Use of 'click chemistry' for the synthesis of tetrazole-containing analogues of the neuroprotective agent glycyl-L-prolyl-L-glutamic acid

Article abstract of DOI:10.1055/s-0028-1088128

Tetrazole-containing analogues of glycyl-L-prolyl-L-glutamic acid (GPE) were prepared by coupling of Cbz-glycyl-L-proline with tetrazole-containing glutamic acids followed by hydrogenation of the resultant tripeptide. Synthesis of the tetrazole-containing glutamic acids involved 1,3-dipolar cycloaddition of sodium azide to nitrile derivatives of the corresponding glutamic acids. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0028-1088128

LETTER
1233
Use of ‘Click Chemistry’ for the Synthesis of Tetrazole-Containing Analogues
of the Neuroprotective Agent Glycyl-L-prolyl-L-glutamic Acid
Synthesis of
T
etraz
u
ole-Contain
o
ing
A
nalogue
-
s
of Gly
y
cyl-
L
-prolyl
u
L
-glutamic
A
a
cid n Hung, Paul W. R. Harris, Margaret A. Brimble*
Department of Chemistry, University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand
Fax +64(9)3737422; E-mail: m.brimble@auckland.ac.nz
Received 11 February 2009
exhibit improved metabolic stability and oral bioavail-
Abstract: Tetrazole-containing analogues of glycyl-L-prolyl-L-
glutamic acid (GPE) were prepared by coupling of Cbz-glycyl-L-
proline with tetrazole-containing glutamic acids followed by hydro-
genation of the resultant tripeptide. Synthesis of the tetrazole-
containing glutamic acids involved 1,3-dipolar cycloaddition of
sodium azide to nitrile derivatives of the corresponding glutamic
acids.
ability.5a,b
3
N
3
4N
N
4N
2
NH
N2
5
N1
N
5
H1
4a
4b
Key words: GPE, 1,3-dipolar cycloaddition, tetrazole, TMSI,
BoPCl
Figure 2 Structure of tetrazole
Thus, the preparation of tetrazole-containing analogues of
GPE by substituting either the a- or g-carboxylic acid (2
or 3) of glutamic acid with a tetrazole ring may afford a
better therapeutic agent for traumatic brain injury.
Glycyl-L-prolyl-L-glutamic acid (GPE, 1, Figure 1) is a
tripeptide derived from insulin-like growth factor I (IGF-
1) upon enzymatic hydrolysis.1 Use of GPE for the treat-
ment of hypoxic-ischemic (HI) brain injury is significant
although the poor pharmacological profiles of this agent
prompts the development of better analogues.2 Several
analogues of GPE have previously been synthesised with
modification at the side chains of either the glutamic
acid,3a,b glycine,3c or proline3d,e residues in order to probe
the structure–activity relationship (SAR) of GPE and to
identify analogues with better neuroprotective activities
than the native tripeptide.
Our strategy to synthesise tetrazole analogues 2 and 3 of
GPE involved coupling of protected glycyl-L-proline 5 to
tetrazole-containing glutamic acids 6 and 7, respectively,
thus affording the tripeptide mimics after deprotection by
hydrogenolysis of the benzyloxycarbonyl groups
(Scheme 1).
