Synthesis of a novel pentagastrin-drug conjugate for a targeted tumor therapy

Article abstract of DOI:10.1002/chem.200701521

The synthesis of the novel pentagastrin seco-CBI conjugate 3, which is based on the highly cytotoxic antitumor antibiotic (+)-duocarmycin SA (1), is reported. A key step in the synthesis is the palladium-catalyzed carbonylation of aryl bromide 7 to give the benzyl ester 16, which is transformed into the new seco-CBI derivative 21 bearing a carboxylic acid ester moiety. Subsequent transformation of 21 into an activated ester followed by the introduction of ss-alanine and tetragastrin led to the new pentagastrin drug 3 that contains a peptide moiety for targeting cancer cells expressing CCK-B/gastrin receptors.

Full text of DOI:10.1002/chem.200701521

FULL PAPER
DOI: 10.1002/chem.200701521
Synthesis of a Novel Pentagastrin-Drug Conjugate for a Targeted Tumor
Therapy
Lutz F. Tietze,* Olaf Panknin, Felix Major, and Birgit Krewer[a]
Dedicated to Professor Peter Hofmann on the occasion of his 60th birthday
Abstract: The synthesis of the novel
pentagastrin seco-CBI conjugate 3,
which is based on the highly cytotoxic
antitumor antibiotic (+)-duocarmycin
SA (1), is reported. A key step in the
synthesis is the palladium-catalyzed
carbonylation of aryl bromide 7 to give
the benzyl ester 16, which is trans-
formed into the new seco-CBI deriva-
tive 21 bearing a carboxylic acid ester
moiety. Subsequent transformation of
21 into an activated ester followed by
the introduction of b-alanine and tetra-
gastrin led to the new pentagastrin
drug 3 that contains a peptide moiety
for targeting cancer cells expressing
CCK-B/gastrin receptors.
Keywords: antibiotics · antitumor
agents · carbonylation · duocarmy-
cins · pentagastrin · targeting
Introduction
triazene alkylating agent.[5] However, the observed receptor-
mediated cytotoxicity was quite low. Better results were ob-
tained with heptagastrin linked to a potently cytotoxic ellip-
ticine derivative.[6] A high receptor-mediated cytotoxicity
could also be achieved with the anthracyclines daunorubicin,
doxorubicin, and 2-pyrrolinodoxorubicin as well as other cy-
totoxic agents such as melphalan, cisplatin, or methotrexate
coupled to analogues of peptides like LHRH,[3,7] bombe-
One of the main problems in anticancer therapy is the usu-
ally low differentiation between normal and malignant cells
by the known antiproliferating agents, thus causing severe
side effects. An approach to overcome this problem is the
antibody-directed enzyme prodrug therapy (ADEPT).[1,2]
Another promising concept is the use of ligands of low mo-
lecular weight for tumor targeting that are able to transport
diagnostic or antineoplastic agents to the tumor. Among
these ligands, small peptides play an important role, which is
a result of their better tumor penetration and lower immu-
nogenicity relative to antibodies as well as their high specifi-
ties and affinities to receptors that are often overexpressed
on certain cancer cells.[3] Some of these peptides that are al-
ready successfully applied in cancer therapy belong to the
gastrin family. For example, radio labeled gastrin derivatives
have shown a high therapeutic and diagnostic potential in
targeting cholecystokinin (CCK-B)/gastrin receptor express-
ing tumors.[4] In addition, a gastrin analogue was linked to a
AHCTREUNG
sin,[3,7a,8] somatostatin,[3,7a,9] and neuropeptide Y.[3,10] This is
in agreement with our observations that only highly potent
anticancer agents are suitable for such an approach.
Here we describe the synthesis of a gastrin derivative
based on the naturally occurring antibiotic (+)-duocarmycin
SA (1), which is a particularly potent cytotoxic compound
with an IC50 value of 10 pm (L1210).[11]
The antiproliferative effect of 1 and its analogues, such as
CBI 2b, derives most probably from a selective alkylation of
the N-3 atom of adenine in DNA by nucleophilic attack at
the spirocyclopropyl–cyclohexadienone moiety as the phar-
macophoric group.[12] To improve the accessibility of a po-
tential prodrug based on this concept, we decided not to use
CBI 2b but the corresponding seco-compound 2a for conju-
gation with the peptide. It has been shown by us and others
in in vitro experiments that seco-compound 2a has nearly
the same cytotoxicity as the corresponding CBI compound
2b as it can easily cyclize under basic conditions in a so-
called Winstein cyclization to give 2b (Scheme 1).[1,12,13] Fur-
thermore, we decided not to use the whole heptadecapep-
tide gastrin but the shorter b-alanine modified pentagastrin
[a] Prof. Dr. L. F. Tietze, Dr. O. Panknin, Dr. F. Major,
Dipl.-Chem. B. Krewer
Institut für Organische und Biomolekulare Chemie
Georg-August-Universität Gçttingen
Tammannstraße 2, 37077 Gçttingen (Germany)
Fax : (+49)551-39-9476
E-mail: ltietze@gwdg.de
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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NaH in THF provided the E-configurated styrene 10 in
74% yield (Scheme 3).
Selective hydrolysis of the tert-butyl ester in 10 with a
TFA/water mixture followed by a Friedel–Crafts acylation
by using Ac2O and NaOAc yielded 12 in 78% via 11 over
two steps. Then, deprotection of the O-acetate moiety in 12
with K2CO3 in EtOH and reprotection under standard con-
ditions by using benzyl bromide, K2CO3, and catalytic
amounts of TBAI in DMF afforded benzyl ether 14 via 13 in
95% yield over two steps. After this, ethyl ester 14 was hy-
drolyzed with LiOH·H2O to give carboxylic acid 7 in 85%
yield. Finally, a Curtius rearrangement of 7 with DPPA and
NEt3 in tBuOH led directly to the protected naphtholamine
15 in 85% yield.
For the introduction of the carboxylic acid moiety in 15,
which is necessary for coupling with pentagastrin, we tried
to perform a palladium-catalyzed carbonylation of 15. How-
ever, this gave only a moderate yield, probably due to the
high electron density of the substrate resulting in a low reac-
tion rate of the oxidative addition as the first step. We,
therefore, used bromide 7 which contains an electron-with-
drawing group (Scheme 4). But when using 7 as the sub-
strate, the new carboxylic ester group had to be introduced
as an easily cleavable ester to enable a differentiation be-
tween the two carboxyl moieties then existing in the mole-
cule. However, a corresponding published carbonylation of
an aryl bromide by using benzyl alcohol gave the corre-
sponding benzyl ester in only 30% yield.[17]
Scheme 1. Structures of (+)-duocarmycin SA (1), seco-CBI 2a, and CBI
2b.
because its b-Ala-Trp-Met-Asp-Phe-NH2 sequence repre-
senting the C-terminal amide of the natural peptides re-
stores the whole biological activity of gastrin in a compara-
ble order of magnitude.[3e,14] As a consequence, the cytotoxic
agent had to be attached to the N-terminal amino function
of pentagastrin. This resulted in the requirement of a seco-
CBI derivative with a carboxylic acid moiety by which an
amide linkage to the peptide could be achieved. This linkage
should be located in a position that neither the receptor af-
finity nor the drugꢁs mode of action should be influenced.
Therefore, we decided to pre-
Fortunately, heating 7 under a CO atmosphere (1 bar)
with [PdBr2ACHTERUGN ACHTREUNG(nBu)3
(PPh3)2] as the catalyst and dppf[18] and N
pare molecule 3, the synthetic
strategy of which is outlined in
Scheme 2.
Thus, coupling of the central
seco-CBI unit
5 with TMI-
CO2H 6 as the DNA-binding
subunit and finally with penta-
gastrin (4) should provide 3.
The introduction of the carbox-
ylic acid moiety in 5 could be
performed by means of a palla-
dium-catalyzed carbonylation
of bromide 7.
Results and Discussion
The synthesis of the necessary
substrate 7 was performed ac-
cording to an approach devel-
oped by Boger et al. for a simi-
lar system.[15] Thus, a Horner–
Wadsworth–Emmons reaction
between
the
commercially
available 4-bromobenzaldehyde
(8) and phosphonate 9[16] with Scheme 2. Retrosynthetic analysis of drug 3.
