Syntheses of [14C] and [2H4]PD0205520, an inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor

Article abstract of DOI:10.1002/jlcr.944

5-(4-Methyl-piperazin-1-yl)-pent-2-ynoic acid [4-(3-chloro-4-fluoro- phenylamino)-pyrido[3,4-d]pyrimidin-6-yl]-amide, PD0205520, was under investigation as a potential inhibitor of the tyrosine kinase (TK) activity of the epidermal growth factor receptor (EGFR) for cancer treatment. Both radio- and stable-isotope-labeled compounds were required for drug absorption, distribution, metabolism and excretion (ADME) and quantitative mass spectrometry bio-analytical studies. PD0205520 ¹⁴C-labeled in the pyrimidine ring system was prepared in seven steps in an overall radiochemical yield of 26% from [¹⁴C]thiourea. PD0205520 ²H-labeled in the piperazine ring was synthesized in four steps in a 32% overall yield. Copyright

Full text of DOI:10.1002/jlcr.944

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 485–496.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.944
Research Article
Syntheses of [14C] and [2H4]PD0205520, an inhibitor of
the tyrosine kinase activity of the epidermal growth
factor receptor
Yinsheng Zhang*, Yun Huang and Che C. Huang
Radiochemistry Group, Chemical R&D, Michigan Pharmaceutical Sciences, Pfizer Inc.,
301 Henrietta St, Kalamazoo, MI 49007, USA
Summary
5-(4-Methyl-piperazin-1-yl)-pent-2-ynoic acid [4-(3-chloro-4-fluoro-phenylamino)-
pyrido[3,4-d]pyrimidin-6-yl]-amide, PD0205520, was under investigation as
a
potential inhibitor of the tyrosine kinase (TK) activity of the epidermal growth
factor receptor (EGFR) for cancer treatment. Both radio- and stable-isotope-labeled
compounds were required for drug absorption, distribution, metabolism and excretion
(ADME) and quantitative mass spectrometry bio-analytical studies. PD0205520 I4C-
labeled in the pyrimidine ring system was prepared in seven steps in an overall
radiochemical yield of 26% from [14C]thiourea. PD0205520 2H-Iabeled in the
piperazine ring was synthesized in four steps in a 32% overall yield. Copyright #
2005 John Wiley & Sons, Ltd.
Key Words: carbon-14; deuterium; [14C]thiourea; [2H]piperazine; [14C]formamidine
acetate; EGFR TK inhibitor; anticancer
Introduction
The epidermal growth factor receptor (EGFR) is a marker for poor prognosis
in a significant proportion of human tumours.1 Selective inhibitors of tyrosine
phosphorylation by EGFR have become an important class of potential
anticancer drugs.2a,b 5-(4-Methyl-piperazin-1-yl)-pent-2-ynoic acid [4-(3-
chloro-4-fluoro-phenylamino)-pyrido[3,4-d]pyrimidin-6-yl]-amide, PD0205520,
was under investigation as a potential EGFR tyrosine kinase (TK) inhibitor
for chronic therapy in adjuvant treatment of cancers and in chemo-prevention
in individuals with a high risk for developing cancer. Both radio- and stable-
isotope-labeled compounds were required for drug absorption, distribution,
*Correspondence to: Yinsheng Zhang, Radiochemistry Group, Chemical R&D, Michigan Pharmaceutical
Sciences, Pfizer Inc., 301 Henrietta Street, Kalamazoo, MI 49007, USA.
E-mail: yinsheng.zhang@pfizer.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 20 December 2004
Revised 15 February 2005
Accepted 25 February 2005

