Synthesis and photophysical properties of a deazaflavin-bridged porphyrinatoiron(III) that mimics the interaction of a deazaflavin inhibitor with the heme-thiolate cofactor of cytochrome P450 3A4

Article abstract of DOI:10.1002/hlca.200690268

Cytochrome P450 3A4 metabolizes a majority of administered therapeutic agents in the human liver. We recently reported the synthesis of a new inhibitor, 1, whose binding to and displacement from the active site of CYP 3A4 can be conveniently followed by t

Full text of DOI:10.1002/hlca.200690268

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Synthesis and Photophysical Properties of a Deazaflavin-Bridged
Porphyrinatoiron(III) That Mimics the Interaction of a Deazaflavin Inhibitor
with the Heme-Thiolate Cofactor of Cytochrome P450 3A4
by Michael A. Müllera), Martin Gaplovskyb), Jakob Wirz*b), and Wolf-D. Woggon*a)
a) Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel
b) Department of Chemistry, University of Basel, Klingelbergstrasse 80, CH-4056 Basel
Cytochrome P450 3A4 metabolizes a majority of administered therapeutic agents in the human liver.
We recently reported the synthesis of a new inhibitor, 1, whose binding to and displacement from the
active site of CYP 3A4 can be conveniently followed by the associated changes in fluorescence intensity.
Here we report the synthesis of a bichromophoric compound, 6, in which deazaflavin was strapped over
the distal side of a porphyrinatoiron(III) complex to mimic the envisaged enzyme–inhibitor interaction
within the active site. Femtosecond pump–probe and fluorescence spectroscopies were used to study the
photophysical processes of 6. Rapid intramolecular energy transfer and enhanced intersystem-crossing
processes induced by the high-spin FeIII central ion are responsible for the complete suppression of dea-
zaflavin fluorescence in 6. Fluorescence quenching is less efficient in the iron-free analogue of 6, i.e., in
21.
1. Introduction. – Cytochrome P450 3A4 (CYP 3A4), a heme–thiolate protein, is
one of the most important P450s in the human liver [1]. The ability of CYP 3A4 to
metabolize a majority of administered therapeutic agents accounts for the large num-
ber of documented drug–drug interactions associated with CYP 3A4 inhibition [2]. It is
therefore of great importance to know the affinity of new drug candidates to CYP 3A4
at an early stage, in order to determine their potential as inhibitors, and to evaluate
their influence on the metabolism of co-administered drugs. Some particular features
of CYP 3A4 make it difficult to achieve this goal. Though X-ray structures are now
available from truncated CYP 3A4 [3], these pictures provide no clues on how the
enzyme accepts substrates as different as, e.g., erythromycin and nifedipine, how the
active site accommodates more than one substrate [4], and why partial inhibition
and even activation is observed when pairs of drugs are co-incubated [5].
Our group has recently reported [6] the synthesis of a new inhibitor 1 that displayed
favourable IC50 values in the range of 1–4 mM for three characteristic substrates of CYP
3A4 such as testosterone (2), midazolam (3), and nifedipine (4) (Fig. 1). The binding of
1 to the active site of CYP 3A4 and its displacement by other substrates can be followed
by the associated change in the intensity of the deazaflavin fluorescence [7], such that a
screening for drugs that might cause drug–drug interactions requires neither the deter-
mination of substrate turnover nor product analyses.
From these experiments, it was suggested [6] that the fluorescence of 1 is quenched
upon binding to CYP 3A4 due to an interaction such as that shown in structure 5 of the
ꢀ 2006 Verlag Helvetica Chimica Acta AG, Zürich

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Fig. 1. The new inhibitor 1, substrates 2–4 [8], and the envisaged orientation of 1 towards the cofactor
of CYP 3A4 as shown in structure 5

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Fig. 1. We now report our efforts to understand the origin of this fluorescence quench-
ing by means of the model system 6 (see Scheme).
2. Results and Discussion. – The target molecule 6 contains a thiolato ligand, which
coordinates to iron(III) and mimics the cysteine Sꢀ ion of native P450s [1]. The opposite
face of the porphyrin is strapped with the deazaflavin chromophore ca. 4.5 Š above the
porphyrin plane; the complete system models the envisaged interaction of the fluores-
cent chromophore of the inhibitor 1 with the cofactor of CYP 3A4, as in 5.
The porphyrin 7 was prepared as described previously [9]. The synthesis of the dea-
zaflavin strap started with commercially available 8 and 9, which were converted to 10
and 11, respectively, by adaptation of known procedures [10], the latter via intermedi-
ates 12–15. Condensation of 10 and 11 furnished 16, which was cyclized to the deaza-
flavin derivative 17. N-Alkylation of 17 led to 18 [11] which, after saponification [12],
gave the diacid 19 that was converted to the activated ester 20, ready for macrocycliza-
tion with the diamino-porphyrin 7. Iron was inserted into the resulting doubly bridged
porphyrin 21 to form the desired product 6 (Scheme).
No fluorescence was detected from a solution of the porphyrinatoiron(III) 6 in
MeCN by using a conventional fluorescence spectrometer. This result, though it did
confirm the concept, nevertheless came as a surprise, because binding of 1 to cyto-
chrome P450 3A4 leaves a residual fluorescence of ca. 20% of the intensity of that of
1 in the absence of enzyme [6]. Three intramolecular processes for fluorescence
quenching may be envisaged to occur in the dyad 6 [13]: i) resonance energy transfer,
ii) electron transfer, and iii) enhanced intersystem crossing (ISC) induced by the high-
spin FeIII centre. The last process is not operative in iron-free 21.
The fluorescence properties of deazaflavin 18, porphyrin 7, and dyad 21 in degassed
MeCN solution were recorded with a streak camera following sub-picosecond excita-
tion at 388 or 310 nm. The measured lifetimes are collected in Table 1 together with
the wavelengths of selected absorption and emission maxima.
Table 1. Absorption and Emission Properties of 18, 7, and 21 in Degassed MeCN
Absorption
Fluorescence
emission
lmax/nm
excitation
lmax/nm
lifetime t/ns
(475 nm)a)
lifetime t/ns
(698 nm)b)
lmax/nm
18
7
21
18+7c)
265, 339, 403
414, 509, 544, 576, 630
348, 414, 510, 544, 577, 634
475
631, 698
635, 700
403
414
414
3.0ꢁ0.2
–
10.3ꢁ0.4
2.3ꢁ0.2
9.4ꢁ0.4
–
2.2ꢁ0.2
a) Deazaflavin unit. b) Porphyrin unit. c) 10ꢀ3 M each.
We introduce the following notation to characterize the electronic states of the
dyads 21 and 6: The letter P is used for the porphyrin moiety, PFe for porphyrinato-
A
locally excited singlet and triplet states of the P and F moieties are designated as P*