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Targeted Tumor Therapy
FULL PAPER
tions in an aqueous HCl/
EtOAc mixture with Et3SiH as
the
cation
scavenger[23]
(Scheme 5). The obtained hy-
drochloride 20 was directly
coupled with TMI-CO2H 6,[24]
employing EDC·HCl as the
coupling reagent to give 21 in
63% yield over two steps. To
connect 21 with the peptide,
the benzyl ester was selectively
cleaved with LiOH·H2O to
afford 22 in 79% yield without
any loss of the primary chlo-
ride.
Coupling with pentagastrin
was performed in a stepwise
manner. Earlier attempts to
couple pentagastrin directly to
the aromatic carboxylic acid 22
Scheme 3. Synthesis of precursors 7 and 15: a) NaH, THF, 0–208C, 22.5 h, 74%; b) TFA/H2O, 20 8C, 4.5 h,
quant.; c) Ac2O, NaOAc, reflux, 1 h, 78%; d) K2CO3, EtOH, reflux, 1.5 h, 95%; e) K2CO3, BnBr, TBAI, DMF,
208C, 1 d, quant.; f) LiOH·H2O, THF/MeOH/H2O, 20 8C, 3 d, 85%; g) DPPA, tBuOH, NEt3, MS (4) Š),
reflux, 2.5 d, 85%. DPPA=diphenylphosphoryl azide; TBAI=tetrabutylammonium iodide; TFA=trifluoro-
acetic acid.
were not successful. To diminish complications, we decided
to couple the seco-CBI scaffold with b-alanine (24) first.
This amino acid is part of the pentagastrin sequence and
should at the same time serve as a spacer unit. To avoid pro-
tection steps we first synthesized the active ester of 22. As
activator HOSu[5] was used, which gave active ester 23 upon
coupling with acid 22 by using EDC·HCl. This ester was re-
acted without further purification with unprotected b-ala-
nine (24), employing NEtiPr2 as the base to give 25 in 69%
yield over two steps. At this stage, the benzyl ether moiety
in 25 had to be cleaved because any attempts to cleave the
ether group after the introduction of the pentagastrin
Scheme 4. Synthesis of seco-CBI compound 5: a) 1 bar CO, 5 mol%
[PdBr2A(PPh3)2], 20 mol% dppf, (nBu)3, BnOH, 85–1308C, 95 min,
C
NACHTREUNG
82%; b) DPPA, tBuOH, NEt3, MS (4) Š, reflux, 3.5 d, 65%; c) NIS,
TsOH·H2O, THF/MeOH, 508C, 40 min, 99%; d) NaH, 1,3-dichloropro-
pene, DMF, 208C, 20.5 h, 85%; e) HSiCATHRE(UNG SiMe3)3, AIBN, benzene, reflux,
3 h, 87%. AIBN=azo-bis-isobutyronitrile; Boc=tert-butoxycarbonyl;
dppf=bis(diphenylphosphino)ferrocene; NIS=N-iodosuccinimide; Ts=
tosyl.
in benzylic alcohol as the solvent afforded the benzyl ester
16 in an excellent yield of 82%.[19] After Curtius rearrange-
ment under the aforementioned conditions to give 17 and
iodination, employing NIS[15,20] and by using TsOH·H2O as
the catalyst, the iodide 18 was obtained in 64% yield over
two steps. N-Alkylation of the generated sodium salt of 18
by using 1,3-dichloropropene afforded an E/Z mixture of
alkene 19. This alkene was submitted to a 5-exo-trig radical
cyclization[21] by using the comparatively untoxic tris(trime-
thylsilyl)silane (TTMSS)[22] as the hydride source and AIBN
as the radical starter, providing seco-CBI derivative 5 in
74% yield over two steps.
Scheme 5. Synthesis of 25: a) HCl/EtOAc, Et3SiH, 208C, 3 h; b) 6,
EDC·HCl, DMF, 208C, 1 d, 63% (two steps); c) LiOH·H2O, THF/
MeOH/H2O, 0–208C, 3.5 h, 79%; d) HOSu, EDC·HCl, THF, 0–208C,
13 h; e) NEtiPr2, H2O/MeCN, 208C, 7 h, 69% (two steps). EDC·HCl=1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOSu=N-
hydroxysuccinimide.
For the coupling reaction with the DNA-binding indole
subunit, the Boc group in 5 was cleaved under acidic condi-
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2813

L. F. Tietze et al.
moiety were not successful. The debenzylation was achieved
under transfer hydrogenolytic conditions with an aqueous
ammonium formate solution and with palladium on charcoal
as the catalyst[25] to afford the phenol 26, which was used in
the next reaction step without any purification. Thus, 26 was
transferred into the active ester 27 with HOSu under the
aforementioned conditions; then, 27 was directly coupled
with the fully unprotected tetrapeptide 28[26] to give penta-
gastrin seco-CBI drug 3 in 27% yield over three steps
(Scheme 6).
Experimental Section
General: All reactions were performed in flame-dried glassware under
an argon atmosphere. Solvents were dried and purified according to stan-
dard procedures and redistilled prior to use. TLC chromatography was
performed on precoated aluminum silica gel SIL G/UV254 plates (Ma-
cherey–Nagel) and silica gel 60 (0.040–0.063 mm) (Merck) was used for
column chromatography. IR spectroscopy: Bruker Vector 22. UV/VIS:
Perkin–Elmer Lambda 2. 1H NMR spectroscopy: Varian Mercury-200,
Unity-300 (300 MHz), Unity Inova-600 (600 MHz). 13C NMR spectrosco-
py: Varian Mercury-200 (50 MHz), Unity-300 (75 MHz), Unity Inova-600
(150 MHz). For 1H and 13C NMR spectroscopy, CDCl3, [D6]DMSO, and
[D7]DMF were used as solvents. Chemical shifts are reported on a d
scale. Signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), mc (centered multiplet), and br (broad). MS: Finnigan
MAT 95, TSQ 7000, LCQ. HRMS was carried out by using, among
others, a modified peak matching technique, error ꢀ2 ppm, with a resolu-
tion of ca. 10,000. Elemental analysis: Mikroanalytisches Labor des Insti-
tutes für Organische und Biomolekulare Chemie der Universität Gçttin-
gen.
tert-Butyl-(E)-3-(ethoxycarbonyl)-4-(4-bromophenyl)-3-butenoate (10):
NaH (13.0 g, 60% in oil, 326 mmol) was washed with dry n-pentane,
dried for ca. 15 min under reduced pressure, and then suspended in THF
(500 mL). Phosphonate 9 (296 mmol, 100 g) was added dropwise at 08C
and stirring was continued at 208C for 4.5 h. The mixture was again
cooled to 08C and added by cannula to a solution of 4-bromobenzalde-
hyde (8) (60.3 g, 326 mmol) in THF (250 mL) also maintained at 08C.
The reaction mixture was warmed to 208C, stirred for a further 18 h, and
the solvent removed under reduced pressure. The ensuing red residue
was dissolved in CH2Cl2 (300 mL), washed with water (3”100 mL), brine
(100 mL), dried (MgSO4), filtered, and concentrated under reduced pres-
sure. The crude product (81.0 g, 74%) was used for the next reaction
without further purification. An analytically pure sample was obtained
after column chromatography by using pentane/Et2O (10:1) as the
eluent. Rf =0.44 (pentane/Et2O 4:1); 1H NMR (200 MHz, CDCl3): d=
1.33 (t, J=7.0 Hz, 3H; OCH2CH3), 1.45 (s, 9H; CACHTREU(GN CH3)3), 3.40 (s, 2H; 2-
Scheme 6. Synthesis of seco-CBI pentagastrin drug 3: a) Pd/C,
NH4HCO2, THF/MeOH, 208C, 2.5 h; b) HOSu, EDC·HCl, THF/CH2Cl2/
DMF, 0–208C, 28.5 h; c) NEtiPr2, DMF, 208C, 57 h, 27% (three steps).
H2), 4.27 (q, J=7.0 Hz, 2H; OCH2CH3), 7.20–7.25 (m, 2H; Ar-H), 7.49–
7.54 (m, 2H; Ar-H), 7.76 ppm (s, 1H; 4-H); 13C NMR (75 MHz, CDCl3):
d=13.85 (OCH2CH3), 27.54 (CACTHRE(UNG CH3)3), 34.36 (C-2), 60.64 (OCH2CH3),
80.52 (C(CH3)3), 122.54 (C-3), 127.17 (C-4’), 130.16, 131.32 (C-2’, C-3’, C-
AHCTREUNG
5’, C-6’), 133.65 (C-1’), 139.34 (C-4), 166.58 (C(O)OEt), 169.48 ppm (C-
1); MS (DCI, NH3, 200 eV): m/z (%): 754 (4) [2M+NH4]+, 386 (100)
[M+NH4]+, 369 (12) [M+H]+.
