Engineered Substrate for Cyclooxygenase-2: A Pentapeptide Isoconformational to Arachidonic Acid for Managing Inflammation

Article abstract of DOI:10.1021/acs.jmedchem.9b00823

Beyond the conventional mode of working of anti-inflammatory agents through enzyme inhibition, herein, COX-2 was provided with an alternate substrate. A proline-centered pentapeptide isoconformational to arachidonic acid, which exhibited appreciable selectivity for COX-2, overcoming acetic acid- and formalin-induced pain in rats to almost 80%, was treated as a substrate by the enzyme. Remarkably, COX-2 metabolized the pentapeptide into small fragments consisting mainly of di- and tripeptides that ensured the safe breakdown of the peptide under in vivo conditions. The kinetic parameter K_cat/K_m for COX-2-mediated metabolism of the peptide (6.3 × 10⁵ M^-1 s^-1) was quite similar to 9.5 × 10⁵ M^-1 s^-1 for arachidonic acid. Evidenced by the molecular dynamic studies and the use of Y385F COX-2, it was observed that the breakage of the pentapeptide has probably been taken place through H-bond activation of the peptide bond by the side chains of Y385 and S530.

Full text of DOI:10.1021/acs.jmedchem.9b00823

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Article
Engineered Substrate for Cyclooxygenase-2: A Pentapeptide iso-
Conformational to Arachidonic Acid for Managing Inflammation
Baljit Kaur, manpreet kaur, Navjot Kaur, Saweta Garg, Rajbir Bhatti, and Palwinder Singh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00823 • Publication Date (Web): 17 Jun 2019
Downloaded from http://pubs.acs.org on June 18, 2019
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Engineered Substrate for Cyclooxygenase-2: A Pentapeptide iso-
Conformational to Arachidonic Acid for Managing Inflammation
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Baljit Kaurψ, Manpreet Kaur, Navjot Kaurψ, Saweta Garg,Ф Rajbir Bhatti,Ф Palwinder Singh*ψ
Department of Chemistry and Centre for Advanced Studies, Guru Nanak Dev University,
Amritsar-143005. India
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ФDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005. India
Abstract: Beyond the conventional mode of working of anti-inflammatory agents through
enzyme inhibition, herein, COX-2 was provided with an alternate substrate. Proline centered
pentapeptide iso-conformational to arachidonic acid, exhibited appreciable selectivity for COX-2
overcoming acetic acid and formalin induced pain in rats to almost 80%, was treated as a
substrate by the enzyme. Remarkably, COX-2 metabolized the pentapeptide into small fragments
consisting mainly of di- and tri- peptides that ensured the safe breakdown of the peptide under
the in-vivo conditions. The kinetic parameter Kcat/Km for COX-2 mediated metabolism of
peptide 6.3 x 105 M-1 s-1 was quite similar to 9.5 x 105 M-1 s-1 for arachidonic acid. Evidenced by
the molecular dynamic studies and the use of Y385F COX-2, it was observed that the breakage
of the pentapeptide has probably been taken place through H-bond activation of the peptide bond
by the side chains of Y385 and S530.
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Introduction
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In addition to the primary source of energy, the fatty acids generate metabolites that play
regulatory role in controlling various biochemical processes.1-8 Arachidonic acid (AA) is one of
those fatty acids that profoundly impinge the physiological state of the organism through its
metabolites in maintaining body temperature, working of cardiovascular system, induction of
labor pain, keeping-up immune system and regulation of inflammation.9-14 These metabolites
including prostaglandins, prostacyclins, prostanoids, and leukotrienes are being generated by the
catalytic activity of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), lipoxygenase
(LOX) and cytochrome P450 (CytP450)15-21 on AA. Irrespective of the 70% structural
homology; COX-1 and COX-2 constitutively generate functionally diverse prostaglandins,
prostacyclins and prostanoids for regulating body temperature, smooth working of
cardiovascular system, and maintenance of the immune system, respectively.22,23 However, under
inflammatory conditions, the inducible enzyme COX-2 synthesizes PGE2 resulting in the
emergence of inflammatory diseases.24,25 Therefore, the natural mode of working of COX-1 and
COX-2, and the consequent generation of metabolites in AA cascade imply that a strategic
design of molecules for selectively controlling the inducible COX-2 must be made to avoid the
side effects of the anti-inflammatory drugs. Overcoming the limitations of non-selective anti-
inflammatory drugs; the launch of selective COX-2 inhibitors (COXIBS) was the result of above
mentioned tactical approach.26-30 But, excessive suppression of COX-2 by the use of COXIBS
relegated the normal immunological functions of this enzyme.31-35 Under these circumstances,
we planned another strategy to minimize the formation of inducible COX-2 mediated metabolites
of AA by providing an alternative substrate to the enzyme. In fact, while keeping in mind the
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selectivity for COX-2 over its isozyme COX-1, we underpinned the COX-2 specificity by
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replacing AA with an iso-conformational substrate– the rationally designed small peptide,
expecting that instead of formation of PGE2 from AA, alternative safe metabolites may get
formed in case these substrates are used as anti-inflammatory drugs.
