5
Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 196–200 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al hom epage: www.elsevier.com/locate/jpba Isolation and structure elucidation of the main UV-A photoproducts of vandetanib Stefano Dall’Acqua , Daniela Vedaldi, Alessia Salvador ∗∗ Department of Sciences of Drug, University of Padova, Italy a r t i c l e i n f o Article history: Received 12 April 2013 Received in revised form 28 May 2013 Accepted 31 May 2013 Available online 18 June 2013 Keywords: Vandetanib Photoproduct Photostability Phototoxicity HPLC a b s t r a c t Exposure of aqueous solutions of the antitumor drug vandetanib to UV-A light results in the pho- tochemical degradation. Two main photodegradation products were identified by HPLC–MS analysis and their structures were elucidated, after their isolation by HPLC, on the basis of LC–MS and NMR spectra. The photoproducts derived from a simple debromination (N-(2-fluorophenyl)-6-methoxy-7- ((1-methylpiperidin-4 yl)methoxy)quinazolin-4-amine, FP3) or from the loss of the bromide atom followed by the solvent addition (N-(4-hydroxy-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4 yl)methoxy)quinazolin-4-amine, FP2). At our knowledge this is the first report about the photodegrada- tion of vandetanib. © 2013 Elsevier B.V. All rights reserved. 1. Introduction In April 2011, the Food and Drug Administration approved the use of vandetanib for the treatment of unresectable or metastatic medullary thyroid cancer (MTC) [1]. Its mechanism of action involves the inhibition of vascular endothelial growth factor recep- tor (VEGFR), of epidermal growth factor receptor (EGFR) and of rearranged during transfection (RET) tyrosine kinase activity [2]. In the majority of cases of MTC, there is activation of the RET proto- oncogene, and both VEGFR and EGFR signaling pathways may also contribute to the pathogenesis. Therefore, vandetanib represents a promising targeted approach for the treatment of patients with MTC [3]. Moreover, vandetanib alone or in combination with other chemotherapeutics or radiotherapy is under many clinical trials for the management of other kinds of tumors [4–6]. Thus, this drug could be prescribed to much more people in the next few years. Vandetanib shows an acceptable safety and tolerability profile with a majority of the adverse events manageable with supportive therapy or dose reduction [7]. As many other multikinase inhibitors [8], skin is one of the main affected organs for its side effects: in fact, at least in part, the cutaneous toxicity can be explained by the Corresponding author at: Department of Sciences of Drug, University of Padova, Via Marzolo 5, 35131 Padova, Italy. Tel.: +39 049 8275344; fax: +39 049 8275366. ∗∗ Corresponding author at: Department of Sciences of Drug, University of Padova, Via Marzolo 5, 35131 Padova, Italy. Tel.: +39 049 8275034; fax: +39 049 8275366. E-mail addresses: [email protected] (S. Dall’Acqua), [email protected] (A. Salvador). fact that the signaling pathways and/or receptors are physiolog- ically expressed in the skin. Photosensitivity skin reactions were also reported with the administration of vandetanib [9–11]. Phototoxicity of drugs can derive from the classical photo- chemical reactions with the formation of radical oxygen species or singlet oxygen [12] but also from the toxic products formed due to photodegradation [13,14]. Drug photodegradation is not only related to phototoxicity but it can also cause loss of efficacy and for these reasons is an event to avoid [15]. Thus, photostability testing must be planned and performed during the develop- ment and the registration process of new pharmaceutical products [16,17]. In the present study, we reported the photodegradation of vandetanib in aqueous solution after UV-A irradiation and we determined the chemical structure of its main photoproducts. 2. Experimental 2.1. Materials Vandetanib, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1- methylpiperidin-4 yl)methoxy)quinazolin-4-amine, was pursued by Selleck Chemicals. Methanol and water for HPLC were obtained from WVR International. 2.2. Photodegradation conditions A solution of vandetanib (2.0 × 10 5 M) was prepared in deionised water, placed in caped quartz cuvette and exposed 0731-7085/$ see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.05.049

Isolation and structure elucidation of the main UV-A photoproducts of vandetanib

  • Upload
    alessia

  • View
    213

  • Download
    0

Embed Size (px)

