A facile two-step synthesis of novel ring-A double substituted tryptophan building blocks for combinatorial chemistry

Article abstract of DOI:10.1055/s-2003-40820

A fast synthesis of ring-A disubstituted Fmoc and Boc protected L-tryptophan analogs was achieved starting from the appropriate 2,4- or 2,3-disubstituted phenylhydrazines and optically active N,N-diprotected L-glutamic acid γ-aldehydes, utilizing a Fischer-indole synthesis as a key step. Unlike most of the previously reported methods, that required the multistep stereoselective generation of a chiral carbon, this fast methodology is useful for generating optically active ring-A disubstituted protected tryptophans starting from a simple and common chiral precursor. These building blocks have a wide application scope in peptide and combinatorial chemistry fields.

Full text of DOI:10.1055/s-2003-40820

LETTER
1411
A Facile Two-Step Synthesis of Novel Ring-A Double Substituted Tryptophan
Building Blocks for Combinatorial Chemistry
Synthesis of
N
o
ovel ring-A
fsia
a
Compugen Ltd., Chemistry Division, 11 Ha’Amal St., P.O.Box 3066, Ashkelon 78785, Israel
b
Laboratory of Peptidomimetics and Genetic Chemistry, Department of Chemistry, Bar Ilan University, 52900-Ramat Gan, Israel
E-mail: garyg@compugen.co.il.
Received 31 March 2003
tryptophan analogs were obtained via a palladium-cata-
lyzed heteroannulation reaction of substituted o-iodo-
anilines with a preformed Schollkopf’s auxiliary.10 An
internal alkyne was prepared via alkylation of the
Abstract: A fast synthesis of ring-A disubstituted Fmoc and Boc
protected L-tryptophan analogs was achieved starting from the ap-
propriate 2,4- or 2,3-disubstituted phenylhydrazines and optically
active N,N-diprotected L-glutamic acid g-aldehydes, utilizing a
Fischer-indole synthesis as a key step. Unlike most of the previous- Schöllkopf’s auxiliary with diphenyl (triethylsilyl)propar-
ly reported methods, that required the multistep stereoselective
gyl phosphate. After the heteroannulation a mixture con-
generation of a chiral carbon, this fast methodology is useful for
taining L-isotryptophan as minor product was obtained.
generating optically active ring-A disubstituted protected tryp-
The desired L-tryptophan was isolated after separation
tophans starting from a simple and common chiral precursor. These
from the minor isomer by column chromatography. In a
building blocks have a wide application scope in peptide and com-
third approach 5-hydroxy derivatives of tryptophan were
prepared via a Fischer indole ring formation mediated by
isolated intermediate hydrazones obtained from the con-
densation of substituted phenylhydrazines and aldehyde
derivatives of protected glutamic acid.15–17
binatorial chemistry fields.
Key words: Boc strategy, combinatorial chemistry, Fischer indole
synthesis, Fmoc strategy, orthogonal protection, tryptophan
Overall, these methods suffer from a number of disadvan-
tages. Some are limited to tryptophan analogs with no
alkyl substituents on the ring A,9,11–13 as the presence of
such alkyl substitutents will likely promote a non-selec-
tive bromination and a mixture of di-halogenated indole
products will be obtained. In general, these methods are
tedious multistep syntheses, involving the problematic
separation of isomers, which are obtained in the alkylation
and annulation reactions respectively, underlining the
main drawbacks of these approaches. Finally, the Fischer-
indole approach, which requires the isolation of the inter-
mediate hydrazone, was limited to a single hydroxyl sub-
stituent on the ring-A and led to the formation of N-acetyl
or N-phtalimido tryptophans, which are not appropriately
protected for peptide or combinatorial chemistry. Never-
theless, this approach seems to be the most versatile and
rapid of the available strategies.