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Scheme. Synthesis of the Doubly Bridged Porphyrinatoiren 6
a) PBr3, Et2O, r.t. ! 508, 1 h. b) NaH, CH2(COOMe)2, DMSO, 08 ! r.t., 45 min, 12, r.t., 45 min. c)
LiI, DMF/H2O 9:1, 160 8, 7 h. d) 5% Pd/C, H2, 1 bar, EtOH, r.t., 4 h. e) NaH, MeI, r.t., 45 min. f)
DMF, POCl3, 08, 15 min, 8, 1008, 4 h. g) CH2Cl2, 1008, 5 min. h) CF3COOH (98%), 608, 1 h. i)
K2CO3, BrCH2COOMe, DMF, r.t., 10 h. j) LiOH·H2O, THF/H2O 3 :2, r.t., 1 h. k) N-[3-(dimethylami-
no)propyl]-N’-ethylcarbodiimide hydrochloride (EDC·HCl), C6F5OH, DMF, r.t., 1 h. l) 20 (2.5
equiv.), dry DMF, 1008, 4 h; 37%. m) 2,6-Lutidine (20 equiv.), FeBr2 (14 equiv.), dry toluene, 1208, 45
min; 89%.

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Scheme (cont.)
and 3P* and 1F* and 3F*, respectively. Charge-transfer states of dyad 21 are denoted as
P+CꢀFꢀC and PꢀCꢀF+C.
The fluorescence lifetimes measured at the two emission maxima of the metal-free
porphyrin 7 (631 and 698 nm) were equal and independent of the excitation wave-
length. Emission from the locally excited deazaflavin chromophore of the iron-free
1
dyad 21, Pꢀ F*, was not detectable. Following excitation of the deazaflavin chromo-
phore of 21, i) resonance energy transfer to the porphyrin forming 1P*ꢀF, or ii) elec-
tron transfer yielding the charge-separated state P+CꢀFꢀC may be considered. The fluo-
rescence emission of deazaflavin 18, lmax 475 nm, overlaps well with the Q-band absorp-
tion of porphyrin 7, lmax 509nm ( Fig. 2). Thus, intramolecular energy transfer between
the two chromophores of 21, i.e., from locally excited deazaflavin to the porphyrin moi-
1
1
ety, Pꢀ F* ! P*ꢀF, is expected to be rapid.

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Fig. 2. Spectral overlap between the Q-band absorbance A (—–) of porphyrin 7 (1·10ꢀ5 M, 1-cm path
length) and the (uncorrected) fluorescence emission (––––) of deazaflavin diester 18. The absorbance
A (······) of 18 (1·10ꢀ5 M, 1-cm path length) is shown on the left.
1
The reverse energy transfer 1P*ꢀF ! Pꢀ F* cannot occur because this would be
highly endothermic. Yet, the fluorescence lifetime of dyad 21 in the porphyrin locally
excited state 1P*ꢀF is much shorter than that of porphyrin 7. Also, diffusional quench-
ing of the fluorescence of 7 is observed when 10ꢀ3 M deazaflavin 18 is added to the sol-
ution (Table 1). These quenching processes are attributed to electron transfer,
1P*ꢀF ! P+CꢀFꢀC. Similar behavior has been reported for porphyrin–quinone systems
[14]; their fluorescence lifetimes increase by an order of magnitude when the quinone
moieties are reduced. To evaluate the energetic feasibility of electron transfer processes
in 21, we determined electrochemical potentials E8 in MeCN (Table 2). The standard
reduction potentials of the quinones in the porphyrin–quinone cyclophanes, E8(Q/
QꢀC)=ꢀ0.72 to ꢀ1.45 V [15], are comparable to that of deazaflavin 18, E8(F/
FꢀC)=ꢀ0.98 V.
Table 2. Standard Electrochemical Potentials E8 (vs. SCE) of Compounds 6, 7, 18, and 21 Determined by
Differential Pulse Voltammetry in MeCNwith 0.1 M LiClO4. P=porphyrin moiety, F=deazaflavin unit.
Redox couple
E8/V
Redox couple
E8/V
18
7
E8(F+C/F)
ca. +1.93a)
ꢀ0.98
21
6
E8(P+C/P)
E8(F/FꢀC)
E8(P/PꢀC)
E8(P+C/P)
E8(F/FꢀC)
E8(P/PꢀC)
+0.73
ꢀ0.91
ꢀ1.31
+0.84
ꢀ1.10
ꢀ1.55
E8(F/FꢀC) [16]
E8(P+C/P)
+0.68
E8(P/PꢀC)
ꢀ1.07
a) Irreversible.

