Cleavable Amide Bond: Mechanistic Insight into Cleavable 4-Aminopyrazolyloxy Acetamide at Low pH

Article abstract of DOI:10.1021/acs.joc.9b00535

The cleavage of amide bonds under mild acidic conditions is a rare chemical event. N-Acetamide bond of peptides is extremely stable even under the strongest organic acid trifluoromethanesulfonic acid. This report mechanistically describes a new cleavable amide bond in 4-aminopyrazolyloxy acetamide peptide analogues under mild acidic conditions such as trifluoroacetic acid (10-20%) or HCl (0.1-4.0 N) at room temperature, and the formation of unusual lactam from 4-aminopyrazolyloxy acetic acid after evaporation of solvent. This is a rare chemical event in peptide bond, which could be explored as acid-sensitive protecting group of free amines.

Full text of DOI:10.1021/acs.joc.9b00535

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Article
Cleavable Amide Bond: Mechanistic Insight of
Cleavable 4-Aminopyrazolyloxy Acetamide at low pH
Amarnath Bollu, and Nagendra K. Sharma
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00535 • Publication Date (Web): 10 Apr 2019
Downloaded from http://pubs.acs.org on April 13, 2019
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Cleavable Amide Bond: Mechanistic Insight of Cleavable 4-Aminopyrazolyloxy Acetamide
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at low pH
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Amarnath Bollu and Nagendra K. Sharma*
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School of Chemical Science, National Institute of Science Education and Research - (NISER)
Bhubaneswar, Jatani-752050, Odisha, India & HBNI-Mumbai, Mumbai-India.
E-mail: nagendra@niser.ac.in
Abstract
The cleavage of amide bonds under mild acidic conditions are rare chemical event. N-
acetamide bond of peptides is extremely stable even under the strongest organic acid TFMSA.
This report mechanistically describes a new cleavable amide bond in 4-aminopyrazolyloxy
acetamide peptide analogues under mild acidic conditions such as TFA (10-20%) or HCl (0.1
– 4.0 N) at room temperature, and the formation of unusual lactam from 4-aminopyrazolyloxy
acetic acid after evaporation of solvent. This is a rare chemical event in peptide bond which
could be explored as acid sensitive protecting group of free amines.
H
+ (Mild)
O
NH2
NHBoc
N
H
NH2
O
O
N
H
H
N
N
N
H+/MeOH
O
N
N
N
N
O
N
O
O
O
N
O
O
O
O
O
H2N-AA-OMe
- Boc
"Only in Solution"
Introduction
Natural amides are extremely stable, and their half-life for spontaneous hydrolysis is estimated
to be 350 to 600 years at neutral pH and room temperature.1 The carbonyl group of amide are
poor electrophiles because of the typical resonance stability. Though amides can readily be
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cleaved/hydrolyzed by enzymes such as proteases. It is difficult to cleave the C-N bond of an
amide selectively using synthetic chemistry. The cleavage/hydrolysis of amide bonds usually
requires heating under strongly acidic or basic conditions. However, the cyclic amides, lactams,
are easily cleavable as compare to the linear amides because of ring strained amides.2 A large
number of the highly strained lactams are synthesized and found as cleavable amide bond under
mild conditions. Since the C=O bond of reactive lactams amide is arising to the strong
electrophile as “ketonic” carbonyl because of the resonance decoupling through N-C=O
torsion. Brown and co-workers have mechanistically confirmed that resonance decoupling
induces the remarkable enhancement in hydrolysis rate by direct nucleophilic attack at the
carbonyl of strained amide.3 The most twisted amide as Trimethylazatricyclodecanone is
highly strained lactam with large resonance decoupling through N-C bond, and found as readily
hydrolysable. This twisted lactam also shows the dual reactivity of amide as nucleophilic
character of amine and electrophilic nature of carbonyl. Booker-Milburn and co-workers have
reported the solvolysis/substitution reaction of acyclic synthetic amides at room temperature
and neutral conditions via the formation of ketene intermediates.4 In literature, the cleavage of
few natural amide bonds are precisely demonsterated through the formation of thiazolinone
derivatives (by Edman degradation), oxazolinium intermediates and chemical modification of
the amide backbone.5 Most Importantly, Edman degradation method for cleavage of amide
bond is foundation for protein sequencing technology.5a The presence of Zinc species in active
sites of metalloproteases have inspired synthetic chemists to cleave amide bond using Zn2+.