H2N
R1
(1) coupling
CO2H
+
N
The tetrazole moiety (4a and 4b, Figure 2) serves as a sur-
rogate for a carboxylic acid functionality.4 Pharmaceuti-
cal agents containing tetrazole rings have been shown to
(2) global deprotection
O
R2
NHCO2Bn
6 R1 = X, R2 = CO2Bn
7 R1 = CO2Bn, R2 = X
5
N
N
H
N
NH
H
α
N
R1
N
N
N
N
N
X =
O
O
NH
O
O
N
H
R2
CO2H
NH2
NH2
N
CO2H
CO2H
2
N
2 R1 = X, R2 = CO2H
3 R1 = CO2H, R2 = X
O
O
H
N
CO2H
NH2
Scheme 1 Synthesis of tetrazole-containing analogues 2 and 3
N
1
O
γ
O
As reported previously, the synthesis of N-benzyloxy-
carbonyl-glycyl-L-proline (5, Scheme 2) was carried out
by coupling N-benzyloxycarbonyl-glycine (8) with N-
hydroxysuccinimide (9) under N2 at 0 °C.6 Without puri-
fication, the resultant compound 10 was then coupled to
L-proline (11) at room temperature followed by acidifica-
tion to afford dipeptide 5 in 77% yield.7
NH2
N
N
N
NH
3
Figure 1 Structures of GPE (1) and a- and g-tetrazole-containing
analogues (2 and 3) of GPE
SYNLETT 2009, No. 8, pp 1233–1236
Advanced online publication: 08.04.2009
DOI: 10.1055/s-0028-1088128; Art ID: D04809ST
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x.
2
0
0
9
a-Tetrazole glutamic acid 12 was obtained by 1,3-dipolar
cycloaddition of nitrile 15 with NaN3 (Scheme 3). Nitrile

1234
K.-y. Hung et al.
LETTER
O
O
O
CO2H
CO2H
(i)
(ii)
N
+
+
HO
N
O N
CO2H
N
NHCO2Bn
O
H
NHCO2Bn
O
O
NHCO2Bn
8
9
10
11
5
Scheme 2 Reagents and conditions: (i) DCC, DME, N2, 0 °C, 3 h; (ii) NaHCO3, H2O, DME, 4 h then HCl (77% over 2 steps).
N
N
BocHN
CO2H
BocHN
CONH2
CO2Bn
BocHN
CN
NH
BocHN
(iii)
(i)
(ii)
N
CO2Bn
CO2Bn
CO2Bn
13
14
15
12
Scheme 3 Reagents and conditions: (i) Boc2O, NH4HCO3, pyridine, dioxane, N2, r.t., 18 h (93%); (ii) cyanuric chloride, DMF, 0 °C to r.t.,
22.5 h (98%); (iii) Et3N, AcOH, NaN3, dry toluene, N2, reflux, 16 h (88%).
BocHN
CO2Bn
BocHN
CO2Bn
CO2H
BocHN
CO2Bn
CONH2
BocHN
CO2Bn
CN
(iii)
(ii)
(i)
N
N
N
NH
17
18
19
16
Scheme 4 Reagents and conditions: (i) Boc2O, NH4HCO3, pyridine, dioxane, N2, r.t., 19 h (79%); (ii) cyanuric chloride, DMF, 0 °C to r.t.,
22.5 h (26%); (iii) Et3N, AcOH, NaN3, dry toluene, N2, reflux, 29 h (82%).
15 in turn was obtained in excellent yield from acid 13 via mechanism giving tert-butyl iodide rather than a tert-
amide 14. Initial synthesis of tetrazole 12 was carried out butyl cation as a byproduct. Formation of tert-butylated
using NaN3 and ZnBr2 in a mixture of H2O–i-PrOH con- byproduct was eliminated in the absence of the
ditions reported by Sharpless et al.8 The synthesis of the electrophic tert-butyl cation.
tetrazole-containing glutamic acid using these conditions
was unsuccessful in our hands despite the synthesis of an
With the synthesis of tetrazoles 6 and 7 in hand, subse-
quent coupling with dipeptide 5 using BoPCl in CH2Cl2
for 3 hours afforded the protected tripeptides 20 and 21 in
26% and 27% yields, respectively, following purification
analogous Fmoc-protected tetrazole-containing glutamic
acid having been reported previously.9
It was suspected in our case that hydrolysis of the ester by reverse-phase HPLC (Scheme 5 and Scheme 6). Tetra-
group by water was occurring under these conditions. zoles 2 and 3 were then obtained via hydrogenation of the
This was confirmed by the observation of benzyl alcohol protected tripeptides 20 and 21.
in the 1H NMR spectrum of the crude product. Use of H2O
was therefore avoided by using Et3N, AcOH, and NaN3 in
In conclusion, the synthesis of two tetrazole-containing
analogues 2 and 3 of the neuroprotective agent GPE (1),
dry toluene under reflux affording a-tetrazole glutamic
acid 12 in 88% yield after purification by flash column
N
N
N
N
N
N
chromatography.10
NH
NH
BocHN
NH2
CO2H
N
g-Tetrazole glutamic acid 16 was next synthesised from
nitrile 19 using the same reaction conditions (Scheme 4).