Citation preview

Io

SD

ARRAA

KVPPPH

1

umitrIocaM

cfc

wt[f

V

V

a

0h

Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 196– 200

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al hom epage: www.elsev ier .com/ locate / jpba

solation and structure elucidation of the main UV-A photoproductsf vandetanib

tefano Dall’Acqua ∗, Daniela Vedaldi, Alessia Salvador ∗∗

epartment of Sciences of Drug, University of Padova, Italy

a r t i c l e i n f o

rticle history:eceived 12 April 2013eceived in revised form 28 May 2013ccepted 31 May 2013vailable online 18 June 2013

a b s t r a c t

Exposure of aqueous solutions of the antitumor drug vandetanib to UV-A light results in the pho-tochemical degradation. Two main photodegradation products were identified by HPLC–MS analysisand their structures were elucidated, after their isolation by HPLC, on the basis of LC–MS and NMRspectra. The photoproducts derived from a simple debromination (N-(2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4 yl)methoxy)quinazolin-4-amine, FP3) or from the loss of the bromide atom

eywords:andetanibhotoproducthotostability

followed by the solvent addition (N-(4-hydroxy-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4yl)methoxy)quinazolin-4-amine, FP2). At our knowledge this is the first report about the photodegrada-tion of vandetanib.

© 2013 Elsevier B.V. All rights reserved.

hototoxicityPLC

. Introduction

In April 2011, the Food and Drug Administration approved these of vandetanib for the treatment of unresectable or metastaticedullary thyroid cancer (MTC) [1]. Its mechanism of action

nvolves the inhibition of vascular endothelial growth factor recep-or (VEGFR), of epidermal growth factor receptor (EGFR) and ofearranged during transfection (RET) tyrosine kinase activity [2].n the majority of cases of MTC, there is activation of the RET proto-ncogene, and both VEGFR and EGFR signaling pathways may alsoontribute to the pathogenesis. Therefore, vandetanib represents

promising targeted approach for the treatment of patients withTC [3].Moreover, vandetanib alone or in combination with other

hemotherapeutics or radiotherapy is under many clinical trialsor the management of other kinds of tumors [4–6]. Thus, this drugould be prescribed to much more people in the next few years.

Vandetanib shows an acceptable safety and tolerability profileith a majority of the adverse events manageable with supportive

herapy or dose reduction [7]. As many other multikinase inhibitors8], skin is one of the main affected organs for its side effects: inact, at least in part, the cutaneous toxicity can be explained by the

∗ Corresponding author at: Department of Sciences of Drug, University of Padova,ia Marzolo 5, 35131 Padova, Italy. Tel.: +39 049 8275344; fax: +39 049 8275366.∗∗ Corresponding author at: Department of Sciences of Drug, University of Padova,ia Marzolo 5, 35131 Padova, Italy. Tel.: +39 049 8275034; fax: +39 049 8275366.

E-mail addresses: [email protected] (S. Dall’Acqua),[email protected] (A. Salvador).

731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jpba.2013.05.049

fact that the signaling pathways and/or receptors are physiolog-ically expressed in the skin. Photosensitivity skin reactions werealso reported with the administration of vandetanib [9–11].

Phototoxicity of drugs can derive from the classical photo-chemical reactions with the formation of radical oxygen speciesor singlet oxygen [12] but also from the toxic products formed dueto photodegradation [13,14]. Drug photodegradation is not onlyrelated to phototoxicity but it can also cause loss of efficacy andfor these reasons is an event to avoid [15]. Thus, photostabilitytesting must be planned and performed during the develop-ment and the registration process of new pharmaceutical products[16,17].

In the present study, we reported the photodegradation ofvandetanib in aqueous solution after UV-A irradiation and wedetermined the chemical structure of its main photoproducts.

2. Experimental

2.1. Materials

Vandetanib, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4 yl)methoxy)quinazolin-4-amine, was pursuedby Selleck Chemicals. Methanol and water for HPLC were obtainedfrom WVR International.

2.2. Photodegradation conditions

A solution of vandetanib (2.0 × 10−5 M) was prepared indeionised water, placed in caped quartz cuvette and exposed

ical and Biomedical Analysis 84 (2013) 196– 200 197

tmiVo

2

saarmdpC(T3

4f(edp

assst(csd2Tw

2

itapia

3

3

aUpdfUa

sd

peak and three other main peaks (Fig. 4) characterized by MSspectrum with intense ion at m/z 112 as vandetanib. Peaks wereindicated as FP1, FP2 and FP3 in increasing retention times and

S. Dall’Acqua et al. / Journal of Pharmaceut

o UV-A irradiation. As UV-A source, HPW 125 Philips lamps,ainly emitting at 365 nm, were used for photochemical exper-

ments. The UV-A dose was determined by a radiometer typeLX-3 W, Vilber Lourmat, with a sensor CX-365, to be each timef 0.25 J cm−2 min−1.