486
Y. ZHANG ET AL.
metabolism and excretion (ADME) and use as an internal standard for
quantitative mass spectrometry (MS) bio-analytical studies. In this paper we
wish to report the efficient syntheses of [14C]PD0205520 and [2H]PD0205520.
Results and discussion
From the retrosynthetic analysis, the target could be prepared from
pyramidine ring system 1 and a side chain, N-methyl piperazine derivative 3
(Figure 1). The former pyramidine ring system 1 might be made from the
condensation of 5-amino-2-fluoropyridine-4-carboxylic acid 4 and formami-
dine acetate 5, both of which may be the radiolabeled starting material. The
latter N-methyl piperazine derivative 3 could be easily labeled with deuterium.
Based on the above analysis, the efficient synthetic approaches to radiolabeled
5-amino-2-fluoropyridine-4-carboxylic acid 4 or formamidine acetate 5, and
[2H]N-methyl piperazine derivative 3 were developed.
Synthesis of [14C]PD0205520
At the beginning of our development work, we attempted to label the
carboxylic group at the 4-position of pyridine by a carboxylation reaction
(Scheme 1). With a large excess CO2 gas, compound 10 was prepared in 64%
yield. However, when CO2 gas was generated from BaCO3 (1.2 equiv.) the
carboxylation reaction gave a poor yield (24%) with various bases (n-BuLi/
TMEDA, s-BuLi/TMEDA, LDA) and solvents (THF, diethyl ether, MTBE).
Figure 1. Retrosynthesis of [14C]/[2H]PD0205520
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

SYNTHESES OF [14C] AND [2H4]
487
Scheme 1. Synthesis of 5-amino-2-fluoropyridine-4-carboxylic acid 4
Therefore, compound 4 was synthesized as a cold intermediate in three steps from
the commertially available material, 5-amino-2-fluoropyridine 8 (Scheme 1).
A detailed literature search revealed that the unlabeled formamidine HCl
salt can be prepared by the reduction of thiourea with Raney-Ni in the
presence of NH4Cl.3 By changing NH4Cl to NH4OAc we were able to prepare
the desired [14C]formamidine acetate (FAA) 12 in nearly quantitative yield
(Scheme 2).
The ring closure of 5-amino-2-fluoropyridine-4-carboxylic acid 4 with 2.2
equiv. formamidine acetate in n-butanol was quite straightforward giving the
desired product in 65% yield,4 while reducing the mole ratio of formamidine
acetate from 2.2 equiv. to 1.5 equiv. resulted in a much lower yield. Several
solvents were screened in hopes of improving the yields (Table 1). By addition
of triethylamine (TEA, 1.1 equv.) to the reaction mixture in methoxylethanol,
Scheme 2. Synthesis of [14C] formamidine acetate 12
Table 1. Solvent selection for ring closure reaction
Entry
Solvents
4/FAA
(mole ratio)
Reaction
time (h)
Yield (%)
1
2
3
4
5
n-Butanol
n-Butanol
DBE
Methoxyethanol
Methoxyethanol/TEA
1:2.2
1:1.5
1:1.5
1:1.5
1:1.2
15
15
15
15
5
65
37
32
47
66
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

488
Y. ZHANG ET AL.
the reaction time was reduced from 15 to 5 h and the yield increased from 47 to
66% with less excess [14C]formamidine acetate (FAA). The addition of
triethylamine changes the pH of the reaction mixture, and might increase the
reactivity of formamidine 12.
[14C]PD0205520 was then prepared in five steps in an overall radiochemical
yield of 39% (Scheme 3). Conversion of fluoropyrido[3,4-d] [2-l4C]pyrimidin-
4(3H)-one 13 to its chloride 14 was performed in a typical manner by reaction
with thionyl chloride. The crude chloride 14 was used directly in the aniline 2
substitution to obtain compound 15 in 83% yield. To convert the 6-fluoro to a
6-amino group in the pyridine ring, a known two-step reaction approach5 was
modified. By addition of phenoxytrimethylsilane to the reaction mixture we
were able to isolate the pure product 16 by simple solvent trituration. Also the
application of triethylsilane for the de-protection reaction allowed us to reduce
the amount of TFA and lower the reaction temperature from 120 to 258C, and
obtain almost quantitative yield of 17. In the final coupling reaction, due to the
instability of the free acid of 3 the excess lithium salt 3 was used to form the
amide 18, [14C]PD0205520.
Scheme 3. Synthesis of [14C]PD0205520
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