COX-2 is a highly conserved enzyme, specific to arachidonic acid.36,37 The catalytic domain
of the enzyme is constituted by Y385, R120, Y355, H206, H386, H388 and S53038-40 wherein
AA is present in U-shaped geometry occupying the large hydrophobic pocket (Figure 1).41
Besides the hydrophobic interaction of the main chain of AA, it exhibits H-bond contacts
through its carboxyl group with S530 and Y385 (Figure 1).42 Mechanistically, Y385 is
responsible for the transfer of radical from heme to AA for initiating the metabolism of AA
(Scheme S1).43-46 In continuation to the previous report on peptide based COX-2 inhibitors,47 it
was hypothesized that a stereo-strained proline centred tetra-/penta- peptide may acquire
geometry similar to that of AA and hence occupy the same space in the COX-2 active site where
AA sits. Accordingly, the peptides 1–3 (Chart 1) were designed. Remarkably, the crystal
coordinates of COX-2 in complex with peptide 2 showed that the peptide has occupied the same
hydrophobic pocket where AA was present (Figure 1). The molecular docking of peptides 1 and
3 in the active site pocket of the enzyme showed that 1 falls short of the size of active site pocket
and do not occupy the whole space as covered by AA (Figure S2) whereas the flexibility in
peptide 3 made its one terminus displaced away from the AA (Figure S3). Therefore, enthused
by the results of molecular docking studies, compounds 1–3 were synthesized along with some
other proline containing peptides. These compounds were screened for anti-inflammatory and
analgesic activity in addition to the investigations about their mode of action. The breakdown of
the peptides under in-vivo and in-vitro conditions was also examined.
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Figure 1. Crystal coordinates of COX-2 in association with AA (pdb ID 1CVU).41 Molecular
docking of peptide 2 (Chart 1) in the active site pocket of the enzyme was performed. The
peptide has occupied same space where AA binds.
NH2
CH
O
O
H3C
O
OCH3
H2C
NH
(S)
NH2
CH
NH2
CH
OCH3
O
H2C
NH
NH
H3CO
O
H3C
H3C
(S)
O
(S)
H2C
HN
O
HC
(S)
CH3
O
CH3
HC
(S)
HN
O
HN
HN
CH3
CH2
CH
(S)
HN
CH2
N
HN
O
O
CH2
O
O
N
O
(S)
O
(S)
N
(S)
1
2
3
Chart 1. Designed peptides as the probable substitutes of AA.
Results and Discussion
Syntheses. N-Cbz-Ala (4) was prepared by the reaction of L-Ala with benzyl chloroformate
(Cbz) and it was coupled with glycine methyl ester by using ethyl chloroformate (ECF) as the
coupling reagent to obtain N-Cbz-Ala-Gly(OMe) (5, Scheme 1). Hydrolysis of dipeptide 5 to 6
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and further coupling with L-Pro(OMe) provided tripeptide 7 and it was hydrolyzed to get N-Cbz-
Ala-Gly-Pro(OH) (8, Scheme 1). Treatment of 8 with alanine methyl ester hydrochloride
followed by the deprotection of Cbz group gave tetrapeptide 1. Ala-gly(OMe) 11 was obtained
by the coupling of Boc-Ala(OH) (9) with glycine methyl ester hydrochloride and then the
removal of Boc from the resulting dipeptide 10. Coupling of tripeptide 8 and dipeptide 11 gave
peptide 12. Treatment of 12 with Pd-C in methanol-acetic acid provided pentapeptide 2 (Scheme
1). Similar to the sequence of reactions for the synthesis of peptide 2, another pentapeptide 20
was prepared by the coupling of tripeptide 16 and dipeptide 19 (Scheme 2). Peptides 21 and 22
were coupled to procure tetrapeptide 23 (Scheme 3) whereas coupling of dipeptides 24 and 25
gave tetrapeptide 26 (Scheme 4). The synthesis of hexapeptide 3 is depicted in Scheme 5. The
percentage purity more than 98% of the compounds was ascertained with q1H NMR spectrum.
Due to the better solubility of compound 12 (in CDCl3 in comparison to its precursor 2 in
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O
O
H
N
O
TFA
equi
O
i
O
(S)
(10
8h
N
H
O
H
OH
v)
N
glyMe
ester
(S)
O
N
(S)
O
O
H2N
H
10
O
O
9
11
O,
Boc2
O
H
N
(2M)
NH2
NaOH
2h
O
(S)
H3CO
HN
H
N
O
(S)
OH
O
CbzCl
NaOH
O
(S)
(S)
i
N
O
H
N
O
H2N
N
(S)
(S)
(S)
OMe
(4M)
NH
O
O
R
2h
N
1
O
H
H
N
O
12, R = COOBn
2, R = H
8
O
(S)
Ala(OMe), i
iii
iii
Bn
OH
glyMe
ester
O
H
N
O
O
O
Bn
H
N
4
(S)
i
(S)
O
L-proMe
ester
OR
N
H
N
(S)
Bn
N
H
O
i
O
O
O
5, R=CH3
6, R=H
OR
O
7, R=CH3
8, R=H
ii
ii
Scheme 1. Reaction conditions:
(i) TEA in THF, ECF, -10 oC-25 oC, 12 h; (ii) NaOH (1M), acetone-H2O (3:2), 45 min;
(iii) Acetic acid- MeOH, Pd/C, H2, 4h
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Ph
O
O
H
N
O
glyMe
ester
Ph
(S)
N
iii
O
O
H
O
Ph
i
OH
18
(S)
N
O
O
H
H
H2N
(S)
N
O
Boc2O
NaOH
O
O
O
(2M)
HO
H
O
O
19
2h
(S)
N
(S)
Bn
N
10
11
12
13
14
15
16
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O
