The use of the 1,2,4-triazine method of pyridine ligand synthesis for the preparation of a luminescent Pt(II) labeling agent

Article abstract of DOI:10.1016/j.tetlet.2012.07.090

The 'triazine' methodology for the synthesis of functionalized pyridine ligands proved to be a convenient method for the preparation of a luminescent Pt(II) complex. The key ligand can be assembled easily starting from readily accessible reagents. Further cycloplatination and post-functionalization led to the ready-to-go luminescent 'tag' 2 for peptide labeling.

Full text of DOI:10.1016/j.tetlet.2012.07.090

Tetrahedron Letters 53 (2012) 5293–5296
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Tetrahedron Letters
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The use of the 1,2,4-triazine method of pyridine ligand synthesis for the
preparation of a luminescent Pt(II) labeling agent
Alfiya F. Suleymanova a, Dmitry N. Kozhevnikov a,b, Anton M. Prokhorov a,
⇑
a Department of Organic Chemistry, Ural Federal University, Mira 19, Ekaterinburg 620002, Russia
b Postovsky Institute of Organic Synthesis, S. Kovalevskoy 22, Ekaterinburg 620990, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
The ‘triazine’ methodology for the synthesis of functionalized pyridine ligands proved to be a convenient
method for the preparation of a luminescent Pt(II) complex. The key ligand can be assembled easily start-
ing from readily accessible reagents. Further cycloplatination and post-functionalization led to the ready-
to-go luminescent ‘tag’ 2 for peptide labeling.
Received 5 June 2012
Revised 28 June 2012
Accepted 19 July 2012
Available online 27 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
1,2,4-Triazine
Pyridine
Ligand
Platinum(II)
Luminescence
Luminescent complexes are of significant interest because of
their applications as molecular probes in biomedical,1 sensing,2
or imaging areas,3 and as light-emitting devices.4 Traditionally,
lanthanide chelates, and in particular, Eu(III) complexes with
long-life intense luminescence in aqueous solutions, have been
widely used as luminescent labels for time-resolved immunoas-
says.5 But recently, due to their greater stability and more intense
emissions, the attention of researchers has turned to cyclometal-
lated platinum(II) and iridium(III) complexes.6–8 These materials
demonstrate high quantum yields, large Stokes shifts, and rela-
tively long lifetimes for easy detection in naturally fluorescent bio-
logical media. In addition, the design of lanthanide chelates is a
real challenge. A ligand should protect the lanthanide center from
quenching by water molecules, provide strong metal binding to
prevent release of the lanthanide ion, and act as a sensitizer to en-
able energy transfer to the metal center.9 To fulfill these require-
ments the ligand should be nine-coordinating with strong
chelating groups (e.g., carboxylic), and also provide participation
in the coordination by the aromatic part, for example, a pyridine
or polypyridine.10 The fine balance between the chromophore
and the coordinating sphere leads to complex ligands such as pyr-
idine derivatives functionalized with polyaminoacetate arms and
also bearing a spacer with a group for bioconjugation (e.g., complex
1). From this point of view, ligands for cyclometallated complexes
of platinum metals seem to be more accessible, as the aromatic
system is simple and is the only metal binding unit. Typical cyclo-
metallated phenylpyridine (ppy) or related complexes of Pt(II) or
Ir(III) with functionalized auxiliary ligands are available in a few
synthetic steps and are easily isolated and purified.11,12 Thus, a
ppy complex of Ir(III) with a bipyridine auxiliary ligand bearing
an amino group was successfully used for the preparation of label-
ing reagents for biological substrates.13
We envisaged that the introduction of a group suitable for pep-
tide labeling directly into the cyclometallating ligand (e.g., 2-thie-
nylpyridine) would make the design of the complex easier. In this
case, a very accessible reagent such as acetylacetone can be used as
an auxiliary ligand in the cyclometallated complex. Only the main
cyclometallating ligand with a 2-arylpyridine fragment bearing a
carboxylic or amino group connected through a spacer is required.
The rational design of such a ligand led us to choose 2-(2-thie-
nyl)pyridine (thpy) as a cyclometallating system. It is reactive to-
ward cyclometallation and the cyclometallated thpy complexes
of Pt(II) exhibit increased lifetimes.14 The same effect can be
achieved by the expansion of the aromatic system by introduction
of an aryl substituent at position 5 of pyridine.15 In addition, it can
act as a spacer-bearing functionality to expedite binding to biolog-
ical targets. For example, the 4-methoxyphenyl substituent can be
easily modified by consecutive demethylation and alkylation.16
This allows introduction of an acetate moiety, which can be further
activated for peptide binding by typical procedures, For example,
formation of an N-succinimide ester.17 In this way, the design led
us to complex 2, and the key feature of this work was the synthesis
of the cyclometallating ligand.