SYNTHESES OF [14C] AND [2H4]
489
Synthesis of [2H4]PD0205520
The stable-isotope-labeled compound could be synthesized using [13C2,
15N]thiourea by following the procedures for the seven-step synthesis of
[l4C]PD0205520. However, a short and less expensive synthesis could be
developed by preparing a deuterium-labeled side chain.
The hydrogenation of N-methyl pyrazine halide salts with deuterium gas in
the presence of catalyst, Rh/Al2O3, was first investigated by modifying a
known procedure6 (Scheme 4). However, changing solvents from ethanol to
acetic acid and pressure from 30 to 50 psi did not improve the yields (38%). It
was also difficult to purify the material due to low boiling point and high water
solubility of N-methyl[2H4]piperazine 22.
The LiAlD4 reduction reaction of l-methyl-3,5-piperazinedione 24 was
straightforward,7 and the pure compound 25 can be isolated as a DCl salt
simply by the addition of 12 M DCl to the solution of the crude compound in
THF (Scheme 5).
N-Alkylation of N-methyl piperazine 25 with tosylate 26 was studied using a
different solvent and a limited amount of the labeled compound 25 (Table 2).
For the preparation of unlabeled version of compound 27, N-methylpiperazine
was used not only as a reactant but also as a solvent. However, using heptane
as a solvent and 1.2:1 mole ratio of 25 and 26, we were able to obtain the
desired labeled compound 27 in 70% yield (Table 2).
Scheme 4. Synthesis of N-methyl[2H4]piperazine 22
Scheme 5. Synthesis of N-methyl[2H4]piperazine 25
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

490
Y. ZHANG ET AL.
Table 2. Alkylation of N-Methyl [2H4]piperazine 27
Entry
Condition (equiv. 25/equiv. 26)
Yield (%)
1
2
3
4
1008C/3 h(10/l)
71
35
45
70
K2CO3/THF/reflux/15 h (1.2/1)
K2CO3/MeCN/reflux/15 h (1.2/1)
K2CO3/Heptane/reflux/15 h (1.2/1)
The carboxylation of the alkyne 27 produced the acid lithium salt 28 which
was isolated instead of the free acid since the latter was not stable and hard to
purify. For the unlabeled synthesis, the final amination reaction was carried
out using a large excess of unlabeled 28 to give 55% yield of PD0205520.
By reversing the mole ratio of 28 (1 equiv.) and 29 (4 equiv.) we were able
to produce the final labeled product 30, [2H4]PD0205520, in 61% yield
(Scheme 6).
Scheme 6. Synthesis of [2H4]PD0205520
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

SYNTHESES OF [14C] AND [2H4]
491
In summary, both [14C] and [2H4]PD0205520 were prepared in good overall
yields from [I4C]thiourea and LiAD4, respectively. The preparation of the key
labeled materials, [14C]formamidine acetate 12 and N-methyl[2H4]piperazine
25, by simple reduction reactions allowed us to make the target compounds
more efficiently.
Experimental
All reactions were carried out under an atmosphere of nitrogen unless
otherwise stated. 1H and 13C-NMR spectra were recorded on a Varian Gemini
200 or 400 MHz. Chemical purity of all labeled compounds was determined by
HPLC and GC-MS or LC-MS. Purifications were done by flash column
chromatography on Biotage Flash 40 system. LiAlD4 (98 at% D) was
purchased from Cambridge Isotope Lab. [14C]Thiourea (600 mCi, 57 mCi/
mmol) was purchased from PerkinElmer Life Sciences. All prepared stable-
isotope-labeled compounds contained less than 0.1% of unlabeled material
based on LC-MS analysis.
tert-Butyl N-(6-fluoro-3-pyridyl)carbamate (9)
To a solution of 5-amino-2-fluoro-pyridine (5.60 g, 51.4 mmol) in MTBE
(12 ml), was added di-tert-butyl dicarbonate (44.8 g, 205.5 mmol) at room
temperature. The mixture was heated to 40–458C. The resulting mixture was
stirred for 10 h. Activated carbon (2.0 g) was added to the reaction mixture,
and then the mixture was filtered. The filtrate was added to hexane (50 ml), to
form the precipitate. The product was filtered and washed with additional
hexane. Drying at 508C under vacuum gave the titled compound 9 as gray-
colored crystals (9.92 g, 91%): m.p. 114–1168C (lit.4113.5–1158C).
2-Fluoro-5-(tert-butoxycarbonylamino)yridine-4-carboxylic acid (10)
To a mixture of the compound 9 (5.0 g, 23.5 mmol), TMEDA (8.75 ml) and
MTBE (70 ml) was added a solution of n-BuLi (2.5 M in hexane, 22.30 ml) at
À708C. After completion of the addition, the mixture was heated to À10 to À158C,
and held at this temperature for 3 h. Dry CO2 gas was sparged at À708C for
2 h. The mixture was heated to 58C, and then water (60 ml) was added. The
aqueous phase was collected and the organic phase was extracted with 1 M
NaOH. To the combined aqueous solution was added MTBE (20 ml). 6 M
HCl was then added slowly to this mixture to adjust the pH to 2.5–3.0. The
product as precipitate was filtered and was washed with MTBE and hexane
(1:1). Drying under vacuum at 508C gave the title compound 10 (3.84 g, 64%):
m.p. 252–2548C (lit.4 m.p. 252–254.58C).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