Both reactions, the activation step and the coupling step,
showed only slow conversion with the last transformation
giving the lowest yields, but no significant formation of side
products was observed, although the phenolic hydroxyl
group was unprotected. We assume that this is due to the
low nucleophilicity of the phenol moiety; however, under
basic conditions, a fast cyclization of the seco-CBI to the
CBI-pharmacophore takes place.
(E)-3-Ethoxycarbonyl-4-(4-bromophenyl)-but-3-enoic acid (11): Crude 10
(75.0 g, 203 mmol) was dissolved in 9:1 TFA/H2O (145 mL) and stirred
for 4.5 h at 208C. The solvent was removed under reduced pressure and
the resulting residue treated with toluene (2”100 mL) and then again
concentrated under reduced pressure. After cooling to 08C, the residue
was treated with NaHCO3 (150 mL, saturated solution), adjusted to pH 1
with HCl (2 n) and extracted with EtOAc (4”100 mL). The combined or-
ganic phases were washed with brine (100 mL), dried (MgSO4), and con-
centrated under reduced pressure. The crude product (76.0 g, quant.) was
afforded as a yellow oil and used for the next reaction without further
purification. An analytically pure sample was obtained after column
chromatography by using pentane/EtOAc 2:1 as the eluent. Rf =0.30
(pentane/EtOAc 2:1); 1H NMR (200 MHz, CDCl3): d=1.35 (t, J=
7.3 Hz, 3H; OCH2CH3), 3.54 (s, 2H; 2-H2), 4.31 (q, J=7.3 Hz, 2H;
OCH2CH3), 7.22–7.26 (m, 2H; Ar-H), 7.53–7.57 (m, 2H; Ar-H), 7.84 (s,
1H; 4-H), 10.05 ppm (brs, 1H; CO2H); 13C NMR (75 MHz, CDCl3): d=
14.15 (OCH2CH3), 33.54 (C-2), 61.53 (OCH2CH3), 123.40 (C-3), 126.12
(C-4’), 130.51, 131.95 (C-2’, C-3’, C-5’, C-6’), 133.58 (C-1’), 141.07 (C-4),
167.14 (C(O)OEt), 176.77 ppm (C-1); MS (DCI, NH3, 200 eV): m/z (%):
642 (2) [2M+NH4]+, 347 (12) [M+NH3+NH4]+, 330 (65) [M+NH4]+,
286 (100) [MꢁCO2+NH4]+.
Conclusions
We have designed the new cytotoxic compound 3 for a se-
lective treatment of cancer based on the assumption that the
pentagastrin moiety allows a selective incorporation of 3
into cancer cells which overexpress the CCK-B/gastrin re-
ceptor. To guaranty a high reactivity of the drug, we used a
seco-duocarmycin analogue with a carboxylic acid moiety at
one of the aromatic rings of the CBI pharmacophore to
allow the introduction of the pentagastrin by an amide
bond. The biological activity of this new type of anticancer
agent is currently being investigated in our cell-culture lab
by using cancer cell lines expressing the gastrin receptor.
Ethyl-1-acetoxy-7-bromo-3-naphthalene carboxylate (12): A solution of
the crude acid 11 (63.6 g, 203 mmol) in acetic anhydride (1.20 l) was
treated with sodium acetate (29.9 g, 305 mmol) and stirred for 1 h under
reflux. The hot reaction mixture was poured into water (2.00 l) and
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Targeted Tumor Therapy
FULL PAPER
cooled to 208C. The precipitated product (54.0 g, 78%) was collected by
filtration and used for the next reaction without further purification. Rf =
0.50 (pentane/EtOAc 4:1); 1H NMR (200 MHz, CDCl3): d=1.44 (t, J=
7.1 Hz, 3H; OCH2CH3), 2.50 (s, 3H; CH3), 4.43 (q, J=7.1 Hz, 2H;
OCH2CH3), 7.65 (dd, J=8.4, 1.9 Hz, 1H; 6-H), 7.85 (d, J=8.4 Hz, 1H; 5-
H), 7.86 (d, J=1.6 Hz, 1H; 2-H), 8.04 (d, J=1.9 Hz, 1H; 8-H), 8.47 ppm
(brs, 1H; 4-H); 13C NMR (75 MHz, CDCl3): d=14.33 (OCH2CH3), 21.00
(C(O)CH3), 61.48 (OCH2CH3), 118.78 (C-2), 123.48, 128.21, 129.97 (C-3,
C-4a, C-8a), 123.83, 128.59, 130.81, 131.02 (C-4, C-5, C-6, C-8), 132.08 (C-
7), 145.62 (C-1), 165.59 (C(O)OEt), 169.08 ppm (C(O)CH3); MS (EI,
70 eV): m/z (%): 336 (12) [M]+, 294 (100) [MꢁC(O)CH3+H]+.
Ethyl-7-bromo-1-hydroxy-3-naphthalene carboxylate (13): A solution of
crude 12 (53.3 g, 158 mmol) in ethanol (650 mL) was treated with K2CO3
(109 g, 790 mmol) and stirred for 1.5 h under reflux. The reaction mixture
was poured into water (1.00 l) and cooled to 208C, adjusted to pH 1 with
HCl (2 n) and extracted with EtOAc (3”150 mL). The combined organic
phases were washed with brine (150 mL), dried (MgSO4) and concentrat-
ed under reduced pressure. The resulting light-brown solid (44.0 g, 95%)
was used for the next reaction without further purification. Rf =0.36
(pentane/EtOAc 4:1); 1H NMR (200 MHz, CDCl3): d=1.35 (t, J=
7.0 Hz, 3H; OCH2CH3), 4.33 (q, J=7.0 Hz, 2H; OCH2CH3), 7.43 (d, J=
1.4 Hz, 1H; 2-H), 7.50 (dd, J=8.8, 2.0 Hz, 1H; 6-H), 7.67 (d, J=8.8 Hz,
1H; 5-H), 7.99 (brs, 1H; 4-H), 8.37 (d, J=2.0 Hz, 1H; 8-H), 9.70 ppm
(brs, 1H; OH); 13C NMR (50 MHz, CDCl3): d=14.20 (OCH2CH3), 60.84
(OCH2CH3), 108.08 (C-2), 120.99, 128.14, 128.35 (C-3, C-4a, C-8a),
121.40, 124.91, 129.94, 130.29 (C-4, C-5, C-6, C-8), 131.93 (C-7), 152.53
(C-1), 166.49 ppm (C(O)OEt); MS (EI, 70 eV): m/z (%): 294 (100) [M]+,
266 (22) [MꢁC2H4]+, 249 (40) [MꢁC2H5O]+.
Ethyl-1-benzyloxy-7-bromo-3-naphthalene carboxylate (14): A magneti-
cally stirred solution of crude 13 (44.3 g, 150 mmol) in DMF (950 mL)
was treated with K2CO3 (31.0 g, 225 mmol) and TBAI (2.2 g, 6.0 mmol).
Benzyl bromide (21.5 mL, 30.8 g, 180 mmol) was added dropwise with
stirring and the resulting mixture was stirred for 1 d at 208C. The reac-
tion mixture was poured into water (1.20 l) and extracted with CH2Cl2
(3”100 mL). The combined organic phases were washed with water (1”
100 mL) and brine (100 mL), dried (MgSO4), and concentrated under re-
duced pressure. The crude product (62.0 g, quant.) was afforded as a
brown oil and used for the next reaction without further purification. An
analytically pure sample was obtained after column chromatography by
using pentane/EtOAc 15:1 as the eluent. Rf =0.59 (pentane/EtOAc 4:1);
1H NMR (200 MHz, CDCl3): d=1.45 (t, J=7.2 Hz, 3H; OCH2CH3), 4.44
(q, J=7.2 Hz, 2H; OCH2CH3), 5.29 (s, 2H; CH2Ph), 7.35–7.58 (m, 6H;
5”Ph-H, 2-H), 7.62 (dd, J=8.6, 1.7 Hz, 1H; 6-H), 7.77 (d, J=8.6 Hz,
1H; 5-H), 8.18 (brs, 1H; 4-H), 8.48 ppm (d, J=1.7 Hz, 1H; 8-H);
13C NMR (50 MHz, CDCl3): d=14.38 (OCH2CH3), 61.28 (OCH2CH3),
70.47 (CH2Ph), 105.10 (C-2), 122.10, 128.33, 128.82 (C-3, C-4a, C-8a),
123.32, 124.88, 128.22, 130.49, 130.61 (C-4, C-5, C-6, C-8, Ph-Cp), 127.69,
128.68 (Ph-Co, Ph-Cm), 131.88 (C-7), 136.30 (Ph-Ci), 153.62 (C-1),
166.50 ppm (C(O)OEt); MS (DCI, NH3, 200 eV): m/z (%): 419 (15)
[M+NH3+NH4]+, 402 (39) [M+NH4]+.