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Combination of these data with the excitation energy of 211) shows that intramolec-
ular electron transfer from the locally excited porphyrin of dyad 21, 1P*ꢀF, to generate
the charge transfer state P+CꢀFꢀC is energetically feasible: {E8(P+C/P)ꢀE8(F/
FꢀC)}eꢀE
0ꢀ0A
1
the reverse electron transfer, P*ꢀF ! PꢀCꢀF+C, would be strongly endothermic:
{E8(F+C/F)ꢀE8(P/PꢀC)}eꢀE
energy diagram for the deactivation processes occurring after excitation of 21 is
shown in Fig. 3.
Fig. 3. Energy diagram and possible deactivation processes following excitation of the deazaflavin-
bridged porphyrin 21. P=porphyrin moiety, F=deazaflavin unit.
1
)
The positions of the first absorption and emission bands (634 and 635 nm, resp., Table 1) define the
energy E0ꢀ0A
(1P*ꢀF) as 1.96 eV.
)
The Coulomb attraction C for stabilization of the charge-transfer complex P+CꢀFꢀC is neglected:
C/eꢂ14.4/(eR) V/pmꢂ0.04 V, assuming an average distance between the charges Rꢂ500 pm and
a dielectric constant e=100 for MeCN.
2

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Fluorescence emission from the charge-transfer state P+CꢀFꢀC is not observed, pre-
sumably due to the low transition moment for radiative back electron transfer. The
pump–probe spectra of 7, 21, and 18 shown in Fig. 4 appear within the laser pulse.
Assignment of the transient spectrum obtained with 21 to the locally excited singlet
1P*ꢀF is strongly supported by its similarity with that obtained with the porphyrin 7
and by the fact that its lifetime of ca. 2.5 ns is consistent with the fluorescence lifetime
of 21.
Fig. 4. Pump–probe spectra taken 1 ps after excitation of porphyrin 7 (—–), 21 (–––), and deazaflavin
18 (–··–··) in MeCN
We now turn to the dyad 6 with the porphyrinatoiron(III) complex, which did not
exhibit any fluorescence, so that information could only be gained from the transient
absorption spectra (Fig. 5). Immediately after the excitation, two main features of
the porphyrin excited state, namely strong bleaching at 405 nm and absorption at 460
nm, appeared. These features were similar to those obtained with the iron-free dyad
21 (Fig. 4). The subsequent changes were, however, complex and differed substantially
from those observed with 21.
Previous studies [17] have shown that excited porphyrinatoiron(III) complexes
relax very rapidly (ca. 50 fs) to their ground states, presumably due to the presence
of several low-lying charge-transfer states. Franzen and co-workers recently reported
that ultra-fast iron-to-porphyrin charge transfer is initiated by excitation of haemoglo-
bins [18]. Porphyrin-to-iron back charge transfer subsequently produces a low-lying
electronic state with a non-equilibrium d-orbital population on the Fe-atom. Such
charge-transfer reactions may also occur in 6. Moreover, the pump pulse initially
excites both chromophores of 6. The spectra detected immediately after the excitation
of 6 are thus attributed to a combination of porphyrin and deazaflavin excited singlet
states with, possibly, a contribution from an iron-to-porphyrin charge-transfer state.
The strong bleaching at 405 nm, which is due to depletion of the ground state of 6,
partially (ca. 50%) recovers rapidly, tꢂ750 fs, and then more slowly, tꢂ11 ps (ca.

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Fig. 5. Pump–probe spectra taken at various delays after excitation of 6 in MeCNat 288 nm
40%). Full recovery of the porphyrin ground state takes longer than 2 ns. The very
broad initial absorption ranging from 430 to 650 nm decays within 200 fs around 500
nm, while a new broad band appears around 570 nm with a risetime of ca. 750 fs. Por-
phyrin triplets are known to exhibit broad absorbance centred around 600 nm [19]. We
therefore attribute the long-lived, 570-nm transient to the porphyrin triplet state of 6,
3PFe*ꢀF, and the 750-fs kinetics to the lowest singlet state of 6, 1PFe*ꢀF, which decays
by a combination of ISC to 3PFe*ꢀF and radiationless conversion [17][18]. No signifi-
cant signal was detected at 680 nm, where the porphyrin radical cation usually displays
its absorbance [19–21], i.e., formation of a PFe+CꢀFꢀC charge-transfer state was not
observed.
All synthetic porphyrinatoiron(III) complexes carrying a thiolato ligand coordi-
nated to the Fe-atom are high-spin [22]. The unusually fast intersystem crossing of
1PFe*ꢀF is, therefore, attributed to a substantial interaction of the d-electrons of FeIII
1
with the unpaired electrons of the porphyrin singlet state PFe*ꢀF.
Our tentative assignments of the observed processes are summarized in the energy
3
diagram of Fig. 6. Here, the energy of the deazaflavin triplet, PFe ꢀ F*, E0ꢀ0 =2.58, was
assumed to be equal to that of 3,10-dimethyl-5-deazaflavin [23] and that of the por-
3
phyrin triplet, PFe*ꢀF, equal to that of tetraarylporphyrinatometals, E0ꢀ0 =1.50 [24].
3. Conclusions. – Compound 6 was studied as a model system for the interaction of
the deazaflavin inhibitor 1 and native CYP 3A4. To mimic the enzyme–inhibitor inter-
action within the active site, deazaflavin was strapped over the distal side of a porphyr-
inatoiron(III). A new synthetic approach for 8-alkylated deazaflavins was developed.

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Fig. 6. Energy diagram and radiationless transitions following excitation of the deazaflavin-bridged
iron porphyrin 6. P=porphyrin moiety, F=deazaflavin unit.
Compound 6 was synthesized in 26 steps comprising the synthesis of diamino–por-
phyrin 7, followed by coupling with deazaflavin active ester 18 to provide the iron-
free compound 21 and, after iron(III) insertion, the model compound 6. A study of
the electrochemical and the time-resolved optical properties of compounds 21 and 6
showed that the mechanism of fluorescence quenching differs strongly in the two
dyads. Radiationless deactivation of the locally excited deazaflavin of the iron-free
1
compound 21, Pꢀ F*, proceeds either by electron transfer forming a charge-separated
1
state, P+CꢀFꢀC, or by energy transfer to the porphyrin unit, P*ꢀF, or both.
The presence of a high spin, paramagnetic iron(III) is responsible for the complete
suppression of fluorescence in model compound 6. It is suggested that the locally
1
excited state PFe*ꢀF, that is formed by excitation of the porphyrin moiety of 6 and,
1
in part, by energy transfer from PFe ꢀ F*, has a lifetime of only 750 fs due to rapid radi-
3
ationless relaxation and enhanced ISC to PFe*ꢀF.
We gratefully acknowledge financial support by the Swiss National Science Foundation.