Combinatorial chemistry is based on the simple assump-
tion that the greater the diversity of compounds tested, the
better the chance of finding one that can be developed into
a drug or other industrial lead. The scope of this approach
is limited to a finite number of molecules as a result of the
limited organic reactions available for synthesis on solid
supports and by the limited availability of multi-reagent
organic reactions. A main goal of the present work is to
develop a new tryptophan-based building block for com-
binatorial chemistry. These compounds can be used as
building blocks for modifications of peptides,1,2 for prep-
aration of new types of natural indole3,4 and bis-indole5,6
alkaloids, for the synthesis of indoleamine 2,3-dioxygen-
ase (IDO)7 or somatostatin8 inhibitors.
In previous work, ring-A double substituted tryptophans
have been reported.9,10 Optically active ring-A mono-sub-
stituted tryptophan analogs were obtained after synthesis
and resolution of racemic compounds or by tedious multi-
step stereoselective alkylation of Schollkopf’s auxiliary
with moderate stereoselectivity. Thus, a regiospecific bro-
mination of N-protected 3-methyl indoles with NBS un-
der free radical conditions (AIBN) led to the precursor 3-
bromomethyl indoles9,11–13 whose reaction with the anion
pre-formed from a Schöllkopf’s auxiliary14 provided
enantiomerically enriched products. The desired trans
diastereomer was subsequently hydrolyzed under acidic
conditions to afford the ring-A mono-substituted
tryprophans. In a similar approach the optically active
The limitations of the current approaches and the lack of
a specific general procedure for accessing di-substituted
A-ring tryptophans, prompted us to develop an improved
method for the preparation of these building blocks,
which would generalize the synthesis, preferably reduce
the number of synthetic steps and produce properly pro-
tected building blocks, that are applicable to SPPS, SPOC
and parallel synthesis. Our approach for the synthesis of
ring-A disubstituted tryptophans is based on a modifica-
tion and extension of the simple Fischer-indole reaction
between 2,4- or 2,3-disubstituted phenylhydrazine hydro-
chlorides and optically active N,N-diprotected glutamic
acid g-aldehydes 4, 12 and 16. Therefore, aldehydes prop-
erly protected for Boc and Fmoc strategies, were required
to achieve our synthetic goal.
Synlett 2003, No. 10, Print: 05 08 2003.
Art Id.1437-2096,E;2003,0,10,1411,1414,ftx,en;D07803ST.pdf.
© Georg Thieme Verlag Stuttgart · New York

1412
S. Gorohovsky et al.
LETTER
O
O
O
O
O
O
(Boc)2O, Et3N, DMAP
DCM, 3d, r.t.
O
HCl·NHMe(OMe), HOBt,
O
N
OtBu
N
OtBu
HO
OtBu
EDC, DIEA, DMF, r.t.
N(Boc)2
NHBoc
3
NHBoc
2
1
DiBAlH
THF dry, –78 °C
O
OtBu
O
O
NHBoc
R1
R2
O
OH
H
OtBu
N
H
N(Boc)2
1) TFA/CH2Cl2 (1:1)
2.5 % TIS/ 2.5% H2O, r.t.
NHFmoc
R1
R2
6
4
R3
∆
O
+
2) Fmoc-OSU, DIEA
ACN/H2O (2:1), r.t.
OtBu
+
iso-propanol
N
H
R1
8
R3
N(Boc)2
R1
R2
NH2 HCl
yield (%)
R2
N
H
a: R1= Me; R2= H; R3= Me
b: R1= OMe; R2=H; R3= Me
c: R1= H; R2= Me; R3= Me
d: R1= Et; R2= H; R3= Ph
46
36
29
45
R3
N
H
7
R3
5
Scheme 1
Evidently, the double protection of the a-amino group in tion in dichloromethane using Fmoc-Cl in the presence of
the g-aldehyde precursors was necessary in order to avoid DIEA (yield after two steps: 65%). The intermediate N,N-
the undesired intramolecular lactamization of the ob- diprotected glutamic acid a-t-butyl ester 10 was converted
tained g-aldehydes,18 preventing racemization of the into hydroxamate 11, which was further reduced with
asymmetric carbon. In the light of this need, we have syn- DiBAlH to afford aldehyde 12.
