Synthesis of 6-acrylamido-4-(2-[18F]fluoroanilino)quinazoline: A prospective irreversible EGFR binding probe

Article abstract of DOI:10.1002/jlcr.903

Acrylamido-quinazolines substituted at the 6-position bind irreversibly to the intracellular ATP binding domain of the epidermal growth factor receptor (EGFR). A general route was developed for preparing 6-substituted-4- anilinoquinazolines from [¹⁸F]fluoroanilines for evaluation as EGFR targeting agents with PET. By a cyclization reaction, 2-[¹⁸F] fluoroaniline was reacted with N′-(2-cyano-4-nitrophenyl)-N,N- dimethylimidoformamide to produce 6-nitro-4-(2-[¹⁸F]fluoroanilino) quinazoline in 27.5% decay-corrected radiochemical yield. Acid mediated tin chloride reduction of the nitro group was achieved in 5 min (80% conversion) and subsequent acylation with acrylic acid gave 6-acrylamido-4-(2-[ ¹⁸F]fluoroanilino)quinazoline in 8.5% decay-corrected radiochemical yield, from starting fluoride, in less than 2h. Copyright

Full text of DOI:10.1002/jlcr.903

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 109–115.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.903
Research Article
Synthesis of 6-acrylamido-4-(2-
[18F]fluoroanilino)quinazoline: a prospective irreversible
EGFR binding probe
Neil Vasdev1, Peter N. Dorff1, Andrew R. Gibbs1, Erathodiyil Nandanan2,
Leanne M. Reid1, James P. O’Neil1 and Henry F. VanBrocklin1,*
1 Department of Nuclear Medicine and Functional Imaging, Lawrence Berkeley National
Laboratory, Berkeley, CA 94720, USA
2 Molecular Therapeutics, Inc. Ann Arbor, MI 48104, USA
Summary
Acrylamido-quinazolines substituted at the 6-position bind irreversibly to the
intracellular ATP binding domain of the epidermal growth factor receptor (EGFR).
A general route was developed for preparing 6-substituted-4-anilinoquinazolines from
[18F]fluoroanilines for evaluation as EGFR targeting agents with PET. By a
cyclization reaction, 2-[18F]fluoroaniline was reacted with N0-(2-cyano-4-nitrophe-
nyl)-N,N-dimethylimidoformamide to produce 6-nitro-4-(2-[18F]fluoroanilino)quina-
zoline in 27.5% decay-corrected radiochemical yield. Acid mediated tin chloride
reduction of the nitro group was achieved in 5 min (80% conversion) and subsequent
acylation with acrylic acid gave 6-acrylamido-4-(2-[18F]fluoroanilino)quinazoline in
8.5% decay-corrected radiochemical yield, from starting fluoride, in less than
2 h. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: EGFR; fluorine-18; PET; quinazoline
Introduction
The epidermal growth factor receptor (EGFR) is a member of the ErbB family
of receptor tyrosine kinases (tk) and is often overexpressed in a variety of
tumors. Inhibition of the EGFR-tk is a focal point in therapeutic development
and considered to be an important target for cancer therapy.1 Recently,
*Correspondence to: H. F. VanBrocklin, Department of Nuclear Medicine and Functional Imaging,
Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS55R0121, Berkeley, CA 94720-8119, USA.
E-mail: hfvanbrocklin@lbl.gov
Contract/grant sponsor: US Department of Energy; contract/grant number: DE-AC03-76SF00098, NIH
grant No. CA94253(HFV)
Contract/grant sponsor: Natural Science and Engineering Research Council of Canada
Copyright # 2005 John Wiley & Sons, Ltd.
Received 30 March 2004
Revised 4 June 2004
Accepted 23 September 2004

110
N. VASDEV ET AL.
6-acrylamido-4-anilino-quinazolines have been developed as a class of
therapeutic agents that bind irreversibly to the intracellular ATP binding
domain of the EGFR-tk and inhibit downstream cell signaling events.2,3
Radiolabeled EGFR-tk probes may be instrumental in understanding tumor
biology or as tools for drug development.
