Teaching old dogs (Fmoc-amine, azodicarboxylate, and phosphine) new tricks (triazolinones)

Article abstract of DOI:10.1016/j.tetlet.2013.06.115

Exposure of Fmoc-amines to an excess of standard Mitsunobu reagents, azodicarboxylate and triphenylphosphine, at ambient temperature yielded triazolinone derivatives in high yield and purity. This transformation appeared to be general and the traditional Mitsunobu reaction can be carried out independently of triazolinone formation because the latter requires a high excess of reagents.

Full text of DOI:10.1016/j.tetlet.2013.06.115

Tetrahedron Letters 54 (2013) 4749–4752
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Teaching old dogs (Fmoc-amine, azodicarboxylate, and phosphine)
new tricks (triazolinones)
a
a
a
a,b,
⇑
ˇ
ˇ
Anna Krchnáková , Patricia A. Miller , Marvin J. Miller , Viktor Krchnák
a Department of Chemistry and Biochemistry, 251 Nieuwland Science Center, University of Notre Dame, Notre Dame, IN 46556, USA
b Department of Organic Chemistry, Faculty of Science, Palacky University, 17. Listopadu 12, 771 46 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Exposure of Fmoc-amines to an excess of standard Mitsunobu reagents, azodicarboxylate and triphenyl-
phosphine, at ambient temperature yielded triazolinone derivatives in high yield and purity. This trans-
formation appeared to be general and the traditional Mitsunobu reaction can be carried out
independently of triazolinone formation because the latter requires a high excess of reagents.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 22 May 2013
Revised 21 June 2013
Accepted 22 June 2013
Available online 29 June 2013
Keywords:
Mitsunobu
Triazolinones
Heterocycle
Fmoc
Azadicarboxylate
Introduction
hydrophilic than expected (shorter retention time on analytical re-
verse phase LC column) and lacked the Fmoc protection group (UV
The traditional outcome of a Mitsunobu reaction1,2 is formation
of a product from two components (unless intramolecular) via car-
bon–oxygen/nitrogen/carbon bond formation between an alcohol
(source of the carbon) and acidic proton containing nucleophile
(source of oxygen/nitrogen/carbon). Azodicarboxylates and phos-
phines serve as reagents without being included in the target prod-
uct. However, numerous reactions involving components of either
of these reagents in competitively formed products were reported,
such as preparation of isocyanates (Scheme 1) from primary
amines and carbon dioxide.3,4 Syntheses of 1,2,4-triazole deriva-
tives (Scheme 1) from isocyanates by Mitsunobu chemistry were
reported by Alizadeh.5 Herein, we report that the reaction of Fmoc
protected amines with excess Mitsunobu reagents results in a
clean formation of triazolinones, thus providing facile access to
an interesting and useful class of heterocycles.
spectrum). We isolated and purified the product and determined
that its structure is triazolinone derivative 3. Both compounds 3a
and 3b, prepared on Wang resin esterified with Fmoc-Ala-OH,
exhibited similar ion patterns in their mass spectra (LC/MS analysis
3a, rt 2.74 min, [M+H]+ 274, [M+NH4]+ 291, [2M+NH4]+ 564,
[2M+Na]+ 569; LC/MS analysis 3b, rt 3.37 min, [M+H]+ 302,
[M+NH4]+ 319, [2M+NH4]+ 620, [2M+Na]+ 625). This pattern was
found to be characteristic for all synthesized triazolinone deriva-
tives. To access the scope and limitation of this simple and clean
transformation under mild conditions we synthesized several
model compounds using Fmoc-protected amino acids attached to
Rink amide6 1a and Wang7 1b resins, respectively (Scheme 2). All
reactions provided triazolinone derivatives (Table 1).
O
O
N
N
O
Results and discussion
O
CO2
R
NH2
+
R
N
C
O
O
Exposure of polymer-supported Fmoc-amines 1 to an excess of
Mitsunobu reagents azodicarboxylates 2 and triphenylphosphine
at ambient temperature led to the formation of an unexpected
product (Scheme 2). The transformation was very clean and
LC/MS analysis revealed that the product was substantially more
PPh3
O
O
O
PPh3
N
N
O
N
R
N
O
C
O
N
O
N
R
O
⇑
Corresponding author.
E-mail address: vkrchnak@nd.edu (V. Krchnák).
ˇ
Scheme 1. Synthesis of isocyanates and triazoles using Mitsunobu reagents.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.06.115