thesized various aldehydes carrying orthogonal protecting
The formation of disubstituted tryptophan esters by Fis-
groups on the a-amino and a-carboxylic moieties starting
cher indole reaction was achieved by reflux in iso-pro-
from the L-glutamic acid counterpart (4, 12 and 16).
panol,23 simplifying significantly the production of the
The synthesis of N,N-di-Boc protected aldehydes 4 and building blocks (previously obtained only after isolation
16 is known.19,20 Our attempts to follow literature proce- of intermediate hydrazones). Thus, 2,3- and 2,4-disubsti-
dures resulted in low yields, therefore aldehydes 4 and 16 tuted phenylhydrazine hydrochlorides24 (5) were refluxed
were synthesized by combination of different strategies. with N,N-di-Boc protected g-aldehyde 4 in iso-propanol
Thus, g-aldehyde 4 (see Scheme 1) was prepared starting (Scheme 1) to give in one step the expected tryptophan
from N-Boc protected L-glutamic acid t-butyl ester 1, analogs. We noticed that the second unstable N-Boc
which was converted into hydroxamate 2 (Weinreb group was partially cleaved in the presence of 1 equiva-
amide) by reaction with N,O-dimethylhydroxylamine hy- lent of HCl during the Fischer indole reaction, affording
drochloride in the presence of EDC/DIEA. The second the mixture of mono-Boc/di-Boc tryptophan esters 6 and
protection with (Boc)2O was performed in dichlo- 7. This partial deprotection appeared to be non-significant
romethane in the presence of triethylamine and a catalytic as the mixture was further treated with TFA/CH2Cl2 (1:1),
amount of 4-dimethylaminopyridine.21 Reduction with leading to the crude unprotected amino acid, which was
DiBAlH at –78 °C in dry THF led to the formation of g- submitted to further protection using Fmoc-OSu in ACN/
aldehyde 4 (final yield after three steps 55%). L-Glutamic H2O (1:1) in the presence of DIEA leading the desired
acid dimethyl ester 14 was simultaneously double protect- Fmoc-tryptophans (8).25
ed with (Boc)2O in dichloromethane in the presence of
In an alternative approach, the synthesis of Fmoc-protect-
triethylamine and a catalytic amount of 4-dimethylami-
ed tryptophan analogs was performed as shown in
nopyridine (see Scheme 3). Reduction with DiBAlH led
Scheme 2. Initially prepared g-aldehyde t-butyl ester 12
was refluxed in iso-propanol with 2,4-disubstituted phe-
to the formation of g-aldehyde 16 (final yield 39%). g-Al-
dehyde 12 was prepared from L-glutamic acid t-butyl ester
nylhydrazine hydrochlorides (5a and 5b) to give the cor-
9 (see Scheme 2). The first protection of 9 was performed
responding protected tryptophan esters (13a and 13b). By
via reductive alkylation of a-amino group with acid labile
this method, the desired disubstituted Fmoc tryptophans
(8a and 8b) were obtained through the acidic removal
(TFA) of t-Bu ester and N-Dmb groups in the final step.
2,4-dimethoxybenzaldehyde in the presence of NaCNBH3
in methanol containing 1% of acetic acid.22 The second
protection to obtain 10 was carried out without purifica-
Synlett 2003, No. 10, 1411–1414 © Thieme Stuttgart · New York

LETTER
Synthesis of Novel Ring-A Double Substituted Tryptophan
1413
O
O
O
O
O
O
1) 2,4-dimethoxybenzaldehyde
NaBH3CN, MeOH (1% AcOH)
HCl·NHMe(OMe), HOBt,
HO
EDC, DIEA, DMF, r.t.
