An efficient synthesis of isotope-labeled PD0331179 and its labeled metabolite

Article abstract of DOI:10.1002/jlcr.2963

4-[1-Methyl-2,4-dioxo-6-(3-phenyl-prop-1-ynyl)-1,4-dihydro-2H-quinazolin-3- ylmethyl]-benzoic acid, PD0331179, was under investigation as a matrix metalloproteinase-13 inhibitor. ¹⁴C-labeled and ²H-labeled PD0331179 and its ²

Full text of DOI:10.1002/jlcr.2963

Research Article
Received 8 June 2012,
Revised 15 July 2012,
Accepted 31 July 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2963
An efficient synthesis of isotope-labeled
PD0331179 and its labeled metabolite
*
Yinsheng Zhang
4-[1-Methyl-2,4-dioxo-6-(3-phenyl-prop-1-ynyl)-1,4-dihydro-2H-quinazolin-3-ylmethyl]-benzoic acid, PD0331179, was under
investigation as a matrix metalloproteinase-13 inhibitor. 14C-labeled and 2H-labeled PD0331179 and its 2H-labeled metabolite
(PD0335699) were required to support its preclinical and clinical studies. [14C] 3-phenyl-1-trimethyl/triphenylsilyl-propyne was
efficiently prepared starting with [14C] benzoic acid and used as a key-labeled reagent for the synthesis of [14C] PD0331179. A
one-pot coupling reaction between aryl iodide and trialkylsilyl propyne was developed to make this synthesis more efficient.
The details of these syntheses are reported.
Keywords: isotope labeling; C-14; H-2; C–C cross-coupling; triphenylsilyl-propyne; MMP-13 inhibitor
carbodiimide, 1-hydroxybenzotriazole, and N-methylmorphine, in
Introduction
DMF provided the amide 4 in 95% yield. Cyclization of 4 with
1,10-carbonyldiimidazole in THF afforded the quinazoline 5 in
Matrix metalloproteinases (MMPs) are a family of zinc-dependent
proteinases that are key enzymes implicated in matrix degrada-
75% yield, which was hydrolyzed with NaOAc to afford the acid
tion and reconstruction. The importance of MMPs in cancer
and inflammation is well known as manifested by the proliferation
6. The final C–C cross coupling between 6 and 2-phenyl-1-propyne
catalyzed by Pd(PPh3)4 and CuI in diisopropylethylamine (DIPEA)
furnished the final API, PD0331179, in 65%. The overall yield for
of publications as well as the number of drug candidates in clinical
trials.1–4 One particular enzyme, MMP-13 (collagenase-3), is mainly
the five-step synthesis was about 20%.
expressed in articular cartilage during osteoarthritis (OA) and in the
Because the C-14 labeling, the highlighted area of PD0331179,
synovium of patients with rheumatoid arthritis and therefore an
shown in Scheme 1, was preferred considering the metabolic
stability, either starting material 1 or 2-phenyl-1-propyne could
attractive drug discovery target.5–8 PD0331179 was selected as a
specific inhibitor of MMP-13 for the indication of OA.9 Both
be chosen as a C-14 labeled chemical. Both labeled analogs were
radioisotope and stable isotope-labeled PD0331179 including its
not commercially available. However, we selected 2-phenyl-1-
labeled metabolite were required to support its absorption,
propyne involved in the last step of the synthesis as our labeled
distribution, metabolism, and elimination, preclinic evaluation
target because it required less steps to prepare. Therefore,
and bioanalytical mass spectrometry clinic studies as an internal
we focused on exploring an effective approach to the labeled
standard, respectively.
3-phenyl-1-propyne.