Mashima and co-workers have reported the cleavage of amides bearing a β-hydroxyethyl group
under mild conditions with Lewis acid Zn(OTf)2.6 Kostic and co-workers have prepared an
artificial peptidase which cleavage of the sequence specific amide bonds as Pro-Met/Pro-His
segments of peptide under mild conditions with Pd-catalyst.7 In repertoire of the making
reactive amide group, Hauk, Garg and co-workers have established the conversion of amide
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functional group into ester group by cleaving C-N bond of amide using Ni-catalyst.8 The
conversion of an amide into another amide with different amine group, transamidation, is
important chemical transformation reaction for ligation or removal of amino functionalized
chemical moieties at carboxylate functionalized molecules.9 Gellman, Stahl and co-workers
have explored the amide groups for transamidation reaction, between secondary amines and
tertiary amines, using Zr/Hf-catalyst.9a Other Metal catalyzed and metal free transamidation
reactions are also reported.9b, c, 10 Recently, we have also established the transamidation reaction
in tropolonylalkylglycinate peptide derivatives with different amino acid/peptides.
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We, recently, have found the instability of amide bonds containing N-
Tropolonylaminoalkylglycinate (Trag) in short peptides under mild acidic conditions. The
formation of stable tropolonium cations followed by the formation of ketene intermediate are
main caused for the cleavage of Trag-amide bonds.11 In repertoire of unnatural heterocyclic
aromatic amino acids, we have explored the syntheses and supramolecular self-assembly
studies of pyrazolone containing amino acid analogues. One of those analogue is 4-
aminopyrazolonyl acetate has been conjugated with natural amino acid/short peptides for
understanding the role of pyrazolonyl moiety in conformational changes of peptides.12
However these conjugated peptides have shown an unusual reactivity under mild acidic
conditions (Figure 1). To understand the role of 4-aminopyrazolonyl acetate in cleveage of
amide bond, herein we report the syntheses and chemical instability of amide bond in simple
non-functional amino acids and di-/tri-peptide containing aminopyrazolonyl acetyl residue
under mild acidic conditions at room temperature by NMR and Mass analyses.
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Previous report
O
O
O
t1/2 = 500 years
pH 6.8; 25oC
O
H
N
OH
H2N
+
N
OH
N
H
OH
H
O
O
1996
, 118, 6105-6109)
(J. Am. Chem. Soc.
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This report
H
O
H2N
N
BocHN
O
O
O
H+ (Mild)
N
O
N AA-OMe
H
OMe
H+/MeOH
N
O
N
N
N
N
Ph
Ph
Ph
"Only in Solution"
4-aminopyrazolyloxy
acetamide
H2N-AA-OMe
Figure 1. Hydrolyzable amide bonds: previous report (a); and this report (b).
Result and discussion
We used previously synthesized 4-aminopyrazolonyl amino acid (APA) ester derivative (1) for
synthesis of conjugated peptides and their chemical behavior under mild acidic conditions.
Ester (1) was hydrolyzed into 4-aminopyrazolonyl amino acid, APA acid (2) with LiOH (1.0N
in THF), and then coupled with amino group of various natural amino acids/peptides (H2N-
AA-OMe) under peptide coupling reaction conditions which produced conjugated peptides 3
(APA-AA-OMe). Natural α-amino acid ester derivatives of Gly-OMe, Ala-OMe, Ile-OMe, and
Phe-OMe produced unnatural respective conjugated dipeptides as BocNH-APA-Gly-OMe
(3a), BocNH-APA-Ala-OMe (3b), BocNH-APA-Ile-OMe (3c), BocNH-APA-Phe-OMe.
Further natural α-amino acid dipeptide esters, Gly-Ala-OMe, Ala-Gly-OMe, Gly-Ile-OMe and
Ile-Gly-OMe gave respective unnatural conjugated tripeptides as BocNH-APA-Gly-Ala-OMe
(3e), BocNH-APA-Ala-Gly-OMe (3f) BocNH-APA-Gly-Ile-OMe (3g), and BocNH-APA-Ile-
Gly-OMe (3h) as shown in Scheme 1. The characterization data (1H-/13C{1H}-NMR/Mass) of
all synthesized peptides are provided in Supporting Information.