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Experimental Part
General. Chemicals were purchased from Aldrich, Sigma, or Fluka and were used without further
purification, if not otherwise stated. Solvents were purified by standard procedures. TLC: Merck pre-
coated silica gel 60 F254 glass plates (0.25-mm layer); Merck RP-18 F254s glass plates (0.25-mm layer). Col-
umn chromatography (CC): Merck silica gel 60 (0.04–0.063 mm); visualization by UV light (254 nm) and
fluorescence (366 nm); FC=flash chromatography. UV/VIS Spectra: Hewlett-Packard 8452A spectro-
photometer; l in nm (e). Fluorescence spectra: ISA Jobin Yvon-Spex Fluoromax-2; l in nm. IR Spectra:
Perkin-Elmer FTIR-1600 spectrophotometer; absorptions in cmꢀ1. 1H- (250, 400, 500, and 600 MHz) and
13C-NMR (400 and 500 MHz) Spectra: in CDCl3, Bruker av250, DPX-NMR, Bruker DRX-500, and
Bruker DRX-600 instruments; at 298 K if not otherwise stated; d in ppm, coupling constants J in Hz.
EI- or FAB-MS: Varian-VG-70-250 or Bruker Esquire3000+ for ESI-MS; in m/z (rel. %). Elemental
analysis: Perkin-Elmer 240 analyzer.
Picosecond Pump–Probe Spectroscopy. Ti :Sa Laser system Clark MXR CPA-2001 (775 nm, pulse
energy 0.8 mJ, full width at half maximum (fwhm) 150 fs, operating frequency 426 Hz). Part of the
beam was fed into a Clark-MXR NOPA. The output at 540 nm was frequency-doubled to 270 nm and,
after compression, provided pump pulses with an energy of 1 mJ and <100 fs pulse width. A probe
beam continuum (300–680 nm) was produced by focusing part of the 775-nm beam 1 mm in front of a
CaF2 crystal of 2 mm path length. The detection system has been described [25]. Two-photon absorption
is observed when the pump and probe pulses coincide at the sample. The fwhm of the two-photon absorp-
tion in H2O was 180 fs, which provides a measure of the time resolution of the detection system. Typically,
several hundred spectra were collected and averaged for each delay. The translation stage allowed for a
maximum time delay of 1.8 ns for the probe beam.
Time-Resolved Fluorescence Spectroscopy. Hamamatsu-C5680 streak camera equipped with a with
Chromex-250IS polychromator unit. Excitation laser pulses (pulse length 140 fs) centred at 310 nm
were provided by frequency doubling of the Clark-MXR-NOPA output. The excitation beam was
focused into the 1-mm sample cell under 458 angle, providing front-side excitation. Incident fluorescence
light was collimated and then focused into the polychromator. The C5680 unit recorded the time and
spectrally resolved fluorescence. The optical grating was chosen to allow recording fluorescence spectra
in 300-nm windows from 250 to 1100 nm. Typically, 500 scans were averaged after electronic jitter and
background corrections. A reflection of the excitation laser beam was recorded for use in a reconvolution
procedure, which improved the lifetime resolution. All samples for the time-resolved spectroscopy were
carefully degassed by four freeze-pump-thaw cycles to a final pressure of ca. 10ꢀ4 Torr.
Differential Pulse Voltammetry. Eco-Chemie-BV-mAutolab type II potentiostat; data were collected
by a three-electrode technique comprising a working electrode (glassy carbon), a counter electrode (Pt-
net), and a reference electrode (Ag-wire). Electrolyte: 0.1M LiClO4 in degassed MeCN. Ferrocene (Fc+/
Fc=+0.41 V vs. SCE (saturated calomel electrode) in MeCN [26]) was used as an internal reference.
Standard electrode potentials E8 are given vs. SCE; for conversion to NHE (normal hydrogen electrode)
add 0.241 V [27]. All compounds except deazaflavin gave reversible waves for the potentials quoted.
2,4,6-Trichloropyrimidine-5-carboxaldehyde (10). To DMF (9.6 ml, 124.8 mmol), POCl3 (48 ml, 524.2
mmol) was added dropwise at 08. After 15 min stirring at 08, 8 (8.02 g, 62.4 mmol) was added. After 4 h
stirring at 1008, the mixture was directly evaporated. The residue was dissolved in ice-cooled H2O. The
precipitate was filtered, washed with H2O, and purified by FC (CH2Cl2): 7.82 g (59%). Colorless crystals.
M.p. 131–1328. Rf 0.48 (CH2Cl2). IR (KBr): 2833.7w, 1706.3s, 1525.9s 1499.0s, 1299.5m, 1118.7m, 885.9m.
1H-NMR (400 MHz, CDCl3): 10.41 (s, 1 H). 13C-NMR (400 MHz, CDCl3): 184.95; 164.433; 123.97. EI-
MS: 208.9(100, [ M+1]+), 173.9(10, [ MꢀCl]+), 146.9(5), 120 (11), 85 (23). Anal. calc. for C HCl3O
5
(211.44): C 28.40, H 0.48, N 13.25, O 7.57; found: C 28.45, H 0.49, N 13.19, O 7.70.
1-(Bromomethyl)-2-methyl-3-nitrobenzene (12). To a soln. of 9 (20.0 g, 120 mmol) in Et2O (200 ml),
PBr3 (18.6 ml, 197 mmol) was added dropwise at r.t. After stirring 1 h at 508, the soln. was cooled to r.t.
and hydrolyzed with H2O (50 ml). The aq. layer was extracted with Et2O, the combined org. phase dried
(Na2SO4) and concentrated, and the residue purified by FC (CH2Cl2): 12 (27.4 g, 99%). Light yellow crys-
1
tals. M.p. 42–438. Rf 0.74 (CH2Cl2). IR (KBr): 3079.5w, 1525.5s, 1454.0m, 1357.2s. H-NMR (400 MHz,
CDCl3): 7.71 (d, J=8.1, 1 H); 7.54 (d, J=7.6, 1 H); 7.30 (t, J=8.1, 1 H); 4.54 (s, 2 H); 2.51 (s, 3 H).