.3. Apparatus and experimental conditions

Photolysis reaction was followed by means of a Cary 50 UV–vispectrophotometer. HPLC–MS measurements were obtained on

Varian 212 series chromatograph equipped with Prostar 430utosampler and MS-500 Ion Trap as detector. MS spectra wereecorded in positive ion mode (50–600 Da). Fragmentation of theain ionic species were obtained during the HPLC run by the turbo

ata depending scanning (tdds) function, yielding in fragmentationattern of eluted compounds. As stationary phase Agilent Zorbax-18 (2.1 × 150 mm) 3.5 �m was used. As mobile phases solvent Awater 0.1% formic acid) and solvent B (methanol) were utilized.he solvent gradient started at 80% A then decreased to 0% A over0 min.

NMR (1D and 2D) spectra were obtained on a Bruker Avance00 spectrometer operating at 400.14 MHz for 1H and 100 MHzor 13C. Spectra were obtained at 298 K using deuterated methanolCD3OD, Sigma Aldrich) as solvent. Collected peaks from HLPC werevaporated to dryness in speedvac and the obtained residue wasissolved in 750 �l of CD3OD. Spectra were obtained using standardulse sequences of Topspin software.

HPLC–UV analyses for kinetic experiments were carried out on Perkin-Elmer instrument series 200 equipped with a quaternaryolvent delivery system and a diode array detector; while HPLCeparations of photoproducts were performed on Agilent 1100ystem equipped with diode array detector, autosampler and frac-ion collector. As stationary phase an Agilent Extend-C18 column150 mm × 4.6 mm; 5 �m particle size) was used. The mobile phaseonsisted of solvent A (ammonium formate buffer, pH 8.5) andolvent B (methanol). The solvent gradient started at 80% A thenecreased to 0% A over 30 min. Chromatogram was recorded at80 nm and UV spectra were recorded in the range 200–400 nm.he flow rate was 1.0 ml min−1, and the sample injection volumeas 20 �l.

.4. Determination of the photodegradation kinetics by HPLC

Twenty microliters of the vandetanib irradiated solution werenjected onto the column at the selected time intervals of irradia-ion. The area of the vandetanib peak at zero time (A0) was takens proportional to the initial concentration; the area of vandetanibeak (At) after irradiation was taken as proportional to the remain-

ng concentration as a function of time (t). A plot of vandetanib peakrea versus time was done.

. Results and discussion

.1. UV-A photolysis of vandetanib aqueous solution

Vandetanib photolysis was first followed irradiating a 10 �Mqueous solution with increasing UV-A doses (0–100 J/cm2) byV–vis spectroscopy. Vandetanib spectrum presents two maineaks: one at 330 nm and the other at 250 nm. After UV-A irra-iation, some changes in its spectrum can be observed (Fig. 1): inact, while the bands at 250 and 330 nm decreased with increasingV-A doses, the onset of a small shoulder between 300 and 260 nm

nd the presence of an isosbestic point at 355 nm can be detected.

Moreover, vandetanib photolysis was followed by HPLC analy-is (Fig. 2). The retention time (tR) of vandetanib with the methodescribed in Section 2.3 is 24 min and its chromatogram did

Fig. 1. Absorption spectra of 10 �M aqueous vandetanib solution after differentUV-A doses (0–100 J/cm2).

not show any presence of decomposition products (Fig. 2). After10 J/cm2 UV-A irradiation, the onset of two different peaks canbe observed: the first one, which corresponds to FP2, had a tR at13 min and the second one, which corresponds to FP3, had a tR at19 min. When UV-A dose was increased up to 100 J/cm2, furtherpeaks appeared (FP1 tR = 10 min and FP4 tR = 23 min) and FP2 andFP3 peak areas increased while the vandetanib peak decreased.

Vandetanib photodegradation kinetic parameters were calcu-lated by plotting the vandetanib peak area after different UV-Airradiation doses versus time. As can be observed in Fig. 3, thephotochemical decomposition of vandetanib in aqueous solutionfollowed a zero-order kinetic reaction: in fact, vandetanib peak areadecreased in a linear way during time. The calculated zero-orderdegradation rate constant was 5046 min−1 and t1/2 was 267 min.

In order to preliminary evaluate the photoproducts, HPLC–MSmeasurements were performed (Fig. 4). The Vandetanib MS spec-trum is characterized by major fragment at m/z 112 (due to themethyl piperidine fragment), which is also found in [18]; more-over, molecular ions with typical isotopic pattern due to bromineat (m/z 475.11 and 477.12; and the doubly charged ions at m/z 238and 239) and fragments due to the quinazoline moiety (m/z 364.28and 366.22) were also observed (see also Fig. S1 for supportinginformation).

The HPLC of the irradiated solution presented the vandetanib

Fig. 2. HPLC chromatograms of vandetanib solution after 0, 10 and 100 J/cm2 UV-Adoses.