ˇ
A. Krchnáková et al. / Tetrahedron Letters 54 (2013) 4749–4752
4750
O
R2
O
O
R2
R1
i, ii
N
R2
N
O
O
N
O
R2
N
O
Fmoc
X
N
H
L
N
O
O
R1
O
2a:
2b:
1a
R
R
2 = Et
: X = NH, L = Rink amide linker
1b: X = O, L = Wang linker
HX
3
2 = i-Pr
Scheme 2. Synthesis of triazolinones from polymer-supported Fmoc-amino acid, azodicarboxylate, and phosphine. Reagents and conditions: (i) PPh3, anhydrous THF, rt, 1 h;
(ii) 50% TFA in dichloromethane (DCM) (v/v), rt, 1 h.
Table 1
Synthesized triazolinones
Mitsunobu reaction with complete conversion of acid to an ester
in 1 h, but did not exhibit the formation of any significant amount
Entry
Product
X
R1
R2
Puritya (%)
Yieldb (%)
of triazolinone even after overnight reaction. Alternatively, the use
of four fold excess (0.4 M solution) quantitatively converted the
Fmoc-Ala-OMe to the triazolinone derivative in 1 h. This experi-
ment confirmed that the traditional Mitsunobu reaction can be
carried out independently and without competition from the for-
mation of triazolinone by avoiding a large excess of reagents.
LC/MS analysis of the reaction of Fmoc-Ala-OH, MeOH, PPh3,
and azodicarboxylates 2c and 2d in solution revealed the presence
of products 3c and 3d (XH@OCH3), thus confirming that formation
of 4 and 5 occurred in the cleavage cocktail used to release prod-
ucts from resin (LC/MS analysis 3c, rt 7.93 min, [M+H]+ 344,
[M+NH4]+ 361, [2M+NH4]+ 704, [2M+Na]+ 709; LC/MS analysis
3d, rt 8.53 min, [M+H]+ 412, [M+NH4]+ 429, [2M+NH4]+ 840,
[2M+Na]+ 845).
1
2
3
4
5
3a
3b
3e
4
O
O
NH
O
O
–CH3
–CH3
–CH3
–CH3
–CH3
–CH2CH3
–CH(CH3)2
–CH(CH3)2
–Bn
87
93
68
61
91
77
77
46
47
78
5
–C(CH3)3
a
Purity of the crude product before purification estimated from LC traces @
230 nm.
b
Total yield after purification of target compounds by semi-preparative reverse
phase HPLC.
Next, we carried out the reaction with dibenzyl (2c) and di-tert-
butyl (2d) azodicarboxylates. Whereas diethyl and di-iso-propyl
azodicarboxylates yielded triazolinone derivatives 3, dibenzyl azo-
dicarboxylate formed, after acid-mediated cleavage from the resin,
triazolinone derivative 4 (Scheme 3). The product prepared using
di-tert-butyl azodicarboxylate 2d decomposed in the cleavage
cocktail (50% TFA in DCM) and it was released from the Wang resin
by NaOH. The HRMS and 1H and 13C NMR spectra were consistent
with triazolinone derivative 5 in which the Boc groups had been
cleaved. To document that the transformations of triazolinone
derivatives prepared from azodicarboxylates 2c and 2d to com-
pounds 4 and 5 occurred during cleavage from the resin, we carried
out experiments in solution.
Solid-phase synthesis is traditionally carried out with several-
(typically three)-fold excess of reagents. Therefore, the triazolinone
synthesis was advantageously carried out on solid phase since the
excess of all components of the reaction mixture, except the resin-
bound product, was simply removed by washing the resin beads.
To address the effect of the use of excess Mitsunobu reagents
and competitive traditional Mitsunobu reaction course (formation
of an ester from acid and alcohol), we carried out experiments in
solution. Fmoc-Ala-OH and an equimolar quantity of MeOH in
anhydrous THF (both 0.1 M solution) were exposed to an excess
of diisopropyl azodicarboxylate (DIAD) and PPh3. Two molar excess
of DIAD and PPh3 (0.2 M solution) caused the expected standard
The high purity (61–91%) and high yield (47–78%) of the trans-
formation to triazolinone derivatives with excess Mitsunobu re-
agents prompted us to explore the potential of combining the
traditional course of Mitsunobu reaction with triazolinone forma-
tion on solid phase. As an example, we chose to use Miller’s Mits-
unobu-mediated b-lactam syntheses.8 Thus, hydroxylamine resin
69,10 was acylated with Fmoc-Ser-OH to yield resin 7 (Scheme 4).
Using an excess of azodicarboxylates 2a and 2b and triphenylphos-
phine on resin 7 led to the formation of b-lactams8 and at the same
time transformation of the Fmoc-protecting group to triazolinone
derivatives 8. It is worth mentioning that the synthesis of b-lac-
tams was previously reported on solid phase; however, the authors
did not mention the formation of triazolinone.11
Due to the inherent instability of N-hydroxy-b-lactams,12,13
especially upon exposure to strong acids and their potential rear-
rangement to five-membered ring isoxazolidin-5-ones
9
(Scheme 5), we acetylated product 8b with acetic anhydride. The
conversion to acetate 10b proceeded quantitatively in 30 min
(LC/MS analysis, rt 5.65 min, [M+H]+ 357, [M+NH4]+ 374,
[2M+NH4]+ 730, [2M+Na]+ 735). Then, we adjusted the pH with so-
dium bicarbonate to pH 8. After overnight exposure the acetyl
group was cleaved to allow clean regeneration of 8b (LC/MS
O
O
O
O
i, ii
O
N
Fmoc
O
N
O
L
N
H
N
O
O
HN
N
O
O
O
OH
4
O
O
2c
L = Wang linker
HN
O
N
i, iii
O
N
Fmoc
N
L
OH
N
H
N
O
O
O
O
5
2d
L = Wang linker
Scheme 3. Polymer-supported synthesis of triazolinone derivatives. Reagents and conditions: (i) PPh3, anhydrous THF, rt, 1 h; (ii) 50% TFA in DCM (v/v), rt, 1 h; (iii) 0.5 M
NaOH, THF/MeOH (1:1; v/v), rt, 30 min.