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Scheme 1. Synthesis of N-Boc-aminopyrazolonyl amino acid and their peptides.
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BocHN
O
BocHN
N
O
BocHN
O
O
O
O
H N-AA-OMe
LiOH
(1 N)
/
2
OMe
OH
N AA-OMe
EDC/HOAT
N
H
N
N
N
N
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Ph
Ph
3
(BocNH-APA-NH-AA-OMe)
1
2
(BocNH-APA-OH)
Peptides
3g
3h
. BocNH-APA-Gly-Ile-OMe
. BocNH-APA-Ile-Gly-OMe
3a
. BocNH-APA-Gly-OMe
3b
3c
3d
. BocNH-APA-Phe-OMe
. BocNH-APA-Ala-OMe
. BocNH-APA-Ile-OMe
3e
. BocNH-APA-Gly-Ala-OMe
3f.
BocNH-APA-Ala-Gly-OMe
For further elongation of peptides at N-terminal of hybrid peptides 3a-3h, the removal of N-
Boc group was necessary. The removal of Boc group of N-Boc-aminopyrazolonyl amino acid
containing peptides 3a-3h was attempted with 20% TFA in DCM, unfortunately we could not
achieve desired aminopyrazolonyl amino acid peptides (Scheme 2). Surprisingly, we noticed
the cleavage of amide bond by ESI-Mass studies. Then we isolated a new crystalline product
and characterized as aminopyrazolyl lactam 4 by NMR & HRMS analysis. This crystal was
also studied by single crystal X-ray diffractometer. The X-ray studies confirmed the structure
of lactam 4, the ORTEP diagram is shown in Scheme 2. The cif file of crystal lactam 4 has
deposited to CCDC with number CCDC 1890879. To ensure this cleavage of amide bond, we
performed the similar acid treatment reaction with other hybrid di-/tri-peptides, 3b-3h and then
characterized their isolated products by NMR and Mass analyses (See Supporting Information).
Irrespective of α-amino acid sequence, aminopyrazolyl amino acid containing amide bonds in
peptides 3b-3h were cleaved and produced the similar cyclized product as lactam 4. In case of
hybrid tripeptides 3e-3h, other natural amide bonds, dipeptides (between two α-amino acids)
were stable under that acidic conditions (20% TFA in DCM). To test the selective cleavage of
4-aminopyrazolyloxy acetamide bond in the presence of Boc group, we treated compound 3e
with 0.1 N HCl/1, 4-dioxane and studied with ESI-MS (Figure S20 in SI), Here the removal of
Boc is very slow, under these conditions there are no significant representative peaks at m/z
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348 (N-Boc acid) or 330 (N-Boc lactam) for selective cleavage products. We noticed the
cleavage of 4-aminopyrazolyloxy acetamide bond is not cleavable until the Boc group is
deprotected. Along with lactam 4, we also observed N-trifluoroacylation with 20% TFA in
ESI-MS, this side reaction was minimized/avoided by using 10% TFA or 4.0 N HCl in 1,4-
dioxane (Supporting Information, Figure S13-S15). These results strongly suggest the cleavage
of specific amide bond containing 4-aminopyrazolonyl amino acid residue at C-terminal of α-
amino acid/peptides.
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Further, we attempted to remove the Boc group of N-Boc-aminopyrazolonyl amino acid ester
1 and its carboxylic acid 2 under similar acidic conditions (10-20% TFA in CDCl3) for
conjugation of aminoprazolonyl moiety at C-terminal of above mentioned di-/tri-peptides.
Surprisingly, we failed to generate amino functionalized amino ester (1-NH2) and acid (2-
NH2). However, we isolated a new product and characterized by NMR and ESI-Mass analyses.
Our NMR and Mass results supported that both ester 1 and its acid 2 gave the common cyclized
product as lactam 4 under mild acidic conditions (Scheme 2). The synthesis of that lactam 4 is
extremely unusual from ester 1/acid 2/amide 3 under acidic conditions at room temperature
which are also unusual in peptide chemistry.