In turn, the synthesis of nitrile from amide 18 proceeded
uneventfully.
(i)
+
O
NHCO2Bn
CO2Bn
CO2Bn
12
6
5
Deprotection of the Boc group from tetrazoles 12 and 16
was next required in order to initiate the subsequent cou-
pling reaction. Sureshbabu et al.9 reported that deprotec-
tion of Boc-Phe tetrazole using TFA was unsuccessful. In
the present work, deprotection of the Boc group using
TFA led to formation of tert-butylated byproducts despite
the use of scavengers (H2O–triisopropylsilane). Fortu-
nately the formation of the tert-butyl byproducts was
eliminated using trimethylsilyl iodide (TMSI).11 Depro-
tection of the Boc group using TMSI proceeds via an SN2
N
N
N
N
N
NH
NH
H
N
H
α
N
N
(ii)
(iii)
N
N
O
O
O
O
CO2Bn
CO2H
NHCO2Bn
NH2
20
2
Scheme 5 Reagents and conditions: (i) TMSI (3 equiv), MeCN, N2,
18 min (ca. 100%); (ii) BoPCl (2 equiv), CH2Cl2, r.t., 3 h (26% over
2 steps from 12); (iii) H2, 10% Pd/C, MeOH–H2O (80:20), r.t., 17.5 h
(60%).
Synlett 2009, No. 8, 1233–1236 © Thieme Stuttgart · New York

LETTER
Synthesis of Tetrazole-Containing Analogues of Glycyl-L-prolyl-L-glutamic Acid
1235
a-Benzyl N-tert-Butyloxycarbonyl-L-glutamate g-Tetrazole (16)
HRMS (EI): m/z [M+] calcd for C17H23N5O4: 361.1750; found:
361.1756. IR: 3344, 2979, 1712, 1671, 1523, 1455, 1367, 1158,
BocHN
CO2Bn
H2N
CO2Bn
CO2H
(i)
N
+
1
754, 698 cm–1. Mp 118–120 °C. H NMR (300 MHz, MeOD):
O
d = 1.70 [9 H, s, C(CH3)3], 2.35–2.44 (m, 1 H, Glub-H), 2.59–2.63
(m, 1 H, Glub-H), 3.26–3.29 (t, J = 5.5 Hz, 2 H, Glug-H2), 4.49–
4.51 (m, J = 3.3 Hz, 1 H, Glua-H), 5.42–5.44 (s, 2 H, OCH2PH),
7.64–7.60 (m, 5 H, Ph). 13C NMR (75 MHz, MeOD): d = 20.36
(CH2, Glug-C), 27.57 [CH3, C(CH3)3], 29.35 (CH2, Glub-C), 53.53
(CH, Glua-C), 66.85 (CH2, OCH2Ph), 79.61 [q, C(CH3)3], 128.10
(CH, Ph), 128.16 (CH, Ph), 128.42 (CH, Ph), 136.05 (q, Ph), 157.50
(q, NCO2), 158.28 (q, C=N), 172.33 (q, Glua-CO)
N
N
N
N
NHCO2Bn
N
NH
N
NH
16
7
5
H
H
N
CO2Bn
N
CO2H
N
N
(ii)
(iii)
O
O
γ
O
O
NHCO2Bn
NH2
N
N
N
N
General Procedure for the Preparation of Tetrazole-Containing
GPE Analogues 2 and 3
N
NH
N
NH
21
3
TMSI (3 equiv) was added to a solution of tetrazole-containing
glutamic acids 6 and 7 in MeCN, and the reaction was stirred under
N2 for 18 min. MeOH was added, and the solution was stirred for 5
min. The solvent was removed under reduced pressure to afford an
oil that was reacted with dipeptide 5 (1 equiv), BoPCl (2 equiv), and
DIPEA (3 equiv) in CH2Cl2 under N2. After completion of the reac-
tion, the solvent was removed under reduced pressure, and the resi-
due was purified by reverse-phase HPLC (Waters C18 Xterra,
19 × 250 mm, 10 mL/min, 1% B to 70% B where B: 0.1% TFA in
MeCN; A: 0.1% TFA in H2O) to afford protected tripeptides as co-
lourless solids. Hydrogenation of the resultant tripeptides afforded
the desired analogues 2 and 3.