ˇ
A. Krchnáková et al. / Tetrahedron Letters 54 (2013) 4749–4752
4751
O
R
O
R
N
O
N
O
H
i
ii, iii
N
N
O
O
O
Fmoc
N
H
L
H2N
L
N
OH
8a
: R = Et
8b: R = i-Pr
O
OH
7
6
Scheme 4. Concurrent synthesis of b-lactams using Miller’s route and formation of triazolinones. Reagents and conditions: (i) Fmoc-Ser-OH (1 equiv), HOBt (1 equiv), N,N0-
diisopropylcarbodiimide (DIC) (1 equiv), DCM/DMF (1:1, v/v), rt, 2 h; (ii) PPh3, azodicarboxylate 2a or 2b, anhydrous THF, rt, 1 h; (iii) 50% TFA in DCM (v/v), rt, 1 h.
O
O
O
N
N
N
N
N
O
N
HN
O
O
O
N
N
N
O
N
O
O
N
i
O
N
ii
iii
N
O
O
O
O
N
N
O
8b
OH
O
OH
O
O
O
OH
10b
8b
11b
O
N
HN
N
O
O
N
O
i
O
N
O
O
N
O
O
N
O
NH
O
9b
12b
Scheme 5. Chemical evidence for structure confirmation. Reagents and conditions: (i) Ac2O, THF, 30 min; (ii) sodium bicarbonate, water/THF, overnight; (iii) 0.5 M NaOH in
MeOH/THF (1:1) to pHꢀ10, 15 min.
analysis, rt 3.22 min, [M+H]+ 315, [M+NH4]+ 332, [2M+NH4]+ 646,
[2M+Na]+ 651). We also adjusted the pH of acetylated product
10b to pH 10 with a 0.5 M solution of NaOH. After 15 min, the solu-
tion was acidified with acetic acid and analyzed. LC/MS traces re-
vealed the presence of de-acetylated product 11b that also had
lost the iso-propyl carbamate group (LC/MS analysis, rt 0.91 min,
[M+H]+ 229, [M+Na]+ 251, [2M+Na]+ 479). The loss of the iso-pro-
pyloxycarbonyl group in NaOH has already been demonstrated in
our previous experiments. This result provided evidence that prod-
uct 8b is the b-lactam, because the N-acetyl derivative of the rear-
ranged compound, 12b, would not be readily de-acetylated under
these conditions.
Table 2
Inhibition activity of synthesized compounds at 20
l
M concentration
PC3 (%)
Entry
Compound
MCF7 (%)
HeLa (%)
1
2
3
4
5
6
7
3a
3b
3e
4
5
8a
8b
4
25
6
15
15
10
0
11
12
11
12
15
16
14
2
0
10
9
4
1
17
A plausible mechanism for the formation of triazolinone is out-
lined in Scheme 6. The presence of PPh3 is essential; in the absence
of PPh3 no transformation to triazolinone derivative was observed.
Synthesis of analogous 1,2,4-triazole derivatives from isocyanates
by Mitsunobu reagents by a similar mechanism has also been
reported.4,5
control were tested for their antibacterial activities against vari-
ous strains of Gram-positive and Gram-negative bacteria using
agar diffusion assays. None of the compounds exhibited signifi-
cant inhibition at 2 mM concentration. Antiproliferative and cyto-
toxic activities were tested using the cell lines MCF7 (breast
cancer cell line), and PC3 (prostate cancer cell line) for antiprolif-
erative effects and HeLa (human cervix carcinoma) for cytotoxic
Because of druglikeness14 of synthesized heterocycles we con-
sidered it meaningful to evaluate their potential antibacterial and
cytotoxic activities. Triazolinone derivatives and ciprofloxacin as a
effects. Only marginal inhibition was observed at 20 lM concen-
tration (Table 2). Details of assays were described in our previous
publication.15
To conclude, Fmoc-amines were converted to 4-alkyl-3-
alkoxy-5-oxo-4,5-dihydro-1H-1,2,4-triazole-1-carboxylates with
an excess of Mitsunobu reagents azodicarboxylate and triphenyl-
phosphine.16 This transformation requires high excess of reagents
and does not interfere with potential formation of traditional Mits-
unobu products. Thus, control of stoichiometry allows controlled
product variation and heterocycle generation.
R2
O O
R2
R2
O O
R2
O
O
O
O
N
N
N
O
NH
Ph3P
O
H
O
Ph3P
O
HN
R1
HN
R1
R2
Acknowledgements
O
O
O
R2
O
O
H
N
R2
R2
R2
O
R2
H
N
N
O
N
N
O
N
O
O
This research was supported by the Department of Chemistry
and Biochemistry, University of Notre Dame, by the projects
P207/12/0473 from GACR and CZ.1.07/2.3.00/20.0009 from the
European Social Fund. We gratefully appreciate the use of the
NMR facility at the University of Notre Dame.
HO N
R1
N
O
N
O
O
R1
R1
H
Ph3PO
Scheme 6. Consistent mechanism for triazolinone formation.