3-Amino-1-benzyloxy-7-bromo-N-(tert-butoxycarbonyl)naphthalene (15):
A suspension of acid 7 (12.4 g, 34.7 mmol) in dry tert-butanol (850 mL)
was treated with activated molecular sieves 4 Š (70 g), triethylamine
(5.80 mL, 4.20 g, 41.6 mmol), and DPPA (9.00 mL, 11.4 g, 41.6 mmol) and
heated for 2.5 d at reflux without stirring. After cooling to 208C, the mo-
lecular sieves were filtered off and washed thoroughly with EtOAc. The
filtrate was concentrated under reduced pressure and the resulting resi-
due was dissolved in EtOAc (300 mL). The organic phase was washed
with HCl (2 n, 2”100 mL), NaHCO3 (1”100 mL, saturated solution),
and brine (100 mL), and then dried (MgSO4) and concentrated under re-
duced pressure. Recrystallization of the resulting residue gave the title
compound along with a brown oil which was purified by column chroma-
tography (pentane/EtOAc 7:1) to yield further product. The protected
amine 18 (12.7 g, 85%) was afforded as a white solid. Rf =0.27 (pentane/
EtOAc 10:1); 1H NMR (200 MHz, CDCl3): d=1.55 (s, 9H; C
ACHTREUNG(CH3)3),
5.22 (s, 2H; CH2Ph), 6.61 (brs, 1H; NH), 7.06 (d, J=1.8 Hz, 1H; 2-H),
7.30–7.60 (m, 8H; 5”Ph-H, 4-H, 5-H, 6-H), 8.34 ppm (d, J=1.8 Hz, 1H;
8-H); 13C NMR (50 MHz, CDCl3): d=28.35 (C
ACTHERN(GU CH3)3), 70.34 (CACHTREUNG(CH3)3),
80.87 (CH2Ph), 99.95 (C-2), 106.66 (C-4), 117.56, 123.52 (C-7, C-8a),
124.50, 128.16, 128.51, 130.39 (C-5, C-6, C-8, Ph-Cp), 127.56, 128.67 (Ph-
Co, Ph-Cm), 133.20 (C-4a), 136.39, 136.64 (C-3, Ph-Ci), 152.65, 154.30 ppm
(C-1, C=O); MS (EI, 70 eV): m/z (%): 427 (9) [M]+, 371 (16)
[MꢁC4H8]+, 91 (100) [C7H7]+, 57 (24) [C4H9]+.
1-Benzyloxy-7-benzyloxycarbonyl-3-naphthalene carboxylic acid (16): A
magnetically stirred and degassed solution of bromide 7 (100 mg, 280
mmol) and
NACHTREUNG
(5.00 mL, 5.20 g, 48.0 mmol) was treated with [PdBr CTHREUNG
14 mmol) and 1,1’-bis(diphenylphosphino)ferrocene (31 mg, 56 mmol). The
reaction mixture was degassed again, set under a carbon monoxide at-
mosphere (1 bar) and stirred for 45 min at 858C and for a further 50 min
at 1308C. The benzylic alcohol and tributylamine were distilled off under
reduced pressure and the resulting residue quenched with HCl (2 n) and
water (pH 2). The water phase was extracted with EtOAc (4”50 mL)
and the combined organic phases washed with brine (50 mL), dried
(MgSO4), and then concentrated under reduced pressure. The resulting
brown oil was adsorbed on silica gel and purified by column chromatog-
raphy (pentane/EtOAc 1:1+0.5% HOAc). The acid 16 (94 mg, 82%)
was obtained as a white solid. Rf =0.29 (pentane/EtOAc 4:1+0.5%
HOAc); 1H NMR (300 MHz, [D6]DMSO): d=5.40 (s, 4H; 2”CH2Ph),
7.35–7.60 (m, 11H; 10”Ph-H, 2-H), 8.07 (dd, J=8.7, 1.5 Hz, 1H; 6-H),
8.18 (d, J=8.7 Hz, 1H; 5-H), 8.26 (s, 1H; 4-H), 8.90 (brs, 1H; 8-H),
13.00 ppm (brs, CO2H); 13C NMR (75 MHz, [D6]DMSO): d=66.37
(CH2Ph-ester), 69.84 (CH2Ph-ether), 105.43 (C-2), 122.47, 124.09, 125.96,
129.72 (C-4, C-5, C-6, C-8), 126.04, 128.11, 131.17, 135.27, 135.94, 136.51
(C-3, C-4a, C-7, C-8a, 2”Ph-Ci), 127.37, 127.80, 127.90, 128.02, 128.43
(10”Ph-C), 154.69 (C-1), 165.29, 167.04 ppm (2”C=O); MS (EI, 70 eV):
m/z (%): 412 (13) [M]+, 91 (100) [C7H7]+.
3-Amino-1-benzyloxy-7-benzyloxycarbonyl-N-(tert-butoxycarbonyl)naph-
thalene (17): A suspension of acid 16 (802 mg, 1.94 mmol) in dry tert-bu-
tanol (50 mL) was treated with activated molecular sieves 4 Š (4 g), trie-
thylamine (327 mL, 236 mg, 2.33 mmol), and DPPA (505 mL, 641 mg,
2.33 mmol) and heated for 3.5 d at reflux without stirring. After cooling
to 208C, the reaction mixture was filtered through a pad of Celite and
then it was thoroughly washed with EtOAc. The filtrate was concentrated
under reduced pressure and the resulting residue dissolved in EtOAc
(100 mL). The organic phase was washed with HCl (2 n, 2”50 mL),
NaHCO3 (1”50 mL, saturated solution), and brine (50 mL), and then
dried (MgSO4) and concentrated under reduced pressure to give a light-
brown oil. This material then underwent column chromatography (pen-
tane/EtOAc 7:1) to give the protected amine 17 (534 mg, 65%) as a
light-yellow solid. Rf =0.22 (pentane/EtOAc 10:1); 1H NMR (300 MHz,
1-Benzyloxy-7-bromo-3-naphthalene carboxylic acid (7): A solution of
crude 14 (57.8 g, 150 mmol) in 3:1:1 THF/MeOH/H2O (1.25 l) was treat-
ed with LiOH·H2O (31.5 g, 750 mmol) and stirred for 3 d at 208C. The re-
sulting mixture was adjusted to pH 1 with HCl (2 n) and the precipitated
crude product was collected by filtration. The filtrate was extracted with
EtOAc (3”100 mL), the combined organic phases dried (MgSO4), and
concentrated under reduced pressure to give an additional amount of
crude product. The combined crude products were recrystallized
(EtOAc) to afford acid 7 (45.7 g, 85%) as a white solid. Rf =0.17 (pen-
tane/EtOAc 2:1); 1H NMR (300 MHz, [D6]DMSO): d=5.34 (s, 2H;
CH2Ph), 7.32–7.45 (m, 3H; 3”Ph-H), 7.51–7.56 (m, 3H; 2”Ph-H, 2-H),
7.70 (dd, J=8.8, 1.8 Hz, 1H; 6-H), 8.01 (d, J=8.8 Hz, 1H; 5-H), 8.20 (s,
1H; 4-H), 8.28 (d, J=1.8 Hz, 1H; 8-H), 13.00 ppm (brs, 1H; CO2H);
13C NMR (75 MHz, [D6]DMSO): d=69.83 (CH2Ph), 105.62 (C-2), 121.30,
127.90, 129.20 (C-3, C-4a, C-8a), 122.89, 123.62, 127.93, 130.22, 131.31 (C-
4, C-5, C-6, C-8, Ph-Cp), 127.51, 128.49 (Ph-Co, Ph-Cm), 131.63 (C-7),
136.53 (Ph-Ci), 152.85 (C-1), 167.08 ppm (C(O)OH); MS (EI, 70 eV):
m/z (%): 356 (11) [M]+, 91 (100) [C7H7]+.