Mishani and coworkers4ꢀ7 have synthesized and evaluated 6-substituted-4-
haloanilinoquinazolines labeled with carbon-11 (half-life=20.4 min) as novel
irreversible positron emission tomography (PET) probes for EGFR-tk. These
tracers have been explored to overcome the ATP competitive binding that may
have interfered with the retention of the carbon-11 labeled reversible probes.4,7
As a longer-lived alternative to the carbon-11 probes, we report herein the
synthesis of 6-acrylamido-4-(2-[18F]fluoroanilino)quinazoline ([18F]7) as a
potential probe for EGFR-tk. The increased tracer lifetime (fluorine-18 half-
life=109.7 min) may provide a better match to the pharmacokinetics and
EGFR turnover.
Our synthetic strategy involves the following steps: (1) preparation of a
radiolabeled aniline; (2) ring closure of N0-(2-cyano-4-nitrophenyl)-N,N-
dimethylimidoformamide, 4, with the labeled aniline to form 6-nitro-4-
anilinoquinazoline;8 (3) reduction of the nitro group to form 6-amino-4-
anilinoquinazoline, a key intermediate for several labeled analogs; and (4)
acylation of the amine with acrylic acid.8,9 This strategy may be extended to
provide a series of labeled 6-substituted-4-anilinoquinazolines.
Results and discussion
Scheme 1 shows the synthetic route to [18F]7. Fluorine-18 labeled 2-
fluoroaniline hydrochloride ([18F]3) was selected for coupling to 4 because it
NO2
NO2
NH2 HCl
18F
18F
NO2
ON
N
O2N
i
ii, iii
+
N(CH3)2
1
[
18F]2
[
18F]3
4
18F
18F
18F
HN
HN
HN
H
N
O2N
H2N
vi
iv, v
N
vii
N
N
O
N
18F]5
N
[
18F]6
N
18F]7
[
[
Scheme 1. Reagents: (i) K18F/K222, DMSO, 1308C, 5 min; (ii) NaBH4/Pd-C, rt,
.
5 min; (iii) HCl; (iv) HOAc, 1008C, 20 min; (v) NaOH; (vi) SnCl2 2H2O, HCl,
.
1008C, 5 min; (vii) acrylic acid, EDCI HCl, pyridine, DMF, rt, 5 min
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F-18 EGFR LIGAND
111
is efficiently prepared10 and potentially possesses suitable metabolic properties
when coupled to quinazolines.11,12 Cyclization provides 6-nitro-4-(2-[18F]fluor-
oanilino)quinazoline ([18F]5), incorporating the labeled 4-anilino moiety in a
single step. Compound [18F]5 was obtained (after HPLC purification) in
27.5% decay-corrected radiochemical yield, relative to the initial 18F-fluoride
activity. Acid mediated nitro group reduction of [18F]5 to [18F]6 with tin
chloride was accomplished in 5 min with 80% conversion. Subsequent
acylation with acrylic acid followed by HPLC purification provided chemically
and radiochemically pure [18F]7 (>95%). The overall radiochemical yield was
8.5% from starting fluoride in less than 120 minutes. The measured specific
activity at the end of synthesis was 12.1 GBq/mmol. The corresponding
fluorine-19 analogs, compounds 5–7, were prepared by the same route
1
(Scheme 1) and were characterized by H NMR spectroscopy and electron
impact mass spectrometry. The identities of [18F]5 - [18F]7 were confirmed by
comparison of the HPLC retention times and co-injection with unlabeled 5–7.
We have recently described the optimized synthesis of 2-, 3- and 4-
[18F]fluoroanilines.10 As the [18F]fluoroaniline isomers are similarly prepared,
one could easily substitute the 3- and 4-[18F]fluoroanilines in the current
approach. Likewise, one could envision extending the series with radiobromo-
or radioiodo-anilines. Additionally, elaboration in the 6 position such as the
acetamido analogs reported by Mishani and coworkers9 expands the series of
potential EGFR-tk probes. Thus, the chosen synthetic approach permits the
preparation of a variety of labeled quinazolines compounds for assessment as
biologic probes.
Experimental section
Materials
Absolute EtOH USP (Aaper), NaBH4 (Spectrum), HPLC grade MeOH
(Burdick and Jackson), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (EDCI ꢁ HCl; Pierce), stannous chloride dihydrate (Mallinckrodt)
and [18O]H2O (Rotem Industries Ltd., Israel, >94.1 atom%) were used
without further purification. All other reagents were purchased from Aldrich
and were used without further purification, with the exception of 1,2-
dinitrobenzene (Aldrich, >99%), which was recrystallized from EtOAc prior
to use.