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Scheme 2. Reaction of APA derivatives under mild acidic conditions.
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H+/ROH
N AA-OMe
H2N-AA-OMe
BocHN
N
O
O
N
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H
Ph
H2N
N
O
H
O
Peptides (3a-3h)
O
N
OR
(BocNH-
APA-AA-OMe)
N
H+/ROH
N
O
Ph
H+/ROH
H+/ROH
N
BocHN
N
O
5
O
"Only in Solution"
OH
N
2
Ph
4 (Lactam)
BocHN
N
O
O
OMe
N
Ph
4
1
1
The progress of amide cleavage reactions, under acidic conditions, are studied by H-NMR
technique. We recorded the time dependent 1H-NMR spectra of peptide 3a under 10% TFA in
1
deuterated solvent CDCl3. The array of the H-NMR spectra with time is provided in
Supporting Information, Figure S25. Most importantly, the gradual disappearance of NMR
signal, singlet at δ 5.0 (ppm), is noticed in peptide 3a with respect to time under that acidic
conditions. A transient increase and decrease of signal at δ 4.5 (ppm) is also observed. Similar
time dependent NMR experiments were performed under acidic conditions with another
peptide 3e. Their NMR data are provided in Supporting Information (Figure S27). After the
NMR study the same samples were subjected to ESI-MS in solvent CH3CN (neat). Their mass
spectra exhibit the mass (m/z) peak at 230, in solvent CH3CN, for both peptides 3a/3e, which
belongs to mass of lactam 4 as [M+H]+ Supporting Information (Figure S26 & S28). These
results (NMR and Mass) strongly support the involvement of α-CH2 of aminopyrazolonyl
residue in the cleavage of peptide 3a/3e under acidic condition (10% TFA in CDCl3) via the
formation of stable lactam 4. Further, we performed the similar NMR and Mass experiments
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with N-Boc-aminopyrazolonyl amino acid ester 1 and its carboxylic acid 2. Their NMR spectra,
NMR profiles and mass spectra are provided in the Supporting Information. Our results suggest
that the formation of lactam 4 is common for both aminopyrazolonyl ester 1 and its carboxylic
acid 2 under mild acidic conditions (10% TFA in CDCl3). We studied the 1H-NMR kinetics of
amide 3a/acid 2/ester 1 hydrolysis in CDCl3. The relative concentrations were calculated in
mole fractions (x) from relative integrations of –O-CH2- peak of amide 3a/acid 2/ester 1 with
gradually increasing lactam 4 with time. The plots of mole fraction (x) with time for APA
amide 3a and APA acid 2 is provided in Figure 2, and APA ester 1 is provided in supporting
information (Figure S35A). We have also generated simulated kinetic plots from the sequential
first-order reaction model with COPASI software program using experimentally obtained k1
and k2 values and then fitted with our experimental kinetic plots (Figure S41-S43).
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1.2
1.0
0.8
0.6
0.4
0.2
0.0
A
(4)
(3a)
(3a without Boc)
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0
50
100
150
200
Time (min)
1
Figure 2. Time dependent H-NMR studies of APA peptide 3a (A) and APA acid 2 (B) in
solvent CDCl3 with 20 mM concentrations.
From the plots of concentration versus time, a transient increase and decrease of intermediate
is also observed, this suggest that the cyclization of amide 3/acid 2/ester 1 is proceeding through
an intermediate. After seeing these plots, we propose that compound 1, 2, & 3a are following
consecutive first order kinetic pathway and the rate laws are given by equations 1-3 in
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supporting information. The plots of integral mole fraction versus time, (ln(x) vs time) are
provided in Supporting Information (Figure S33-S35) and gave a straight line with negative
slope which are consistent with literature reports for consecutive reaction pathway.13 The rate
constants k1, k2 and their respective half-lives (t1/2) are calculated from these integral plots and
provided in Table 1. The observed rate constants and half-lives from Table 1 suggest that the
acid 2 is hydrolysed relatively faster than amide 3a followed by ester 1. This is possibly due to
the intramolecular hydrogen bonding in APA peptide 3a and acid 2, between H-atom (-COOH-
) or amide NH with O-atom (–O-CH2-) of pyrazolonyl residue, which provide rigidity at
carbonyl group and enhance the nucleophilic addition elimination reaction (Figure S44). Such
hydrogen bonding is not possible with APA ester 1.