i
H
(S)
O
N
Ph
NH2
O
O
(S)
Ph
HN
H
(S)
N
O
16
O
Ph
Bn
H
N
O
O
O
Ph
O
20
Bn
OH
(S)
N
H
(S)
L-pheMe
ester
O
N
H
(S)
O
O
N
L-proMe
ester
O
H
N
4
i
(S)
Bn
(S)
O
O
(S)
i
N
H
OR
OR
O
O
15, R=CH3
16, R=H
Ph
13, R=CH3
14, R=H
ii
ii
o
o
Scheme 2. Reaction conditions
: (i) TEA in THF, ECF, -10 C-25 C, 12 h
(ii) NaOH (1M), acetone - H2O (3:2), 45 min; (iii) TFA (10 equiv), 12h
Bn
O
O
O
S
Ph
O
Ph
O
H
NH
(S)
(S)
H
O
HO
N
O
(S)
N
O
O
N
Bn
(S)
HN
O
O
H
HN
(S)
i
O
O
O
+
O
HN
(S)
N
O
S
22
21
23
Scheme 3. Reaction conditions: (i) TEA in THF, ECF, -10 oC - 25 oC, 12 h
Ph
O
O
O
S
(S)
N
S
(S)
H
O
O
O
NH2
(S)
N
HN
N
N
(S)
i
O
(S)
H
(S)
+
O
O
O
NH
N
O
Ph
O
O
H
O
25
24
O
26
Scheme 4. Reaction conditions: (i) TEA in THF, ECF, -10 oC-25 oC, 12 h
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8
O
L-glyMe
ester
O
N
H
(S)
O
N
TFA(10
8h
O
N
(S)
O
N
H
i
(S)
O
O
eq)
R
O
(S)
O
O
N
2
9
O
O
HN
H
O
H
(S)
O
N
27, R=CH3
28, R=H
(S)
L-alaMe ester
N
H
ii
O
H
O
i
N
O
N
30
9
N
(S)
O
N
O
i
H
O
(S)
10
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12
13
14
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(S)
NH
NH
O
OH
H3C
(S)
R
O
NH
O
O
O
33, R=COOBn
3, R=H
O
O
H
N
H
iii
H
N
O
(S)
N
OH
O
N
Bn
Bn
(S)
H
N
O
H
O
O
O
O
O
L-glyMe ester
H
N
H
32
OH
6
(S)
N
O
N
H
Bn
i
ii
O
O
31
O
OCH3
Scheme 5. Reaction conditions:
(i) TEA in THF, ECF, -10 oC-25 oC, 12 h;
(ii) NaOH (1M), acetone-H2O (3:2), 45 min;
(iii) Acetic acid- MeOH, Pd/C, H2, 4h
DMSO, MeOH), its structure was established with the help of various 2D NMR experiments
(Figure S31-S38). The observation of nOe between the terminal NH of one arm and ester CH3 of
the second arm (Figure 2) indicated the closeness of the two arms acquiring a U-shaped
geometry by molecule 12 (Figure 3) similar to the conformation attained by peptide 2 in the
active site pocket of COX-2 (Figure 1).
Figure 2. 1H-1H nOe spectrum of N-Cbz AGPAG-OMe (12).
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3
Bn
4
O
O
5
6
7
8
1.35-1.36
H3C
H3C
3.60
NH
6.21
O
4.37-4.39
(S)
H
9
O
4.13-4.24
7.38
HN
10
11
12
13
14
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H
O
7.27
HN
H
4.13-4.24
H
H
O
3.64-3.75
3.64-3.75
O
H
4.5
N
H
3.91
(S)
HN
H
H
7.07
O
3.55
H
(S)
CH3
1.42-1.44
H
2.00-2.09
H
H
2.17-2.20
Figure 3. Configuration of compound 12 as depicted from the nOe (black arrows) and COSY
cross peaks (brown arrows, Figure S35).
COX-1 and COX-2 assays
The potency of the synthesized peptides against COX-1 and COX-2 was screened using
enzyme immunoassay kits.48 Excitingly, in concurrence with the results of molecular docking
studies, pentapeptide 2 exhibited superior enzyme inhibitory profile in comparison to the other
peptides screened in the present investigation. Although it is difficult to draw a conclusive
structure activity relationship but a comparison of the IC50 and selectivity of peptide 2 for COX-
2 was made with the other small peptides (Table 1). Peptide 12, the Cbz protected 2, with IC50
0.4 μM for COX-2 was relatively less potent than 2. Presence of Phe residues in 20 increased its
IC50 for COX-2 to 5 μM in comparison to that of 12. This may be attributed to the increase in the
size of the peptide 20 than that of peptide 12. The hexapeptide 3 and tetrapeptides 1, 23 and 26
exhibited poor inhibition of COX-2. These results made us to choose compounds 2, 12, 20 for
further investigations on animal models. Peptides 23 and 26 were included for the comparisons.
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Table 1. COX-1 and COX-2 inhibitory activities and selectivity index (SI) of the compounds.
5
6
compd
Structure
IC50 (µM) ψ
SI*
7
8
9
COX-1
COX-2
0.6
1
Ala-Gly-Pro-Ala(OMe)
0.8
40
1.3
400
5
10
11
12
13
14
15
16
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20
21
22
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2
Ala-Gly-Pro-Ala-Gly(OMe)
Ala-Gly-Gly-Pro-Ala-Gly(OMe)
N-Cbz-Ala-Gly-Pro(OMe)
0.1
3
10
2
7
-
0.3
-
8
N-Cbz-Ala-Gly-Pro(OH)
0.05
50
0.02
0.4
25
12
N-Cbz-Ala-Gly-Pro-Ala-Gly(OMe)
N-Cbz-Ala-Phe-Pro-Phe-Gly(OMe)
N-Cbz-Met-Gly- Pro-Phe(OMe)
N-Boc- Pro-Phe(OH)
125
120
5
20
600
0.5
-
5
23
24
0.1
0.08
0.4
-
26
N-Boc- Pro-Phe-Met-Gly(OMe)
50
125
375
0.08
celecoxib
indomethacin
15
0.04
0.96
0.08
ψAverage of the three values with deviation <2%. *IC50(COX-1)/IC50(COX-2)
Human Whole Blood Assay48,49
The desirable selectivity of compound 2 for COX-2 over COX-1, as observed in the enzyme
immunoassays, was further supported with the help of human whole blood assay in which LPS
induced expression of COX-2 was reversed (Figure 4B) but no effect was observed on calcium
ionophore induced expression of COX-1 (Figure 4A).