CDCl3): d=1.56 (s, 9H; CACTHRE(UGN CH3)3), 5.24 (s, 2H; CH2Ph-ether), 5.42 (s,
2H; CH2Ph-ester), 6.81 (brs, 1H; NH), 7.10 (d, J=1.8 Hz, 1H; 2-H),
7.30–7.55 (m, 11H; 10”Ph-H, 4-H), 7.69 (d, J=8.7 Hz, 1H; 5-H), 8.06
(dd, J=8.7, 2.1 Hz, 1H; 6-H), 9.04 ppm (brs, 1H; 8-H); 13C NMR
(125 MHz, CDCl3): d=28.30 (CACTHRE(UNG CH3)3), 66.52 (CH2Ph-ester), 70.22
(CH2Ph-ether), 80.98 (C(CH3)3), 99.56 (C-2), 106.32 (C-4), 121.56, 125.00,
AHCTREUNG
136.28, 136.40, 137.27 (C-4a, C-7, C-8a, 2”Ph-Ci), 125.70, 126.62, 126.93,
127.26, 127.96, 127.99, 128.05, 128.53, 128.60 (C-5, C-6, C-8, 10”Ph-C),
Chem. Eur. J. 2008, 14, 2811 – 2818
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2815

L. F. Tietze et al.
138.88 (C-3), 152.53, 156.21 (C-1, C=O), 166.76 ppm (C(O)OBn); MS
(EI, 70 eV): m/z (%): 483 (14) [M]+, 427 (18) [MꢁC4H8]+, 91 (100)
[C7H7]+, 57 (15) [C4H9]+.
13C NMR (50 MHz, CDCl3): d=28.42 (C
10), 53.14 (C-2), 66.57 (CH2Ph-ester), 70.38 (CH2Ph-ether), 81.06 (C-
(CH3)3), 97.00 (C-4), 121.48, 124.33, 132.51 (C-3a, C-5a, C-7, C-9a, C-9b),
ACHTRE(UNG CH3)3), 41.45 (C-1), 46.36 (C-
AHCTREUNG
121.81 (C-9), 127.14 (C-6), 127.27 (C-8), 127.43, 127.94, 128.04, 128.08,
128.56, 128.62 (10”Ph-C), 136.24, 136.33 (2”Ph-Ci), 152.42 (C-5), 157.27
(C=O), 166.57 ppm (C(O)OBn); UV/Vis (CH3CN): lmax (lg e)=205
(4.496), 272 (4.757), 355 nm (4.184); IR (KBr): n˜ =3403 (NH), 2976, 1704
(C=O), 1373, 1327, 1248, 1141, 848, 761 cmꢁ1; MS (EI, 70 eV): m/z (%):
557 (16) [M]+, 501 (25) [MꢁC7H7+H]+, 91 (60) [C7H7]+, 56 (76) [C4H8]+
, 41 (100) [CH3CN]; elemental analysis calcd (%) for C33H32ClNO5
(558.06): C 71.02, H 5.78; found: C 70.98, H 5.98.
2-Amino-4-benzyloxy-6-benzyloxycarbonyl-N-(tert-butoxycarbonyl)-1-io-
donaphthalene (18):
A magnetically stirred solution of 17 (1.56 g,
3.23 mmol) in 1:1 THF/MeOH (44 mL) was treated with a solution of
TsOH·H2O (61 mg, 0.32 mmol) in THF (2 mL) and N-iodosuccinimide
(1.45 g, 6.46 mmol). The resulting mixture was warmed to 508C, and
stirred for a further 40 min. The reaction mixture was quenched with
NaHCO3 (20 mL, saturated solution) and water and extracted with
EtOAc (2”40 mL). The combined organic phases were washed with
Na2S2O3 (1”50 mL, saturated solution) and brine (50 mL), dried
(MgSO4) and then concentrated under reduced pressure. The afforded
pale-yellow solid (1.94 g, 99%) was used for the next reaction without
AHCTRE(GNU 1R/S)-5-Benzyloxy-1-chloromethyl-3-(5,6,7-trimethoxyindole-2-carbon-
yl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid benzyl ester (21): A
solution of the protected amine 5 (275 mg, 493 mmol) in CH2Cl2 (2 mL)
was treated with HCl (14 mL of a 4m solution in EtOAc) and Et3SiH
(80.0 mL, 57.0 mg, 493 mmol) and stirred for 3 h at 208C. The solvent was
removed under reduced pressure and the ensuing residue was treated
with toluene (2”10 mL) and then concentrated under reduced pressure
again.
1
further purification. Rf =0.40 (pentane/EtOAc 10:1); H NMR (300 MHz,
CDCl3): d=1.60 (s, 9H; CACTHRE(UGN CH3)3), 5.31 (s, 2H; CH2Ph-ether), 5.42 (s,
2H; CH2Ph-ester), 7.32–7.59 (m, 10H; 10”Ph-H), 8.04 (dd, J=8.7,
0.6 Hz, 1H; 8-H), 8.12 (dd, J=8.7, 1.8 Hz, 1H; 7-H), 8.14 (s, 1H; 3-H),
9.01 ppm (d, J=1.8 Hz, 1H; 5-H); 13C NMR (125 MHz, CDCl3): d=
28.29 (C
ACHTREUNG(CH3)3), 66.67 (CH2Ph-ester), 70.46 (CH2Ph-ether), 79.14 (C-
The resulting crude hydrochloride 20 was dried under reduced pressure
and then treated with 5,6,7-trimethoxyindole-2-carboxylic acid (6)
(136 mg, 543 mmol), EDC·HCl (284 mg, 1.48 mmol), and DMF (12 mL)
and stirred for 1 d at 208C. The reaction mixture was adjusted to pH 2
with HCl (2 n) and extracted with EtOAc (4”15 mL). The combined or-
ganic phases were washed with water (3”20 mL), brine (20 mL), dried
(MgSO4) and concentrated under reduced pressure to give a green solid.
This material was adsorbed on silica gel and purified by column chroma-
tography (pentane/EtOAc 2:1) to yield TMI-derivative 21 (214 mg, 63%
ACHTREUNG(CH3)3), 81.57 (C-1), 100.13 (C-3), 122.71, 125.67, 136.13, 136.17, 137.18
(C-4a, C-6, C-8a, 2”Ph-Ci), 125.94, 127.67, 127.97, 128.09, 128.10, 128.12,
128.57, 128.59, 131.45, (C-5, C-7, C-8, 10”Ph-C), 140.69 (C-2), 152.50,
156.58 (C-4, C=O), 166.28 ppm (C(O)OBn); UV/Vis (CH3CN): lmax (lg
e)=224 (4.099), 268 (4.477), 340 nm (3.557); IR (KBr): n˜ =3396 (NH),
1720 (C=O), 1602, 1518, 1385, 1235, 1155, 731 cmꢁ1; MS (EI, 70 eV): m/z
(%): 609 (38) [M]+, 553 (26) [MꢁC4H8]+, 426 (6) [MꢁC4H8ꢁI]+, 91
(100) [C7H7]+, 57 (16) [C4H9]+.
