Expedient synthesis of a novel class of pseudoaromatic amino acids: Tetrahydroindazol-3-yl- and tetrahydrobenzisoxazol-3-ylalanine derivatives

Article abstract of DOI:10.1016/j.tetlet.2003.11.133

A concise synthesis of novel homochiral aromatic amino acid surrogates comprising a tetrahydroindazole or a benzisoxazole system was developed via the acylation of a cyclic 1,3-diketone by the side-chain carboxyl functionality of either Boc-Asp-OtBu or Boc-Glu-OtBu followed by regioselective condensation with hydrazine, N-benzylhydrazine and hydroxylamine. The tetrahydroindazole nucleus was also constructed by the condensation of Boc-Asp-OtBu with the enamine, 1-pyrrolidino-1-cyclohexene followed by acid-hydrolytic treatment and reaction with hydrazines. Further functional group transformations gave N ^α-Fmoc-protected derivatives as useful building blocks for solid-phase peptide assembly.

Full text of DOI:10.1016/j.tetlet.2003.11.133

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 1237–1242
Expedient synthesis of a novel class of pseudoaromatic amino acids:
tetrahydroindazol-3-yl- and tetrahydrobenzisoxazol-3-ylalanine
derivatives
Richard J. Middleton,a Sarah L. Mellor,a Siri Ram Chhabra,a Barrie W. Bycrofta
and Weng C. Chana,b,
*
aSchool of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK
bInstitute of Infection, Immunity and Inflammation, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Received 8 October 2003; revised 14 November 2003; accepted 27 November 2003
Abstract—A concise synthesis of novel homochiral aromatic amino acid surrogates comprising a tetrahydroindazole or a benzis-
oxazole system was developed via the acylation of a cyclic 1,3-diketone by the side-chain carboxyl functionality of either Boc-Asp-
OtBu or Boc-Glu-OtBu followed by regioselective condensation with hydrazine, N-benzylhydrazine and hydroxylamine. The tet-
rahydroindazole nucleus was also constructed by the condensation of Boc-Asp-OtBu with the enamine, 1-pyrrolidino-1-cyclohexene
followed by acid-hydrolytic treatment and reaction with hydrazines. Further functional group transformations gave Na-Fmoc-
protected derivatives as useful building blocks for solid-phase peptide assembly.
ꢀ 2003 Elsevier Ltd. All rights reserved.
In recent years, there has been considerable interest in
the synthesis of amino acid surrogates with conforma-
tionally constrained or unnatural side-chain function-
alities, which when incorporated into biological peptides
have the capacity to impart unique properties. These
unnatural amino acids can also be incorporated effi-
ciently into proteins in response to a nonsense or amber
codon in vivo. Furthermore, an amino acid residue with
isosteric chemical functionality allows the development
of molecular probes for the elucidation of peptide con-
formation and the spatial elements required for optimal
peptide-receptor recognition.1 In this context, we have
recently established new methods for the synthesis of
novel amino acid surrogates based on the 4-azalysine
template.2 Herein, we report the synthesis of a novel
class of homochiral heterocyclic amino acids, b-(tetra-
hydroindazol-3-yl)- and b-(tetrahydrobenzisoxazol-3-yl)-
alanine. The synthetic strategy developed is amenable to
the construction of structurally diverse derivatives.
In our previous studies on the design of the novel amine-
protecting group, N-[1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene)ethyl] (N-Dde) and its variants,3;4 the forma-
tion of the heterocyclic structures was an important
consideration with respect to the deprotection mecha-
nism. The N-Dde group is readily introduced by the
condensation of the primary amine with 2-acetyldime-
done, which in turn is efficiently removed by a solution
of 2% NH2NH2ÆH2O in DMF with the concomitant
formation of 3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-
indazole as the byproduct. The latter can also be directly
obtained by the condensation of 2-acetyldimedone with
hydrazine. Moreover, the 2-acyldimedones4 are conve-
niently prepared by the C-acylation of dimedone. We
envisaged that the acylation of dimedone or other cyclic
1,3-diketones by the carboxylic acid functionality in the
side chain of
L-Asp or L-Glu 3 would furnish the
homochiral a-amino acid bearing b-triketone moiety 2.
