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									CHAPTER




Structure elucidation of aplidine metabolites formed in vitro by human
liver microsomes using triple quadrupole mass spectrometry.

Esther F.A. Brandon, Ronald D. van Ooijen, Rolf W. Sparidans,
Luis López Lázaro, Albert J.R. Heck, Jos H. Beijnen, and Jan H.M. Schellens.


Abstract

    The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four
metabolites of this compound were found after incubation with pooled human microsomes
using gradient HPLC with ultraviolet detection. After chromatographic isolation, the
metabolites have been identified using nano-electrospray triple quadrupole mass
spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the
depsipeptide ring and cleavage of the amino acid residues gives sequence information when
activated by collision-induced dissociation in the second quadrupole. The aplidine molecule
could undergo the following metabolic reactions: hydroxylation at the isopropyl group
(metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite
apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group
(metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The
identification of these metabolites formed in vitro may greatly aid the elucidation of the
metabolic pathways of aplidine in humans.




                                            67
Chapter 4


Introduction

    Aplidine (figure 1) is a cyclic depsipeptide isolated from the Mediterranean tunicate
Aplidium albicans. It was previously named dehydrodidemnin B, because of its structural
relation to didemnin B (figure 1) and other didemnins harvested from the tunicate
Trididemnum solidum. Aplidine showed the strongest in vitro anti-tumor activity of 42 tested
didemnins [1]. Aplidine has demonstrated in vitro anti-proliferative activity against a variety
of human tumor cell lines, but specifically against non-small cell lung and colon tumor cells
[2]. In in vivo studies in mice significant tumor growth inhibition by aplidine was observed in
human lymphomas and gastric, prostate, and bladder tumors [3, 4]. Aplidine is believed to
down-regulate the flt-1 receptor for the vascular endothelial growth factor, which is involved
in angiogenesis and tumor vascularization, arrest of the cell cycle in the G1 phase, and
induction of apoptosis in cancer cells [2, 5-7]. It is also a potent inhibitor of the protein
synthesis [8].

                                         [3S,4R,5S-Ist]                     [2S,4S-Hip]

                                                          OH     O
       aplidine       R =O
                                                                                               O
       didemnin B R -OH                                                 O
                                             O      N                           O         NH
                                         O                                       O
                                                          [1S,2R-Thr]
                                                          O      O
                                             N                          O                          [(S)-Leu]
             [(R)-N(Me)-Leu]
                                                                                N
                           O        N
                       O                                         N
                                                                             [(S)-Pro]
                           N
          [Pyr]   R

                             [(S)-Pro]                         [(S)-Me2-Tyr]
                                                          O


Figure 1. Chemical structure of aplidine and didemnin B [10, 11]. Hip - hydroxyisovalerylpropionyl, Ist -
isostatine, Leu - leucine, Me – methyl, Pro - proline, Pyr - pyruvoyl, Thr - threonine, Tyr - tyrosine.

    Four phase I metabolites were discovered by us after recent in vitro investigations of the
aplidine biotransformation using pooled mixed gender human liver microsomes. This chapter
presents the mass spectrometric investigations for the identification of the four metabolites of
aplidine. The structure elucidation of didemnin B and its sodium adduct using
collision-induced dissociation (CID) mass spectrometry was previously reported by Ngoka et
al. (1999) [9]. The in vitro metabolites of aplidine were identified using similar structure
assignments by MS/MS after liquid chromatographic isolation.