492
Y. ZHANG ET AL.
5-Amino-2-fluoropyridine-4-carboxylic acid (4)
To a solution of the compound 10 (6.0 g, 23.4 mmol) in CH2Cl2 (20 ml) was
added trifluoroacetic acid (10.8 ml) at 08C. The resulting solution was stirred
at room temperature for 3 h. The mixture was concentrated under vacuum to
an oily residue. MTBE (16 ml) was added to the residue to form a slurry, which
was then filtered and washed with MTBE. Drying under vacuum at 508C gave
the title product 4 as an off-white solid (3.12 g, 85%): m.p. 2598C (lit.4 m.p.
2598C decomp).
[14C]formamidine acetate (12)
[l4C]Thiourea (600 mCi, 57 mCi/mmol) and NH4OAc (1.2 g, 15.7 mmol) were
suspended in dry EtOH (15 ml), and then heated at 858C for 15 min to form a
homogenous solution. To this solution was added Raney-Ni (5 g, wet in
EtOH) rapidly in small portions. The resulting mixture was stirred at 90–958C
for 3 h. A little charcoal was added to the mixture, and then the solid was
filtered off, washed with hot EtOH (20 ml). The filtrate was evaporated to
dryness. The residue was extracted with dry EtOH (10 ml). Any solid was
removed by filtration. The filtrate was evaporated with toluene (10 ml) to
dryness. Drying the residue under vacuum for 15 h at room temperature gave
the product 12 as a greenish solid (1.21 g, 100%). TLC RCP>98%, Rf=0.11,
silica gel, EtOAc/MeOH/NH4OH (40/4/1).
6-Fluoropyrido[3,4-d] [2-14C]pyrimidin-4(3H)-one (13)
The compound 4 (1.72 g, 8.80 mmol) and [14C] formamidine acetate 12 (1.10 g,
10.52 mmol) were dissolved in 2-methoxylethanol (10 ml) and triethylamine
(1.5 ml). The resulting solution was stirred at 1408C for 1.5 h, and then
evaporated to remove a half of the solvent. After cooling to 258C, the
precipitate was filtered off, and washed with 2-methoxylethanol (2 Â 2 ml), and
then suspended in saturated NaHCO3 solution (10 ml). The suspension was
stirred for 30 min at 258C, and then cooled at 08C. The slurry was filtered and
the cake was washed with water. Drying under vacuum at 508C for 15 h gave
the title product 13 as a rusty red solid (0.957 g, 66%). TLC RCP>98%,
Rf=0.33, silica gel, EtOAc/hexane (2:1), m.p. 2858C (lit.4 2878C decomp).
4-(3-Chloro-4-fluoro-phenylamino)-6-fluoro-pyrido [3,4-d] [2-l4C]pyrimidine (15)
A suspension of the compound 13 (0.491 g, 2.98 mmol) in SOCl2 (4 ml) and
DMF (0.1 ml) was heated at 65–758C for 20 h. The reaction mixture was
evaporated to remove the excess SOCl2 and HCl. The residue was dissolved in
toluene (5 ml), and the solution was evaporated again to dryness (0.54 g). To a
solution of the above crude product in dry toluene (7 ml), was added 3-chloro-
4-fluoroaniline (0.472 g, 3.2 mmol), and then diisopropylethylamine (0.52 ml).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