Scheme 6 Reagents and conditions: (i) TMSI (3 equiv), MeCN, N2,
18 min (ca. 100%); (ii) BoPCl (2 equiv), CH2Cl2, r.t., 3 h (28% over
2 steps from 16); (iii) H2, 10% Pd/C, MeOH–H2O (80:20), r.t., 19.5 h
(63%).
is reported herein. Additionally, protected tetrazole-
containing glutamic acids 12 and 16 were successfully
prepared by 1,3-dipolar cycloaddition of sodium azide
with glutamic acid derived nitriles 15 and 19. Deprotec-
tion of the Boc group using TMSI was important to avoid
unwanted tert-butylation of the deprotected tetrazoles 6
and 7. Coupling of tetrazoles 6 and 7 with dipeptide 5
gave tripeptides 21 and 22 which then underwent hydro-
Glycyl-L-prolyl-L-glutamate a-Tetrazole (2)
HRMS (EI): m/z [M+] calcd for C12H20N7O4: 326.1571; found:
326.1570. IR: 2947, 1648, 1402, 1353, 1246, 1201, 1119 cm–1. Mp
genation to afford the desired tetrazole-containing ana- not measured as compound was hydroscopic. 1H NMR (300 MHz,
D2O): d = 1.77–2.36 (m, 8 H, Prog-H2, Prob-H2, Glug-H2, Glub-
H2), 3.36–3.48 (m, 2 H, Prod-H2), 3.82–3.94 (m, 2 H, Glya-H2),
4.32–4.37 (m, 1 H, Proa-H), 5.31–5.18 (m, 1 H, Glua-H). 13C NMR
(75 MHz, D2O): d = 21.96* (CH2, Prog-C), 24.18 (CH2, Prog-C),
29.38 (CH2, Prob-C), 31.77* (CH2, Prob-C), 28.65* (CH2, Glub-C),
28.90 (CH2, Glub-C), 31.42 (CH2, Glug-C), 31.77* (CH2, Glug-C),
40.19* (CH2,Glya-C), 40.38 (CH2,Glya-C), 45.25 (CH, Glua-C),
45.66* (CH, Glua-C), 46.83 (CH2, Prod-C), 47.52* (CH2, Prod-C),
59.93* (CH, Proa-C), 60.45 (CH, Proa-C), 145.79 (q, C=N),165.59
(q, Gly-CO), 165.90* (q, Gly-CO), 172.90* (q, NCO), 173.62 (q,
NCO), 179.05 (q, Glug-COOH). The product was shown to be a
75:25 mixture of trans/cis conformers and the chemical shifts for
the minor cis-conformer are denoted by an asterisk (*).
logues 2 and 3. More importantly, the tetrazole-modified
glutamic acids 6 and 7 are useful building blocks for the
preparation of bioisosteres of glutamic acid containing
peptides thus providing an attractive tool for the genera-
tion of peptidomimetics.