                                                   68
                                                 Structure elucidation of aplidine metabolites


Materials and methods

    Chemicals. Aplidine was kindly donated by PharmaMar (Tres Cantos, Madrid, Spain).
Acetonitrile (gradient grade) was purchased from Biosolve (Valkenswaard, The Netherlands)
and formic acid (p.a.), MgCl2.6H2O (p.a.) and dimethyl sulfoxide (DMSO, synthesis grade)
from Merck (Darmstadt, Germany). Water was purified on a multi-laboratory scale by
reversed osmosis. Pooled human liver microsomes (mixed gender) were provided by Gentest
(Becton Dickinson, Woburn, MA, USA). All other chemicals were purchased from Sigma
Chemical Company (St. Louis, MO, USA) and were of analytical grade.
    Sample Preparation. The incubation procedure of aplidine with human liver microsomes
was a modification of the method described by Sparidans et al. (2001) for ecteinascidin 743
[10]. Twenty-five µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a
polypropylene micro tube on ice and 50 µl NADP regenerating system (NRS: 1.5 U/ml
glucose-6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 4.0 mg/ml D-glucose-6-phosphate
in 0.6 % (w/v) NaHCO3), 7.5 µl of 20 mg/ml MgCl2.6H2O solution, and 50 µl of an aqueous
aplidine solution (final concentration of 30 µg/ml in the microsomes suspension) were added.
After briefly vortex-mixing, the tubes were incubated for 2 min at 37°C in a shaking water
bath. Next, 5 µl pooled human liver microsomes (mixed gender, lot number 21) were added.
The tube was vortex-mixed briefly again and the mixture was incubated at 37°C in a shaking
water bath for 3 h. The reaction was terminated by adding 125 µl acetonitrile and
vortex-mixing. The sample was centrifuged at approximately 15,000 g and 4°C for 1 min to
remove proteins and the supernatant was injected for HPLC analysis. Control experiments
were performed without aplidine and without liver microsomes, respectively. The extracts
were purified by injection of 100 µl of the sample into the chromatographic system and
manual collection of the individual metabolites. Pooled and single fractions of each
metabolite were evaporated by a Gyrovap centrifugal evaporator (Howe, Banburry, UK)
under vacuum (approximately 32 Pa) and temperature was kept at -50°C by a Cryocool
CC-100 II (Neslab Instuments Inc., Portsmouth, NH, USA). For mass spectrometric analysis
the residues were dissolved in 5 to 10 µl of 0.1% (v/v) formic acid in water/acetonitrile
(1/1 (v/v)).
    High performance liquid chromatography. The chromatographic assay was a
modification of the method described by Waterval et al. (2001) [11]. The supernatants of the
incubated mixtures were analyzed on an HPLC system consisting of two LC-10ATVP pumps,
a SIL-10ADVP autoinjector (equipped with a 500 µl sample loop), a SCL-10AVP system
controller, and a SPD-M10AVP photodiode array detector (all from Shimadzu, Kyoto, Japan).
Data acquisition and processing were performed using a Class-VP 5.032 data system
(Shimadzu). Injections were made on a Symmetry C18 column (4.6 x 100 mm, dp=3.5 µm,
Waters Chromatography, Milford, MA, USA) with a Sentry Guard Symmetry C18 pre-
column (3.9 x 20 mm, dp=5 µm, Waters). The column temperature was kept at room
temperature. A gradient program was used with eluent A comprising 10 mM formic acid in
water and eluent B comprising 10 mM formic acid in acetonitrile. After injection, elution
started with 45% B and the eluent composition was raised linearly to 75% B during 20 min.
This percentage was maintained for 2 min before conditioning with 45% B for 8 min. The
eluent flow rate was 1.0 ml/min, the UV detection array was operated between 190 and
300 nm (resolution is 2 nm) and the peak areas were determined at 225 nm.




                                            69
Chapter 4

    Mass spectrometry. Mass spectrometric measurements were performed on a triple
quadrupole instrument (Quattro Ultima, Micromass, Manchester, UK) equipped with a
Z-nano electrospray source. Data acquisition and processing were performed using
MassLynx NT 3.5 data system (Micromass). The protonated aplidine and metabolite ions
[M+H]+ or the sodium adduct ions [M+Na]+ were formed via nano-electrospray ionization
(ESI+). The compounds entered the mass spectrometer through an electrospray capillary set at
1.1 - 1.2 kV and nitrogen was used as nebulizing gas at a flow rate of 70 l/h. The cone
voltage was set at 20 to 30 V and the ion-source parameters were optimized with respect to
the analyzed ions. The MS spectra, recorded between m/z 500 – 1200, were obtained at a scan
speed of ~350 m/z units s-1 with a mass resolution corresponding to 0.5 m/z unit at half peak
height. Argon was used as collision gas for CID at a pressure of 2.3 * 10-3 mbar and the
collision energy was set at 20 to 30 V. The product ion spectra, recorded between 100 and
1200 m/z (varied depending on the compound), were obtained at a scan speed of
~550 m/z units s-1, with mass resolution corresponding to 0.5 m/z unit at half peak height. The
instrument was previously calibrated with sodium iodide.