SYNTHESES OF [14C] AND [2H4]
493
The resulting mixture was stirred at 80–858C for 7 h. The solvent was
evaporated, and then co-evaporated again with MeCN (3 ml) to dryness under
vacuum. The residue was stirred in MeCN (4 ml) and water (4 ml) at 508C for
20 min. After cooling to room temperature, the precipitates were collected and
washed with MeCN/H2O (1:1, 1 ml). Drying under vacuum at room
temperature for 10 h gave the product 15 as a yellowish solid (0.725 g,
83%): TLC RCP>98%, Rf=0.46, silica gel, EtOAc/hexane (2/1); m.p. 267–
2698C (lit.5 m.p. 267–2698C).
4-[3-Chloro-4-fluoro-phenylamino]-6-(4-methoxylbenzylamino)-pyrido [3,4-d]
[14C]pyrimidine (16)
A mixture of the compound 15 (0.725 g, 2.47 mmol), p-methoxyl-benzylamine
(0.563 g, 4.10 mmol) and phenoxytrimethylsilane (0.342 g) in DMSO (3.6 ml)
was stirred at 1208C for 20 h. The solid in the reaction mixture was filtered off
and washed with DMSO (1 ml) and MeOH (3 ml). The filtrate was evaporated
under vacuum to dryness. The residue was dissolved in EtOAc (1 ml), and
cooled to room temperature. The precipitates were collected and washed with
EtOAc/Hexane (2:1, 2 ml). Drying the solid under vacuum at room
temperature for 10 h gave the product 16 as a yellowish solid (0.80 g, 79%):
TLC RCP>98%, Rf=0.33, silica gel, EtOAc: m.p. 196–1978C (lit.5 196–
1978C).
4-[3-Chloro-4-fluoro-phenylamino]-6-amino-pyrido [3,4-d] [14C]pyrimidine (17)
To a suspension of the compound 16 (0.80 g, 1.95 mmol) and triethylsilane
(0.64 ml) in CH2Cl2 (12 ml), was added trifluoroacetic acid (3.1 ml) slowly at
08C. The resulting solution was stirred at room temperature for 15 h, and then
evaporated under vacuum to dryness. The residue was triturated with hexane
(8 ml) at 558C for 30 min. The solid was stirred in NH3/MeOH (2.0 M, 8 ml) at
508C for 30 min and then cooled to room temperature. The solid was filtered
off. Drying under vacuum at 608C for 20 h gave the product 17 as a yellow
solid (0.56 g, 99%). TLC RCP>98%, Rf=0.36, silica gel, EtOAc/MeOH
(1:1); m.p. 263–2658C (lit.5 263–2668C).
5-(4-Methyl-piperazin-1-yl)-pent-2-ynoic acid [4-(3-chloro-4-fluorophenylami-
no)-pyrido [3,4-d] [14C]pyrimidin-6-yl]-amide ([14C]PD0205520) (18)
A suspension of the compound 17 (0.425 g, 1.46 mmol), pyridine-HBr (1.62 g,
10.1 mmol), pyridine (6.5 ml) and 4-methylpiperazin-1-yl)-pent-2-ynoic acid
lithium salt 3 (1.742 g, 8.6 mmol) in CH2Cl2 (6.5 ml) was stirred at 258C for
10 min. To this suspension was added EDAC (1.68 g, 8.76 mmol) at 08C. The
resulting mixture was stirred at 58C for 6.5 h. After water (10 ml) was added to
the reaction mixture, the mixture was evaporated to remove CH2Cl2. The
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