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13C-NMR (400 MHz, CDCl3 ): 151.96; 139.07; 134.31; 131.97; 127.24; 124.78; 30.99; 14.87. EI-MS 229.0 (6,
M+), 214.0 (6, [MꢀMe]+), 183.0 (1, [MꢀNO2]+), 150.1 (100, [MꢀBr]+), 132.1 (7), 103.1 (42), 77.1 (20),
51.0 (11). Anal calc. for C8H8BrNO2 (230.06): C 41.77, H 3.51, N 6.09; found: C 41.76, H 3.30, N 6.04.
Dimethyl 2-[(2-Methyl-3-nitrophenyl)methyl]propanedionate (13). To a soln. of NaH (13.1 g, 328
mmol) in DMSO (150 ml), CH2(COOMe)2 (38 ml, 328 mmol) was added dropwise under cooling in an
ice bath. Then 12 (15.1 g, 65.6 mmol) was added at r.t. within 45 min. The soln. was poured into ice-cooled
H2O (200 ml). The precipitate was filtered, washed with H2O, and dried in vacuo: 13 (10.7 g, 58%). Col-
orless crystals. M.p. 81–838. Rf 0.40 (CH2Cl2). IR (KBr): 2953.1w, 2360.2m, 1731.0s, 1531.3s, 1434.9s,
1
3
5
1355.6s, 1293.3m, 1228.0m, 1159.6m, 1020.7m. H-NMR (400 MHz, CDCl3): 7.63 (dd, J=8.1, J=1.0,
1 H); 7.38 (d, J=7.1, 1 H); 7.24 (d, J=7.8, 1 H); 3.71 (s, 6 H); 3.65 (t, J=7.6, 1 H); 3.33 (d, J=7.6, 2
H); 2.45 (s, 3 H). 13C-NMR (400 MHz, CDCl3): 169.16; 151.95; 139.23; 134.04; 130.90; 126.81; 123.23;
53.17; 52.20; 32.67; 15.10. FAB-MS 282 (100, [M+H]+), 265 (7), 250 (14), 218 (36), 150 (20), 137 (30).
Anal. calc. for C13H15NO6: C 55.51, H 5.38, N 4.98, O 34.13; found: C 55.55, H 5.25, N 4.95, O 33.97.
Methyl 2-Methyl-3-nitropbenzenepropanoate (14). To a soln. of 13 (9.3 g, 33 mmol) in DMF (50 ml)
and H2O (5 ml), LiI (13.2 g, 98 mmol) was added. After stirring 7 h at 1608, the soln. was cooled to r.t. and
hydrolyzed with H2O (300 ml). The aq. layer was extracted with CH2Cl2, the combined org. phase dried
(Na2SO4) and concentrated, and the residue purified by FC (AcOEt/hexane 2 :1): 14 (5.6 g, 76%). Yellow
oil. Rf 0.37 (AcOEt/hexane 2 :1). IR (NaCl): 3088.9w, 3000.0w, 2952.9m, 1737.5s, 1605.6w, 1527.4s,
1438.1m, 1355.7s, 1294.4m, 1261.1m, 1211.1m, 1171.9m, 1088.1m, 1027.0w, 988.9w, 905.6w, 877.8w,
1
3
5
833.3w, 806.7w, 772.0w, 733.4m. H-NMR (400 MHz, CDCl3): 7.52 (dd, J=8.1, J=1.0, 1 H); 7.33 (d,
J=7.1, 1 H); 7.18 (t, J=7.8, 1 H); 3.62 (s, 3 H); 2.98 (t, J=8.1, 2 H); 2.55 (t, J=8.1, 2 H); 2.35 (s, 3 H).
13C-NMR (400 MHz, CDCl3): 172.99; 151.83; 141.92; 133.19; 130.42; 126.78; 122.53; 52.06; 40.32;
28.93; 14.89. EI-MS: 223.0 (3, M+), 206.1 (19), 192.1 (17, [MꢀOMe]+), 174.1 (76), 164.1 (41,
[MꢀCOOMe]+), 146.1 (49), 132.1 (100), 115.1 (28), 103.1 (30), 91.1 (44), 77.1 (25), 59.1 (17), 39.1
(14). Anal. calc. for C11H13NO4 (223.23): C 59.19, H 5.87, N 6.27, O 28.67; found: C 58.96, H 5.82, N
6.26, O 28.81.
Methyl 3-Amino-2-methylbenzenepropanoate (15). A mixture of 5% Pd/C, 14 (5.4 g, 24.3 mmol), and
95% EtOH (100 ml) was shaken for 4 h under H2 at r.t. The soln. was filtered over Celite, the latter
washed with EtOH, and the filtrate diluted with H2O. The aq. layer was extracted with CH2Cl2, the com-
bined org. phase dried (Na2SO4) and concentrated, and the residue purified by FC (AcOEt/hexane 1:1):
15 (3.6 g, 76%). Colorless crystals. M.p. 45–468. Rf 0.33 (AcOEt/hexane 1:1). IR (KBr): 3452.9m,
3374.2m, 3002.9w, 2950.4m, 1731.1s, 1635.1m, 1587.1m, 1472.9m, 1437.0m, 1369.0m, 1291.0m, 1250.0m,
1
1195.1m, 1167.1m, 1083.0m, 782.8m. H-NMR (400 MHz, CDCl3): 6.96 (t, J=7.6, 1 H); 6.63 (d, J=7.6,
1 H); 6.60 (d, J=8.1, 1 H); 4.00–3.50 (br., 2 H); 3.69( s, 3 H); 2.94 (t, J=8.3, 2 H); 2.56 (t, J=8.3, 2
H); 2.12 (s, 3 H). 13C-NMR (400 MHz, CDCl3): 173.88; 144.94; 139.81; 126.82; 120.94; 120.20; 114.29;
52.01; 35.35; 29.54; 12.85. EI-MS 193.1 (100, M+), 162.1 (16, [MꢀOMe]+), 134.1 (68, [MꢀCOOMe]+),
120.1 (65, [MꢀCH2COOMe]+), 106.1 (34, [MꢀCH2CH2COOMe]+), 77.1 (11). Anal calc. for C11H15NO2
(193.25): C 68.37, H 7.82, N 7.25, O 16.56; found: C 68.25, H 7.77, N 7.15, O 16.60.
Methyl 2-Methyl-3-(methylamino)benzenepropanoate (11). To a suspension of 15 (5.0 g, 25.9mmol)
and NaH (683 mg, 28.5 mmol) in dry DMF (75 ml), MeI (2 ml, 31 mmol) was added within 30 min at r.t.
After stirring further 15 min at r.t., the sol. was diluted with H2O, and 1M NaOH was added until a pH of
ca. 11 was reached. The aq. layer was extracted with CH2Cl2, the combined org. phase dried (Na2SO4) and
concentrated, and the residue purified by FC (AcOEt/hexane 1:1): 11 (2.6 g, 64%). Light yellow oil. Rf
0.47 (AcOEt/hexane 1:1). IR (NaCl): 3438.4m, 2948.0m, 2360.8w, 1733.9s, 1590.2s, 1511.1m, 1477.9m,
1433.3m, 1361.1w, 1287.1m, 1160.3m, 1105.6w, 1027.8w, 778.2w. 1H-NMR (250 MHz, CDCl3): 7.14 (t,
J=7.8, 1 H); 6.65 (d, J=7.7, 1 H); 6.56 (d, J=8.1, 1 H); 3.80–3.70 (br., 1 H); 3.73 (s, 3 H); 3.01 (t,
J=7.5, 2 H); 2.93 (s, 3 H); 2.60 (t, J=7.7, 2 H); 2.12 (s, 3 H). 13C-NMR (400 MHz, CDCl3): 173.97;
147.75; 139.10; 127.07; 120.20; 118.72; 108.45; 52.04; 35.54; 31.56; 29.77; 12.59. EI-MS 207.1 (100, M+),
176.1 (15, [MꢀOMe]+), 160.0 (6), 148.1 (51, [MꢀCOOMe]+), 134.1 (32), 120.0 (36), 91.0 (9). Anal.
calc. for C12H17NO2 (207.27): C 69.54, H 8.27, N 6.76, O 15.44; found: C 69.06, H 8.30, N 6.81, O 15.15.
Methyl 3-[(5-Formyl-2,6-dichloropyrimidin-4-yl)methylamino]-2-methylbenzenepropanoate (16). To
a soln. of 11 (1.00 g, 4.8 mmol) in dry CH2Cl2 (25 ml), 10 (1.02 g, 4.8 mmol) was added in portions. After
stirring for 5 min at 1008, the soln. was cooled to r.t., and H2O was added, followed by 1M NaOH until a