Results and discussion

    Chromatograms of aplidine incubated without and with pooled human liver microsomes
are shown in figure 2. Two conformations of aplidine (aplidine A and B) can be observed.
The two conformers are the cis and trans isomers of the pyruvoyl - proline amide bond and
the plateau between the peaks is caused by the inter-conversion of the aplidine isomers during
the chromatographic run [11-14]. The trans conformer of aplidine is eluted before the cis
conformer [13]. The observed products are metabolites of aplidine, because they are only
formed in the presence of human liver microsomes with the drug. Furthermore, the spectra of
aplidine and the observed products recorded online with a photodiode array detector are
almost identical (figure 3).




                                              70
                                                                                                              Structure elucidation of aplidine metabolites



                    45

                    40
                                                                                                                                                                                                2
                    35




                                                                                                                                           apli-dm
                                                                                                                                                                                                1




                                                                                                                                apli-h2
                    30
Absorbance (mAU)




                                                                                                                 apli-h1
                                                                                                                 apli-da
                    25
                   mAU




                                                                                     apli-da/h
                    20

                    15

                    10




                                                                                                                                                       aplidine A



                                                                                                                                                                          aplidine B
                     5

                     0

                    -5
                         0                               2         4      6          8                   10                12             14                16             18              20
                                                                                                       Minutes

                                                                                           Time (min)

 Figure 2. Chromatogram at 225 nm of aplidine after incubation without pooled human liver microsomes (1) and
 aplidine after incubation with microsomes (2).
                             relative absorbance




                                                                                                                                                                                       7
                                                                                                                                                                                       6


                                                                                                                                                                                       5
                                                                                                                                                                                       4
                                                                                                                                                                                       3
                                                                                                                                                                                       2
                                                                                                                                                                                       1

                                                   190       200   210   220   230               240        250        260         270           280                290    300

                                                                                       wavelenght (nm)

 Figure 3. UV spectra (190-300 nm) of the aplidine metabolites apli-da/h (1), apli-h 1 and 2 (2 and 3
 respectively), apli-da (4), and apli-dm (5) and aplidine A and B (6 and 7).




                                                                                                       71
Chapter 4

    The structural assignments of aplidine (dehyrodidemnin B) and its metabolites, are based
on the work of Ngoka et al. (1999) on the MS/MS identification of cyclodepsipeptides, e.g.
didemnin B [9]. The protonated ions are very stable under our (CID) conditions where only
the side chain fragment (296 m/z) and the cyclic fragment (817 m/z) are found for
didemnin B. However, the sodium ion selectively binds to ester bonds causing ring opening.
In didemnins, it exclusively binds to the ester group at threonine and not at
hydroxyisovalerylpropionyl group. The selective cleavage (figure 4) leads to a linear acylium
ion and under CID conditions a sequence specific fragmentation occurs from the C-terminus.
However, the ester linkage between leucine and hydroxyisovalerylpropionyl group is not
broken and they will be ejected simultaneous. To elucidate the structure of the aplidine
metabolites, the atomic mass unit (amu) gains or losses were compared with the [M+H]+
(changes in the side chain) and [M+Na]+ (changes in the ring structure) product ion CID
spectra of aplidine.


                                                                                  A
                                         OH      O
                                                                          O
                                                     O
                             O       N                       O        NH
                                             +
                                         Na                  O
                        R                O       O
                             N                       O
                                                             N                                                 O
                                                 N
                                                                                          R=
                                                                                                   O   N
                                                                                               O

                                         O                                                         N
                                                         ring opening                     O


                                 +
        295.4    R       O Na
                                                                                                       B
                 N               575.7                                845.0       942.1                            1133.4

      418.4
                HN       O               O           O
                                 O                                    N           N
                                                         N
                                                                                      +
                     OH      O                                    O           O       C
                                                                                  O                        O



Figure 4. The sodium adduct of aplidine (A) and its selective scission of the depsipeptide ring (included in the
figure are fragment ions that may be observed under MS/MS conditions after collision-induced dissociation)
(B). R = side chain.