Standard techniques
Preparative HPLC was performed at room temperature using a Beckman
Ultrasphere C-18 5-mm column (250 mm ꢂ 10 mm) with 65 : 35 (v/v) CH3OH :
H2O (1% Et3N in H2O adjusted to pH ¼ 7:4 with H3PO4) as the mobile phase.
A flow rate of 6 ml minꢀ1 was used (pump: Waters model 590) and the eluent
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was monitored for UV absorbance at 254 nm (detector: Linear UV-106) and
radioactivity (detector: Carroll and Ramsey Associates Model 105S).
Analytical HPLC was performed with the same solvent system on a
Phenomenex Hypersil C18 (ODS) 5-mm column (250 mm ꢂ 4.6 mm) with a
flow rate of 1 ml minꢀ1 and the eluent was monitored for UV absorbance at
1
254 nm (detector: Linear UVIS 200). The H NMR spectra were recorded in
CD3OD on a Bruker AV-300 (300 MHz) spectrometer at 308C. The mass
spectrometry analyses were conducted using a VG ProSpec spectrometer
equipped with an electron impact (EI) source operated at 70 eV.
No-carrier-added 18F-fluoride was produced by 10 MeV proton irradiation
of 30% enriched [18O]H2O at low pressure in a silver target by the 18O(p,n)18F
nuclear reaction on the Biomedical Isotope Facility CTI RDS 111-DV01
cyclotron. The [18O]H2O/18Fꢀ (100–450 ml) was transferred into a conical glass
vial containing Kryptofix1 222 (5 mg, 13.2 mmol), K2CO3 (0.5 mg, 3.62 mmol)
in 500 ml of CH3CN. The water was removed by azeotropic distillation with
anhydrous CH3CN (1 ml) at 1008C in vacuo, under a stream of nitrogen. The
azeotropic drying process was repeated.
Radiochemistry
6-nitro-4-(2-[18F]fluoroanilino)quinazoline ([18F]5). Fluorine-18 labeled 2-
fluoroaniline hydrochloride ([18F]3) was prepared as previously described10
and subsequently reacted with 400 ml of a saturated solution of N0-(2-cyano-4-
nitrophenyl)-N,N-dimethylimidoformamide (1 ml of glacial AcOH added to
50 mg of 4) at 1008C for 20 min to provide 6-nitro-4-(2-[18F]fluoroanilino)-
quinazoline ([18F]5). The reaction mixture was made basic with NaOH (1 ml,
1 M) and diluted to 4 ml with H2O. The mixture was passed sequentially
through a cartridge loaded with Celite1 521 filtering agent (Aldrich), an
activated C18 solid phase extraction cartridge (Sep-Pak1, Waters) and a
45 mm nylon disc-filter (Acrodisc Premium, Pall), connected in series, and
rinsed with 4 ml H2O. [18F]5 was eluted with MeOH (3 ml) into a separate vial,
diluted with 1 ml of H2O and injected onto the semi-preparative HPLC system.
[18F]5 (tR ¼ 6:5 min) was well separated from excess 4 (tR=4.0 min). [18F]5
was identified by the semi-preparative (tR ¼ 6:5 min) and analytical
(tR ¼ 6:9 min) HPLC retention times and co-injection with authentic
compound 5. The HPLC peak containing [18F]5 was collected, diluted to
20 ml with H2O, concentrated on a C18 cartridge and eluted with 2.5 ml EtOH.
Isolated [18F]5 was obtained in 27.5 ꢃ 1.5% (n ¼ 3) decay-corrected radio-
chemical yield, relative to the initial 18F-fluoride activity.
6-amino-4-(2-[18F]fluoroanilino)quinazoline ([18F]6). The ethanolic solution
of [18F]5 was added to a vial containing SnCl2 ꢁ 2H2O (8 mg, 35 mmol),
followed by the addition of 20 ml conc. HCl. The reaction vessel was sealed
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F-18 EGFR LIGAND
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with a Teflon lined screw cap and heated at 1008C for 5 min to yield 6-amino-
4-(2-[18F]fluoroanilino)quinazoline ([18F]6), which was used without further
purification. In separate experiments, semi-preparative HPLC was used to
determine the reduction efficiency. The conversion from [18F]5 to [18F]6 was
found to be 80%. [18F]6 was identified by the semi-preparative (tR ¼ 3:2 min)
and analytical (tR ¼ 4:1 min) HPLC retention times and co-injection with
authentic compound 6.