In this paper, we report the synthesis of C-14 labeled
From our literature search, we found that p-chloro-substituted
PD0331179, H-2 labeled PD0331179, and H-2 labeled metabolite
phenylpropyne 9a was prepared by coupling p-cloro-benzylchloride
PD0335699 (Figure 1). [14C] PD0331179 was synthesized in six
steps starting with [14C] benzoic acid, whereas [2H7]PD0331179
was prepared in four steps from [2H7] benzylbromide. An efficient
synthesis for key-labeled intermediates, [14C] and [2H7]3-phenyl-
trialkylsilyl-1-propyne, was developed through a convenient
two-step reaction sequence. In addition, one-pot C–C cross-coupling
reaction between aryl iodide and labeled 1-triphenylsilylpropyne
was achieved. The labeled metabolite [2H7]PD0335699 was
prepared by convenient treatment of the [2H7]PD0331179 with
an organic base, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
with Grignard reagent.10 By adapting this method, we can synthe-
size the labeled phenylpropyne 9b. However, it was found that
phenylpropyne was very volatile and not easy to isolate in a small
scale (Scheme 2). Also, it will not be safe to use so volatile radiola-
beled compound for the synthesis of [14C]PD0331179.
Therefore, we proposed to synthesize a protected phenylpropyne
that should not be so volatile and can be readily isolated and
the protection group of which can be removed as needed later.
In literatures, both bis(diisopropylamino)borane 11 and trimethylsilyl
(TMS)12,13 groups were utilized before as a protection group of an
alkyne (Scheme 2). The former can be removed under aqueous
Results and discussion
The synthesis of unlabeled PD0331179 involves a total of five steps
from a commercially available starting material, 2-methylamino-
benzoic acid 1 (Scheme 1). The iodo substitution of 1 with iodine
in acetic acid and water gave 2-methylamino-5-iodobenzoic acid
2 in 56% yield. Reaction of methyl 4-(aminomethyl)benzoate 3 with
Chemical R&D, Groton Lab, Pfizer, Inc. Groton, CT 06340, USA
*Correspondence to: Yinsheng Zhang, Chemical R&D, Groton Lab, Pfizer, Inc.
Groton, CT 06340, USA.
2 using coupling reagents, 1-ethyl-3-(3-dimethylaminopropyl) E-mail: Yinsheng.zhang@yahoo.com
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

Y. Zhang
Figure 1. Structures of labeled compounds.
Scheme 1. Synthetic route of PD0331179.
Scheme 2. Known synthesis of 3-phenyl-1-propyne.
acidic condition, such as 3N HCl,11 whereas the later can be
One of the metabolites of PD0331179 was allene derivative,
performed in DMF under a basic condition such as KF.12 However, PD0335699. It was known that isomerization between alkyne
the basic removal of TMS group is our better choice because and allene took place under basic treatment. Normally, either
KF could be a suitable base for the final C–C cross-coupling reaction n-BuLi or KOH with the help of a phase transfer catalyst
of our synthesis. So, our proposal for a new approach to the promotes the conversion of alkyne to allene. However, in our case,
labeled PD0331179 was investigated using stable isotope-labeled both bases gave very poor yield (10–20%), and purification was
chemicals first (Scheme 3).
very difficult. We found that DBU can promote the isomerization
Several methods are reported in the literatures for the synthesis and afford a descent chemical yield (45%). It is interesting to point
of 3-aryl-1-propynes.14–16 However, the procedures are often out that 91% deuterium remained at the highlighted position.
cumbersome, unsafe to be performed, and give quite low yields. That might further indicate that the isomerization between the
Therefore, a new general method to prepare 3-aryl-1-propynes in alkyne and allene takes place through a hydrogen shift instead of
high yields from commercially available reagents was needed. a stepwise mechanism.
We found that 3-phenyl-1-propyne can be prepared in situ very
Having established conditions for the synthesis of PD0331179
conveniently and in high overall yields. The synthesis started with and [2H7]PD0331179, it was readily adapted to synthesize the
1-trimethylsilylethyne or triphenylsilylethyne. The treatment of radiolabeled PD0331179 (Schemes 4 and 5). Our radiosynthesis
trialkylsilylethyne 13 a,b with n-BuLi in THF produced trialkylsily- started with a commercially available radioactive chemical,
lethynyl lithium 14a,b. Without isolation and purification of [14C]benzoic acid. So, the labeled acid 17 was reduced by LiAlH4
the lithium compound, it was immediately reacted with benzylbro- in THF to form [14C]benzyl alcohol 18. Without further purifica-
mide or [2H7]benzylbromide in the same pot in the presence tion, 18 was treated with PPh3 and CBr4 in methylene chloride
of copper(I) bromide to give pure [2H7]3-phenyl 1-trialkylsilyl-1- to afford [14C]benzyl bromide 19 in 82% yield after purification.