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Table 1. Kinetic data of 4-aminopyrazolyloxy derivatives from 1H-NMR spectrum.
Compound
3a (Amide)
2 (Acid)
k1/min-1
k2/min-1
k2/k1
0.36
0.55
0.4
t1/2 /min (for k1)
30.34
t1/2 /min (for k2)
83.00
2.28 x 10-2
1.97 x 10-2
1.95 x 10-2
0.83 x 10-2
1.09 x 10-2
0.78 x 10-2
35.09
63.35
1 (Ester)
35.43
88.74
Interestingly, during the hydrolysis of amide bond in peptides 3a-3h, we observed
methanolysis of amide bond in lactam in mass spectrum and exist in methyl ester form in
presence of MeOH (Supporting Information Figure S13-S18). To further understanding the
reactivity of this amide bond, the lactam 4 is monitored in several aspects using mass
spectroscopy (Supporting Information Figure S37), from these studies it is apparent that, acid
and MeOH both are needed for the amide bond cleavage in lactam 4. Moreover, there is no
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complete disappearance of lactam in any of the cleavage conditions implemented during this
studies. This cleavage is also monitored by time dependent 1H-NMR in CD3CN, CD3OD and
PTSA (~ 3.0 equivalents) at 293 K, from the array of spectra the reaction proceeds with the
formation of hydrolysed methyl ester and remains constant after ~500 min without complete
conversion of lactam 4. The relative concentrations were calculated in mole fractions (x) from
relative integrations of –O-CH2- peak of lactam with gradually increasing methyl ester 4-OCD3
with time, the resultant plots indicate the existence of equilibrium (Figure 3) and the rate law
for this equilibrium is given by equation 4 in supporting information. The equilibrium constant
(K) from the plot of mole ratio vs time has a value of 2.72 (Supporting Information Figure
S36B). After NMR study, the same sample is subjected to ESI-MS, we observed the mass of
both deuterated methyl ester (m/z 265) and lactam (Supporting Information Figure S37h).
Importantly, we were not able to isolate this methyl ester. From these studies, we assume that
the lactam 4 and its methyl ester 4-OCH3 forms are in equilibrium in solution state in presence
of acid and MeOH.
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= 4.4

NH2
H
H
N
PTSA (3.5 eq)
CD3CN (0.4ml)
CD3OD (0.1ml)
O
H
H
H
1.0
0.8
0.6
0.4
0.2
0.0
N
N
OCD3
N
O
N
O
O
 

293K
4-OCD3
4
mole fraction of 4
mole fraction of 4-OCD3
0
100
200
300
400
500
Time (min)
Figure 3. Time dependent 1H-NMR studies of lactam 4 in CD3CN, CD3OD and PTSA.
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Since aminopyrazolone is aromatic compound and UV active in nature. Thus we attempted to
monitor the cleavage of lactam 4 into its amino ester derivative 4-OCH3 under acidic condition
by UV studies. We found that the UV spectrum of pure lactam 4 exhibited two characteristic
peaks at wavelength (λ) 240 nm and 280 nm under neutral pH conditions (Supporting
Information Figure S40). Then we recorded UV spectra of lactam 4 at different time intervals
of times under acidic conditions (0.1 N HCl in MeOH) which exhibit the significant depletion
of absorption intensity of lactam 4 at λ240 nm and λ280 nm (Figure 4). Such changes in UV spectra
strongly support the formation of new species under that mild acidic conditions.
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0.6
Time dependent UV spectra
H
NH2
O
0.1N HCl
in MeOH
N
O
0.5
0.4
0.3
0.2
0.1
0.0
N
N
OMe
N
N
O
O
(4-OCH3)
(4)
(time interval,
in min)
1
4
5
10
15
20
25
30
35
40
45
50
4-OCH3
260
220
240
280
300
320
340
Wavelength (nm)
Figure 4. Time dependent UV-Spectra of lactam 4 under acidic conditions, time intervals in
min. (right panel).