Figure 4. (A) TXB2 and (B) PGE2 inhibition in whole blood by indomethacin (1 μM) and
compound 2 (1 μM). TXB2 and PGE2 formed in each experiment are mentioned over the bars.
Calcium ionophore and LPS were used as stimulants of COX-1 and COX-2, respectively.
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In-vivo studies on rat models
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Effect of compounds treatments in acetic acid induced pain model
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Intraperitoneal administration of acetic acid induced abdominal contraction, trunk twisting
and extension of hind legs (writhing) in rats. Treatment with vehicle (control), indomethacin (10
mg kg-1) and compounds 2, 12, 20, 23, 26 at a dose of 5 mg kg-1 and 10 mg kg-1 significantly
reduced the number of writhings as compared to the acetic acid treated groups. Compounds 2
and 12 were found to be the most efficacious with almost 80% reduction in acetic acid induced
writhings (Figure 5A).
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Effect of compound 2 treatment on formalin induced pain
Formalin was injected to the intraplantar region of rat and was found to produce biphasic
hyperalgesia. Increase in the number of flinching in the neurogenic phase (0-5 min) i.e. first
phase and the inflammatory hyperalgesia (20-30 min) i.e. the second phase was observed. A
significant reduction in the number of flinchings in the inflammatory phase was occurred after
the treatment with standard drug indomethacin but no significant effect was observed in
neurogenic phase. Compound 2 reduced the number of flinching by 56% and 80% in both
neurogenic and inflammatory phases, respectively as compared to the control groups (Figure
5B).
Effect of substance P treatment on the analgesic effect of 2 in neurogenic and inflammatory
phases of formalin induced pain
Reverse in the effect of compound 2 in both the phases was found to be significant with pre-
treatment of substance P, a neurokinin receptor agonist and stimulator of COX-2. Thus the
results are indicative of the involvement of cyclooxygenase pathway in reversing inflammation
by compound 2 (Figure 5C).
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Figure 5. (A) Effect of compounds 2, 12, 20, 23 and 26 on acetic acid induced writhings. Ten groups,
each having 5 rats, were used. (B) Effect of compound 2 on formalin induced pain; 4 groups, each
having 5 rats, were used (C) Effect of Substance P on analgesic effect of 2 in formalin induced pain in
three groups each having 5 rats. (D) Effect of L-NAME and L-arginine on analgesic effect of 2 in
formalin induced Pain using three groups each with 5 rats. (E) Effect of compound 2 on carrageenan
induced paw inflammation in rats taking three groups with 5 animals in each group. All the values
are expressed as Mean±SEM (n=5). Statistical significance was calculated using one way ANOVA
followed by Tukey’s multiple range comparison test*p<.05 vs. control. All the experiments were
performed with Male wistar rats weighing 250-300 g.
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Effect of L-NAME and L-arginine on analgesic effect of 2 in neurogenic and inflammatory
phases of formalin induced pain
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Pre-treatment with L-arginine, a NO precursor reversed the analgesic effect of compound 2
as evidenced by a significant increase in the number of flinching in both neurogenic and
inflammatory phases as compared with formalin treated group. However, pre-treatment with L-
NAME, non-selective nitric oxide synthase inhibitor did not alter the effect of 2 in either phase
in formalin induced hyperalgesia (Figure 5D). The anti-inflammatory effect of compound 2
against carrageenan induced paw inflammation in mice was similar to that of indomethacin
(Figure 5E). Therefore, the screening of compound 2 over the animal models clearly supported
its appreciable analgesic and anti-inflammatory effect that was mainly emanating through
targeting COX-2.
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Acute toxicity Studies. Toxicity of compound 2 at a dose of 50, 300, and 2000 mg kg-1 was
checked according to OECD guidelines.50 Four groups (three in each group) of rats were taken.
After 4h of fasting, first group was given vehicle; second, third and fourth groups were treated
with 50, 300, 2000 mg of compound 2, respectively. All the animals were observed for the next
14 days and then, one animal from group with higher dose and control was sacrificed for
histological studies. The tissue histology did not show any lesion confirming no toxicity of the
compound even at 2000 mg kg-1 dose (Figure S164).
Study of peptide 2 metabolism
As per the hypothesis made during the design of the peptides, it was worth to check how
peptide 2 undergoes breakdown after exhibiting the anti-inflammatory and analgesic effect. The
blood serum samples of compound 2 treated animals were collected at different time intervals.