propyne 15a in 81% yield after purification. Then, 15a was treated Similarly, 19 was coupled with triphenylsilylethynyl lithium
with an excess of potassium fluoride in DMF at 60 ꢀC for 1 h to form prepared in situ to give [14C] 3-phenyl-1-triphenylsilylethyne 20
the desired [2H7]3-phenyl-1-propyne 16. Again, without isolation in 80% after purification. Because 3-phenyl triphenylsilylethyne
of the alkyne, it was coupled with the iodide 6 in the presence of has much higher boiling point and is nonvolatile compared with
Pd(PPh3)4, CuI, and DIPEA in the same pot to furnish the final phenylpropyne and trimethylsilylethyne, the radio-safety issue
target, [2H7]PD0331179, in a 78% isolated yield.
was well addressed.
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Y. Zhang
Scheme 3. Synthesis of [2H7]PD0331179 and its labeled metabolite [2H7]PD0335699.
Scheme 4. Synthesis of [3-14C] 3-phenyl-1-triphenylsilylpropyne.
Scheme 5. Synthesis of [14C]PD0331179.
[14C] 3-Phenyl-1-triphenylsilylpropyne 20 was treated with metabolite, PD00335699. A new approach to C-14 and deuter-
KF in DMF at 60 ꢀC for 1 h, and HPLC assay showed 100% of ium-labeled key intermediate, 3-phenyl-1-propyne, was developed
conversion of 20 to [14C]3-phenyl-1-propyne 21. However, the by using trimethylsilane or triphenylsilane as a protection group
final cross-coupling reaction between 21 (prepared in situ) and of the alkyne. This approach avoided potential loss of volatile
the iodide 6 under the same condition as used in the prepara- radioactive material and simplified the purification process. It
tion of [2H7]PD0331179 gave a lower yield (45% vs. 78%). One can be easily extended to synthesize various substituted
possible explanation for the lower yield is that under basic alkynes. In addition, we discovered a one-pot C–C cross-
condition, the radiolabeled 3-phenyl-1-propyne 21 might be coupling reaction of the aryl iodide with the trialkylsilyl-
partially isomerized to allene derivative 22 that lost the reactivity protected alkyne. Further study on the scope and limitation of
of C–C cross-coupling reaction.
this kind of C–C cross coupling is in progress.
We came up to a solution to increase the yield by addition of all
chemicals and reagents including reactants 20 and 6, reagents KF
and DIPEA, and catalysts Pd(PPh3)4 and CuI at the same time
instead of stepwise addition and by reduction of the reaction
temperature from 60 to 40 ꢀC. In this way, [14C]3-phenyl-1-
propyne 21 produced in situ was coupled with the iodide 6
immediately before the isomerization took place. As a result,
the new procedure for the removal of silyl protection group
and cross-coupling reaction offered a better radiochemical yield
(61% vs. 45%). The overall radiochemical yield from [1-14C]
benzoic acid 17 was 38% with a radiochemical purity of 99.4%
and a specific activity of 53.8 mCi/mmol.