The latter when reacted with a range of dinucleophiles
would deliver a diverse array of pseudoaromatic
a-amino acid derivatives 1 (Scheme 1). A somewhat
related approach was recently reported, in which acyclic
b-triketones were reacted with dinucleophiles to afford
heterocyclic substituted a-amino acids.5
Keywords: Unnatural amino acids; Pseudoaromatic; Tetrahydroinda-
zoles.
Thus, the commercially available Boc-Asp-OtBu 4a was
an ideal suitably protected amino acid precursor for the
* Corresponding author. Tel.: +44-115-9515080; fax: +44-115-95150-
78; e-mail: weng.chan@nottingham.ac.uk
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.133

1238
R. J. Middleton et al. / Tetrahedron Letters 45 (2004) 1237–1242
While standard analysis confirmed the structures of the
desired tetrahydroindazoles 7a(i) and 8a(i), it remained
to determine unambiguously the integrity of the ste-
reochemistry of the homochiral starting material fol-
lowing the chemical transformations. Specifically, we
were concerned with the potential for enantiomerisation
due to the moderately basic reagents, DMAP and
hydrazine, used in the two condensation steps. Quanti-
tative assessment of the extent of enantiomerisation was
performed by reaction of 7a(i) with O-tetraacetyl-b-D-
glucopyranosyl isothiocyanate to form the correspond-
ing thiourea derivative 9 (Scheme 3).9 In addition, the
diastereomer 10 was synthesised using the commercially
Scheme 1.
synthesis of the desired heterocyclic alanine derivative
8a(i). C-Acylation of dimedone with amino acid 4a was
accomplished using carbodiimide-mediated activation in
the presence of 4-(dimethylamino)pyridine (DMAP),3;4
to yield the 2-acyldimedone 5a in high yield (Scheme 2).
While previous syntheses of 2-acyl-1,3-diketones have
required Fries-type rearrangements of the preformed O-
acyl species,6 this one-step protocol, which is envisaged
as proceeding via the N-acylpyridinium intermediate
appears to facilitate direct C-acylation. Dinucleophile
condensation across the triketone 5a was then investi-
gated using a slight molar excess of hydrazine mono-
hydrate, and was found to proceed smoothly at room
temperature to yield the tetrahydroindazole derivative
6a(i).7 The novel heterocyclic amino acid was thus
readily available in two steps, in an overall yield of 76%.
available Boc-D-Asp-OtBu. RP-HPLC analysis of 9 and
10 demonstrated the diastereomeric purity10 of each and
thus, no enantiomerisation (<1%) was observed to occur
during the transformation of protected aspartic acid to
the novel heterocyclic alanine derivatives.
Following the synthesis of the tetrahydroindazole 8a(i),
attention was turned to the use of alternative dinucleo-
philes for the construction of other heterocyclic deriva-
tives. The reaction of hydroxylamine with 1,3-diketones
is well documented as a general approach for the syn-
thesis of isoxazoles.11 While inherent electronic and
steric effects may significantly influence the regiochemi-
cal outcome of such reactions, the corresponding con-
densation across the triketone 5a was envisaged to be
regiospecific, by nature of the previously observed
preferential nucleophilic attack at the exocyclic carbonyl
position and the greater nucleophilicity of nitrogen over
oxygen. Thus, the reaction of 5a with hydroxylamine
yielded benzisoxazole 6a(ii) in 70% yield. RP-HPLC and
1H NMR spectrometric analyses confirmed the presence
of only one isomer. Subsequent protecting group
manipulations afforded the N-Fmoc derivative 8a(ii).12
Quantitative removal of the N-Boc and tert-butyl ester
protecting groups was effected by TFA-mediated acido-
lysis to afford the amino acid 7a(i). Alternatively, the
deprotection can be accomplished by treatment with
3 M HCl/dioxane
solution to furnish the amino
ðaqÞ
acidÆHCl that is easily isolated and purified. The desired
N-Fmoc amino acid 8a(i)8 was finally obtained as a
white solid following reaction of 7a(i) with Fmoc-OSu
under basic conditions.
For further illustration of the structural diversity
accessible via this methodology, the N-benzyl tetra-
hydroindazole 8a(iii) was synthesised. However, the use
of an unsymmetrical dinucleophile, N-benzylhydrazine
Scheme 2. Reagents and conditions: (i) Dimedone, EDC, DMAP,
CH2Cl2, 24 h; (ii) NH2NH2ÆH2O or NH2OHÆHCl, DIEA or
NH2NHBnÆ2HCl, DIEA, EtOH, 4–36 h; (iii) TFA or 3 M HCl in
dioxane; (iv) FmocOSu, NaHCO3ðaqÞ, dioxane.