6-acrylamido-4-(2-[18F]fluoroanilino)quinazoline ([18F]7). The ethanolic re-
action mixture containing [18F]6 was evaporated to dryness at 1008C in vacuo.
To the dry residue, a DMF solution (300 ml) containing EDCI ꢁ HCl (6 mg,
31 mmol), followed by pyridine (50 ml) and acrylic acid (10 ml of a 20% acrylic
acid solution in DMF, 0.03 mmol) were added. The reaction vessel was sealed
with a Teflon lined screw cap and the acylation proceeded at room
temperature for 5 min. The reaction mixture was diluted to 5 ml with H2O
and loaded onto a C18 cartridge, then rinsed with an additional 2 ml
H2O. [18F]7 was eluted with 3 ml MeOH into a separate vial, diluted with
1 ml H2O and injected onto the semi-preparative HPLC system. The collected
fraction containing compound [18F]7 was diluted to 20 ml with H2O,
concentrated onto a C-18 cartridge and extracted into a test tube with
methanol. The methanol was evaporated at 1008C in vacuo and [18F]7 was
obtained in 8.5 ꢃ 2% decay-corrected radiochemical yield (n ¼ 3) in less than
2 h with greater than 95% chemical and radiochemical purity as measured by
analytical HPLC. [18F]7 was identified by the semi-preparative (tR ¼ 5:5 min)
and analytical (tR ¼ 6:0 min) HPLC retention times and co-injection with
authentic compound 7. [18F]7 is resolubilized by heating with 20 ml of EtOH at
1008C for 10 s, followed by the addition of saline or aqueous buffer (e.g.
Krebs–Henseleit buffer) to achieve the desired final concentration. The specific
activity was determined by analytical HPLC.
Syntheses of non-radioactive standard compounds
6-nitro-4-(2-fluoroanilino)quinazoline (5). N0-(2-cyano-4-nitrophenyl)-N,N-di-
methylimidoformamide,8 4, (2.4 g, 11.0 mmol) and 2-fluoroaniline (1.41 g,
13.0 mol) were stirred in AcOH (100 ml) at reflux for 2 h. The resulting solid
was filtered hot and washed with ether, followed by drying at 508C in vacuo to
1
give 1.86 g (60%) of 5 as a yellow solid. H NMR spectrum, d (ppm, relative
to TMS):9.58 (bs, 1H ), 8.61 (s, 1H), 8.57 (dd, J ¼ 2:5 Hz, J ¼ 9:2 Hz, 1H),
7.94 (d, J ¼ 9:2 Hz, 1H), 7.54 (t, J ¼ 7:5 Hz, 1H), 7.25–7.37 (m, 3H); MS (EI)
m/z 284.
6-amino-4-(2-fluoroanilino)quinazoline (6). A mixture of 6-nitro-4-(2-fluor-
oanilino)quinazoline, 5, (1.2 g, 4.33 mmol) was stirred in EtOH (7.5 ml) with
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Fe (1.2 g, 21.5 mmol) and AcOH (2 ml). Upon heating to reflux, additional
EtOH (2 ml) and AcOH (0.5 ml) were added and heating was continued for
3 h. After cooling the mixture to room temperature, the solution was filtered
and the residue washed with H2O (20 ml), then MeOH (3 ꢂ 20 ml). The
combined filtrates were neutralized with NH4OH and extracted with CHCl3.
The organic layer was dried (Na2SO4), decolorized with charcoal, and
the solvent was removed in vacuo. The residue was purified by chromato-
graphy (10% MeOH–CHCl3, silica gel) to provide 0.5 g (45%) of 6, a yellow
1
solid. H NMR spectrum, d (ppm, relative to TMS):9.25 (bs, 1H), 8.19
(bs, 1H), 7.56 (t, J ¼ 7:19 Hz, 1H), 7.51 (d, J ¼ 8:7 Hz, 1H), 7.20–7.28
(m, 4H); MS (EI) m/z 254.