Helvetica Chimica Acta – Vol. 89 (2006)
2999
pH of ca. 11 was reached. The aq. layer was extracted with CH2Cl2, the combined org. phase dried
(Na2SO4) and concentrated, and the residue purified by FC (AcOEt/hexane 1:1): 16 (1.55 g, 85%). Col-
orless crystals. M.p. 83–858. Rf 0.54 (AcOEt/hexane 1:1). IR (KBr): 2992.9w, 2949.9w, 2844.0w, 1739.8s,
1700.1s, 1437.0m, 1396.8m, 1341.1m, 1279.1m, 1234.6m, 1134.1m, 1091.3m, 1045.5m, 1021.8m, 982.3m,
1
885.5m. H-NMR (500 MHz, CDCl3): 9.20 (br., 1 H); 7.20–7.10 (m, 2 H); 6.86 (br., 1 H); 3.69( s, 3 H);
3.41 (br., 3 H); 2.99 (td, J=7.1, 2.6, 2 H); 2.62 (td, J=7.9, 3.4, 2 H); 2.18 (s, 3 arom. H). 13C-NMR (500
MHz, CDCl3): 185.48; 173.06; 162.16; 159.74; 143.74; 142.20; 140.05; 134.05; 134.00; 129.48; 127.82;
124.49; 51.81; 40.51; 34.13; 28.68; 13.98. EI-MS 381.0 (100, M+), 366.0 (70, [MꢀMe]+), 352.0 (80),
346.0 (43, [MꢀCl]+), 322.0 (58, [MꢀCOOMe]+), 306.0 (34), 294.0 (40, [MꢀCH2CH2COOMe]), 278.0
(52), 264.0 (36), 115.0 (32), 91.1 (31). Anal. calc. for C17H17Cl2N3O3 (382.25): C 53.42, H 4.48, N 10.99,
O 12.56; found: C 53.48, H 4.45, N 10.93, O 12.27.
Methyl 8,9-Dimethyl-10-deazaisoalloxazine-7-propanoate (=Methyl 2,3,4,10-Tetrahydro-9,10-
dimethyl-2,4-dioxopyrimido[4,5-b]quinoline-8-propanoate; 17). To 16 (1.42 g, 3.7 mmol), 98%
CF3COOH (10 ml) was added. After stirring for 1 h at 608, the soln. was cooled to r.t. and poured on
ice-cooled H2O (200 ml). The aq. layer was extracted with CH2Cl2 until the layer was colorless, the com-
bined org. phase dried (Na2SO4), and evaporated, and the residue purified by FC (CH2Cl2/MeOH 9:1):
17 (1.18 g, 98%). Yellow crystals. M.p. >3008. Rf 0.31 (CH2Cl2/MeOH 9:1). UV/VIS (CHCl 3): 269(100),
343 (43), 404 (41). Fluor. (CHCl3): 474. IR (KBr): 3144.4w, 3012.2w, 2800.0w, 2366.7w, 1737.1w, 1704.5m,
1664.4m, 1623.0s, 1594.4m, 1552.0m, 1527.7m, 1482.1w, 1413.7w, 1377.8w, 1344.4w, 1228.8m, 1175.2w. 1H-
NMR (250 MHz, CDCl3): 8.84 (s, 1 H); 8.46 (br., 1 H); 7.71 (d, J=8.2, 1 H); 7.40 (d, J=8.1, 1 H); 4.15 (s, 3
H); 3.74 (s, 3 H); 3.22 (t, J=7.9, 2 H); 2.75 (t, J=7.5, 2 H); 2.67 (s, 3 H). 13C-NMR (500 MHz, CH3-
COOD): 174.73; 164.34; 161.33; 160.91; 151.75; 145.50; 145.31; 130.56; 128.31; 127.32; 123.74; 114.77;
52.68; 43.81; 34.54; 30.45; 18.98. EI-MS 327.1 (37, M+), 296.0 (6, [MꢀOMe]+), 268.1 (100,
[MꢀCOOMe]+), 254.0 (10, [MꢀCH2COOMe]+), 225.1 (9). Anal. calc. for C17H17N3O4 (327.34): C
62.38, H 5.23, N 12.84; found: C 62.00, H 5.31, N 12.26.
Methyl 3-[(Methoxycarbonyl)methyl]-8,9-dimethyl-10-deazaisoalloxazine-7-propanoate (=Methyl