Scheme 3.

R. J. Middleton et al. / Tetrahedron Letters 45 (2004) 1237–1242
1239
raised concerns since the electronic and steric factors
governing the regiochemistry of the condensation step
were less defined. Nevertheless, we postulated that the
b-(1-benzyltetrahydroindazol-3-yl)alanine 6a(iii) would
be favoured over the isomeric b-(2-benzyl-
tetrahydroindazol-3-yl)alanine due to the formation of
the hydrogen bond stabilised linear intermediate 11
(Scheme 4). In fact, the desired derivative 6a(iii) was
synthesised in 63% yield, and NOE difference analysis
confirmed the hypothesised regioselectivity of the con-
densation reaction. Thus, irradiation of the benzyl CH2
enhanced one of the methylene groups of the tetra-
hydroindazole, which in conjunction with no observed
NOE enhancement of the CbH2 group strongly suggests
structure 6a(iii) (see Scheme 4). Acidolysis of the N-Boc
and tert-butyl ester groups and N-Fmoc derivatisation
was then performed as previously outlined to yield the
N-Fmoc-b-(1-benzyltetrahydroindazolyl)alanine 8a(iii).
The successful synthesis of the novel amino acid deriv-
atives 8a(i)–(iii) from
then extended to the corresponding
L
-aspartic acid derivative 4a was
-glutamic acid 4b.
L
Hence, the heterocyclic amino acids 8b(i)–(iii) were
readily synthesised in three steps from the triketone 5b,13
which was in turn prepared by the C-acylation of
dimedone (Scheme 2).
We have shown that the C-acylation of the cyclic 1,3-
diketone, dimedone by the side-chain carboxyl func-
tionality of L-Asp and L-Glu yields the corresponding
Scheme 5. Reagents and conditions: (i) 1-Pyrrolidino-1-cyclohexene,
DIPCDI, DMAP, CH2Cl2, 4 h; (ii) NH2NH2ÆH2O, EtOH, 4 h; (iii) 3 M
b-triketone appended amino acid derivatives. The regio-
specific condensation of a range of dinucleophiles across
the b-triketone has been established for the synthesis of
six novel pseudoaromatic amino acids. The construction
of further series of heterocyclic alanine and a-amino-
butanoic acid analogues by employing alternative
dinucleophiles is self-evident. To complement these
amino acids derived from dimedone, we have further
exploited the a-acylation of cyclic ketones and the con-
densation of dinucleophiles across the resultant b-dike-
tone. Such methodology generates alternative motifs to
the heterocyclic framework, supplementing the diversity
that can be introduced by means of dinucleophiles.
HCl in dioxane/H2O; (iv) FmocOSu, NaHCO3ðaqÞ
, dioxane; (v)
NH2OHÆHCl, DIEA, EtOH; (vi) NH2NHBnÆ2HCl, DIEA, EtOH, 4 h.
5). The 1H NMR spectrum of the product predomi-
nantly displayed the characteristic low-field enol signal
at ꢀd 15, which together with RP-HPLC analysis con-
firmed the diketone as an equilibrium mixture of tau-
tomers 12a and 12b. Condensation of 12a with a slight
molar excess of hydrazine at room temperature yielded
the tetrahydroindazole 13 in 85% yield, which was fol-
lowed by protecting group manipulation to afford the
N-Fmoc derivative 15.15
Thus, the a-acylation of cyclohexanone with Boc-Asp-
OtBu 4a was performed with the commercially available
enamine, 1-pyrrolidino-1-cyclohexene.14 Following car-
bodiimide/DMAP-mediated activation of the acid,
reaction with the enamine and subsequent hydrolysis
yielded the desired diketone 12a in 76% yield15 (Scheme
Unexpectedly, there was no observable reaction between
12a and hydroxylamine in ethanol at room temperature.
However, upon refluxing the solution, two products
were formed, of very similar chromatographic retention
factor. Indeed, LC-MS identified both components as
the desired tetrahydrobenzisoxazolylalanine. It is pos-
tulated that under the forcing conditions employed, and
in the absence of substantial electronic directing groups
in 12a, indiscriminate N-mediated condensation at both
the endo- and exo-cyclic carbonyls had occurred, gen-
erating the isomeric 1,2- and 2,1-tetrahydrobenzisoxaz-
oles 16a,b (Scheme 5).