Experimental
General methods
All reactions were carried out under an atmosphere of nitrogen unless
otherwise stated. LC-MS data were obtained on a Waters Micromass
LCT mass spectrometer with flow injection analysis and electrospray
ionization (ESI). 1 H and 13C NMR spectra were recorded on a Varian
Gemini 400 MHz instrument. Chemical purity of all compounds was
determined by HPLC and LC-MS. Purifications were done by flash
column chromatography on Biotage Flash 40 system. Quantitation of
radioactivity of C-14 labelled compounds was performed using a Packard
2200CA liquid scintillation analyzer, with Scintiverse BD cocktail used
In conclusion, we have reported the efficient synthesis of
14C-labeled and 2H-labeled PD0331179 and its 2H-labeled throughout. Commercial reagents and solvents were purchased from
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

Y. Zhang
Sigma-Aldrich and used as-received unless otherwise noted. [14C]benzoic the titled product (0.838 g, 80%, 121 mCi) as a colorless oil. 1 H NMR
acid (200 mCi, 54 mCi/mmol) was purchased from American Radiolabeled (CDCl3): d 7.64 (d, 6 H), 7.42-7.25 (m, 14 H), 3.82 (s, 2 H); 13C NMR (CDCl3):
Chemicals, Inc. [2 H7]benzylbromide (>98% 2 H7 enrichment) was d 136.4 (1C), 135.3 (6C), 134.2 (3C), 130.7 (3C), 128.9 (1C), 128.4 (2C), 128.2
purchased from Sigma-Aldrich. Intermediate, 4-(6-iodo-1-methyl-2, (2C), 128.0 (6C), 112.9 (1C), 84.2 (1C), 25.9 (1C); LCMS: m/z 375 (M + H)
4-dioxo-1,4-dihydro-2 H-quinazolin-3-ylmethyl)-benzoic acid 6 and final
4-[1-Methyl-2,4-dioxo-6-(3-phenyl-[3-14C]prop-1-ynyl)-1,4-dihydro-2 H-
API, PD0331179, were provided by Chemical R&D, Ann Arbor Lab, Pfizer
Inc. All known compounds were identified by comparison of NMR spectra
to those reported in the literature or the authentic samples.
quinazolin-3-ylmethyl]-benzoic acid, [14C]PD0331179
A mixture of [14C]1-triphenyllsilyl-3-phenyl-1-propyne 20 (0.4g, 1.07 mmol,
57.8mCi), KF (124 mg, 2.14 mmol), compound 6 (436mg, 1.0mmol), CuI
(21mg, 0.11 mmol) and DIPEA (0.5mL) in DMF (5mL) were purged with
N2 for 10 min. Pd(PPh3)4 (62 mg, 0.052mmol) was added to the mixture at
room temperature. The resulting mixture was stirred at 40ꢀC for 4hr and
4-[1-Methyl-2,4-dioxo-6-(3-[2 H5]phenyl-[2-2 H2]prop-1-ynyl)-1,4-dihydro-
2 H-quinazolin-3-ylmethyl]-benzoic acid, [2 H7]PD0331179
A mixture of [2 H7] 1-trimethylsilyl-3-phenyl-1-propyne 15a (0.80 g,
4.1 mmol), KF (0.476 g, 8.2 mmol) and DMF (15 mL) was stirred at 60 ꢀC
then quenched with water (20 mL). The suspension was stirred at room
for 1.5 hr. After cooling to room temperature, compound 6 (1.65 g,
temperature for 30min and then filtered to give the crude product as a
3.8 mmol), CuI (78 mg, 0.41 mmol) and DIPEA (2.0 ml) were added to
brown solid. The solid was dissolved in THF (15 mL) and heated to reflux
with charcoal (0.5g) for 30 min. The charcoal was removed by filtration
and washed with THF (2 x 5mL). The filtrate was evaporated under vacuum
to give a off-white solid. Further purification by flash chromatography (2%
the reaction mixture. After the mixture was purged with N2 gas for
5 min, Pd(PPh3)4 (236 mg, 0.2 mmol) was added to the mixture at room
temperature. The resulting mixture was stirred at room temperature for