⇑
Corresponding author. Tel.: +7 9122805967; fax: +7 3433740458.
E-mail address: aprohor@yandex.ru (A.M. Prokhorov).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.090

5294
A. F. Suleymanova et al. / Tetrahedron Letters 53 (2012) 5293–5296
ene as the main building blocks. Cyclization of the
O
N
bromoacetophenone with 2 equiv of the hydrazide26 resulted in
the formation of 1,2,4-triazine 5. Next, cycloaddition of 2,5-norbor-
nadiene to the triazine led to the substituted pyridine 6 via a cas-
O
O
O
N
C
S
O
N
cade involving
a Diels-Alder reaction with inverse electron
N
N
N
Eu
S
demand, a retro Diels-Alder reaction with elimination of a nitrogen
molecule, and a retro Diels-Alder reaction with elimination of
cyclopentadiene. Phenol 7 was obtained by demethylation of the
methyl ether 6 either with boron tribromide or pyridine hydro-
chloride. In the first case, the conditions were milder and the yield
was higher. The final ligand 8 was prepared by alkylation of phenol
7 with ethyl bromoacetate in acetonitrile (Scheme 1).27
N
O
O
O
O
O
O Pt
O
O
O
1
2
O
The ligand was initially cyclometallated according to the typical
procedure in refluxing acetic acid containing potassium tetrachlo-
roplatinate. The reaction was complicated by significant reduction
of platinum with the formation of platinum black, and therefore
proceeded in low yield to give dimeric complex 9. Next, we chan-
ged the solvent to acetonitrile. The reaction was clean and pro-
ceeded without any trace of platinum black in spite of the longer
reaction time, yielding 70% of complex 9 (Scheme 2). The dimeric
complex was cleaved with dimethylsulfoxide to form an interme-
diate monomeric DMSO complex, PtL(DMSO)Cl. Neither the di-
meric dichloro complex nor the monomeric DMSO complex was
emissive, but the DMSO ligand was very labile and could be easily
substituted by another auxiliary ligand. Hence the DMSO complex
was not purified and was directly converted into complex 10 by
treatment with an excess of sodium acetylacetonate in acetone.28
Hydrolysis of the ethyl ester in 1 N LiOH solution gave the corre-
sponding acid 11.29 Finally, this was activated with N-hydroxy-
succinimide in the presence of dicyclohexylcarbodiimide
according to the standard protocol to give the target platinum label
2.30
A convenient method for the preparation of substituted pyridines
involves transformation of 1,2,4-triazines in cycloaddition reactions
with various nucleophilic dienophiles.18–20 We previously reported
a 1,2,4-triazine method of bipyridine ligand synthesis for the prep-
aration of new luminescent Eu(III) labeling reagents, for example,
1.21 The methodology included an easy synthesis of substituted
1,2,4-triazine followed by transformation into the corresponding
pyridine via an inverse electron demand Diels-Alder reaction, and
a further relatively long sequence of modifications. We now pro-
pose an extension of the methodology for a simple preparation of
a pyridine ligand for luminescent Pt(II) labeling agent 2, starting
from very accessible reagents in a few steps. The usual approach
for the synthesis of cyclometallating pyridine ligands includes con-
secutive introduction of aromatic substituents into the pyridine
core by cross-coupling reactions with the stepwise formation of
an aromatic system,22 in contrast, the ‘1,2,4-triazine’ method allows
rapid formation of the entire aromatic system with the required
substituents.23–25
The aromatic part of the ligand was assembled according to the
‘1,2,4-triazine’ strategy starting from 2-bromo-40-methoxyaceto-
phenone (3), thiophene-2-carbohydrazide (4), and 2,5-norbornadi-
The absorption and luminescence spectra of 2 in aerated CH2Cl2
at room temperature are presented in Figure 1. The photophysical
O
O
RO
N
O
O
S
i
N
ii
+
S
S
HN
NH2
N
Br
N
3
4
6
, R = Me, 89%
5
60%
iii
iv
7, R = H, 92%
8, R = CH2COOEt, 93%
Scheme 1. Ligand synthesis. Reagents and conditions: (i) NaOAc, AcOH/EtOH, reflux, 10 h; (ii) 2,5-norbornadiene, o-xylene, reflux, 48 h; (iii) BBr3, CH2Cl2, 0 °C, 4 h or PyꢀHCl,
150 °C, 10 h; (iv) BrCH2COOEt, K2CO3, MeCN, reflux, 10 h.