In spite of this apparent absence of regiocontrol with
hydroxylamine, however, the condensation of 12a with
benzylhydrazine surprisingly afforded a single isomer
of the b-(N-benzyltetrahydroindazolyl)alanine 1716 in
Scheme 4.

1240
R. J. Middleton et al. / Tetrahedron Letters 45 (2004) 1237–1242
Figure 1. Structures of amino acid derivative 17 obtained by X-ray diffraction analysis, showing two conformers.
moderate yield (Scheme 5). Analysis by NOE difference
spectrometry was used to ascertain the position of the
N-benzyl substituent. Crucially, the structure of 17 was
unambiguously established by X-ray diffraction analysis
(Fig. 1),17 which clearly shows the tetrahydroindazole
nucleus with the benzyl substituent at the N2-position.
Two conformers of 17 were in fact observed in the
crystal lattice, in which they differ in the spatial orien-
tation of the tetrahydroindazole ring as a result of
rotation around the Ca–Cb bond. Further chemical
transformation of 17 to the N-Fmoc derivative 1916 was
achieved using the protocols developed above.
cil (BBSRC), UK and the University of Nottingham for
the funding of studentships.
References and notes
1. For example: (a) Kawahata, N. H.; Goodman, M.
Tetrahedron Lett. 1999, 40, 2271–2274; (b) Gibson, S. E.;
Guillo, N.; Kalindjian, S. B.; Tozer, M. J. Bioorg. Med.
Chem. Lett. 1997, 7, 1289–1292; (c) Salvadori, S.; Balboni,
G.; Guerrini, R.; Tomatis, R.; Bianchi, C.; Bryant, S. D.;
Cooper, P. S.; Lazarus, L. H. J. Med. Chem. 1997, 40,
3100–3108; (d) Wang, L.; Brock, A.; Herberich, B.;
Schultz, P. G. Science 2001, 292, 498–500; (e) Atkinson,
G. E.; Cowan, A.; McInnes, C.; Zheleva, D. I.; Fischer, P.
M.; Chan, W. C. Bioorg. Med. Chem. Lett. 2002, 12, 2501–
2505; (f) Hruby, V. J. J. Med. Chem. 2003, 46, 4215–4231;
(g) Chin, J. W.; Cropp, T. A.; Anderson, J. C.; Mukherji,
M.; Zhang, Z.; Schultz, P. G. Science 2003, 301, 964–967.
2. Chhabra, S. R.; Mahajan, A.; Chan, W. C. J. Org. Chem.
2002, 67, 4017–4029.
In summary, the sequential acylation of cyclic 1,3-di-
ketones or cyclic enamines by the side-chain carboxyl
functionalities of appropriately protected aspartic or
glutamic acids, followed by regioselective cycloaddition
with dinucleophiles such as hydrazines has led to a novel
class of pseudoaromatic a-amino acids. These novel
homochiral amino acids with tetrahydroindazole or
tetrahydrobenzisoxazole appended as side-chain func-
tionalities offer unique opportunities, not only as
structural surrogates of tryptophan, but also as novel
amino acid building blocks for the design of molecular
probes. The Na-Fmoc-protected derivatives are useful
tools for the assembly of peptides using established
solid-phase methodologies, and the incorporation of
these amino acids into biologically active cyclic peptides
are currently in progress.
3. (a) Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Hone, N.
D. J. Chem. Soc., Chem. Commun. 1993, 778–779; (b)
Nash, I. A.; Bycroft, B. W.; Chan, W. C. Tetrahedron Lett.
1996, 37, 2625–2628.
4. Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.;
Bycroft, B. W.; Chan, W. C. Tetrahedron Lett. 1998, 39,
1603–1606.
5. Adlington, R. M.; Baldwin, J. E.; Catterick, D.; Pritchard,
G. J. J. Chem. Soc., Perkin Trans. 1 2000, 299–302.
6. (a) Smith, H. J. Chem. Soc. 1953, 803–810; (b) De Buyck,
Acknowledgements
R.J.M. and S.L.M. would like to acknowledge the
Biotechnology and Biological Sciences Research Coun-
ꢀ
L.; Schamp, N.; Verhe, R. Tetrahedron Lett. 1975, 29,

R. J. Middleton et al. / Tetrahedron Letters 45 (2004) 1237–1242
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2491–2492; (c) Akhrem, A. A.; Lakhvich, F. A.; Budai,
S. I.; Khlebnicova, T. S.; Petrusevich, I. I. Synthesis 1978,
925–927; (d) Tabuchi, H.; Hamamoto, T.; Ichihara, A.