Recently, we have reported an unusual syn conformation between -O-CH2–CO-NH- in crystal
structures of peptides 3b/3h and a weak intramolecular hydrogen bonding between H-atom of
amide (-CONH-) and O-atom (–O-CH2-).12 Similar syn conformation is also noticed in crystal
structure of APA ester 1 (Figure 5). Structural analyses of APA peptides 3b/3h reveal that the
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APA amide bond is twisted with tau () ~ 5.5o (Supporting Information, Figure S39). We also
noticed intramolecular hydrogen bonding between H-atom (-COOH-) and O-atom (–O-CH2-)
in APA acid 2 from NMR studies (Supporting Information, Figure S1-S4). The strong
synergetic effects, such as electronegative –O-CH2-, twisted amide bond ( ~ 5.5o) and
unfavourable syn conformation (ψ) weaken the stability of that amide bond. As a resultant, the
favourable intramolecular attack of APA amine (6-exo-trig) led to the cleavage of that amide
bond under mild acidic conditions.
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(a)
2
acid
1
ester
3b 3h
amide
/
 value
o
3b
: -12.5
O
3.9o

(  o for
/
)
O
O
H
3b 3h
H
H
o
3h
: 26.5



N
Pyr
Pyr
Pyr
H
O
H
H
O
O
O
O

O
H
H
H3C
O
From crystal strcuture
From crystal strcuture
proposed strcuture
(b)
O
O
O
H
H
H
H
H
H
H-bond angle
H-bond length
o
3b
3h
: 2.25Å
: 2.30Å
3b
: 108.4
: 105.0
o
3h


O
O
O
Pyr
Pyr
Pyr
N
O
O
peptide
H
H3C
H
strong H-bonding &
more stabilization
weak H-bonding &
less stabilization
no H-bonding &
no stabilization
Figure 5. (a) Structures of amide 3b/3h, ester 1, acid 2; (b) Their Newman Projection.
The removal of Boc group of aminopyrazolonyl derivatives 1/2/3 occurs under acidic
conditions (10-20% TFA in DCM/CH3CN) and generates the protonated amine intermediates
(a) which may protonate the intramolecular carbonyl group of ester/acid/amide derivatives as
intermediates (b). Subsequently the amine nucleophile possibly attacks at the protonated
carbonyl electrophile as typical intramolecular nucleophilic addition-elimination reaction to
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facilitate the formation of cyclic amide (lactam) derivative 4 (Figure 6). Most importantly, this
lactam is stable in solvent free conditions and its structure is confirmed by single crystal X-ray
analyses. The stability of this lactam 4 is further investigated under protic nucleophilic solvent
(MeOH) under acidic conditions. Importantly, this lactam 4 is unstable in nucleophilic solvents
(H2O/MeOH) under acidic conditions and proceeds to amide hydrolysis possibly due to
protonation of amide bond N-atom under acidic conditions. As resultant, lactam 4 hydrolyzes
into acid/ester derivatives (f) with respective solvents H2O/MeOH.
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H
O
BocHN
N
H+
Y
O
H
O
Boc
Y
O
N
Y
O
H
O
H
Y
H2N
H2N
HN
N
O
O
Ph
N
N
N
Y= OMe (1)
Y= OH (2)
Y= HN-AA-OMe (3)
N
N
Ph
Ph
Ph
(c)
(a)
(b)
H
H
O
Y
H
N
H
N
H
H H
N
O
N
O
f
O
H+/Nu
HN
N
O
O
N
N
Nu
O
N
Nu
O
e
N
N
Ph
N
Y-H
Ph
Ph
Ph
(4)
d
Figure 6. Plausible mechanism of amide hydrolysis.
Conclusion
In summary, 4-aminopyrazolyloxy acetate are successfully incorporated at N-terminal of
natural di-/tri-peptides aminopyrazolyloxy acetamide derivatives which are hydrolysable under
mild acidic conditions and such amide hydrolyses are rare. This unique character of amide
bond is elaborated by NMR/UV/Mass/X-ray studies which helped to demonstrate the
mechanism of reactions. Since aminopyrazoly rings and its derivatives are chromophores. Thus
4-aminopyrazolyloxy acetate could be employed in protection of free amine via acid sensitive
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chromophoric amide bond unlike Boc group. Our future works are in progress to demonstrate
the utility of this process, and have planned to study with other amino acids/longer peptides
including functionalized amino acid/peptide derivatives.