The proteins were removed by centrifugation and the mass spectra of the supernatants were
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recorded. Intriguingly, the LC-MS (Figure 6A, Figure S150) corresponding to various fragments
of the peptide as depicted in Figure 6D (Figure S151-S156, Scheme S2) were observed. In order
to confirm that in in-vivo conditions, the peptide 2 was broken under the influence of COX-2
(though the anti-inflammatory effect of 2 and its targeting and selectivity for COX-2 is
established above), we incubated a solution of peptide 2, COX-2 in HEPES buffer at pH 7.0. The
LC-MS of this solution (Figure 6B, Figure S158) were also showing the similar fragmentations
as observed for the in-vivo samples. The kinetic constants of COX-2 mediated breakage of
peptide 2, calculated from the LC-MS data (monitoring the disappearance of peptide 2 under in-
vitro conditions), were Km 2.1x10-5 M and Kcat 13.2 s-1 having Kcat/Km 6.3 x 105 M-1 s-1. Km and
Kcat for COX-2 mediated metabolism of AA (disappearance of AA) were 2.1 x 10-4 M and 2.0 x
102 s-1, respectively making Kcat/Km 9.5 x 105 M-1 s-1 (Figure S159). Apparently, the peptide was
metabolized by COX-2. In order to have an insight of the mode of interaction of the peptide and
COX-2 and rationalizing its enzyme mediated breakdown; in addition to the docking studies,
molecular dynamics of COX-2 – peptide 2 complex was performed.
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Molecular Dynamics (MD)
The co-crystallized structure obtained after the molecular docking of peptide 2 into the active
site of COX-2 was subjected to molecular dynamic simulations for 50 ns (Figure S160). The
analysis of the simulation results showed a number of interactions between the enzyme and the
ligand. The peptide 2 exhibited direct long-lived H-bond interactions with S530, Y385 and F529
(Figure 6E). The interactions of 2 with S530, V116 and R120 through H2O were also visible
besides the direct interaction of H2O with carbonyl oxygen of glycine. Almost all the NH and
CO groups of the pentapeptide were involved in H-bond interactions with the enzyme. Y385 was
involved for more than 90% of the time through both polar and non-polar interactions with the
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O
D
H2N
H2N
O
O
b
H
a
H3C
O
H2N
N
N
2
O
O
NH
a
N
O
a, b
O
d
d
O
O
O
d
O
HO
NH
HN
c
O
N
NH
H
N
HO
O
N
H
C12H20N4O5
C13H22N4O5
m/z 301.1521 [M+H]+
calcd m/z 301.1506
C16H27N5O6
m/z 313.1498 [M-H]-
calcd m/z 313.1506
c
b
m/z 386.2025 [M+H]+
calcd m/z 386.2034
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O
H
O
H
a
H2N
N
H2N
N
CH3
H2N
O
N
N
O
O
O
O
O
e
c
f
O
O
O
N
O
HN
HO
NH
OH
N
N
H
H
O
C10H16N3O4
m/z 244.1286 [M+H]+
calcd m/z 244.1292
C13H22N4O5
m/z 315.1656 [M+H]+
calcd m/z 315.1663
C15H25N5O6
m/z 372.1872 [M+H]+
calcd m/z 372.1878
Figure 6. LC stacked plots of: (A) Blood serum samples of compound 2 treated rats taken at (i)
30, (ii) 60, (iii) 90, (iv) 120 min; the proteins were removed by centrifugation and the
supernatants were subjected to LC-MS. After 6 h (trace iv), compound 2 was completely broken
to small peptides. (B) Solution of 2, COX-2 in HEPES buffer at pH 7.5 (i) in the absence of 2;
after (ii) 30 min, (iii) 60 min, (iv) 120 min of adding 2. (C) solution of 2, Y385F COX-2 in
HEPES buffer, pH 7.5. HRMS of LC peaks a, c, d, e and f is shown. (D) Fragments of 2
identified with LC-MS. (E) 2d view of COX-2–peptide 2 complex after MD and the probable
cleavage marks (pdb ID 1CVU was used for MD).
ligand whereas S530 interacted for 100% of the time. R120 showed ionic interactions for 30% of
the time whereas F205, F518, L352, V349 exhibited hydrophobic interactions for more than 20%
of the time (Figure S161). Supporting the results of MD studies that the H-bond interactions
between the peptide 2 and the enzyme seems responsible for the breakage of the peptide, only a
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fraction of peptide 2 was broken from ‘a’ under the enzymatic action of Y385F COX-2 (Figure
6C, S162). The MD of Y385F COX-2–peptide 2 eliminated the H-bond activation of C=O
(S163) and hence the ‘c’ and ‘d’ cleavages were not occurred. Hence, the results of MD studies
and the LC-MS data obtained with the use of Y385F variant of COX-2 were in favor of COX-2
mediated H-bond activated cleavage of the peptide.
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Chemical shift and relaxation time based studies for COX-2 – peptide 2 interactions
1H NMR chemical shift and inversion recovery NMR experiments with compound 2, in the
absence and presence of COX-2, were recorded to further confirm the protein – ligand
interactions. 1H NMR spectrum of compound 2 in the absence of COX-2 was recorded in 0.6 mL
DMSO-d6 at 25 °C. Addition of 5 μL of COX-2 to the compound solution resulted in the
downfield shift of NH protons of alanine and glycine residues. The changes in the chemical
shifts were probably due to the H-bond interactions of the NH groups with the active site
residues of COX-2. Similarly, it was observed that the T1 of NH protons of compound 2 was
increased in comparison to its CH protons when COX-2 was added to the solution of the
compound indicating that compound 2 interacts with COX-2 preferably through its amino
protons (Figure 7).
1
Figure 7. H T1 relaxation times of compound 2 in the absence (red dots) and in the presence
(blue dots) of COX-2.