                                                             72
                                                                                                                             Structure elucidation of aplidine metabolites

    Aplidine A and B. The product ion spectra for aplidine A and B as [M+H]+ and [M+Na]+
in the mass range of m/z 200 – 1200 amu are illustrated in figure 5A and B, respectively. In
the [M+H]+ product ion spectra, the side chain (295 m/z) and the ring (815 m/z) are found
(table 1). The first fragmentation step for aplidine in the [M+Na]+ product ion spectra is the
loss of the (S)-Me2-tyrosine moiety (-191.2 m/z) and the fragmentation cascade is followed
by the proline (-97.1 m/z), leucine and hydroxyisovalerylpropionyl (- 269.3 m/z), and the
isostatine (-157.2 m/z) moieties (table 2) and finally the threonine group (-123.1 m/z).
Sequence specific fragmentation occurs and aplidine behaves the same as didemnin B.
Therefore, the [M+Na]+ spectra can be used to elucidate the metabolite structures. Aplidine A
and B have identical MS spectra, which agrees with their isomeric structures.


    100
                                                                                                                    A                                                                                  1111




     %




                             295


                                                                                                                                                                                                1083
                                                                                                                                         799



                                                                                                                                               817

            220                   307
                                             349

      0                                                                                                                                                                                                                m /z
      200          250           300         350          400     450            500        550         600       650     700    750     800           850      900     950       1000   1050     1100        1150




    100
          x10
                                                                                                              B                                                        941.7




                                                                                                                                                                                                  1132.8




     %                                                                                                                                                                   942.6                         1133.8


                                         210.2




                                 194.3                                                                        575.4



                   113.1

                         140.1                                                  407.3
                                                                        374.3                                                                                                                          1134.8
                                                 226.2                              418.3
                                                                                                                                                                         943.7
                                                                  365.2                                           576.4
                                 175.3            238.3                                         487.4
                                                                                        443.4                                                           844.6
                97.1                                      292.3 336.3                                                                          798.6
                                                                                                    515.4 557.4                  713.5

      0                                                                                                                                                                                                                m /z
                  100                  200                 300              400                   500              600          700             800              900             1000       1100                1200




Figure 5. Product ion CAD spectra of aplidine A and B as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on
the mass spectrometer. In the [M+H]+ spectrum the 1083 m/z is [M+H]+ with loss of C2H5 from isostatine and
the 799 m/z is the ring with loss of H2O.


                                                                                                                        73
Chapter 4

    Metabolite apli-h 1 and 2. The product ion spectra for apli-h 1 and 2 as [M+H]+ and
[M+Na]+ are illustrated in figure 6A and B respectively. The [M+H]+ exhibits a 16 amu
higher mass in the ring compared to aplidine (table 1). This indicates the gain of an
OH-group in the ring. From the product ions of the [M+Na]+ it can be seen that the OH-group
is attached to the leucine-hydroxyisovalerylpropionyl group (table 2). The product ion of the
leucine-hydroxyisovalerylpropionyl group was further investigated with MSMS of the
product ion. The fragmentation pattern showed that the OH-group was attached to the
isopropyl group of hydroxyisovalerylpropionyl (results not shown). Apli-h 1 and 2 have
identical MS spectra, which can be explained by the cis and trans isomerism as for aplidine.

