Note
Received 29 May 2009,
Revised 29 July 2009,
Accepted 14 September 2009
Published online 3 December 2009 in Wiley Interscience
A facile and efficient synthesis of d6-labeled
PU-H71, a purine-scaffold Hsp90 inhibitor
Ã
Tony Taldone, Danuta Zatorska, Yanlong Kang, and Gabriela Chiosis
PU-H71 is a purine-scaffold Hsp90 inhibitor currently undergoing late stage preclinical evaluation for the treatment of
cancer. In this investigation, we present a simple method for the synthesis of d6-labeled PU-H71 for use as an internal
standard to accurately quantitate the drug in biological matrices based on an LC-MS-MS method. PU-H71-d6 was
synthesized in five steps using readily available 1,3-dibromopropane-d6 and is an important compound for the
advancement of our clinical program.
Keywords: deuterium labeled PU-H71; purine; heat shock protein 90; inhibitor; cancer
sulfate (8). Cyclocondensation with CS2 gives 8-mercaptoadenine
(9) in quantitative yield and is used directly without further
Introduction
Molecular chaperone heat shock protein 90 (Hsp90) is essential
for the proper folding and conformation of proteins and has
recently emerged as an attractive target for the development
of drugs against a variety of cancers.1,2 Hsp90 has arisen to
prominence as a cancer target because it plays a vitally
important role in regulating clinically validated oncogenic
proteins such as Her-2, ER and Bcr-Abl, as well as other
signaling proteins that play a role in malignancy, such as Raf-1,
p-Akt, Cdk4, mutant p53 and Flt-3.3–6 Hsp90 functions as a
chaperone through its ATPase activity and inhibition of this
results in proteasomal degradation of its client proteins. The
first synthetic small molecules to be discovered and translated
into clinic in patients with advanced cancers are the purine-
scaffold Hsp90 inhibitors,1,2 and one such derivative, CNF-
2024/BIIB021 (1), is currently in Phase I/II clinical trials for a
variety of cancers (Figure 1). There are also non-purine
synthetic Hsp90 inhibitors currently in clinical trials including
the isoxazole VER-52296/NVP-AUY922 (2) and the 6,7-dihydro-
indazol-4-one SNX-5422 (3). 17-AAG (4, tanespimycin) and IPI-
504 (5, retaspimycin) are both derivatives of the natural
product geldanamycin, and despite promising initial results
for 17-AAG (4) clinical trials have recently been halted.
PU-H71 (6) is a second generation purine-scaffold Hsp90
inhibitor which our laboratory is currently developing for clinical
trials. In support of our clinical program, we needed to develop a
rapid and sensitive method for determining drug levels in
biological matrices such as plasma and urine. One method
evaluated for this purpose was LC-MS-MS utilizing an isotopi-
cally labeled analog, with identical physical and chromato-
graphic properties to the unlabeled compound, as an internal
standard. In this paper, we present the synthesis and
characterization of PU-H71-d6 (7).
purification in the next step, to result in the 8-arylsulfanylpurine
(10) in 60% yield via a copper catalyzed coupling to 1-iodo-3,4-
methylenedioxybenzene. Reaction with N-iodosuccinimide gives
11 in 85% yield and alkylation with 1,3-dibromopropane results in
12 in 37% yield. PU-H71 (6) is obtained in the final step by reaction
of 12 with excess isopropylamine in 90% yield.
Based on this synthetic sequence the simplest, most efficient
and direct method for introducing the isotopic label would be
during the final step using isopropylamine-d7, which is readily
available. Unfortunately, this moiety is not particularly stable
as it is prone to fragment from the molecule on ionization in
LC-MS-MS to yield identical product ions to the unlabeled
compound 6. The next logical step for the introduction of
the label was in the 9-propyl chain in the penultimate step of
the sequence and which remains intact during LC-MS-MS. The
compound 11 was reacted with 1,3-dibromopropane-d6 (13) to
give 14 in 33% yield which was subsequently reacted with
excess isopropylamine to give PU-H71-d6 (7) in 92% yield
1
(Scheme 2). The compound 7 was fully characterized by H and
13C NMR, HRMS and LCMS. The low yield obtained in the
formation of 14 was expected from our previous experience
with the unlabeled compound 12, and can be primarily
attributed to unavoidable competing 3-alkylation. Five equiva-
lents of 1,3-dibromopropane is typically used in the preparation
of 12 to ensure complete reaction of 11 and to limit other
undesirable side-reactions such as dimerization. In an attempt to
decrease the amount of 1,3-dibromopropane-d6 (13) consumed
we found that 3.7 equivalents can be used to obtain 14 in
Program in Molecular Pharmacology and Chemistry and Department of
Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Results and discussion
*Correspondence to: G. Chiosis, Program in Molecular Pharmacology and
Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer
Center, 1275 York Avenue, Box 482, New York, NY 10021, USA.
E-mail: chiosisg@mskcc.org
PU-H71 (6) can be obtained from a five-step synthetic sequence as
shown in Scheme 1, commencing from 4,5,6-triaminopyrimidine
J. Label Compd. Radiopharm 2010, 53 47–49
Copyright r 2009 John Wiley & Sons, Ltd.