A
over two steps) as a yellow solid. Rf =0.39 (pentane/EtOAc 2:1);
A
1H NMR (600 MHz, CDCl3): d=3.44 (dd, J=11.4, 10.2 Hz, 1H; 10-Hb),
3.86–4.06 (m, 11H; 3”OCH3, 1-H, 10-Ha), 4.58 (dd, J=10.2, 9.0 Hz, 1H;
2-Hb), 4.72 (dd, J=10.2, 1.8 Hz, 1H; 2-Ha), 5.25–5.31 (m, 2H; CH2Ph-
ether), 5.37–5.42 (m, 2H; CH2Ph-ester), 6.86 (s, 1H; 4’-H), 6.97 (d, J=
2.4 Hz, 1H, 3’-H), 7.33–7.52 (m, 10H; 10”Ph-H), 7.62 (d, J=9.0 Hz, 1H;
9-H), 8.10 (dd, J=9.0, 1.8 Hz, 1H; 8-H), 8.21 (brs, 1H; 4-H), 9.08 (d, J=
1.8 Hz, 1H; 6-H), 9.65 ppm (d, J=2.4 Hz, 1H; indole-NH); 13C NMR
(50 MHz, CDCl3): d=42.80 (C-1), 45.91 (C-10), 55.15 (C-2), 56.19, 61.08,
61.43 (3”OCH3), 66.63 (CH2Ph-ester), 70.36 (CH2Ph-ether), 97.58 (C-4’),
98.95 (C-4), 106.81 (C-3’), 116.03, 123.52, 125.69, 131.94, 144.57 (C-3a, C-
5a, C-7, C-9a, C-9b), 122.14 (C-9), 122.57, 125.14, 129.48, 138.79, 140.65
(C-2’, C-3a’, C-6’, C-7’, C-7a’), 126.94 (C-6), 127.29 (C-8), 127.31, 127.92,
128.00, 128.09, 128.54, 128.59 (10”Ph-C), 136.12, 136.26 (2”Ph-Ci),
150.20 (C-5’), 156.82 (C-5), 160.58 (C=O), 166.36 ppm (C(O)OBn); UV/
Vis (CH3CN): lmax (lg e)=208 (4.618), 229 (4.450), 272 (4.409), 315
(4.384), 365 nm (4.453); IR (KBr): n˜ =3459 (NH), 2932 (CH2), 1714 (C=
O), 1624, 1526, 1458, 1407, 1308, 1107, 834, 746 cmꢁ1; MS (ESI): m/z (%):
713 (100) [M+Na]+, 1403 (44) [2M+Na]+, 689 (100) [MꢁH]ꢁ; elemental
analysis calcd (%) for C40H35ClN2O7 (691.17): C 69.51, H 5.10; found: C
69.68, H 5.19.
stirred solution of 18 (1.51 mmol, 921 mg) in DMF (22 mL) was treated
with NaH (151 mg, 60% in oil, 3.78 mmol). Stirring was continued for
1.5 h at 208C. (E/Z)-1,3-Dichloropropene (279 mL, 335 mg, 3.02 mmol)
was then added dropwise and the mixture was stirred for a further 19 h
at 208C. The ensuing mixture was then quenched with NH4Cl (10 mL, sa-
turated solution) and extracted with EtOAc (4”20 mL). The combined
organic phases were washed with water (3”25 mL) and brine (25 mL),
dried (MgSO4), and concentrated under reduced pressure to give a red
oil. Purification of this material by column chromatography (pentane/
EtOAc 7:1) gave iodide 19 (875 mg, 85%) as a pale-yellow foam. Rf =
0.28, 0.36 (pentane/EtOAc 10:1); 1H NMR (300 MHz, CDCl3): d=1.30/
1.58 (2”s, 9H; CACHTREUNG(CH3)3), 3.70–4.00 (m, 1H; 1’-Ha), 4.40–4.65 (m, 1H; 1’-
Hb), 5.31 (brs, 2H; CH2Ph-ether), 5.45 (brs, 2H; CH2Ph-ester), 5.90–6.15
(m, 2H; 2’-H, 3’-H), 6.65–6.85 (m, 1H; 3-H), 7.32–7.54 (m, 10H; 10”Ph-
H), 8.19 (d, J=9.0 Hz, 1H; 8-H), 8.25 (dd, J=9.0, 1.8 Hz, 1H; 7-H),
9.04–9.15 ppm (m, 1H; 5-H); 13C NMR (75 MHz, CDCl3): d=28.18/28.44
(C
ACHTREUNG
(CH2Ph-ether), 80.93 (C
ACHTREUNG
122.01 (C-3’), 124.72/124.77, 127.67/127.76, 137.63 (C-4a, C-6, C-8a),
125.71, 127.03/127.07, 127.93/127.98, 128.05, 128.15, 128.22, 128.42/128.59,
128.73/128.76, 133.15/133.17 (C-5, C-7, C-8, C-2’, 10”Ph-C), 135.89/135.95
(2”Ph-Ci), 145.12 (C-2), 153.35/153.57 (C-4), 156.01 (C=O), 166.07/
166.10 ppm (C(O)OBn); MS (EI, 70 eV): m/z (%): 683 (3) [M]+, 500
(38) [Mꢁ2”C7H7ꢁH]+, 91 (100) [C7H7]+, 57 (36) [C4H9]+.
AHCTRE(GNU 1R/S)-5-Benzyloxy-1-chloromethyl-3-(5,6,7-trimethoxyindole-2-carbon-
yl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid (22): A magnetically
stirred solution of 21 (176 mg, 255 mmol) in 3:1:1 THF/MeOH/H2O
(4.5 mL) at 08C was treated with LiOH·H2O (32.0 mg, 765 mmol). The re-
action mixture was warmed to 208C and stirring was continued for 3.5 h.
The ensuing solution was then adjusted to pH 1–2 with HCl (2 n) and ex-
tracted with EtOAc (4”10 mL). The combined organic phases were
washed with brine (10 mL), dried (MgSO4), and concentrated under re-
duced pressure. The resulting brownish solid was adsorbed on silica gel
and purified by column chromatography (CH2Cl2/MeOH 30:1+0.5%
HOAc) to afford acid 22 (121 mg, 79%) as a yellow solid. Rf =0.58
(CH2Cl2/MeOH 10:1+0.5% HOAc); 1H NMR (300 MHz, [D6]DMSO):
d=3.80–3.94 (m, 10H; 3”OCH3, 10-Hb), 4.04 (dd, J=10.8, 2.9 Hz, 1H;
10-Ha), 4.22–4.27 (m, 1H; 1-H), 4.52 (d, J=9.9 Hz, 1H; 2-Hb), 4.75 (dd,
J=10.5, 9.9 Hz, 1H; 2-Ha), 5.27 (brs, 2H; CH2Ph), 6.97 (s, 1H; 4’-H),
7.08 (d, J=1.5 Hz, 1H; 3’-H), 7.35–7.54 (m, 5H; 5”Ph-H), 7.94–8.05 (m,
3H; 4-H, 8-H, 9-H), 8.84 (d, J=0.9 Hz, 1H; 6-H), 11.49 ppm (brs, 1H;
indole-NH); 13C NMR (75 MHz, [D6]DMSO): d=40.81 (C-1), 47.40 (C-
10), 55.06 (C-2), 55.94, 60.89, 61.05 (3”OCH3), 69.87 (CH2Ph), 98.07 (C-
ACHTREUNG(1R/S)-5-Benzyloxy-3-(tert-butoxycarbonyl)-1-chloromethyl-2,3-dihydro-
1H-benz[e]indole-7-carboxylic acid benzyl ester (5): Through a magneti-
cally stirred solution of iodide 19 (471 mg, 688 mmol) in benzene (20 mL)
was bubbled argon for 1.5 h. The oxygen-free solution was then treated
with tris(trimethylsilyl)silane (229 mL, 183 mg, 736 mmol) and AIBN
(26.0 mg, 158 mmol) and stirred for 3 h under reflux. The ensuing mixture
was adsorbed on silica gel and purified by column chromatography (pen-
tane/EtOAc 7:1) to afford CBI system 5 (336 mg, 87%) as a colorless
foam. Rf =0.27 (pentane/EtOAc 10:1); 1H NMR (200 MHz, CDCl3): d=
1.61 (s, 9H; CACHTREUNG(CH3)3), 3.45 (dd, J=10.4, 9.6 Hz, 1H; 10-Hb), 3.84–4.05
(m, 2H; 1-H, 10-Ha), 4.15 (dd, J=11.4, 9.6 Hz, 1H; 2-Hb), 4.21–4.35 (m,
1H; 2-Ha), 5.29 (s, 2H; CH2Ph-ether), 5.41 (s, 2H; CH2Ph-ester), 7.32–
7.60 (m, 10H; 10”Ph-H), 7.65 (d, J=8.8 Hz, 1H; 9-H), 7.88 (brs, 1H; 4-
H), 8.11 (dd, J=8.8, 1.6 Hz, 1H; 8-H), 9.09 ppm (d, J=1.6 Hz, 1H; 6-H);
2816
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2811 – 2818

Targeted Tumor Therapy
FULL PAPER
4’), 98.68 (C-4), 106.37 (C-3’), 116.69, 123.13, 125.88, 131.66, 144.37 (C-3a,
C-5a, C-7, C-9a, C-9b), 121.53, 125.52, 130.58, 139.03, 140.01 (C-2’, C-3a’,
C-6’, C-7’, C-7a’), 123.15 (C-9), 125.25 (C-6), 126.95 (C-8), 127.59 (2”Ph-
C), 128.07 (Ph-Cp), 128.56 (2”Ph-C), 136.41 (Ph-Ci), 149.24 (C-5’), 155.52
(C-5), 160.48 (C=O), 167.38 ppm (C(O)OH); MS (ESI): m/z (%): 623
(100) [M+Na]+, 645 (68) [MꢁH+2Na]+, 599 (100) [MꢁH]ꢁ.