O
N
HO
OtBu
OtBu
OtBu
2) Fmoc-Cl, DIEA, DCM
N
N
NH2
Dmb
Fmoc
11
Dmb
Fmoc
9
10
DiBAlH
Dmb
THF dry, –78 °C
O
O
O
O
OH
OtBu
H
OtBu
Fmoc
N
+
NHFmoc
R1
Fmoc
N
R1
TFA
∆
12
Dmb
2.5 % TIS/ 2.5% H2O, r.t.
iso-propanol
N
R1
N
H
H
R2
R2
NH2 HCl
8
13
N
yield (%)
H
R2
a: R1= Me; R2= Me
b: R1= OMe; R2=Me
38
35
5
Scheme 2
Based on this successful route to produce disubstituted 19.26 In this approach the formation of undesired di-Boc
tryptophans for Fmoc solid-phase chemistry, we decided trypthophan ester reduces the yield of the final product 19.
to develop a general approach for synthesis of the tryp- Attempts to optimize the reaction conditions are in
tophans for combinatorial synthesis via Boc chemistry on progress. Although, the generation of this mixture during
solid support and as well as in solution. For this purpose, the synthesis could not be prevented, both building blocks
g-aldehyde methyl ester 16 was prepared and refluxed in are suitable for the same synthetic methods and will bring
iso-propanol with 2,4-dimethylphenylphenylhydrazine about the same products in a combinatorial approach after
hydrochloride (5a), as described (Scheme 3). As expect- the Boc groups are cleaved.
ed, the mixture of mono and di-Boc products (17 and 18)
was obtained again. After separation by flash chromato-
We have shown a simple and convenient two-step synthe-
sis of disubstituted tryptophan building blocks for combi-
graphy, the resultant methyl ester of mono-Boc-5,7-dime-
natorial chemistry. This general methodology allows the
thyl tryptophan 17 was hydrolyzed by heating in ethanol
quick production of a variety of protected tryptophans and
with 1 N NaOH to give the corresponding final product
O
O
O
O
(Boc)2O, Et3N, DMAP
CH2Cl2, Ar, r.t.
MeO
OMe
MeO
OMe
NH2
N(Boc)2
14
15
O
O
DiBAlH
O
THF dry, –78 °C
O
OMe
OMe
NHBoc
+
N(Boc)2
H3C
H3C
H
OMe
N(Boc)2
+
∆
16
N
H
N
H
iso-propanol
CH3
CH3
H3C
18 (yield: 38%)
17 (yield: 51%)
NH2 HCl
N
H
O
1N NaOH
CH3
5a
∆, EtOH
OH
NHBoc
H3C
N
H
CH3
19 (yield: 86%)
Scheme 3
Synlett 2003, No. 10, 1411–1414 © Thieme Stuttgart · New York

1414
S. Gorohovsky et al.
LETTER
with 0.35 M KHSO4 (3 × 50 mL) and water (3 × 50 mL). The
solvent was evaporated under reduced pressure. The product
3 was purified by MPLC on RP18 to give 15.35 g (72%). 1H
NMR (CDCl3) (d ppm): 4.82 (m, 1 H), 3.66 (s, 3 H), 3.16 (s,
3 H), 2.47 (m, 3 H), 2.14 (m, 1 H), 1.50 (s, 9 H), 1.47 (s, 9
H), 1.45 (s, 9 H). MS (m/z): 447 (MH+).
simplifies scale up procedure. The key step involves the
standard Fischer-indole reaction between the disubstitut-
ed phenylhydrazine hydrochlorides and N,N-diprotected
glutamic acid g-aldehydes that we have improved and
simplified for adaption to SPPS and SPOS. The further
simple treatment of the protecting groups, affords the for-
mation of ring-A disubstituted a-nitrogen Boc or Fmoc
protected tryptophans. These important synthons are use-
ful in peptidomimetics and combinatorial syntheses for
the discovery of novel bioactive compounds.
(22) Gellerman, G.; Elgavi, A.; Salitra, Y.; Kramer, M. J. Peptide
Res. 2001, 57, 277.
(23) 5.11 g (13.2 mmol) of 4 and 2.28 g (13.2 mmol) of 2,4-
dimethylphenyl hydrazine hydrochloride 5a were refluxed
in 100 mL of iso-propanol for 5 h. The reaction mixture was
cooled to r.t. and the solvent was evaporated under vacuum
to give 4.5 g of crude mixture. Yields (according to HPLC):
26.9% of mono-Boc product 6 and 50.5% of di-Boc product
7. The mixture of 6 and 7 was dissolved in 40 mL
Acknowledgment
We wish to thank to Mr. Alex Rozovsky for analytical assistance.