ˇ
4752
A. Krchnáková et al. / Tetrahedron Letters 54 (2013) 4749–4752
12. Zercher, C. K.; Miller, M. J. Tetrahedron Lett. 1989, 30, 7009–7012.
13. Singh, J.; Kissick, T. P.; Fox, R.; Kocy, O.; Mueller, R. H. J. Heterocycl. Chem. 1989,
26, 17–21.
14. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3–25.
15. Krchnak, V.; Waring, K. R.; Noll, B.; Moellmann, U.; Dahse, H.-M.; Miller, M. J. J.
Org. Chem. 2008, 73, 4559–4567.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.tetlet.2013.06.115.
16. Triazolinone synthesis: Resin 1 (250 mg) in a 5 mL plastic reaction vessel was
washed 3ꢁ with anhydrous THF. A solution of PPh3 (2 mmol, 525 mg) in 2.5 mL
of anhydrous THF was then added to the reaction vessel followed by addition
of azodicarboxylate (2 mmol). The resin slurry was shaken at rt for 1 h. The
resin was washed 5ꢁ with THF and 3ꢁ with DCM. Cleavage and isolation: Acid-
mediated cleavage: Triazolinones on Wang or Rink resins (250 mg) were treated
with 3 mL of 50% TFA in DCM at rt for 1 h. The TFA solution was collected. The
resin was washed 3ꢁ with 50% TFA in DCM, and the combined extracts were
evaporated by a stream of nitrogen gas. The residual products were purified by
semi-preparative reverse phase HPLC in a gradient of acetonitrile (10–40%) in
10 mM ammonium acetate buffer. NaOH-mediated cleavage: Triazolinones on
Wang resin (250 mg) were treated with 2.5 mL of a 0.5 M solution NaOH in
MeOH/THF (1:1; v/v) for 30 min. The solution was collected. The resin was
washed 3ꢁ with MeOH and the combined extracts were neutralized by glacial
acetic acid, concentrated and the products were purified by semi-preparative
References and Notes
1. Mitsunobu, O.; Yamada, M. Bull. Chem. Soc. Jpn. 1967, 40, 935–939.
2. Mitsunobu, O. Synthesis-Stuttgart 1981, 1–28.
3. Saylik, D.; Horvath, M. J.; Elmes, P. S.; Jackson, W. R.; Lovel, C. G.; Moody, K. J.
Org. Chem. 1999, 64, 3940–3946.
4. Horvath, M. J.; Saylik, D.; Elmes, P. S.; Jackson, W. R.; Lovel, C. G.; Moody, K.
Tetrahedron Lett. 1999, 40, 363–366.
5. Alizadeh, A. Helv. Chim. Acta 2005, 88, 2777–2780.
6. Rink, H. Tetrahedron Lett. 1987, 28, 3787–3790.
7. Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328–1333.
8. Miller, M. J.; Mattingly, P. G.; Morrison, M. A.; Kerwin, J. F. J. Am. Chem. Soc.
1980, 102, 7026–7032.
9. Krchnak, V.; Slough, G. A. Tetrahedron Lett. 2004, 45, 4649–4652.
10. Stanger, K. J.; Krchnak, V. J. Comb. Chem. 2006, 8, 435–439.
11. Meloni, M. M.; Taddei, M. Org. Lett. 2001, 3, 337–340.
reverse phase HPLC in
ammonium acetate buffer.
a gradient of (10–40%) acetonitrile in 10 mM