2,3,4,10-Tetrahydro-3-(2-methoxy-2-oxoethyl)-9,10-dimethyl-2,4-dioxopyrimido[4,5-b]quinoline-8-prop-
anoate; 18). To a suspension of 17 (1.13 g, 3.5 mmol) and K2CO3 (2.67 g, 19.3 mmol) in dry DMF (15 ml),
BrCH2COOMe (3.2 ml, 34.5 mmol) was added. After stirring for 10 h at r.t., the clear soln. was diluted
with H2O. The aq. layer was extracted with CH2Cl2, the org. phase dried (Na2SO4) and concentrated, and
the residue purified by FC (CH2Cl2/MeOH 9:1): 18 (900 mg, 65%). Yellow crystals. M.p. 218–2208. Rf
0.39(CH Cl2/MeOH 95 :5). UV/VIS (CHCl3): 267 (100), 343 (35), 405 (31). Fluor. (CHCl3): 477. IR
2
(KBr): 2927.0m, 2367.2w, 1752.2s, 1733.3s, 1699.5m, 1650.0m, 1616.6s, 1531.7s, 1433.4s, 1369.8m,
1
1322.6w, 1209.5s, 1066.7w, 966.7w, 933.0w, 796.2m. H-NMR (500 MHz, CDCl3): 8.77 (s, 1 H); 7.65 (d,
J=8.1, 1 H); 7.33 (d, J=8.1, 1 H); 4.83 (s, 2 H); 4.07 (s, 3 H); 3.74 (s, 3 H); 3.69( s, 3 H); 3.17 (t,
J=8.0, 2 H); 2.71 (t, J=8.0, 2 H); 2.62 (s, 3 H). 13C-NMR (500 MHz, CDCl3): 172.54; 168.6; 161.69;
159.18; 156.39; 149.50; 144.03; 143.13; 129.10; 126.42; 125.21; 121.85; 52.38; 52.00; 42.71; 42.14; 33.85;
29.54; 18.96. EI-MS: 399.1 (57, M+), 368.1 (12, [MꢀOMe]+), 340.1 (100, [MꢀCOOMe]+), 280.1 (19,
[MꢀCOOMe]+), 225.1 (8). Anal. calc. for C20H21N3O6 (399.41): C 60.14, H 5.30, N 10.52, O 24.04;
found: C 57.88, H 5.24, N 10.02, O 24.61.
3-(Carboxymethyl)-8,9-dimethyl-10-deazaisoalloxazine-7-propanoic Acid (=3-(Carboxymethyl)-2,3,
4,10-tetrahydro-9,10-dimethyl-2,4-dioxopyrimido[4,5-b]quinoline-8-propanoic Acid; 19). To a suspension
of 18 (400 mg, 1 mmol) in THF/H2O 6 :4 (10 ml), LiOH·H2O (210 mg, 5 mmol) was added. After stirring
for 1 h at r.t., the soln. was diluted with H2O (10 ml). Then 1M HCl was added until a pH of ca. 3 was
reached and the soln. was concentrated. The residue was directly purified by reversed-phase CC
(H2O/MeCN 3 :1): 19 (252 mg, 68%). Yellow crystals. M.p. 291–2928. Rf 0.74 (H2O/MeCN 3 :1). UV/
VIS (MeOH): 229(82), 264 (100), 343 (32), 401 (26). Fluor. (MeOH): 476. IR (KBr): 2933.3 w, 2361.1s,
1727.8s, 1694.4s, 1670.3s, 1586.4s, 1536.4s, 1483.3m, 1452.4m, 1411.1m, 1377.8w, 1338.9w, 1311.1w,
1250.0w, 1208.3s, 927.4w. 1H-NMR (250 MHz, CDCl3): 13.00–12.00 (br., 2 H); 8.98 (s, 1 H); 7.91 (d,
J=7.9, 1 H); 7.47 (d, J=8.0, 1 H); 4.55 (s, 2 H); 3.98 (s, 3 H); 3.08 (t, J=7.4, 2 H); 2.65 (t, J=8.0, 2
H); 2.62 (s, 3 H). 13C-NMR (500 MHz, (D6)DMSO): 173.7; 169.6; 161.3; 158.9; 155.5; 150.3; 143.4;
143.0; 128.9; 126.2; 125.4; 121.5; 112.9; 42.1; 41.8; 33.6; 29.3; 18.4. EI-MS 371.2 (100, M+), 340.1 (12),