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Conclusions
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Alternative to the conventional mode of working of the therapeutic agents through the
inhibition of the enzyme, a proline centered pentapeptide was identified as the suitable
replacement of the arachidonic acid- the natural substrate of COX-2 for the treatment of acetic
acid and formalin induced inflammation. The pentapeptide 2 was found to occupy the same
space in the active site pocket of COX-2 as covered by AA. The peptide significantly reversed
the analgesic and inflammatory effect in rats and as evidenced by the LC-MS data, it was also
broken into small fragments under the influence of COX-2. Overall, a small peptide, iso-
conformational to the natural substrate of COX-2 was identified as an appropriate candidate for
pursuing further investigations.
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EXPERIMENTAL SECTION
General. All the solvents were chromatography grade and used without further purification. The
chemicals were purchased either from sigma-aldrich or S.d. fine/Spectrochem. Thin layer
chromatography (TLC) was performed using silica plates precoated with silica gel GF-254
(Qualigens, India). Column chromatography was performed with silica 60-120 mesh. LC-MS
and high resolution mass spectra were recorded on a Bruker micrOTOF Q II Mass spectrometer
1
using +ve/-ve ion mode. Melting points were determined in capillaries and are uncorrected. H
and 13C NMR spectra were recorded on a Bruker 500 and 125 MHz NMR spectrometer,
respectively, in CDCl3 and/or DMSO-d6. Chemical shifts are given in ppm with TMS as an
internal reference. Coupling constants (J) are given in Hz. Signals are abbreviated as singlet, s;
doublet, d; double−doublet, dd; triplet, t; and multiplet, m. In 13C/DEPT-135 data, +ve sign
corresponds to signals due to CH3, CH groups while -ve signs symbolize signals of CH2 groups
and signals of quaternary carbon are absent (ab) in DEPT-135 spectra. IR and UV-vis spectral
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data were recorded on FTIR Agilent CARY 630 and BIOTEK Synergy H1 Hybrid Reader
instruments, respectively. Percentage purity of the compounds was ≥98% determined by q1H
NMR spectra.
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General Synthetic procedure
Procedure A. For the protection of amino acid at the amino group, L-amino acid (0.01 mole)
was dissolved in 4M sodium hydroxide solution (0.01 mol) and cooled to 0 °C. While stirring at
0 °C, addition of 0.01 mole of benzyl chloroformate and 0.01 mole of 4 M sodium hydroxide
solution was done alternatively and portion-wise over 30 min. Then reaction mixture was
allowed to stir at room temperature for 1 h. The contents of the reaction mixture were washed
with 2 × 15 mL ether, acidified with dil HCl to pH 2 and extracted with ether. After passing over
anhydrous Na2SO4, solvent was removed to obtain the desired N-Cbz-AA-OH (90 %).
Procedure B. At room temperature, a suspension of L-amino acid (0.01 mol) in 10 mL of
dioxane : water (2:1) was dissolved in 0.01 mole of sodium hydroxide (1 M). After stirring for 5
min at room temperature, 0.01 mole of Boc2O was added. The mixture was stirred for 1.5 h.
Then dioxane was evaporated under vacuum and aqueous part was acidified with dil HCl to pH
2. The liberated oil was extracted with ethyl acetate, dried over anhydrous sodium sulphate and
concentrated under vacuum to obtain the desired N-Boc-AA-OH (90%).
Procedure C. To a solution of N-Cbz-AA or N-Boc-AA (1 mmol) in THF (50 mL) at -10 °C,
triethylamine (0.021 mol) was added, followed by the addition of ethylchloroformate (0.014
mol). After stirring for 15 min at -10 °C, cooled solution of the neutralized AA methyl ester
o
hydrochloride (1.2 mmol) in 5 mL THF was added to the reaction mixture. 0 C to -10 °C
temperature of reaction mixture was maintained for 1 h and then it was stirred at room
temperature for 12 h. After completion of the reaction (TLC), THF was evaporated under
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vacuum and the residue was dissolved in ethyl acetate (100 mL) and washed with water (30 mL).
Then organic layer was washed with 5% Na2CO3 solution, 5% citric acid solution, dried over
anhydrous Na2SO4, evaporated to dryness under vacuum, and purified through column
chromatography using ethyl acetate−hexane as eluent to obtain pure N-Cbz-AA-AA methyl ester
(70%).
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Procedure D. 1N NaOH (0.01 mol) was added slowly to a solution of N-Cbz-AA−AA methyl
ester or N-Boc-AA-AA methyl ester (0.01 mol) in acetone-water (3:2). The reaction mixture was
stirred at room temperature. After completion of the reaction (TLC), acetone was evaporated
under vacuum, followed by acidification with dil HCl. The product was extracted in ethyl acetate
(4 x 25 mL) and obtained in pure form after removal of the solvent under vacuum.
Procedure E. N-Cbz-AA-AA was dissolved in MeOH and 10% Pd-C (0.1 mol%) was added.
While stirring at room temperature, hydrogen gas was passed through reaction mixture. After
completion of the reaction (TLC), the catalyst was removed by filtration, and solvent was
evaporated under vacuum to obtain the desired peptide.
Procedure F. Trifluoroacetic acid (10 equiv of the peptide) was added to the solution of N-Boc-
AA-AA in DCM and stirred at room temperature for 8h. After completion of the reaction (TLC),
the reaction mixture was concentrated in vacuum followed by washing with diethyl ether (3x10
mL) to obtain the desired peptide.