10 hr and then quenched with water (80 mL). The suspension was stirred
MeOH in CH2Cl2) gave the product (263 mg, 62%, 53.8mCi/mmol, 33.3mCi)
at room temperature for 30 min and then filtered to give the crude
as a white solid. HPLC condition: column, YMC ODS-AQ, 5mm, 250 x 4.6mm;
product as a brown solid. The solid was dissolved in THF (50 mL) and
mobile phase, A = 0.1% TFA in H2O, B = CH3CN; 60% B linear gradient
heated to reflux with charcoal (2.0 g) for 30 min. The charcoal was
to 80% B over 15min, hold A:B 20:80 to 45 min; flow rate, 1.0 mL/min; UV
removed by filtration and washed with THF (2 x 5 mL). The filtrate
detection, 218 nm, retention time= 11.8min; TLC: 6% MeOH in CH2Cl2,
was evaporated under vacuum to give a off-white solid. Further purifica-
Rf = 0.45; 1HNMR (DMSO-d6): d 12.78 (bs, 1 H), 8.04 (d, J = 2.0 Hz, 1 H), 7.83
tion by flash chromatography (2% MeOH in CH2Cl2) gave the titled
(d, J=8.3Hz, 2H), 7.82 (dd, 1H), 7.21-7.53 (m, 8H), 5.17 (s, 2H), 3.54 (s, 2H),
product (1.25 g, 78%) as a white powder. 1 H NMR (DMSO-d6): d 8.01
3.51 (s, 3 H); 13C NMR (DMSO-d6): d 168.2 (1C), 160.8(1C), 150.9 (1C), 141.6
(d, J = 2.0 Hz, 1 H), 7.84 (d, J = 8.3 Hz, 2 H), 7.82 (dd, 1 H), 7.43 (d, J = 8.5,
(1C), 139.2 (1C), 135.5 (1C), 132.2 (1C), 129.9 (2C), 128.9 (1C), 128.6 (2C),
Hz, 1 H), 7.38 (d, J = 8.3 Hz, 2 H), 5.17 (s, 2 H), 3.51 (s, 3 H); 13C NMR
128.2 (1C), 128.1 (2C), 127.3 (2C), 117.6 (1C), 114.3(1C), 113.3 (1C), 87.8(1C),
(DMSO-d6): d 168.1 (1C), 160.6 (1C), 150.9 (1C), 141.6 (1C), 139.2 (1C),
81.8 (1C), 43.9 (1C), 29.8 (1C), 25. 5 (1C); LC-MS: m/z 425 (M + H).
135.4 (1C), 132.1 (1C), 129.7 (2C), 128.8 (m, 1C), 128.6 (m, 2C), 128.2
(1C), 128.1 (m, 2C), 127.3 (2C), 117.6 (1C), 114.3 (1C), 113.3 (1C), 87.8
(1C), 81.8 (1C), 43.8 (1C), 29.8 (1C), 25. 4 (m, 1C); LC-MS: m/z 432 (M + H,
Acknowledgements
2 H7 enrichment >98%).
4-[1-Methyl-2,4-dioxo-6-(3-[2 H5]phenyl-[2 H2]propa-1,2-dieynl)-1,4-
The authors would like to thank Dr Che C. Huang and Dr Laura
Greenfield for their support necessary to successfully complete
this project.
dihydro-2 H-quinazolin-3-ylmethyl]-benzoic acid, [2 H7] PF-0335699
A solution of [2 H7]PD0331179 (500 mg, 1.18 mmol) and DBU (450 mg,
1.0 mmol) in THF (8 mL) was stirred at room temperature for 8 hr. The
reaction mixture was diluted with water (5 mL) and extracted with diethyl
Conflict of Interest
ether (2 x 5 mL). The aqueous layer was separated and acidified by 2 M
HCl, then extracted with ethyl acetate (3 x 6 mL). The combined organic
layers were washed with brine (2 x 5 mL), dried over MgSO4 and concen-
trated under vacuum to give the crude product. Purification by flash
chromatography (5% MeOH in CHCl3) afforded the titled product
(225 mg, 45%) as a yellow solid. 1 H NMR (DMSO-d6) d 3.53 (s, 3 H), 5.37
(s, 2 H), 7.15 (d, 1 H), 7.32 (t, 2 H), 7.35 (d, 1 H), 7.54 (2 H), 7.61 (dd, 1 H),
7.98 ( d, 2 H), 8.22 (s, 1 H); 13C NMR (DMSO-d6): d 203.6 (1C), 168.2 (1C),
162.3 (1C), 151.5 (1C), 142.6 (1C), 135.6 (1C), 130.7 (1C), 129.8 (2C), 128.
3 (m, 2C), 127.3 (2C), 126.7 (m, 2C), 123.3 ( m, 1C), 120.5 (1C), 102.7
(m 1C), 98.3 (m, 1C), 44.5 (1C), 29.6 (1C); LC-MS: m/z 432 (M + H, 2 H7
enrichment 97.1%).
The authors did not report any conflict of interest.
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