Synlett 1993, 651–652; (e) Katritzky, A. R.; Pastor, A.
J. Org. Chem. 2000, 65, 3679–3682.
12. N-Fmoc-b-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1,2-
benzisoxazol-3-yl)-
L
-alanine (8a(ii)). Mp 134–136 ꢁC; RP-
25
HPLC tR 11.6 min; ½aꢂ )55.3ꢁ (c 0.993, MeOH); dH 1.10,
D
1.12 (6H, 2 · s), 2.41, 2.81 (4H, 2 · s), 3.36–3.55 (2H, m),
4.13–4.36 (3H, m), 4.81–4.89 (1H, m), 6.15 (1H, d, J
8.4 Hz), 7.31–7.78 (8H, m); HRMS (FAB) calcd for
C27H27N2O6 (MHþ): 475.186912, found: 475.188635.
13. tert-Butyl (S)-2-tert-butoxycarbonylamino-5-oxo-5-(4,4-
dimethyl-2,6-dioxocyclohex-1-yl)pentanoate (5b). RP-
HPLC tR 15.4 min; dH 1.05 (6H, s), 1.41, 1.45, 1.46, 1.49
(18H, 4 · s), 1.83–2.20 (2H, m), 2.32, 2.51 (4H, 2 · s), 3.09
(2H, t, J 7.4 Hz), 4.13–4.49 (1H, m), 5.16 (1H, d, J 7.7 Hz);
HRMS (FAB) calcd for C22H36NO7 (MHþ): 426.249178,
found: 426.249062.
7. tert-Butyl (S)-2-tert-butoxycarbonylamino-4-oxo-4-(4,4-
dimethyl-2,6-dioxocyclohex-1-yl)butanoate (5a). To
a
solution of Boc- -Asp-Ot-Bu (1.480 g, 5.0 mmol), dime-
L
done (0.701 g, 5.0 mmol) and DMAP (0.611 g, 5.0 mmol)
in CH2Cl2 (15 mL) was added EDC (1.054 g, 5.5 mmol)
and the reaction mixture was stirred for 24 h. The solvent
was removed and the residue dissolved in EtOAc (25 mL).
After successive washes with 1 M KHSO4ðaqÞ (3 · 20 mL)
and brine solution (2 · 20 mL), the organic layer was dried
and evaporated, to yield a golden oil (2.5 g). The oil was
purified by filtration through a short silica column
(EtOAc/hexane, 3:1) and dried under high vacuum to
yield 5a as a glassy yellow solid (1.871 g, 91%): m=z (+ES)
412.3 (MHþ), calcd 412.2; RP-HPLC tR 15.4 min; dH 1.05
(6H, s), 1.39, 1.40 (18H, 2 · s), 2.32, 2.51 (4H, 2 · s), 3.46
(1H, dd, J 18.7, 4.4 Hz), 3.65 (1H, dd, J 18.7, 5.2 Hz),
4.50–4.57 (1H, m), 5.33 (1H, d, J 9.0 Hz).
14. Smith, A. B., III, Akaishi, R.; Jones, D. R.; Keenan, T. P.;
Guzman, M. C.; Holcomb, R. C.; Sprengeler, P. A.;
Wood, J. L.; Hirschmann, R. Biopolymers (Pept. Sci.)
1995, 37, 29–53.
15. tert-Butyl (S)-2-tert-butoxycarbonylamino-4-hydroxy-4-
(2-oxocyclohexylidene-1-yl)butanoate (12a). To a solution
of Boc-L-Asp-Ot-Bu (1.447 g, 5.0 mmol) and DMAP
(611 mg, 5.0 mmol) in CH2Cl2 (10 mL) was added N,N0-
diisopropylcarbodiimide (783 lL, 5.0 mmol). After 5 min,
1-pyrrolidino-1-cyclohexene (805 lL, 5.0 mmol) was added
and the resultant mixture was stirred for 4 h. A mixture of
1 M KHSO4ðaqÞ/brine solution, 1:1; 5 mL) was then added
and the biphasic mixture was stirred vigorously for a
further 30 min. After dilution with brine (30 mL), the
organic layer was separated and evaporated to dryness.