6-acrylamido-4-(2-fluoroanilino)quinazoline (7). Acrylic acid (91 ml, 1.1 mmol)
was added dropwise into an ice-cold solution of 6-amino-4-(2-fluoroanilino)-
quinazoline, 6, (0.127 g, 0.5 mmol) in pyridine (0.5 ml) and DMF (4 ml). After
a few minutes EDCI ꢁ HCl (0.239 g, 1.25 mmol) was added. The reaction was
warmed to room temperature and stirred for 1 h. The solvent was removed in
vacuo and the residue was purified by chromatography (5% MeOH–CHCl3,
silica gel) to provide 0.2 g (8.5%) of 7. 1H NMR spectrum, d (ppm, relative to
TMS): 8.75 (d, J ¼ 2:2 Hz, 1H), 8.39 (s, 1H), 7.84 (dd, J ¼ 2:2 Hz, J ¼ 8:9 Hz,
1H), 7.75 (d, J ¼ 8:9 Hz, 1H), 7.67 (dt, J ¼ 1:8 Hz, J ¼ 7:8 Hz), 7.35 – 7.20
(m, 3H), 6.47 (d, J ¼ 3:8 Hz, 1H), 6.46 (d, J ¼ 8:1 Hz, 1H), 5.83 (dd,
J ¼ 3:8 Hz, J ¼ 8:1 Hz, 3H); MS (EI) m/z 308.
Conclusions
The first prospective fluorine-18 labeled irreversibly-binding EGFR-tk tracer,
6-acrylamido-4-([18F]2-fluoroanilino)quinazolines, ([18F]7), has been prepared.
The synthetic strategy reported herein may be applied to develop a series of
labeled compounds through acylation of the amine at position 6 and/or
coupling with substituted anilines for assessment as EGFR-tk probes.
Acknowledgements
This work was supported by the Director, Office of Science, Office of
Biological and Environmental Research, Medical Science Division of the US
Department of Energy under Contract No. DE-AC03-76SF00098 and NIH
grant #CA94253 (HFV). We thank the Natural Sciences and Engineering
Research Council of Canada for a Postdoctoral Fellowship (NV). We also
thank Chris Ramsey for technical expertise and production of [18F]fluoride.
References
1. Carpenter G (ed.). Exp Cell Res (review issue) 2003; 284: 1–159.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 109–115

F-18 EGFR LIGAND
115
2. Fry DW, Bridges AJ, Denny WA, Doherty A, Greis KD, Hicks JL, Hook KE,
Keller PR, Leopold WR, Loo JA, McNamara DJ, Nelson JM, Sherwood V,
Smaill JB, Trumpp-Kallmeyer S, Dobrusin EM. Proc Nat Acad Sci USA 1998; 95:
12022–12027.
3. Smaill JB, Palmer BD, Rewcastle GW, Denny WA, McNamara DJ, Dobrusin
EM, Bridges AJ, Zhou H, Schowalter 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.
4. Ben-David I, Rozen Y, Ortu G, Mishani E. Appl Radiat Isot 2003; 58: 209–217.
5. Ortu G, Ben-David I, Rozen Y, Freedman NMT, Chisin R, Levitzki A, Mishani
E. Int J Cancer 2002; 101: 360–370.
6. Mishani E, Levitzki A, Abourbeh G, Ortu G, Rozen Y, Ben-David I, Lester H,
Freedman N, Chisin R. J Nucl Med 2003; 44: 50.
7. Mishani E, Abourbeh G, Rozen Y, Jacobson O, Laky D, Ben David I, Levitzki A,
Shaul M. Nuc Med Biol 2004; 31: 469–476.
8. Tsou H-R, Mamuya N, Johnson BD, Reich MF, Gruber BC, Ye F, Nilakantan
R, Shen R, Discafani C, DeBlanc R, Davis R, Koehn FE, Greenberger LM,
Wang Y-F, Wissner A. J Med Chem 2001; 44: 2719–2734.
9. Mishani E, Abourbeh G, Ortu G, Rozen Y, Ben-David I, Froimovici S, Dissoki
S, Gazit A, Levitzki A. J Label Compd Radiopharm 2003; 46: S115.
10. VanBrocklin HF, Vasdev N, Dorff PN, O’Neil JP, Chin FT, Gibbs AR,
Nandanan E. J Label Compd Radiopharm, submitted for publication.
11. VanBrocklin HF, Lim JK, Coffing SL, Hom DL, Negash K, Ono MY, Hanrahan
SM, Taylor SE, Vanderpoel JL, Slavik SM, Morris AB, Riese II DJ. J Med Chem,
accepted for publication.
12. VanBrocklin HF, Vasdev N, Dorff PN, O’Neil JP, Taylor SE. J Label Compd
Radiopharm 2003; 46: S290.
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J Label Compd Radiopharm 2005; 48: 109–115