LC–MS
analysis; this behaviour was not observed for the authentic metabolite. The attempted cleavage

8833
of (12) by a b-glucuronidase (in an NMR tube experiment) did not furnish Trocade™, but the
cyclic compound (13) [Ro 32-4779] which must be derived from the Trocade™ epimer (14), Ro
32-6436 (Scheme 4).9 The epimerisation almost certainly occurred at the acid chloride stage, the
low solubility of (10) suggesting the origin may be crystallisation induced.10 This epimerisation
has also been observed independently by colleagues at F. Hoffmann-La Roche, Basel.11
Scheme 5. Reagents: (i) (9)/EDAC/HOBT/N-ethyl morpholine/CH2Cl2; (ii) NaOMe (cat.)/MeOH; (iii) Duolite
C255(H+) resin (92%, two steps); (iv) 1 M NaOH (aq.); (v) Duolite C255(H+) resin followed by prep. HPLC and
lyophilization (60%, two steps)
1
Figure 4. 400 MHz H NMR

8834
The synthesis was now repeated, using an HOBT/EDAC mediated coupling between the sugar
(9) and acid (7). Subsequent steps were performed as before and the final product (2) purified
by preparative HPLC (Scheme 5). The product was identical (Fig. 4) in all respects to the
authentic metabolite (1H NMR, m/z, LC) and did not show any tendency for intramolecular
cyclization. This confirmed the metabolite possessed structure (2), with the aglycone linked to
the sugar through oxygen.
In summary, the structure of the glucuronide adduct of Trocade™ has been shown to be 2 by
means of an unambiguous chemical synthesis. A practical route to 9 has been demonstrated, an
intermediate which should prove useful for the preparation of glucuronide adducts of various
hydroxamic acids.
References
1. For a review see: Watson, S. A.; Tierney, G. BioDrugs 1998, 9, 325–335.
2. Mulder, G. J. Annu. Rev. Pharmacol. Toxicol. 1992, 32, 25.
3. See for example: van Boeckel, C. A. A.; Delbressine, L. P. C.; Kaspersen F. M. Recl. Trav. Chim. Pays Bas 1985,
104, 259.
4. Schmidt, R. R.; Grundler, G. Synthesis 1981, 885.
5. Mu¨ller, T.; Schneider, R.; Schmidt, R. R. Tetrahedron Lett. 1994, 35, 4763.
6. Cao, S.; Tropper, D.; Roy, R. Tetrahedron 1995, 51, 6679.
7. CAS [32449-92-6]; Lancaster product no. 4643
8. Professor R. Stoodley, University of Manchester Institute of Science and Technology (UMIST), personal
communication.
1
9. Reference H NMR data was available for pure (13) and (14), obtained from previous independent syntheses.
10. For a review of Crystallisation-Induced Dynamic Resolution (CIDR) see: Caddick, S.; Jenkins, K. Chem. Soc.
Rev. 1996, 25, 447.
11. Dr H. Hilpert, F. Hoffmann-La Roche, Basel, personal communication.
.