9.9 Hz, 1H; 2-Ha), 6.93/6.96 (2”s, 1H; 4’-H), 7.06 (mc, 1H; 3’-H), 7.85–
8.06 (m, 3H; 4-H, 8-H, 9-H), 8.64 (t, J=5.0 Hz, 1H; NH), 8.68 (brs, 1H;
6-H), 11.35/11.40 ppm (2”brs, 1H; indole-NH); 13C NMR (150 MHz,
[D6]DMSO): d=34.35 (C-2’’), 35.86 (C-1’’), 40.86 (C-1), 47.42 (C-10),
55.08 (C-2), 55.97, 60.87, 61.03 (3”OCH3), 98.07/98.10 (C-4’), 100.65 (C-
4), 106.16/106.19 (C-3’), 114.93, 123.11, 125.41, 130.86, 143.75 (C-3a, C-5a,
C-7, C-9a, C-9b), 121.16, 128.64, 131.05, 139.02, 139.91 (C-2’, C-3a’, C-6’,
C-7’, C-7a’), 122.64/122.76 (C-9), 123.02 (C-6), 125.46 (C-8), 149.21 (C-5’),
155.31 (C-5), 160.31 (C=OTMI), 166.25 (C-7-C=O), 173.64 ppm
(C(O)OH); MS (ESI): m/z (%): 604 (100) [M+Na]+, 1185 (26)
[2M+Na]+, 544 (100) [MꢁHꢁHCl]ꢁ, 580 (32) [MꢁH]ꢁ.
ACHTREUNG(1R/S)-5-Benzyloxy-1-chloromethyl-3-(5,6,7-trimethoxyindole-2-carbon-
yl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid (N-hydroxysuccini-
mide) ester (23): A magnetically stirred solution of acid 22 (121 mg,
200 mmol) and N-hydroxysuccinimide (34.5 mg, 300 mmol) in THF
(14 mL) at 08C was treated with EDC·HCl (57.5 mg, 300 mmol). The re-
action mixture was warmed to 208C and stirring was continued for 13 h.
The ensuing solution was then adjusted to pH 1–2 with HCl (2 n) and ex-
tracted with EtOAc (3”10 mL). The combined organic phases were
washed with brine (10 mL), dried (MgSO4), and concentrated under re-
duced pressure to give active ester 23 as orange solid which was used for
the next reaction without further purification. Rf =0.89 (CH2Cl2/MeOH
10:1+0.5% HOAc); 1H NMR (200 MHz, CDCl3): d=2.92 (brs, 4H; 2”
CH2C(O)), 3.48 (dd, J=11.0, 10.6 Hz, 1H; 10-Hb), 3.90–4.17 (m, 11H;
3”OCH3, 1-H, 10-Ha), 4.69 (dd, J=10.7, 8.6 Hz, 1H; 2-Hb), 4.80 (dd, J=
10.7, 1.8 Hz, 1H; 2-Ha), 5.27–5.40 (m, 2H; CH2Ph), 6.88 (s, 1H; 4’-H),
7.03 (d, J=1.6 Hz, 1H; 3’-H), 7.30–7.56 (m, 5H; 5”Ph-H), 7.75 (d, J=
9.0 Hz, 1H; 9-H), 8.12 (dd, J=9.0, 1.7 Hz, 1H; 8-H), 8.26 (brs, 1H; 4-H),
9.18 (d, J=1.7 Hz, 1H; 6-H), 9.45 ppm (brs, 1H; indole-NH); MS (ESI):
m/z (%): 720 (100) [M+Na]+, 1417 (50) [2M+2Na]+, 696 (100)
[MꢁH]ꢁ.
AHCTRE(UGN 1R/S)-1-Chloromethyl-5-hydroxy-3-(5,6,7-trimethoxyindole-2-carbonyl)-
2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(N-succinimidyloxycar-
bonyl)ethyl] amide (27): A magnetically stirred solution of crude acid 26
(119 mg, 204 mmol) and N-hydroxysuccinimide (141 mg, 1.22 mmol) in
4:1:1 THF/CH2Cl2/DMF (30 mL) at 08C was treated with EDC·HCl
(117 mg, 612 mmol). The reaction mixture was warmed to 208C and stir-
ring was continued for 18 h. Another batch of N-hydroxysuccinimide
(47.0 mg, 408 mmol) and EDC·HCl (78.0 mg, 408 mmol) was added and
stirring was continued for a further 10.5 h at 208C. The ensuing solution
was then adjusted to pH 1–2 with HCl (2n) and extracted with EtOAc
(4”15 mL). The combined organic phases were washed with water (2”
15 mL) and brine (15 mL), dried (MgSO4), and concentrated under re-
duced pressure to afford 27 as orange solid which was used for the next
reaction without further purification. Rf =0.55 (CH2Cl2/MeOH 10:1+
0.5% HOAc); MS (ESI): m/z (%): 701 (35) [M+Na]+, 733 (44)
[M+MeOH+Na]+, 677 (44) [MꢁH]ꢁ, 709 (100) [MꢁH+MeOH]ꢁ.
ACHTREUNG(1R/S)-5-Benzyloxy-1-chloromethyl-3-(5,6,7-trimethoxyindole-2-carbon-
AHCTRE(UGN 1R/S)-1-Chloromethyl-5-hydroxy-3-(5,6,7-trimethoxyindole-2-carbonyl)-
yl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid b-alanyl amide (25):
A magnetically stirred solution of b-alanine (24) (27.0 mg, 300 mmol) in
water (2 mL) was treated with NEtiPr2 (52.0 mL, 39.0 mg, 300 mmol) and
dropwise with a solution of crude 23 (140 mg, 200 mmol) in MeCN
(5 mL). The reaction mixture was stirred for 7 h at 208C, then adjusted
to pH 2 with HCl (2 n) and extracted with EtOAc (3”10 mL). The com-
bined organic phases were washed with brine (10 mL), dried (MgSO4)
and concentrated under reduced pressure. The resulting brownish solid
was adsorbed on silica gel and purified by column chromatography
(CH2Cl2/MeOH 30:1+0.5% HOAc) to afford amide 25 (93 mg, 69%
over two steps) as a pale-yellow solid. Rf =0.54 (CH2Cl2/MeOH 10:1+
0.5% HOAc); 1H NMR (300 MHz, [D6]DMSO): d=2.56 (t, J=7.0 Hz,
2H; 2’’-H2), 3.50–3.56 (m, 2H; 1’’-H2), 3.80–3.94 (m, 10H; 3”OCH3, 10-
Hb), 4.05 (mc, 1H; 10-Ha), 4.26 (mc, 1H; 1-H), 4.51 (d, J=9.9 Hz, 1H; 2-
Hb), 4.76 (dd, J=10.3, 9.9 Hz, 1H; 2-Ha), 5.30 (brs, 2H; CH2Ph), 6.98 (s,
1H; 4’-H), 7.08 (brs, 1H, 3’-H), 7.31–7.55 (m, 5H; 5”Ph-H), 7.95–8.01
(m, 3H; 4-H, 8-H, 9-H), 8.70 (t, J=5.1 Hz, 1H; NH), 8.74 (brs, 1H; 6-
H), 11.47 ppm (brs, 1H; indole-NH); 13C NMR (75 MHz, [D6]DMSO):
d=33.94 (C-2’’), 35.75 (C-1’’), 40.81 (C-1), 47.42 (C-10), 54.98 (C-2),
55.95, 60.87, 61.01 (3”OCH3), 69.73 (CH2Ph), 98.08 (C-4’), 98.68 (C-4),
106.24 (C-3’), 116.64, 123.11, 125.45, 130.65, 143.59 (C-3a, C-5a, C-7, C-
9a, C-9b), 121.66, 129.49, 130.85, 139.00, 139.96 (C-2’, C-3a’, C-6’, C-7’, C-
7a’), 122.64/122.91 (C-9), 125.21/125.59 (C-6), 127.34 (2”Ph-C), 127.91/
128.09 (C-8), 128.50 (2”Ph-C), 128.79 (Ph-Cp), 136.50 (Ph-Ci), 149.21 (C-
5’), 155.27 (C-5), 160.39 (C=OTMI), 166.16 (C-7-C=O), 172.92 ppm
(C(O)OH); MS (ESI): m/z (%): 694 (58) [M+Na]+, 717 (100)
[MꢁH+2Na]+, 670 (100) [MꢁH]ꢁ.