                                                                                                                                                                                                                                1127

                                                                                                                         A
    100




      %




                             295




                                                                                                                                                                833
                                                                                                                                                          815


                                                                                                                                                                                                                         1099




                               307
                                                                                                                                                                      859
             220                              365
                                                                                                                                                                                                      1013       1069
                                                                                    535 557                                                             797
                                                                                                                         684 707     735                                            917

       0                                                                                                                                                                                                                                      m /z
       200           250      300       350           400       450           500     550           600           650         700         750           800         850        900          950     1000     1050        1100      1150




    aplidine305 53 (4.543) C m (37:146)                                                                                                                                                                                  T O F M S M S 1148.70E S +
           x10                                                                                                                                                        899.6                                                                 2.20e3
     100
                                                                                                                              B
                                                                                                                                                                                                                         1148.8



                                                                                                                                                                                                             1090.7




                                                                                                                                                                                    957.7
                                                                                                                                                                                                                            1149.8



      %
                                                                                                                                                                          900.6                                 1091.7
                                         365.3                                            575.4



                                                    400.3



                                                                                                                                                                                      958.7

             210.3
                                                       418.3

                                                                                                                                                                                                                            1150.7
                       268.3                                          487.4                                                                                                                                     1092.7
             226.2         277.3                                                                              671.4
                                                                                            576.4                                               802.5
                                                            443.5                                         645.5                     756.6                                   901.7
                                                                                    557.4
                                      347.3                               515.4                                                                                                       959.7
                              310.3                              469.4                                             672.4              757.6       803.6


       0                                                                                                                                                                                                                                      m /z
       200           250     300       350          400        450       500        550           600      650          700         750         800           850      900          950      1000    1050      1100      1150          1200




Figure 6. Product ion CAD spectra of apli-h 1 and 2 as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the
mass spectrometer. In the [M+H]+ spectrum the 1099 m/z is [M+H]+ with loss of C2H5 from isostatine and the
815 m/z is the ring with loss of H2O. In the [M+Na]+ spectrum the 1091 m/z and 900 m/z are losses of C4H9
from leucine.



                                                                                                                        74
                                                                                                                     Structure elucidation of aplidine metabolites

    Metabolite apli-da. The [M+H]+ and [M+Na]+ product ion spectra, illustrated in figure
7A and B respectively, show the loss of 56 amu units compared to aplidine. From the [M+H]+
spectra, it appears that the loss is specifically in the side chain (table 1). The loss of 56 amu
units can be explained by the loss of an isopropyl group, the only group which leads to
-56 m/z in the side chain. Therefore, the suggested structure of the metabolite is aplidine
C-dealkylated at the N(Me)-leucine group (apli-da).


    100
                                                                                                             A                                                                                             1055




                                                                                                                                                                                                    1027
     %




                                                                375
                                                  307
                                     239
                  138          210
                                                                                     479
                                                                                                                                                          804                         958
                        164                               349                                                                                749            817                 930

      0                                                                                                                                                                                                                         m /z
       50   100    150        200       250      300      350         400      450     500       550         600         650         700     750          800     850     900     950         1000     1050       1100   1150



                                                                                                                                                 885.30
    100
                                                                                                                   B



                                                                                                                                                    885.68




     %




                                                                                                                                                                                      1076.95



                                                                                                                                                                                1076.13


                                                                                                                                                                                            1077.64
                                                                518.91

                                                                                                                         788.00                                                             1078.08
                                                                                                                                                    887.44
                                               431.41    502.51    520.35
                               362.25

      0                                                                                                                                                                                                                         m /z
      200   250     300       350        400       450      500          550     600       650         700         750         800         850      900         950     1000    1050         1100     1150        1200   1250




Figure 7. Product ion CAD spectra of apli-da as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass
spectrometer. In the [M+H]+ spectrum the 1027 m/z is [M+H]+ with loss of C2H5 from isostatine and the 804 m/z
is the ring with loss of CH.