494
Y. ZHANG ET AL.
precipitate was filtered off, and washed with water (3 ml). The crude product
was subjected to flash chromatography (eluent: MeOH/EtOAc/NH3 3:1:0.1)
to obtain the product (0.415 g, 61%). TLC RCP>99%, Rf=0.34, silica gel,
1
MeOH/EtOAc/NH4OH: 4/1/0.1; m.p. 210–2128C; H-NMR (DMSO-d6) d
9.00 (s, 1H), 8.750 (s, 1H), 8.630 (s, 1H), 8.12 (d, 1H), 7.780 (m, 1H), 7.450 (t,
1H), 2.550 (s, 4H), 2.42 (b, 4H), 2.280 (b, 4H),2.11 (s, 3H); 13C-NMR (DMSO-
d6) d 157.57, 155.62, 155.15, 153.20, 151.64, 148.02, 142.48, 136.60, 125.09,
123.90 (d), 121.49, 119.47 (d), 117.23 (d), 105.16, 89.1277.14, 56.12, 55.20, 52.
69, 46.23, 17.17; MS (ESI): 468 [M+H]. Radiochemical purity: 98.63%;
Chemical purity: 99.25%; Specific activity: 61.72 mCi/mg; Total activity:
25.6 mCi. HPLC Column: HyPurity Advance 5 m, 250 Â 4.6 mm; Mobile
phase: A=[0.025 M NH4H2PO4 & 0.05 M TFA] pH 3.0 w/H3PO4,
B=CH3CN. Initial 25% B, gradient to 70% B over 14 min, hold A:B 30:70
to 30 min; Flow rate: 1.0 ml/min; UV detection: 240 nm.
N-methyl[2H4]piperazine dihydrochloride (25)
To a stirred suspension of LiAlD4 (3.8 g) in dry THF (25 ml), was added
dropwise a solution of l-methylpiperazine-3,5-dione 24 (3.8 g, 29.6 mmol,
prepared by following Houghton’s procedure8) in dry THF (60 ml) at room
temperature. The resulting mixture was stirred at 858C for 2.5 h, and then at
room temperature for 12 h. To the reaction mixture was added ether (40 ml),
D2O (3.8 ml), 15% NaOD (3.8 ml) and D2O (5 ml) at 08C. The mixture was
stirred at 08C for 1 h. The precipitate was filtered off, and extracted with ether
(150 ml) for 2 h using soxhlet. The combined ether layers were dried with
MgSO4, and then concentrated to dryness. The residue was dissolved in dry
THF. To this THF solution was added concentrated DCl to make the solution
strongly acidic. The acidic solution was evaporated under vacuum to dryness.
The residue was lyophilized overnight. Further drying at 808C under vacuum
gave the product 25 as a white solid (4.72 g, 90%): m.p. 240–2428C (lit.7 241.5–
2438C).
3-(N-methyl[2H4]peperazine)-prop-1-yne (27)
A mixture of N-methyl-[2H4]piperazine DCl salt 25 (3.67 g, 20.27 mmol) and
K2CO3 (9.80 g, 70.96 mmol) was finely ground, and then heated in heptane
(50 ml) at 1008C for 15 h. To the above suspension was added 3-hydroxy-prop-
1-yne tosylate 26 (4.84 g, 21.5 mmol) at 508C. The resulting mixture was heated
at 1108C for 15 h. The solid was filtered off and washed with heptane (25 ml
 3). The combined heptane solution was evaporated under vacuum to give a
yellowish residue, which was subjected to flash chromatography (eleunt:
EtOAc/hexane, 7/3). The product 27 was obtained as a colorless liquid (2.20 g,
70%). 1H-NMR (CDCl3) d 2.599 (t, 2H), 2.479 (bm, 4H), 2.364 (t, 2H), 2.307
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