198 S. Dall’Acqua et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 196– 200

F

Mcwppc

3

atsama

ig. 3. Zero-order kinetics of the photodegradation of vandetanib aqueous solution.

S data suggest that the FP1 and FP2 (m/z 207) are isomers thatan be formed by the bromine loss and addition of hydroxyl grouphile the FP3 (m/z 199) can be derived by bromine loss. To com-letely characterize the two main photoproducts (FP2 and FP3), weroceeded with HPLC separation of the photoproducts in order toomplete structure assignments by NMR spectroscopy.

.2. FP2 characterization

The ESI-MS spectrum of the compounds FP1 and FP2 showed molecular ion [M+2H]+ at m/z 207 with base peak at m/z 112,hus supporting that the bromine of the Vandetanib was lost and

ubstituted by a hydroxyl group. The FP1 MS spectrum presentedlso a fragment at m/z 130 that supports the modification of theethyl piperidine fragment. The low amount of FP1 did not allow

complete structure assignment.

Fig. 4. HPLC–MS spectra of 100 J/cm2 irradiated vandetanib solution. Mass s

Fig. 5. 1H NMR spectra of the aromatic region of FP2 and FP3.

The 1H NMR of FP2 (Fig. 5) was characterized by three singletsin the aromatic proton region at ı 8.25, 7.70 and 7.15 integrat-ing one proton each and a singlet due to a methoxy group at ı4.03 (3H). Comparing the spectrum with Vandetanib, such signalswere assigned to the quinazoline moiety. In the aromatic region,the triplet integrating for one proton due to the coupling with fluo-rine atom was observed but with a shift compared with vandetanib(ı 7.29 vs ı 7.58, respectively), confirming chemical modificationof the benzyl ring in the FP2. A multiplet at ı 6.70 integrating fortwo protons was assigned to the positions 5 and 6 of the benzyl

ring. The signals due to methyl piperidine fragment in FP2 werealmost unchanged compared to vandetanib, namely the doubletat ı 4.07 (J = 6.2), and the multiplets at ı 2.96, 2.12, 1.95 and 1.56.

pectra of FP1, FP2 and FP3. The fragmentation scheme is also included.

S. Dall’Acqua et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 196– 200 199

hotod

Fs

mThy

3

usssta(tom

4

tpst

Fig. 6. Vandetanib p

urthermore, the methyl and methoxy groups were also present asinglets at ı 4.01 and 2.31, respectively.

Such data indicated a modification in the fluorophenyloiety and confirm the indications from the MS spectrum.

hus, the structure of FP2 can be assigned to N-(4-ydroxy-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4l)methoxy)quinazolin-4-amine.

.3. FP3 characterization

The ESI-MS spectrum of the compound FP3 showed a molec-lar ion [M+2H]+ at m/z 199 with base peak at m/z 112 thusupporting the loss of the bromine atom from the Vandetanibtructure. The H-NMR of FP3 (Fig. 5) was characterized by similarignals due to the quinazoline and piperazine moieties, in par-icular the triplet due to fluorine atom was observed at ı 7.62nd signals due to four aromatic protons were observed at ı 7.272H) and 6.69 (2H), supporting the loss of the bromine atom pos-ulated on the basis of MS measurements. Thus, the structuref FP3 can be assigned to N-(2-fluorophenyl)-6-methoxy-7-((1-ethylpiperidin-4 yl)methoxy)quinazolin-4-amine.

. Conclusion

This paper reports the first evidence of the Vandetanib pho-

odegradation due to the exposure of 100 J/cm2. Two mainhotoproducts were observed by HPLC–MS analysis. Isolation andtructural characterization of the two main photoproducts revealedhat the part of the compound that is subjected to photoreaction is

egradation scheme.

the fluorophenyl moiety. A scheme of the supposed photochem-ical reaction was introduced in Fig. 6: after UV absorption, thetypical C Br bond homolysis can occur, leading to an aryl radi-cal. The resulting radical can undergo electron transfer to form anaryl cation, which can be readily attacked by nucleophiles such aswater (FP2). The simple photodehalogenation led to FP3. Furtherstudies about the toxicological aspects of the two photoproductsare in progress.

Acknowledgements

This research was carried out in the frame of the program “Tar-get molecolari e cellulari coinvolti nell’attività di farmaci” grantedby Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR),Rome.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jpba.2013.05.049.