O
O
O
O
O
RO
S
N
i
ii
8
Pt
Cl
S
Cl
Pt
N
Pt
O
N
O
S
10,
R = Et, 70%
11, R = H, 80%
2, R = N-succinimide, 65%
iii
iv
O
9
,
O
70%
O
Scheme 2. Complex preparation. Reagents and conditions: (i) K2PtCl4, AcOH, reflux, 24 h or K2PtCl4, MeCN, reflux, 48 h; (ii) (1) DMSO, reflux, 5 min; (2) Na-acac, acetone,
reflux, 5 h; (iii) 1 N LiOH, H2O/THF, reflux, 10 h; (iv) N-hydroxysuccinimide (NHS), DCC, Et3N, DMF, 60 °C, 24 h.

A. F. Suleymanova et al. / Tetrahedron Letters 53 (2012) 5293–5296
5295
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
07.090.
References and notes
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4. Bakker, B. H.; Goes, M.; Hoebe, N.; Van Ramesdonk, H. J.; Verhoeven, J. W.;
Werts, M. H. V.; Hofstaat, J. W. Coord. Chem. Rev. 2000, 208, 3.
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Cheung, K.-K. Coord. Chem. Rev. 2005, 249, 1434.
7. Wieczorek, B.; Lemcke, B.; Dijkstra, H. P.; Egmond, M. R.; Klein Gebbink, R. J. M.;
VanKoten, G. Eur. J. Inorg. Chem. 2010, 1929.
Figure 1. Absorption and emission spectra of
solution.
2 in aerated dichloromethane
8. Lo, K. K.-W.; Leung, S.-K.; Pan, C.-Y. Inorg. Chim. Acta 2012, 380, 343.
9. Armelao, L.; Quici, S.; Barigelletti, F.; Accorsi, G.; Bottaro, G.; Cavazzini, M.;
Tondello, E. Coord. Chem. Rev. 2010, 254, 487.
10. Pandya, S.; Yu, J.; Parker, D. Dalton Trans. 2006, 2757.
11. Donnio, B.; Williams, J. A. G.; Bruce, D. W. J. Am. Chem. Soc. 2011, 133, 5248.
12. Prokhorov, A. M.; Slepukhin, P. A.; Rusinov, V. L.; Kalinin, V. N.; Kozhevnikov, D.
N. Chem. Commun. 2011, 47, 7713.
13. Lo, K. K.-W.; Ng, D. C.-M.; Chung, C.-K. Organometallics 2001, 20, 4999.
14. Brooks, J.; Babayan, Y.; Lamansky, S.; Djurovich, P. I.; Tsyba, I.; Bau, R.;
Thompson, M. E. Inorg. Chem. 2002, 41, 3055.
Table 1
Photophysical parameters of 2
a
c
d
kabs (nm)
eb (dm3 molꢁ1 cmꢁ1
)
kem (nm)
Uem
237
320
415
8793
8594
3500
597
0.01
15. Kozhevnikov, D. N.; Kozhevnikov, V. N.; Ustinova, M. M.; Santoro, A.; Bruce, D.
W.; Koenig, B.; Czerwieniec, R.; Fischer, T.; Zabel, M.; Yersin, H. Inorg. Chem.
2008, 48, 4179.
16. Prokhorov, A. M.; Santoro, A.; Williams, J. A. G.; Bruce, D. W. Angew. Chem. Int.
Ed. 2012, 51, 95.
a
b
c
kabs — absorption band maximum, in aerated CH2Cl2 solution at 298 K.
— molar absorptivity ( 5%).
kem — emission maximum, in aerated CH2Cl2 solution at 298 K.
e
d
Uem — photoluminescence quantum yield relative to Ru(bipy)3Cl2 in aerated
water solution ( 20%).31
17. Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. Soc. 1839, 1964,
86.
18. Raw, S. A., Taylor, R. J., Katritzky, A. R., Eds.Advances in Heterocyclic Chemistry;
Academic Press: Oxford, 2010; Vol. 100, p 75.
19. Stanforth, S. P.; Tarbit, B.; Watson, M. D. Tetrahedron Lett. 2003, 44, 693.
20. Hajbi, Y.; Suzenet, F.; Khouili, M.; Lazar, S.; Guillaumet, G. Tetrahedron 2007, 63,
8286.
21. Prokhorov, A. M.; Kozhevnikov, V. N.; Kopchuk, D. S.; Bernard, H.; Le Bris, N.;
Tripier, R.; Handel, H.; Koenig, B.; Kozhevnikov, D. N. Tetrahedron 2011, 67, 597.