2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [b-alanyl-l-tryptophyl-l-
methionyl-l-aspartyl-l-phenylalanineamidyl] amide (3): A magnetically
stirred suspension of tetragastrin (28)[26] (114 mg, 192 mmol) in DMF
(3 mL) was treated with NEtiPr2 (33.5 mL, 24.8 mg, 192 mmol) and drop-
wise with a solution of crude 27 (147 mg, 216 mmol) in DMF (12 mL).
Stirring was continued for 57 h at 208C. The ensuing mixture was adjust-
ed to pH 2 with HCl (2n) and extracted with EtOAc (5”10 mL). The
combined organic phases were washed with water (2”10 mL) and brine
(10 mL), treated with toluene (10 mL), and concentrated under reduced
pressure. The resulting brownish solid was adsorbed on silica gel and sub-
jected to column chromatography (CH2Cl2/MeOH 30:1+0.5% HOAc!
5:1+0.5% HOAc) to give 3 (67 mg, 27% over three steps, 33% based
on recovery, 1:1 mixture of both diastereomeres) as a light-yellow solid.
Further purification was achieved by HPLC. Rf =0.13 (CH2Cl2/MeOH
10:1+0.5% HOAc); 1H NMR (600 MHz, [D7]DMF): d=1.90–1.98 (m,
1H; 2c-Hb), 2.01–2.06 (m, 4H; 2c-Ha, SCH3), 2.37–2.42 (m, 1H; 3c-Hb),
2.44–2.50 (m, 1H; 3c-Ha), 2.59–2.70 (m, 2H; 1e-H2), 2.75 (mc, 1H; 2b-
Hb), 2.81 (mc, 1H; 2b-Ha), 3.02 (dd, J=13.7, 9.2 Hz, 1H; 2a-Hb), 3.20–
3.26 (m, 2H; 2d-Hb, 2a-Ha), 3.33 (dd, J=15.0, 4.8 Hz, 1H; 2d-Ha), 3.67
(mc, 2H; 1f-H2), 3.88 (s, 3H; OCH3), 3.90 (s, 3H; OCH3), 3.93 (dd, J=
11.2, 8.1 Hz, 1H; 10-Hb), 4.03 (s, 3H; OCH3), 4.11 (dd, J=11.2, 2.9 Hz,
1H; 10-Ha), 4.29 (mc, 1H; 1-H), 4.39 (mc, 1H; 1c-H), 4.55 (mc, 1H; 1a-
H), 4.64–4.68 (m, 3H; 1b-H, 1d-H, 2-Hb), 4.84 (t, J=9.9 Hz, 1H; 2-Ha),
6.98 (t, J=7.8 Hz, 1H; 5d’-H), 7.04 (s, 1H; 4’-H), 7.07 (t, J=7.8 Hz, 1H;
6d’-H), 7.14–7.17 (m, 1H; 1”Ph-H), 7.18 (d, J=1.5 Hz, 1H; 3’-H), 7.26
(mc, 2H; 2”Ph-H), 7.32–7.33 (m, 3H, 2d’-H; 2”Ph-H), 7.39 (d, J=
7.8 Hz, 1H, 7d’-H), 7.61 (d, J=7.8 Hz, 1H; 4d’-H), 7.83 (mc, 1H; NH),
7.95 (d, J=8.9 Hz, 1H; 9-H), 8.00–8.02 (m, 3H; 8-H, 2”NH), 8.06 (brs,
1H; 4-H), 8.21 (d, J=7.2 Hz, 1H; NH), 8.37 (mc, 2H; 2”NH), 8.66 (mc,
1H; NH), 8.84 (s, 1H; 6-H), 10.84 (mc, 2H; indole-NHTrp, C(O)OH),
11.31 ppm (brs, 1H; indole-NHTMI); 13C NMR (150 MHz, [D7]DMF): d=
14.93 (SCH3), 27.84 (C-2d), 30.52 (C-3c), 31.52 (C-2c), 36.11 (C-2b),
36.53 (C-1e), 37.03 (C-1f), 38.12 (C-2a), 42.26 (C-1), 47.97 (C-10), 51.37
(C-1b), 54.15 (C-1c), 55.45 (C-1a), 55.88/55.93 (C-1d), 55.98 (C-2), 56.48,
61.34, 61.42 (3”OCH3), 98.87 (C-4’), 101.66 (C-4), 107.07 (C-3’), 110.83/
110.84 (C-3d’), 111.98 (C-7d’), 115.94, 124.33, 126.53, 131.85, 144.96 (C-
3a, C-5a, C-7, C-9a, C-9b), 118.95 (C-4d’), 119.04 (C-5d’), 121.60 (C-6d’),
122.27, 129.70, 132.15, 139.94, 141.09 (C-2’, C-3a’, C-6’, C-7’, C-7a’),
123.38 (C-9), 123.91 (C-6), 124.57/124.59 (C-2d’), 126.15/126.17 (C-8),
126.83 (Ph-Cp), 128.35 (C-3ad’), 128.75 (2”Ph-C), 129.91 (2”Ph-C),
137.33 (C-7ad’), 138.94 (Ph-Ci), 150.51 (C-5’), 156.42 (C-5), 161.26 (C=
ACHTREUNG(1R/S)-1-Chloromethyl-5-hydroxy-3-(5,6,7-trimethoxyindole-2-carbonyl)-
2,3-dihydro-1H-benz[e]indole-7-carboxylic acid b-alanyl amide (26): A
magnetically stirred solution of benzyl ether 25 (101 mg, 150 mmol) in 3:1
THF/MeOH (5.6 mL) was treated with 10% Pd/C (41 mg) and dropwise
with NH4HCO2 (393 mL of a 25% solution in water, 1.56 mmol) and stir-
ring was continued for 2.5 h at 208C. The reaction mixture was filtered
through a pad of Celite and was then thoroughly washed with MeOH
and THF. The filtrate was treated with toluene (10 mL) and concentrated
under reduced pressure to give 26 as light-yellow solid which was used
for the next reaction without further purification. Rf =0.39 (CH2Cl2/
MeOH 10:1+0.5% HOAc); 1H NMR (300 MHz, [D6]DMSO): d=2.53
(t, J=7.1 Hz, 2H; 2’’-H2), 3.49–3.56 (m, 2H; 1’’-H2), 3.80–3.91 (m, 7H;
2”OCH3, 10-Hb), 3.94 (s, 3H; OCH3), 4.01 (dd, J=10.8, 2.6 Hz, 1H; 10-
Ha), 4.19 (mc, 1H; 1-H), 4.46 (d, J=9.9 Hz, 1H; 2-Hb), 4.72 (dd, J=10.8,
Chem. Eur. J. 2008, 14, 2811 – 2818
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L. F. Tietze et al.
OTMI), 162.33 (C=OAla), 167.59 (C-7-C=O), 171.20, 172.72/172.75, 173.24/
173.27, 173.70, 173.94 ppm (4”C=O, (C(O)OH); MS (ESI): m/z (%):
1182 (29) [M+Na]+, 1204 (64) [MꢁH+2Na]+, 1158 (100) [MꢁH]ꢁ;
HRMS: calcd for C58H62ClN9O13S: 1182.37685 [M+Na]+; found:
1182.37679; HPLC (preparative): column: Kromasil 100 C18; eluent:
75% MeOH, 25% H2O+0.05% TFA; flow: 12 mLminꢁ1; Rt: 14–20 min.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie. F.M. thanks the Studienstiftung des
deutschen Volkes (German National Academic Foundation) for a PhD
scholarship. B.K. is grateful to the Deutsche Telekom Foundation for a
PhD scholarship.
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Received: September 25, 2007
Published online: January 23, 2008
2818
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2811 – 2818