AGPA-OME (1). Compound 1 was synthesized according to general procedure C and E as
yellowish liquid; yield 30%;
= -90o (0.4, methanol); IR (ATR): 3280, 2929, 1736, 1640,
1
o
1543, 1446, 1162, 1326, 1207, 998 cm-1; H NMR (500 MHz, DMSO-d6, 25 C, TMS): δ 1.28-
1.31 (m, 6H, 2 x CH3Ala), 1.77- 1.89 (m, 2H, CH2Pro), 2.00- 2.20 (m, 1H, CH2Pro), 2.20- 2.32 (m,
1H, CH2Pro), 3.41- 3.48 (m, 2H, CH2Pro), 3.76 (s, 3H, OCH3), 3.87-4.00 (m, 2H, CH2Gly), 4.20-
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4.21 (m, 1H, CHAla), 4.30- 4.34 (m, 1H, CHAla ), 4.47 (m, 1H, CHPro)), 8.35 (d, J= 5.41 Hz, 1H,
5
6
13
NH), 8.45 (br, 1H, NH), 8.65-8.66 (br, 1H, NH); C NMR (125 MHz, normal/DEPT-135): δ
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9
17.28 (+ve, CH3Ala), 18.83 (+ve, CH3Ala), 22.46 (-ve,CH2Pro), 24.58 (-ve,CH2Pro), 29.57 (-
ve,CH2Pro), 32.17 (-ve,CH2Pro), 41.63 (-ve, CH2Gly), 46.31 (-ve, CH2Pro), 47.24 (-ve, CH2Pro),
47.97 (+ve, CHAla), 49.01(+ve, CHAla), 52.27 (+ve, OCH3), 59.17 (+ve, CHPro), 59.61 (+ve,
CHPro); 166.82 (C=O), 171.68 (C=O), 171.97 (C=O), 173.42 (C=O); MS: Calcd for [M+H]+)
329.18 found 329.10 .
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AGPAG-OMe (2). Compound 2 was obtained from 12 by using procedure E. White solid; yield
o
20%, mp 132 C;
= -32.5o (0.4, DMSO); IR (ATR): 3278, 3056, 2981, 2937, 2359, 2340,
2132, 1645, 1541, 1520, 1457, 1395, 1340 cm-1; Two isomers of 2 in the ratio 6:1 (1H NMR
spectrum) were seen that were probably arising due to the different configuration around proline.
However, 2 showed one set of peaks only in MeOH-d4 (SI). 1H NMR (500 MHz, methanol-d4, 25
oC, TMS): δ 1.41-1.44 (m, 6H, CH3Ala), 2.04-2.06 (m, 3H, CH2Pro), 2.21-2.30 (m, 1H, CH2Pro),
3.60-3.65 (m, 1H, CH2Pro), 3.69-3.71 (m, 1H, CH2Pro), 3.74 (s, 3H, OCH3), 3.89-4.01 (m, 2H,
CH2Gly), 4.05-4.14 (m, 2H, CH2Gly), 4.39-4.44 (m, 2H, CHAla), 8.00 (br,1H, NH), 8.09-8.12 (m,
1H, NH), 8.26- 8.27 (d, J= 7.26 Hz, 1H, NH), 8.33 (br, 1H, NH); 13C NMR (125 MHz,
normal/DEPT-135): δ 16.24 (+ve, CH3Ala), 18.09 (+ve, CH3Ala), 24.35 (-ve, CH2Pro), 29.19 (-ve,
CH2Pro), 40.14 (-ve, CH2Gly), 46.54 (-ve,CH2Pro), 48.90 (+ve, CHAla), 49.46 (+ve, CHAla), 51.25
(+ve, OCH3), 60.63 (+ve, CHPro), 168.64 (C=O), 170.25 (C=O), 170.40 (C=O), 172.82 (C=O),
173.93 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for C16H27O6N5 ([M+H]+) 386.2034
found 386.1973.
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AGGPAG-OMe (3). Compound 3 was synthesized according to general procedure C and E as a
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thick oil; yield 52%;
= -57.5o (0.4, methanol); IR (ATR): 3220, 3078, 2109, 1640, 1535,
8
9
1401, 1207, 1148, 1021, 521 cm-1; 1H NMR (400 MHz, methanol-d4, 25 oC) δ 1.28- 1.30 (d, J =
6.4 Hz, 3H, CH3Ala), 1.39-1.50 (s, 3H, CH3Ala), 1.68-1.92 (m, 3H, 1H of CH2Pro + CH2Pro), 2.01-
2.27 (m, 1H, CH2Pro), 3.38-3.67 (m, 5H, OCH3 + CH2Pro), 3.83-4.15 (m, 7H, 3xCH2Gly + CHAla),
4.19-4.40 (s, 2H, CHAla+CHPro); 13C NMR (101 MHz, normal/DEPT-135): δ 16.34 (+ve,
CH3Ala), 17.10 (+ve, CH3Ala), 24.45 (-ve, CH2Pro), 29.27 (-ve, CH2Pro), 40.55 (-ve, CH2Gly), 41.58
(-ve, CH2Gly), 42.14 (-ve, CH2Gly), 46.63 (-ve, CH2Pro), 49.00 (+ve, CHAla), 49.20 (+ve, CHAla),
51.35 (+ve, OCH3), 60.82 (+ve, CHPro), 168.85 (C=O), 170.26 (C=O), 170.33 (C=O), 170.48
(C=O), 172.93 (C=O), 174.122 (C=O); MS: Calcd for C18H30O7N6 ([M+H]+) 443.20, found
443.20.
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N-Cbz Ala-OH (4). Compound 4 was synthesized according to general procedure A as a white
solid; yield 90%; mp 82 oC.