The residual material was taken up in EtOAc (25 mL) and
washed with 1 M KHSO4ðaqÞ (3 · 15 mL), brine solution
(2 · 15 mL), dried and evaporated to yield a yellow oil
(2.5 g). The N,N0-diisopropylurea by-product was removed
by filtration of the crude product through a silica pad
(EtOAc/hexane, 1:3) to yield 12a as a yellow oil (1.411 g,
76%): RP-HPLC tR 14.0 min; dH 1.40, 1.41 (18H, 2 · s),
1.63–1.66 (4H, m), 2.25–2.29 (4H, m), 2.84 (1H, dd, J 17.9,
4.3 Hz), 3.10 (1H, dd, J 17.9, 4.1 Hz), 4.38–4.45 (1H, m),
5.52 (1H, d, J 8.9 Hz), 15.28 (1H, s); HRMS (FAB) calcd
for C19H32NO6 (MHþ): 370.222963, found: 370.224449.
RP-HPLC was performed on a Hypersil Pep5-C18e
column (4.6 · 150 mm). Eluent was monitored at 220 nm.
The linear elution gradient was 50–100% B in 20 min at
1.20 mL minꢁ1 (A ¼ 0.06% aqueous TFA, B ¼ 0.06% TFA
in 90% aqueous acetonitrile).
N-tert-Butoxycarbonyl-b-(1H-6,6-dimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-3-yl)- -alanine a-tert-butyl ester (6a(i)).
L
To a solution of 5a (1.871 g, 4.55 mmol) in EtOH (12 mL)
was added hydrazine monohydrate (441 lL, 9.10 mmol)
and the solution stirred at room temperature for 4 h. The
solvent was removed and the residue dissolved in EtOAc
(20 mL). After successive washes with 1 M KHSO4ðaqÞ
(3 · 20 mL) and brine solution (2 · 20 mL), the organic
layer was dried and evaporated to yield a foamy yellow
solid. This was purified by flash silica chromatography
(EtOAc/hexane, 9:1) to yield the title compound as a
glassy yellow solid (1.563 g, 84%): m=z (+ES) 407.9
25
D
(MHþ), calcd 408.2; RP-HPLC tR 9.2 min; [aꢂ )10.7ꢁ
(c 0.88, MeOH); dH 1.06, 1.08 (6H, 2 · s), 1.35, 1.37
(18H, 2 · s), 2.33, 2.73 (4H, 2 · s), 3.36–3.38 (2H, m), 4.56
(1H, m), 5.89 (1H, m), 8.86 (1H, br s); HRMS (FAB)
calcd for C21H34N3O5 (MHþ): 408.249847, found:
408.249374.
b-(1H-4,5,6,7-Tetrahydroindazol-3-yl)-L-alanine hydro-
chloride (14). Mp 201–205 ꢁC; m=z (+ES) 210.4 (MHþ),
calcd 210.1; dH ([d6]-DMSO) 1.69 (4H, m), 2.40 (2H, m),
2.61 (2H, m), 3.13 (2H, d, J 6.8), 4.21 (1H, m), 8.52 (2H,
br s).
8. b-(1H-6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydroindazol-3-yl)-
N-Fmoc-b-(1H-4,5,6,7-tetrahydroindazol-3-yl)-L-alanine
L
-alanine HCl (7a(i)). Mp 174–176 ꢁC; dH ([d6]-DMSO)
(15). Mp 131–134 ꢁC (decomposed); m=z (+ES) 432.8
24
1.01, 1.03 (6H, 2 · s), 2.27, 2.69 (4H, 2 · s), 3.31 (2H, d, J
6.9 Hz), 4.34–4.39 (1H, m), 8.66, 8.67 (3H, 2 · br s);
HRMS (FAB) calcd for C12H18N3O3 (MHþ): 252.134817,
found: 252.135678.
N-Fmoc-b-(1H-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydroind-
azol-3-yl)-
(MHþ), calcd 432.2; RP-HPLC tR 17.2 min; ½aꢂ )29.6ꢁ (c
D
0.992, MeOH); dH 1.58 (4H, br s), 2.31 (2H, br s), 2.44
(2H, br s), 3.15 (2H, t, J 6.2 Hz), 4.26–4.33 (1H, m), 4.63–
4.74 (3H, m), 6.26 (1H, d, J 7.6 Hz), 7.31–7.39, 7.49–7.61,
7.69–7.74 (8H, 3 · m).