Dr. Gerardo Byk is indebted to the ‘Marcus Center for Medicinal
Chemistry’.
dichloromethane. 100 mL of tri-iso-propyl silane and 40 mL
of trifluoroacetic acid were added at 0 °C. The reaction
mixture was stirred for 30 min at 0 °C and for 3 h at r.t. The
solvent was evaporated and the residue was dissolved in 40
mL ACN/H2O (2:1) and DIEA (to pH = 9). Fmoc-OSu (3.14
g) was then added and the final mixture was stirred overnight
at r.t. The reaction mixture was acidified with 5% HCl.
Acetonitrile was evaporated and the product was extracted
with EtOAc (3 × 30 mL). The organic phase was washed
with NaHCO3 (3 × 30 mL), water and dried over MgSO4.
The pure product was isolated after elution from a self-
packed column RP-MPLC (LiChroprep RP-18, 40-63 mL
Merck) using acetonitrile and water (0.1% of TFA), 2.7 g
(46%).
References
(1) Kobayashi, J.; Ishibashi, M. Chem. Rev. 1993, 93, 1753.
(2) Braekman, J. C.; Daloze, D.; Moussiaux, B. J. Nat. Prod.
1987, 50, 99.
(3) Safdy, M. E.; Kurchasova, E.; Schut, R. N.; Vidro, H.; Hong,
E. J. Med. Chem. 1982, 25, 723.
(4) Wright, C. W.; Allen, D.; Cai, Y.; Phillipson, J. D.; Said, I.
M.; Kirby, G. C.; Warhurst, D. C. Phytother. Res. 1992, 6,
121.
(24) (a) Commercially available 2,4-dimethylphenylhydrazine
hydrochloride(5a) and 2,3-dimethylphenylhydrazine
hydrochloride(5c) were purchased from Aldrich. (b) 2-
Methyl-4-methoxyphenylhydrazine hydrochloride(5b) was
synthesized according to the procedure from: Bare, T. M.;
Chapdelaine, M. J.; Davenport, T. W.; Empfield, J. R.;
James, R.; Garcia-Davenport, L. E.; Jackson, P. F.;
McKinney, J. A.; McLaren, C. D.; Sparks, R. B. PCT Int.
Appl., CODEN: PIXXD2 WO 9615127, 1996. (c) 5-Ethyl-
biphenyl-2-yl-hydrazine(5d) was prepared according to the
procedure from: Katritzky, A.; Wang, Z. J. Heterocycl.
Chem. 1988, 25, 671. (d) 5-Ethyl-biphenyl-2-ylamine was
prepared according to procedure from: Bumagin, N. A.;
Luzikova, E. V. J. Organomet. Chem. 1997, 532, 271.
(25) Selected data for compounds 8a and 8d. 8a: 1H NMR (d6-
DMSO) (d ppm): 12.65 (br s, COOH), 10.66 (br s, NH), 7.87
(d, 2 H, J = 7.5 Hz), 7.66 (d, 2 H, J = 7.2 Hz), 7.62 (s, NH),
7.38–7.42 (m, 2 H), 7.26–7.33 (m, 2 H), 7.14 (s, 1 H), 7.12
(d, 1 H, J = 1.8 Hz), 6.70 (s, 1 H), 4.18–4.21 (m, 4 H), 3.12–
3.18 (m, 1 H), 2.96–3.01 (m, 1 H), 2.38 (s, 3 H), 2.33 (s, 3
H). HRMS (m/z): 455.1964 (MH+, calculated 455.1971 for
C28H27N2O4). 8d: 1H NMR (d6-DMSO) (d ppm): 12.75 (br s,
COOH), 10.65 (br s, NH), 7.87 (d, 2 H, J = 7.5 Hz), 7.60–
7.75 (m, 5 H), 7.51 (t, 2 H, J = 7.5Hz), 7.37–7.40 (m, 4 H),
7.20–7.34 (m, 2 H), 7.17 (d, 1 H), 6.98 (d, 1 H, J = 1.2 Hz),
4.15–4.28 (m, 4 H), 3.19–3.25 (m, 1 H), 3.00–3.15 (m, 1 H),
2.72 (q, 2 H, J = 7.5 Hz), 1.28 (t, 3 H, J = 7.5Hz). HRMS
(m/z): 531.2281 (MH+, calculated 531.2284 for
(5) Cook, J. M.; LeQuesne, P. W. Phytochemistry 1971, 10, 437.