                                                                                                             75
Chapter 4

    Metabolite apli-da/h. The product ion spectra for apli-da/h as [M+H]+ and [M+Na]+ are
illustrated in figure 8A and B, respectively. The product ions of the [M+H]+ show a loss of
56 amu in the side chain (table 1) and from the product ions of the [M+Na]+ a gain of 16 amu
in the ring structure (table 2) can be seen. This indicates that an isopropyl group has been lost
and an OH-group is gained and thus a combination of the two metabolic conversions
previously mentioned. Thus, aplidine is hydroxylated at the isopropyl group and
C-dealkylated at the N(Me)-leucine group (apli-da/h).
                                                                                                                                                                                                                     1072
    100




                                                                                                                        A




     %




                                                                         375
                                                        307
                                                                                                                                                                                                              1043
                                           239
                                 210
                                                                                                                                                                                                985
                   138
                               192
                                                                                              479
                                                                                                                                                           803
                         164                     268
                                                                                                                                       706
             113                                                                                    497
                                                                                            461                                              741
                                                                   347                                              594             688               786            858 885            946           1013

      0                                                                                                                                                                                                                               m /z
       50   100     150        200          250        300         350         400      450         500     550     600     650       700     750          800      850        900      950          1000     1050      1100   1150




                                                                                                                                              843.5
    100




                                                                                                                    B                                                                       1034.6




                                                                                                                                                                                                        1092.6




                                                                                                                                                                                               1035.6



     %
                                                                                                                                                                                                              1093.7


                                                                                                                                                   844.5


                                                                                                                                                            901.6




                                                                                                                                                                 902.5

                                                                                                                                                                                               1036.6         1094.7
                                                                           519.3
                                                                                           589.4    615.3
                                                                                                                            746.4
                                                                                                                                                   845.5
                                                                                520.3                                                                            903.6
                   288.4                                     457.4                                          673.4   700.4     747.4                                                                  1091.8


      0                                                                                                                                                                                                                               m /z
      200   250      300             350         400         450         500         550           600      650     700      750      800      850           900         950         1000      1050         1100       1150    1200



Figure 8. Product ion CAD spectra of apli-da/h as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass
spectrometer. In the [M+H]+ spectrum the 1043 m/z is [M+H]+ with loss of C2H5 from isostatine, the 985 m/z is
[M+H]+ with loss of C4H9 from leucine, and the 803 m/z is the ring with loss of CH2O. In the [M+Na]+ spectrum
the 1035 m/z and 844 m/z are losses of C4H9 from leucine.




                                                                                                                    76
                                                                                                                              Structure elucidation of aplidine metabolites

    Metabolite apli-dm. The [M+H]+ and [M+Na]+ product ion spectra of metabolite
apli-dm are shown in figure 9A and B. This metabolite shows a loss of 14 amu in the ring
structure (table 1), indicating the loss of a methyl-group. From the comparison of the
[M+Na]+ product ions of aplidine and this metabolite (table 2), it can be concluded that the
demethylation has occured in the last group sequentially deleted from the ring. Therefore,
aplidine is C-demethylated at the threonine group (apli-dm).


    100
                                                                                                                      A                                                                                                      1097




                   295




     %
                                                                                                                                                                                                                      1069




                                                                                                                                              785




                        307

                                                                                                                                                    803
                                    349   375
                                                                              522
                                                                                                                                            773                  845
            268                                                                            575                          683                                                                           999
                                                          440                       547             612                                                                         912
                                                394 413                                      587           637                        749                  827     860                         972             1034
                                                                       496                                                      715                                       889          940

      0                                                                                                                                                                                                                             m /z
      250           300             350          400       450         500          550        600              650           700     750          800           850        900          950          1000        1050       1100




                                                                                                                                                                                                            1118.97
    100

                                                                                                                                B




                                                                                                                                                                                                               1119.84




     %




                                                                                                                                                                       927.45




                                                                                              561.56


                                                                                                                                                   830.91

      0                                                                                                                                                                                                                             m /z
      100         150         200     250        300      350    400         450     500      550         600         650      700    750    800          850      900     950        1000     1050    1100      1150    1200



Figure 9. Product ion CAD spectra of apli-dm as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass
spectrometer. In the [M+H]+ spectrum the 1069 m/z is [M+H]+ with loss of C2H5 from isostatine and the 785 m/z
is the ring with loss of H2O.