L
-alanine (8a(i)). Mp 139–141 ꢁC; RP-HPLC tR
16. N-tert-Butoxycarbonyl-b-(2-benzyl-4,5,6,7-tetrahydroind-
25
11.6 min; ½aꢂ )43.5ꢁ (c 0.994, MeOH); dH 1.04, 1.09 (6H,
azol-3-yl)-L-alanine a-tert butyl ester (17). A white foamy
D
2 · s), 2.41, 2.65 (4H, 2 · s), 3.46–3.62 (2H, m), 4.19–4.31
(3H, m), 4.71–4.84 (1H, m), 7.05 (1H, d, J 6.1 Hz), 7.24–
7.80 (8H, m); HRMS (FAB) calcd for C27H28N3O5
(MHþ): 474.2074, found: 474.2029.
solid. Recrystallisation from petroleum ether (bp 100–
120 ꢁC) yielded a sample suitable for X-ray determination:
mp 128–129 ꢁC; RP-HPLC tR 12.0 min; dH 1.36, 1.39 (18H,
2 · s), 1.61–1.82 (4H, m), 2.42 (2H, t, J 5.8 Hz), 2.64 (2H, t,
J 6.0 Hz), 2.86 (2H, d, J 7.4 Hz), 4.26–4.35 (1H, m), 4.92
(1H, d, J 8.3 Hz), 5.13–5.35 (2H, m), 7.10–7.13, 7.19–7.31
(5H, 2 · m); HRMS (FAB) calcd for C26H38N3O4 (MHþ):
456.286232, found: 456.285056.
9. Chan, W. C.; Micklewright, R.; Barrett, D. A. J. Chro-
matogr. A 1995, 697, 213–217.
10. RP-HPLC (35% B for 5 min, then 35–65% B in 30 min).
GITC-derivatives 9: tR 17.2 min; m=z (+ES) 641.4 (MHþ),
calcd 641.7; 10: tR 18.0 min; m=z (+ES) 641.4 (MHþ), calcd
641.7.
11. (a) Wiley, R. H.; Hexner, P. E. Org. Synth., Coll. 1963, IV,
351–353; (b) Baraldi, P. G.; Barco, A.; Benetti, S.;
Manfredini, S.; Pollini, G. P.; Simoni, D. Tetrahedron
1987, 43, 235–242; (c) Joule, J. A.; Mills, K. Heterocyclic
Chemistry; Blackwell Science: Oxford, 2000. pp 431–448.
N-Fmoc-b-(2-benzyl-4,5,6,7-tetrahydroindazol-3-yl)-L-
alanine (19). Mp 194 ꢁC (decomposed); m=z (+ES) 522.1
24
(MHþ), calcd 522.2; RP-HPLC tR 9.2 min; ½aꢂ )36.9ꢁ (c
D
0.101, MeOH); dH ([d6]-DMSO) 1.55 (4H, m), 2.31 (2H,
m), 2.39 (2H, m), 2.67–2.96 (2H, m), 3.96–4.20 (4H, m),
5.05–5.23 (2H, m), 6.97–7.04, 7.15–7.37 (10H, 2 · m), 7.61,
7.82 (4H, 2 · d, J 7.3 Hz).

1242
R. J. Middleton et al. / Tetrahedron Letters 45 (2004) 1237–1242
17. Crystal description: colourless column, 0.50 · 0.20 ·
R indices [I > 2rðIÞ]: R1 ¼ 0:0770, wR2 ¼ 0:159; Final R
indices (all data): R1 ¼ 0:118, wR2 ¼ 0:195. The crystallo-
graphic data for the structure of compound 17 have been
deposited in the Cambridge Crystallographic Data Centre
as supplementary publication CCDC 224296. Copies
of the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
ꢁ
0.12 mm. Unit cell dimensions: a ¼ 10:476ð5Þ A,
ꢁ
a ¼ 65:83ð4Þꢁ, b ¼ 10:827ð6Þ A, b ¼ 88:04ð4Þꢁ, c ¼
3
ꢁ
ꢁ
12:814ð7Þ A, c ¼ 81:17ð4Þꢁ. Volume, 1309.6(12) A . Reflec-
tions for cell refinement, 16. Dcalcd ¼ 1:155 mg mꢁ3. Reflec-
tions collected, 5130; Independent reflections, 5076
(Rint ¼ 0:095); Observed reflections, 3518 [I > 2rðIÞ]. Final