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EXPERIMENTAL SECTION
General methods. All required materials were obtained from commercial suppliers and used
without any further purification. Dimethylformamide (DMF) was distilled over calcium
hydride. Reactions were monitored by thin layer chromatography, visualized by UV and
ninhydrin. Column chromatography was performed in 230-400 mesh silica. Mass spectra and
HRMS were obtained from Bruker micrOTOF-Q II Spectrometer. H NMR, C{1H} NMR,
were recorded on Bruker AV-400 or 700 MHz at 298 K. 1H and 13C{1H} NMR chemical shifts
were recorded in ppm downfield from tetramethylsilane or residual solvent peak. Splitting
patterns are abbreviated as: s, Singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet;
dq, doublet of quartet; m, multiplet.
1
13
Experimental procedure. Experimental procedure and their characterization data for
compounds 1, 3b-3d, 3g-3h are previously reported from our lab.12
N-phenyl, 4-aminopyrazolyloxy acetic acid, BocNH-APA-OH (2). Compound 1 (1.26 g, 3.52
mmol) is dissolved in THF (25 mL) and cooled to 0 oC, then added aqueous LiOH (1 M, 25mL)
and at the same temperature stirred for 20 min. The solvents are evaporated under vacuum to
half of its volume and adjusted the pH to 6-7 with 1 M HCl. The resulting aqueous layer
extracted with EtOAc (3*30 mL) and dried over anhydrous Na2SO4 and concentrated under
vacuum. The product is precipitated with chloroform and hexane, dried the solid to produce
1.0 g (90%) of title compound. 1H NMR (400 MHz, CDCl3) δ 7.90 (OH, s, 1H), 7.63 (d, J =
7.8 Hz, 2H), 7.40 (t, J = 7.8 Hz, 2H), 7.27 (dd, J = 6.5, 4.7 Hz, 1H), 6.02 (NH, s, 1H), 4.67 (s,
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2H), 2.15 (s, 3H), 1.49 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3) δ 172.6, 158.3, 147.4, 146.9,
138.2, 128.9, 126.7, 122.5, 101.2, 82.9, 67.8, 28.3. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C17H22N3O5 348.1554; Found 348.1567.
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General experimental peptide coupling procedure for compounds, 3a-3h:
Compound 2, corresponding amine (TFA or HCl salt form) (1.5 equivalent), HOAT (1.5
equivalent) was dissolved in Dry DMF (1.5 M). After stirring for 10 min, N-methyl morpholine
o
(3 equivalent) was added drop wise and cooled the temperature to 0 C and added EDC.HCl
(1.5 equivalent). After 20 min allowed to warm to rt followed by heating at 60 oC for 8 h. The
crude reaction mixture was evaporated under reduced pressure. The resultant crude was
purified by column chromatography with MeOH in CH2Cl2 (1-3%).
methyl
2-(2-((4-((tert-butoxycarbonyl)amino)-3-methyl-1-phenyl-1H-pyrazol-5-
1
yl)oxy)acetamido)acetate, BocNH-APA-Gly-OMe (3a): Rf 0.25 (3% MeOH in CH2Cl2); H
NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.8 Hz, 2H), 7.44 (t, J = 7.9 Hz, 2H), 7.30 (t, J = 7.4
Hz, 1H), 6.94 (t, 1H), 6.15 (s, 1H), 4.65 (s, 2H), 4.07 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H), 2.19 (s,
3H), 1.48 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3) δ 170.0, 167.8, 155.1, 147.1 (two peaks
merged), 138.1, 129.2, 126.9, 122.6, 102.6, 80.6, 70.7, 52.5, 40.5, 28.2, 11.8. HRMS (ESI-
TOF) m/z: [M + H] + Calcd for C20H27N4O6 419.1925; Found 419.1914.