                                                                                                                  77
Chapter 4

    The loss of the isopropyl group from the side chain as in apli-da and apli-da/h may result
in the loss of the cis-trans isomerism. The metabolite apli-dm with an intact isopropyl group
will most likely show cis-trans isomerism, but both isomers may not show separation or
possibly one elutes around aplidine A or aplidine B. The attachment of the OH to the
hydroxyisovalerylpropionyl group leads to a more unstable structure in the [M+Na]+.
Furthermore, the structure variations of the metabolites all correspond with their
chromatographic elution order and thus supports the suggested structures of the metabolites.
The gain of an OH-group and the loss of an alkyl group will give a more polar compound,
which will elude before aplidine.

            Table 1. [M+H]+ mass spectrometric results of aplidine and its metabolites.
                                                product ions [M+H]+
                compound           parent ion         side chain             ring
                aplidine A             1111               295                817
                aplidine B             1111               295                817
                  apli-h 1             1127               295                833*
                  apli-h 2             1127               295                833*
                  apli-da              1055              239**               817
                                                             **
                 apli-da/h             1072              239                803***
                  apli-dm              1097               295              803****
       *
              gain of OH-group in 2S,4S-hydroxyisovalerylpropionyl
       **
              loss of C4H8-group from (R)-N(Me)-leucine
       ***
              gain of OH-group in 2S,4S-hydroxyisovalerylpropionyl   and loss of CH2O-group from
              (S)-Me2-tyrosine
       ****
              loss of CH2-group from 1S,2R-threonine



        Table 2. [M+Na]+ mass spectrometric results of aplidine and its metabolites.
                                              product ions [M+Na]+
                                                       loss of
                 parent                                    (S)-Leu3 and
                                      1              2
 compound         ion     (S)-Me2-Tyr        (S)-Pro        2S,4S-Hip4      3S,4R,5S-Ist5
 aplidine A      1132.8       941.7           844.6            575.4             418.3
 aplidine B      1132.8       941.7           844.6            575.4             418.3
   apli-h 1      1148.8       957.7           861.0            575.4             418.3
   apli-h 2      1148.8       957.7           861.0            575.4             418.3
   apli-da       1077.0       885.3           788.0            518.9             362.3
  apli-da/h      1092.6       901.6           804.9            519.3             362.3
   apli-dm       1119.0       927.5           830.9            561.6             404.4
   1
     (S)-Me2-tyrosine
   2
     (S)-proline
   3
     (S)-leucine
   4
     2S,4S-hydroxyisovalerylpropionyl
   5
     3S,4R,5S-istostatine




                                                 78
                                                                 Structure elucidation of aplidine metabolites


Conclusions

    The chemical structures of four metabolites of aplidine generated in vitro have been
identified by mass spectrometric methods. Aplidine hydroxylated at the isopropyl group
(figure 10 A), aplidine C-dealkylated at the N(Me)-leucine group (figure 10 B), aplidine with
both these converted amino acid residues (figure 10 C), and aplidine C-demethylated at the
threonine group (figure 10 D) have been found. Investigation of their presence in clinical
samples can now be started, preferably using LC-MS/MS.


                                                  OH
        (A)                                                        (B)
                                 OH       O                                                   OH       O
                                                        O                                                           O
                                              O                                                            O
                            O   NH                O    NH                                O   NH                O   NH
                        O                                                            O                         O
                                                  O
                                 O        O                                                   O        O
                            N                 O                                          N                 O
                            H                     N                                      H                     N
                O   N                                                        O   N
            O                             N                              O                             N

                N                                                            N
       O                                                            O

                                 O                                                            O




                                                  OH

      (C)                            OH   O                          (D)
                                                                                                  OH   O
                                                            O                                                           O
                                              O                                                            O
                            O   NH                O    NH                                O   NH                O   NH
                        O                         O                                  O                         O
                                     O    O                                                       O    O
                            N                 O                                          N                 O
                            H                     N                                      H                     N
                O   N                                                        O   N
            O                             N                              O                             N

                N                                                            N
        O                                                           O

                                     O                                                            O




Figure 10. Proposal for the structures of discovered metabolites of aplidine: apli-h1 and 2 (A), apli-da (B),
apli-da/h (C), and apli-dm (D).




                                                            79
Chapter 4


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