22. Kozhevnikov, D. N.; Kozhevnikov, V. N.; Shafikov, M. Z.; Prokhorov, A. M.;
Bruce, D. W.; Williams, J. A. G. Inorg. Chem. 2011, 50, 3804.
23. Prokhorov, A. M.; Slepukhin, P. A.; Rusinov, V. L.; Kalinin, V. N.; Kozhevnikov, D.
N. Tetrahedron Lett. 2008, 49, 3785.
24. Kozhevnikov, D. N.; Kozhevnikov, V. N.; Prokhorov, A. M.; Ustinova, M. M.;
Rusinov, V. L.; Chupakhin, O. N. Tetrahedron Lett. 2006, 47, 869.
25. Stanforth, S. P.; Tarbit, B.; Watson, M. D. Tetrahedron Lett. 2002, 43, 6015.
26. Kozhevnikov, V. N.; Ustinova, M. M.; Slepukhin, P. A.; Santoro, A.; Bruce, D. W.;
Kozhevnikov, D. N. Tetrahedron Lett. 2008, 49, 4096–4098.
27. Preparation of ligand 8: The starting phenol 7 (600 mg, 2.37 mmol) and K2CO3
(3.2 g, 23.68 mmol) were suspended in MeCN (35 mL) and ethyl bromoacetate
(435 mg, 2.6 mmol) was added. The mixture was stirred for 10 h at reflux. The
solvent was removed, H2O (50 mL) was added to the residue, and the white
data are collected in Table 1. The absorption spectrum is typical for
cyclometallated arylpyridine complexes of platinum(II).14 The
moderate absorption band at 415 nm was assigned to a metal-to-
ligand charge transfer (MLCT) transition. More intense absorption
bands at 280–320 nm corresponded to ligand-centered (LC)
p, -
p⁄
transitions. Upon photoexcitation at the lowest energy band, the
complex was moderately emissive in aerated CH2Cl2 solution at
room temperature and exhibited orange luminescence with a
Stokes shift of about 180 nm. The emission band at 597 nm showed
some resolved vibrational modes with 1200 cmꢁ1 progression.
Based on previous studies of complexes of this type, the emission
origin was assumed to be a result of ligand-centered 3p p⁄-transi-
,
tion from a triplet state.15 The emission lifetime in ambient condi-
tions corresponds to tens of microseconds. The quantum yield of
the phosphorescence was about 0.01 at room temperature in aer-
ated CH2Cl2 solution.
solid product
8 was filtered and recrystallized from EtOH. Yield 750 mg,
2.2 mmol, 93%. 1H NMR (400 MHz, CDCl3, ppm): 1.30 (t, J = 7.1 Hz, 3H, Et), 4.27
(q, J = 7.1 Hz, 2H, Et), 4.65 (s, 2H, CH2), 6.99 (m, 2H, Ar), 7.11 (dd, J1 = 4.8 Hz,
J2 = 3.7 Hz, 1H, thienyl), 7.38 (dd, J1 = 4.8 Hz, J2 = 1.1 Hz, 1H, thienyl), 7.51 (m,
2H, Ar), 7.58 (dd, J1 = 3.7 Hz, J2 = 1.1 Hz, 1H, thienyl), 7.67 (d, J = 8.2 Hz, 1H, Py),
7.81 (d, J1 = 8.2 Hz, J2 = 2.2 Hz, 1H, Py), 8.74 (d, J = 2.2 Hz, 1H, Py). Found: C,
67.15; H, 5.01; N, 3.95. Calcd for C19H17NO3S: C, 67.24; H, 5.05; N, 4.13.
28. Preparation of complex 10: The starting ligand 8 (750 mg, 2.2 mmol) was
suspended in MeCN and pulverized potassium tetrachloroplatinate powder
(917 mg, 2.2 mmol) was added with vigorous stirring. The mixture was stirred
and refluxed for 2 d. The solvent was removed and the resulting complex 9 was
treated with 3 ml of DMSO. The mixture was heated for 5 min at reflux, and
then the DMSO was removed under vacuum. The residue was dissolved in
acetone (50 mL) and sodium acetylacetonate (3.1 g, 22.10 mmol) was added,
and the suspension stirred under reflux for 5 h. The solvent was removed, and
the product 10 was isolated by column chromatography on silica with CH2Cl2
as the eluent. The crude product was recrystallized from acetone to give orange
crystals of 10. Yield 680 mg, 1.07 mmol, 49%. 1H NMR (400 MHz, CDCl3, ppm):
1.31 (t, J = 7.1 Hz, 3H, Et), 1.97 (s, 3H, acac), 1.99 (s, 3H, acac), 4.28 (q, J = 7.1 Hz,
2H, Et), 4.67 (s, 2H, CH2), 5.48 (s, 1H, acac), 7.00 (m, 2H, Ar), 7.19 (d, J = 4.5 Hz,
1H, thienyl), 7.31 (d, J = 8.2 Hz, 1H, Py), 7.49 (m, 3H, Ar + thienyl), 7.82 (d,
J1 = 8.2 Hz, J2 = 2.2 Hz, 1H, Py), 9.00 (dd, J = 2.2 Hz, JHPt = 43 Hz, 1H, Py). HRMS
(ESI): C24H23NO5PtS requires M+H, 633.1023, found 633.1082.