(6) Yamazaki, M.; Suzuki, K.; Fijimoto, H.; Akijama, T.;
Sankawa, U.; Iitaka, Y. Chem. Pharm. Bull. 1980, 28, 861.
(7) Botting, N. Chem. Soc. Rev. 1995, 24, 401.
(8) Berk, S. C.; Rohrer, S. P.; Degrado, S. J.; Birzin, E. T.;
Mosley, R. T.; Hutchins, S. M.; Pasternak, A.; Schaeffer, J.
M.; Underwood, D. J.; Chapman, K. T. J. Comb. Chem.
1999, 1, 388.
(9) Liu, R.; Zhang, P.; Gan, T.; Cook, J. M. J. Org. Chem. 1997,
62, 7447.
(10) Ma, C.; Liu, X.; Li, X.; Flippen-Anderson, J.; Yu, S.; Cook,
J. M. J. Org. Chem. 2001, 66, 4525.
(11) Allen, M. S.; Hamaker, L. K.; La Loggia, A. J.; Cook, J. M.
Synth. Commun. 1992, 22, 2077.
(12) Zhang, P.; Cook, J. M. Synth. Commun. 1995, 25, 3883.
(13) Zhang, P.; Liu, R.; Cook, J. M. Tetrahedron Lett. 1995, 36,
9133.
(14) Schöllkopf, U.; Lonsky, R.; Lehr, P. Liebigs Ann. Chem.
1985, 413.
(15) Masumi, F.; Takeuchi, H.; Kondo, S.; Suzuki, K.; Yamada,
S. Chem. Pharm. Bull. 1982, 30, 3831.
(16) Irie, K.; Ishida, A.; Nakamura, T.; Ohishi, T. Chem. Pharm.
Bull. 1984, 32, 2126.
(17) Schirlin, D.; Gerhart, F.; Hornsperger, J. M.; Haman, M.;
Wagner, J.; Jung, M. J. J. Med. Chem. 1988, 31, 30.
(18) Bold, G.; Steiner, H.; Moesch, L.; Walliser, B. Helv. Chim.
Acta 1990, 73, 405.
(19) Kokotos, G.; Padron, J. M.; Martin, T.; Gibbons, W. A.;
Martin, V. S. J. Org. Chem. 1998, 63, 3741.
(20) Constantinou-Kokotou, V.; Magrioti, V.; Markidis, T.;
Kokotos, G. J. Peptide Res. 2001, 58, 325.
(21) 17 g (49.13 mmol) of 2 were dissolved in 300 mL of CH2Cl2.
53.60 g (245.65 mmol) of (Boc)2O, 147 mL (1.1 mol) of
Et3N and 0.6 g (4.91 mmol) of DMAP were added under Ar
atmosphere. The reaction mixture was stirred under Ar
atmosphere for 3 days. The CH2Cl2 solution was washed
C34H31N2O4).
(26) Selected data for compound 19: 1H NMR (d6-DMSO) (d
ppm): 12.50 (br s, COOH), 10.66 (br s, NH), 7.10 (s, 1 H),
7.07 (d, NH), 6.92 (d, 1 H, J = 7.8 Hz), 6.69 (s, 1 H), 4.09–
4.18 (m, 1 H), 3.04–3.11 (m, 1 H), 2.87–2.95 (m, 1 H), 2.36
(s, 3 H), 2.34 (s, 3 H), 1.33 (s, 9 H). HRMS (m/z): 333.1831
(MH+, calculated 333.1814 for C18H25N2O4).
Synlett 2003, No. 10, 1411–1414 © Thieme Stuttgart · New York