SYNTHESES OF [14C] AND [2H4]
495
(s, 3H), 1.963 (t, 1H); 13C-NMR (CDCl3) d 82.81, 69.32, 56.95, 54.91, 52.20
(m), 46.06, 16.95 (d); MS (ESI): 157 [M+H].
5-(4-Methyl[2H4]piperazin-1-yl)-pent-2-ynoic acid Li salt (28)
To a solution of the compound 27 (1.62 g, 10.38 mmol) in THF (32 ml), was
added a solution of n-BuLi (2.5 M in hexane, 4.15 ml) at À788C. The resulting
solution was stirred at À788C for 2 h. Dry CO2 gas was sparged at À708C for
2 h. The mixture was stirred at room temperature for 24 h, and then
concentrated to dryness. The residue was then stirred with THF (2.5 ml) and
MTBE (25 ml) at room temperature for 5 h. The white precipitate was
collected by filtration under N2 gas. Drying under vacuum at 608C for 17 h
1
gave the product 28 (1.89 g, 90%). H-NMR (D2O) d 2.433 (t, 2H), 2.40–2.30
(b, 4H), 2.306 (t, 2H), 2.035 (s, 3H), 1.963 (t, 1H); 13C-NMR (D2O) d 161.46,
82.13, 77.59, 55.21, 53.07, 50.73 (m), 44.44, 15.51; MS (ESI): 201 [M+H].
5-(4-Methyl[2H4]piperazin-1-yl)-pent-2-ynoic acid [4-(3-chloro-4-fluoro-phe-
nylamino)-pyrido [3,4-d]pyrimidin-6-yl]-amide ([2H4]PD0205520) (30)
A suspension of 4-[3-Chloro-4-fluorophenylamino]-6-aminopyrido [3,4-d]
pyrimidine 29 (4.50 g, 15.53 mmol), pyridine-HBr (1.0 g, 6.25 mmol), pyridine
(5 ml) and 4-(N-methyl[2H4]piperazin-1-yl)-pent-2-ynoic acid lithium salt 28
(0.80 g, 3.88 mmol) in CH2Cl2 (5 ml) was stirred at 258C for 20 min. To the
suspension was added EDAC (1.40 g, 7.33 mmol) at 08C. The resulting mixture
was stirred at 58C for 15 h. After water (6 ml) was added to the reaction
mixture, the mixture was evaporated to dryness under vacuum. The residue
was triturated with water (8 ml), and then the precipitate was collected, and
subjected to flash chromatography (eluent: MeOH/EtOAc/NH4OH, 3:1:0.1)
1
to obtain the title product 30 (1.11 g, 61%). H-NMR (DMSO-d6) d 11.43 (s,
1H), 10.32 (s, 1H), 8.99 (s, 1H), 8.76 (s, 1H), 8.61 (s, 1H), 8.09 (s, 1H), 7.77 (s,
1H), 7.44 (t, J=9.10 Hz, 1H), 2.55 (s, 4H), 2.36–2.16 (m, 4H), 2.12 (s, 3H);
13C-NMR (DMSO-d6) d 157.55, 154.38 (d, J=243.2 Hz), 155.09, 151.69,
151.55, 148.02, 142.41, 136.71, 125.05, 123.89 (d, J=6.90 Hz), 121.57, 119.54
(d, J=18.41 Hz), 117.24 (d, J=21.48 Hz), 105.25, 89.18, 77.12, 56.07, 55.14,
52.07 (m), 46.45, 17.17; MS (ESI): 472 [M+H].
Acknowledgements
The author thanks Dr Laura Greenfield for helpful discussions and valuable
remarks concerning this work.
References
1. Hickey K, Grehan D, Reid IM. Cancer 1994; 74: 1693–1698.
2. (a) Bridges AJ. Curr Med Chem 1996; 3: 167–194; (b) Fry DW. Annu Rep Med
Chem 1996; 31: 151–160.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496

496
Y. ZHANG ET AL.
3. Tabor H, Rabinowitz JC. Biochem Prep 1957; 5: 100–105.
4. Rewcastle GW, Denny WA, Winters RT, Colbry NL, Schowalter HDH. J Chem
Soc Perkin Trans 1 1996; 18: 2221–2226.
5. Smaill JB, Palmer BD, Rewcastle GW, Denny WA, McNamara DJ, Dobrusin EM,
Bridges AJ, Zhou H, Showalter HDH, Winters RT, Leopold WR, Fry DW,
Nelson JM, Slintak V, Elliot WL, Roberts BJ, Vincent PW, Patmore SJ. J Med
Chem 1999; 42: 1803–1815.
6. Paulis T, Davis DA, Smith HE. J Label Compd Radiopharm 1988; 25(9):
1027–1033.
7. Shetty HU, Hawes EM, Midha KK. J Label Compd Radiopharm 1980; 28:
1633–1640.
8. Houghton RP, Williams E. J Chem Soc Perkin Trans 1 1982; 2693–2695.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 485–496