In conclusion, an efficient and straightforward ‘1,2,4-triazine’
methodology for the synthesis of a pyridine ligand has been success-
fully applied for the preparation of a luminescent cyclometallated
platinum(II) labeling agent. The methodology allows easy construc-
tion of the platinum(II) luminophore bearing an NHS-activated car-
boxylic group for peptide binding, starting from readily available
reagents in a few steps. The key step is the preparation of an appro-
priately substituted cyclometallating pyridine ligand via the corre-
sponding 1,2,4-triazine. The preliminary results revealed that the
luminophore 2 prepared in this way was a ‘ready-to-go’ luminescent
label, which can be excited with cheap lasers and possesses suffi-
ciently intense long-lived luminescence with a strong Stokes shift
for time-resolved detection. Further studies of bioconjugation and
luminescent visualization are in progress.
29. Preparation of complex 11: Complex 10 (198 mg, 0.312 mmol) was suspended
in THF (2.5 mL) and 1 N aq LiOH solution was added. The mixture was refluxed
for 10 h. The solvents were condensed in vacuo, and the solids filtered. The
remaining solution was acidified with 1 N HCl to pH 1–2. A yellow precipitate
of 11 was formed and removed by filtration. This was dried and used for next
Acknowledgements
The authors thank Ural Federal University and the President of
Russian Federation (grant MR-1103.2012.3) for financial support.

5296
A. F. Suleymanova et al. / Tetrahedron Letters 53 (2012) 5293–5296
reduced pressure and the final product
step without further purification. Yield 56 mg, 0.250 mmol, 80%. 1H NMR
2
was isolated by column
(400 MHz, DMSO-d6, ppm): 1.95 (s, 3H), 2.01 (s, 3H), 4.63 (s, 2H), 5.54 (s, 1H),
7.01 (d, 3J = 4.8 Hz, 1H), 7.02 (m, 2H), 7.44 (d, 3J = 8.4 Hz, 1H), 7.52 (d,
3J = 4.8 Hz, 1H), 7.57 (m, 2H), 8.09 (dd, 3J = 8.4 Hz, 4J = 2 Hz, 1H), 8.93 (d,
4J = 2 Hz, 1H). HRMS (ESI): C22H19NO5PtS requires M+H, 605.0710, found
605.0716.
chromatography (silica, CH2Cl2, Rf = 0.5) as orange crystals. Yield 135 mg,
0.19 mmol, 65%. The product was unstable toward water and should be kept
cold under an inert atmosphere. 1H NMR (400 MHz, CDCl3, ppm): 1.97 (s, 3H,
Acac), 2.00 (s, 3H, Acac), 2.86 (s, 4H, NHS), 5.03 (s, 2H, CH2), 5.48 (s, 1H, Acac),
7.06 (m, 2H, Ar), 7.20 (d, J = 4.5 Hz, 1H, thienyl), 7.32 (d, J = 8.2 Hz, 1H, Py), 7.49
(d, J = 4.5 Hz, 1H, thienyl), 7.55 (m, 2H, Ar), 7.83 (d, J1 = 8.2 Hz, J2 = 2.2 Hz, 1H,
30. Preparation of the labeling agent 2: The complex 11 (180 mg, 0.30 mmol) was
dissolved in dry DMF 30 mL and then dicyclohexylcarbodiimide (38 mg,
0.33 mmol) and N-hydroxysuccinimide (68 mg, 0.33 mmol) were added. The
Py), 9.03 (dd, J = 2.2 Hz, JHPt = 43 Hz, 1H, Py). HRMS (ESI): C26H22N2O7PtS
requires M+H, 702.0874, found 702.0802.
mixture was stirred at 60 °C for 24 h under
reaction progress was monitored by TLC. The solvent was removed under
a
nitrogen atmosphere. The
31. Suzuki, K.; Kobayashi, A.; Kaneko, S.; Takehira, K.; Yoshihara, T.; Ishida, H.;
Shiina, Y.; Oishi, S.; Tobita, S. Phys. Chem. Chem. Phys. 2009, 11, 9850.