References

[1] A. Mullard, FDA drug approvals, Nat. Rev. Drug Discov. 11 (2012) 91–94.[2] A.J. Ryan, S.R. Wedge, ZD6474—a novel inhibitor of VEGFR and EGFR tyrosine

kinase activity, Br. J. Cancer 92 (2005) S6–S13.[3] P.B. Langmuir, A. Yver, Vandetanib for the treatment of thyroid cancer, Clin.

Pharmacol. Therap. 91 (2012) 71–80.[4] S. Leboulleux, L. Bastholt, T. Krause, C. de la Fauchardiere, J. Tennvall, A. Awada,

J.M. Gómez, F. Bonichon, L. Leenhardt, C. Soufflet, M. Licour, M.J. Schlumberger,

2 ical an

[

[

[

[

[

[

[

[

00 S. Dall’Acqua et al. / Journal of Pharmaceut

Vandetanib in locally advanced or metastatic differentiated thyroid cancer: arandomised, double-blind, phase 2 trial, Lancet Oncol. 13 (2012) 897–905.

[5] Y.Y. Xiao, P. Zhan, D.M. Yuan, H.B. Lv, Y. Shi, Y. Song, Chemotherapy plus van-detanib or chemotherapy alone in advance non-small cell lung cancer: a metaanalysis of four randomised controlled trials, Clin. Oncol. 25 (2013) e7–e15.

[6] J. Drappatz, A.D. Norden, E.T. Worng, L.M. Doherty, D.C. LaFrankie, A. Ciampa,S. Kesari, C. Sceppa, M. Gerard, P. Phan, D. Schiff, T.T. Batchelor, K.L. Ligon, G.Young, A. Muzikansky, S.E. Weiss, P.Y. Wen, Phase I Study of vandetanib withradiotherapy and temozolomide for newly diagnised gliobastoma, Int. J. Rad.Oncol. Biol. Phys. 78 (2010) 85–90.

[7] N. Degrauwe, J.A. Sosa, S. Roman, H.A. Desphande, Vandetanib for the treat-ment of metastatic medullary thyroid cancer, Clin. Med. Insights Oncol. 6 (2012)243–252.

[8] P.L. Myskowski, A.C. Halpern, Skin reactions to the new biologic anticancerdrugs, Curr. Opin. Palliat. Care 3 (2009) 294–299.

[9] H.H. Kong, H.A. Fine, J.B. Stern, M.L. Chanco Turner, Cutaneous pigmantationafter photosensitivity induced by vandetanib therapy, Arch. Dermatol. 145

(2009) 923–925.

10] P. Fava, P. Quaglino, M.T. Fierro, M. Novelli, M.G. Berengo, A rare vandetanib-induced photo-allergic drug eruption, Dermatol. Ther. 23 (2010) 553–555.

11] C.H. Chang, J.W. Chang, C.Y. Hui, C.H. Yang, Severe photosensitivity reaction tovandetanib, J. Clin. Oncol. 27 (2009) e114–e115.

[

d Biomedical Analysis 84 (2013) 196– 200

12] G.M.J. Beijersbergen van Henegouwen, Medicinal photochemistry: photo-toxic and phototherapeutic aspects of drugs, Adv. Drug Res. 29 (1997)79–170.

13] G. Viola, P. Grobelny, M.A. Linardi, A. Salvador, G. Basso, J. Mielcarek, S.Dall’Acqua, D. Vedaldi, F. Dall’Acqua, The phototoxicity of fluvastatin, anHMG.CoA reductase inhibitor, is mediated by the formation of benzocarbazole-like photoproduct, Toxicol. Sci, 118 (2010) 236–250.

14] G. Viola, P. Grobelny, M.A. Linardi, A. Salvador, S. Dall’Acqua, L. Sobotta,J. Mielcarek, F. Dall’Acqua, D. Vedaldi, G. Basso, Arch. Toxicol 86 (2012)483–496.

15] G. Miolo, C. Marzano, V. Gandin, A.C. Palozzo, D. Dalzoppo, A. Salvador, S.Caffieri, Photoreactivity of 5-fluorouracil under UVB light: photolysis and cyto-totoxicity studies, Chem. Res. Toxicol. 24 (2011) 1319–1326.

16] CH International Conference on Harmonisation of Technical Requirements forRegistration of Pharmaceuticals for Human Use, Photostability Testing of NewDrug Substances and Products, Q1B, 1996.

17] W. Aman, K. Thoma, The influence of formulation and manufacturing process

on the photostability of tablets, Int. J. Pharm. 28 (2008) 33–41.

18] F. Bai, J. Johnson, F. Wang, L. Yang, A. Broniscer, C.F. Stewart, Determinationof vandetanib in human plasma and cerebrospinal fluid by liquid chromatog-raphy electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS), J.Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 879 (2011) 2561–2566.