General Procedure for the Preparation of Tetrazole-Containing
Glutamic Acids 12 and 16
To a solution of distilled Et3N (4 equiv) in dry toluene was added
glacial AcOH (4 equiv), and the solution was stirred under N2 for 5
min. The solution was transferred to a flask containing the appropri-
ate nitrile (1 equiv) and NaN3 (4 equiv), and the reaction was stirred
under N2 at reflux for 16 h. The solid was filtered, and the filtrate
was concentrated under reduced pressure to afford an oil which was
purified by flash column chromatography (hexane–EtOAc, 1:1 with
1% AcOH) to afford the desired products as colourless solids.
Glycyl-L-prolyl-L-glutamate g-Tetrazole (3)
HRMS–FAB: m/z [M + H]+ calcd for C12H20N7O4: 326.1577;
found: 326.1570. IR: 2959, 1648, 1536, 1428, 1353, 1246, 1200,
1
1128 cm–1. Mp not measured as compound was hydroscopic. H
NMR (300 MHz; D2O): d = 1.91–2.28 (m, 6H, Prog-H2, Prob-H2,
Glub-H2), 2.88–2.99 (m, 2 H, Glug-H2), 3.41–3.49 (m, 2 H, Prod-
H2), 3.82–3.99 (m, 2 H, Glya-H2), 4.12–4.16 (m, 1 H, Glua-H),
4.37–4.41 (m, 1 H, Proa-H). 13C NMR (75 MHz, D2O): d = 19.63
(CH2, Glug-C), 20.09* (CH2, Prog-C), 22.03* (CH2, Glug-C), 24.26
(CH2, Prog-C), 29.27 (CH2, Glub-C), 29.36 (CH2, Prob-C), 29.77*
(CH2, Prob-C), 31.73* (CH2, Glub-C), 40.24* (CH2, Glya-C),
40.40 (CH2, Glya-C), 46.92 (CH2, Prod-C), 47.54* (CH2, Prod-C),
54.10 (CH, Glua-C), 60.10* (CH, Proa-C), 60.58 (CH, Proa-C),
147.47 (q, C=N), 165.63 (q, Gly-CO), 166.11* (q, Gly-CO),
173.22* (q, NCO), 173.50 (q, NCO), 177.15 (q, Glua-COOH). The
product was shown to be a 80:20 mixture of trans/cis conformers
and the chemical shifts for the minor cis-conformer are denoted by
an asterisk (*).
g-Benzyl N-tert-Butyloxycarbonyl-L-glutamate a-Tetrazole (12)
HRMS (EI): m/z [M+] calcd for C17H23N5O4: 361.1750; found:
361.1752. IR: 3322, 2919, 1716, 1685, 1516, 1438, 1393, 1147,
1
749, 700 cm–1. Mp 144–146 °C. H NMR (300 MHz, MeOD):
d = 1.43 [9 H, s, C(CH3)3], 2.11–2.23 (m, J = 2.1, 6.9 Hz, 1 H, Glub-
H), 2.29–2.40 (m, J = 6.9 Hz, 1 H, Glub-H), 2.51–2.56 (t, J = 7.5
Hz, 2 H, Glug-H2), 5.04–5.09 (m, J = 4.2 Hz, 1 H, Glua-H), 5.12 (s,
2 H, OCH2PH), 7.29–7.37 (m, 5 H, Ph). 13C NMR (75 MHz,
MeOD): d = 27.24 [CH3, C(CH3)3], 28.20 (CH2, Glub-C), 29.63
(CH2, Glug-C), 45.27 (CH, Glua-C), 66.06 (CH2, OCH2Ph), 79.60
[q, C(CH3)3], 127.80 (CH, Ph), 127.92 (CH, Ph), 128.13 (CH, Ph),
136.10 (q, Ph), 156.24 (q, NCO2), 158.28 (q, C=N), 173.10 (q,
Glug-CO).
Synlett 2009, No. 8, 1233–1236 © Thieme Stuttgart · New York

1236
K.-y. Hung et al.
LETTER
González-Muñiz, R.; Herranz, R.; Martín-Martínez, M.;
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Acknowledgment
We thank the Maurice Wilkins Centre for Molecular Biodiscovery
for financial support of this work.
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