3000
Helvetica Chimica Acta – Vol. 89(2006)
326.2 (57, [MꢀCOOH]+), 312.2 (29, [MꢀCH2COOH]+), 283.2 (28), 266.1 (45), 252.1 (9), 237.1 (9). Anal.
calc. for C18H17N3O6 (371.35): C 58.22, H 4.61, N 11.32, O 25.85; found: C 57.62, H 4.73, N 11.05, O 26.32.
Pentafluorophenyl 3-{[(Pentafluorophenoxy)carbonyl]methyl}-8,9-dimethyl-10-deazaisoalloxazine-
7-propanoate (=Pentafluorophenyl 2,3,4,10-Tetrahydro-9,10-dimethyl-2,4-dioxo-3-[2-oxo-2-(penta-
fluoroACHTERpUNG henoxy)ethyl]pyrimido[4,5-b]quinoline-8-propanoate; 20). To 19 (170 mg, 0.46 mmol) and
EDC·HCl (263 mg, 1.37 mmol) in dry DMF (5 ml), C6F5OH (253 mg, 1.37 mmol) was added. After stir-
ring for 1 h at r.t., the soln. was diluted with H2O. The aq. layer was extracted with CH2Cl2, the combined
org. phase dried (Na2SO4) and concentrated, and the residue recrystallized from CH2Cl2/hexane: 20 (267
mg, 83%). Yellow crystals. Rf 0.85 (CH2Cl2/MeOH 9:1). UV/VIS (CHCl 3): 266 (100), 342 (3), 406 (33).
Fluor. (CHCl3): 472. IR (KBr): 2926.7w, 1782.5m, 1700.3m, 1654.6s, 1628.6m, 1603.8w, 1559.7w, 1520.4s,
1419.8w, 1366,7w, 1314.5w, 1252.5w, 1148,3m, 1111.1m, 1001.2s, 796.3w. 1H-NMR (400 MHz, CDCl3): 8.85
(s, 1 H); 7.72 (d, J=8.1, 1 H); 7.41 (d, J=8.1, 1 H); 5.20 (s, 2 H); 4.10 (s, 3 H); 3.32 (t, J=7.6, 2 H); 3.10 (t,
J=7.6, 2 H); 2.68 (s, 3 H).
19-(tert-Butyl)-46-mercapto-41,43-dimethyl-45,47-(2,8,12,18-tetrabutyl-3,7,13,17-tetramethyl-21H,
23H-porphine-5,15-diyl)-13,25-dioxa-2,4,7,31,41-pentaazaheptacyclo[33.5.3.14,40.18,12.117,21.126,30.038,42]hep-
tatetraconta-1,8,10,12(47),17,19,21(46),26,28,30(45),35,37,39,42-tetradecaene-3,6,32,44-tetrone (21). To a
soln. of 7 (58 mg, 0.055 mmol) in dry and degassed DMF (1ml), 20 (97 mg, 0.138 mmol) was added.
After stirring for 4 h at 1008, the soln. was cooled to r.t. and diluted with CH2Cl2 and sat. NaHCO3
soln. The aq. layer was extracted with CH2Cl2, the combined org. phase dried (Na2SO4) and evaporated,
and the crude product purified by CC (CH2Cl2/MeOH 95 :5+1% Et3N): 28.0 mg 21 (37%). Pink solid. Rf
0.33 (CH2Cl2/MeOH 95 :5+1% Et3N). UV/VIS (CHCl3): 269(47), 346 (31), 415 (100), 513 (9), 546 (4),
583 (4). Fluor. (MeOH): 476. 1H-NMR (600 MHz, toluene, 345 K): 10.14 (br., 2 H); 9.05 (d, J=8.8, 1 H);
8.93 (d, J=8.8, 1 H); 8.77 (s, 1 H); 7.95 (s, 1 H); 7.70 (m, 2 H); 6.70 (m, 2 H); 6.15–6.05 (m, 2 H); 5.80 (d,
J=7.7, 1 H); 4.46 (s, 2 H); 4.41 (s, 1 H); 4.18 (d, J=7.9, 1 H); 4.10–3.80 (m, 8 H); 3.27 (t, J=4.6, 2 H);
3.17 (t, J=4.4, 2 H); 2.74 (br., 12 H); 2.50–2.40 (m, 2 H); 2.40–2.10 (m, 8 H); 2.12 (s, 3 H); 1.90–1.70
(m, 8 H); 1.18 (t, J=7.1, 6 H); 1.10 (t, J=7.3, 6 H); 1.05–0.95 (m, 2 H); 0.84 (s, 9H); 0.70 ( m, 2 H);
0.57 (m, 2 H); 0.28 (s, 3 H); 0.26 (m, 2 H); 0.17 (m, 2 H); ꢀ2.57 (s, 1 H); ꢀ2.87 (s, 1 H); ꢀ3.02 (s, 1
H). ESI-MS (MeOH): 1386.5 (100, M+).
[19-(tert-Butyl)-46-(mercapto-kS)-41,43-dimethyl-45,47-(2,8,12,18-tetrabutyl-3,7,13,17-tetramethyl-
21H,23H-porphine-5,15-diyl-kN21,kN22,kN23,kN24)-13,25-dioxa-2,4,7,31,41-pentaazaheptacyclo[33.5.3.14,40
.
18,12.117,21.126,30.038,42]heptatetraconta-1,8,10,12(47),17,19,21(46),26,28,30(45),35,37,39,42-tetradecaene-3,6,32,
44-tetronato(3-)]iron (6). To 21 (7.8 mg, 5.6 mmol) in dry and degassed toluene (3 ml), 2,6-lutidine (=2,6-
dimethylpyridine; 13 ml, 0.112 mmol) was added. After heating to 1208, dry and degassed FeBr2 (17.2 mg,
0.078 mmol) was added. After stirring for 45 min at 1208, the soln. was cooled to r.t., poured on Celite, and
washed until the filtrate was colorless. The soln. was evaporated and the crude product purified by CC
(toluene/THF 2 :1): 6 (6.5 mg, 89%). Brown solid. Rf 0.41 (toluene/THF 2 :1). UV/VIS (CHCl3): 269
(68), 346 (49), 406 (100), 510 (br., 15). ESI-MS (MeOH): 1439.5 (50, M+), 1462.5 (100, [M+Na]+).
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Received June 26, 2006