(S)-methyl
2-(2-(2-((4-((tert-butoxycarbonyl)amino)-3-methyl-1-phenyl-1H-pyrazol-5-
yl)oxy)acetamido)acetamido)propanoate BocNH-APA-Gly-Ala-OMe (3e): Rf 0.18 (3% MeOH
in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.7 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H),
7.30 (d, J = 7.3 Hz, 1H), 7.16 (s, 1H), 6.69 (s, 1H), 6.48 (s, 1H), 4.63 (s, 2H), 4.62 – 4.53 (m,
1H), 3.98 (d, J = 5.3 Hz, 2H), 3.74 (s, 3H), 2.18 (s, 3H), 1.79 (d, J = 7.0 Hz, 2H), 1.46 (s, 9H),
1.41 (d, J = 7.1 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ 173.3, 168.3 (two peaks merged
together), 155.3, 147.2 (two peaks merged together), 138.1, 129.1, 126.9, 122.5, 102.8, 80.4,
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+
70.7, 52.5, 48.2, 42.0, 28.2, 17.9, 11.9. HRMS (ESI-TOF) m/z: [M + H] Calcd for
5
6
C23H30N5O7sNa 512.2116; Found 512.2121.
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(S)-methyl
2-(2-(2-((4-((tert-butoxycarbonyl)amino)-3-methyl-1-phenyl-1H-pyrazol-5-
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yl)oxy)acetamido)propanamido)acetate BocNH-APA-Ala-Gly-OMe (3f): Rf 0.32 (3% MeOH
in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.8 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H),
7.30 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 6.8 Hz, 1H), 6.78 (s, 1H), 6.32 (s, 1H), 4.63 (s, 2H), 4.59
– 4.49 (m, 1H), 4.09 – 3.92 (m, 2H), 3.74 (s, 3H), 2.18 (s, 3H), 1.47 (s, 9H), 1.34 (d, J = 7.0
Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ 172.3, 170.3, 167.7, 155.3, 147.3, 147.2, 138.1,
129.3, 127.1, 122.7, 102.8, 80.6, 70.8, 52.4, 48.2, 41.2, 28.3, 17.9, 11.9. HRMS (ESI-TOF)
m/z: [M + H] + Calcd for C23H31N5O7 490.2296; Found 490.2295.
3-methyl-1-phenyl-4,6-dihydropyrazolo[3,4-b][1,4]oxazin-5(1H)-one, (4): 20% TFA in
CH2Cl2 (5 mL) was added to 1 (0.50 g, 1.38 mmol) at room temperature and stirred for 1 h,
then refluxed for an additional 1.5 h at 45 oC. The solvents were removed under vacuum and
resulting pale orange residue was partitioned between EtOAc (25 mL) and saturated NaHCO3
(10 mL). The aqueous layer was extracted with EtOAc (2 * 25 mL). The combined organic
extracts were washed with saturated aqueous NaCl (10 mL), dried over anhydrous Na2SO4 and
concentrated under vacuum. The residual viscous oil was purified via chromatography with
3% MeOH in CH2Cl2 to produce 0.293 g (92%) of 4 as a crystalline solid. Rf 0.2 (0.3:9.7
1
MeOH/CH2Cl2); H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 7.72 – 7.58 (m, 2H), 7.45
(dd, J = 10.8, 5.2 Hz, 2H), 7.25 (t, J = 7.4 Hz, 1H), 4.84 (s, 2H), 2.15 (s, 3H). 13C{1H} NMR
(101 MHz, DMSO-d6) δ 162.8, 140.2, 138.5, 136.0, 129.8, 126.3, 119.5, 106.5, 70.0, 12.2.
HRMS (ESI-TOF) m/z: [M + H] + Calcd for C12H12N3O2 230.0924; Found 230.0933. mp: 216-
218 oC.
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ASSOCIATED CONTENT
Supporting Information
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NMR (1H-/13C{1H})/HRMS/Time dependent NMR spectra of peptides; kinetic profiles of
amide/acid/ester 3a/3h/1/2; X-ray crystallography data, Crystallographic Information
Framework (CIF) file and UV-spectra of lactam 4 are provided in the Supporting Information.
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AUTHOR INFORMATION
Corresponding Author
Phone no. +91-674-249-4141
E-mail: nagendra@niser.ac.in
ACKNOWLEDGMENTS
AB thanks to UGC for JRF/SRF fellowship. We also thank to Tiwari Ranjay Kumar and Dr.
C. S. Purohit for helping in X-ray studies.
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