N-Cbz Ala-Gly-OMe (5). Compound 5 was synthesized according to general procedure C as a
white solid; yield 50%, mp 98 oC;
= -15o (0.4, CHCl3); IR (ATR): 3295, 3065, 2987, 2957,
1
2357, 2338, 1764, 1688, 1653, 1533 cm-1; H NMR (500 MHz, CDCl3, 25 oC, TMS): δ 1.42 (d,
J=7.10 Hz, 3H, CH3), 3.77 (s, 3H, OCH3), 4.05-4.06 (m, 2H, CH2), 4.31-4.32 (m, 1H, CH), 5.1-
5.15 (m, 2H, CH2), 5.35 (br, 1H, NHAla), 6.62 (br, 1H, NHGly), 7.33-7.37 (m, 5H, ArH); 13C
NMR (125 MHz, normal/DEPT-135): δ 18.39 (+ve, CH3Ala), 41.19 (-ve, CH2Gly), 50.47 (+ve,
CHAla), 52.40 (+ve, OCH3), 67 (-ve, CH2), 128.1 (+ve, ArCH), 128.2 (+ve, ArCH), 128.5 (+ve,
ArCH), 136.1 (ab, ArC), 156.0 (C=O), 171.0 (C=O), 172.4 (C=O); HRMS (microTOF-QII, MS,
ESI): Calcd for C14H18O5N2 ([M+Na]+) 317.1107, found 317.1110.
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N-Cbz Ala-Gly-OH (6). Compound 6 was synthesized according to general procedure D as a
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white solid; yield 80%, mp 121 C;
= +5o (0.4, DMSO); IR (ATR): 3298, 3085, 2987,
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2937, 2340, 2357, 2128, 1768, 1742, 1718, 1653, 1688 cm-1; 1H NMR (500 MHz, DMSO-d6, 25
oC, TMS): δ 1.22 (d, J=7.21 Hz, 3H, CH3Ala), 3.60 –3.78 (m, 2H, CH2Gly), 4.06-4.12 (m, 2H,
CHAla), 5.0 –5.05 (m, 2H, CH2), 7.31-7.45 (m, 5H, ArH), 7.43 (d, J=7.72 Hz, 1H, NHAla), 8.12-
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8.14 (t, J=5.5 Hz, 1H, NHGly), 12.58-12.61 (br, 1H, OH); C NMR (125 MHz, normal/DEPT-
135): δ 18.67 (+ve, CH3Ala, 41.08 (-ve, CH2Gly), 50.38 (+ve, CHAla), 65.86 (-ve, CH2), 128.2
(+ve, ArCH), 128.8 (+ve, ArCH), 137.47 (ab, ArC), 156.12 (C=O), 171.58 (C=O), 173.28
(C=O); HRMS (microTOF-QII, MS, ESI): Calcd for C18H23O6N3 ([M+H]+) 281.1131, found
281.1129.
N-Cbz Ala-Gly-Pro-OMe (7). Compound 7 was synthesized according to general procedure C
o
as white solid; yield 35%, mp 99 C;
= -60o (0.4, CHCl3); IR (ATR): 3289, 3039, 2974,
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2357, 2340, 1761, 1746, 1684, 1634, 1436 cm-1; H NMR (500 MHz, CDCl3, 25 C, TMS): δ
1.39 (d, J=7.17 Hz, 3H, CH3Ala), 2.01-2.05 (m, 2H, CH2Pro), 2.09-2.24 (m, 2H, CH2Pro) 3.44-3.49
(m, 2H, CH2Pro), 3.73 (s, 3H, OCH3), 4.01- 4.10 (m, 2H, CH2Gly), 4.29-4.32 (m, 1H, CHAla), 4.52
(dd, J=3.5 Hz, J=8.9 Hz, 1H, CHPro), 5.05- 5.15 (m, 2H, CH2), 5.45- 5.46 (br, 1H, NH), 6.96 (br,
1H, NH), 7.30-7.35 (m, 5H, ArH); 13C NMR (125 MHz, normal/DEPT-135): δ 18.9 (+ve,
CH3Ala), 24.5 (-ve, CH2Pro), 29.2 (-ve, CH2Pro), 42.3 (-ve, CH2Gly), 46 (-ve,CH2Pro), 50 (+ve,
CHAla), 52.40 (+ve, OCH3), 58.9 (+ve, CHPro), 67.02 (-ve, CH2), 128.12 (+ve, ArCH), 128.15
(+ve, ArCH), 128.53 (+ve, ArCH), 136.23 (ab, ArC), 155.83 (C=O), 166.77 (C=O), 172.24
(C=O), 172.3 (C=O); HRMS (microTOF-QII ESI): Calcd for C19H25O6N3 ([M+Na]+) 392.1816,
found 392.1810.
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N-Cbz Ala-Gly-Pro-OH (8). Compound 8 was synthesized according to general procedure C
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and D as white solid; yield 80%, mp 102 C;
= -35o (0.4, DMSO); IR (ATR): 3354, 3309,
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2981, 2506, 2359, 2340, 1716, 1645, 1602 cm-1; 1H NMR (500 MHz, DMSO-d6, 25 oC, TMS): δ
1.83-1.94 (m, 2H, CH2Pro), 2.09-2.15 (m, 2H, CH2Pro) 3.47-3.53 (m, 2H, CH2Pro), 3.81 (dd, J=4.5
Hz, J=17.36 Hz, 1H, CHAla), 4.05 (dd, J=5.9 Hz, J=17.81 Hz, 2H, CH2Gly), 4.25 (dd, J=3.78 Hz,
J=8.6 Hz, 1H, CHPro), 4.99-5.05 (m, 2H, CH2), 7.31- 7.38 (m, 5H, ArH), 7.49-7.51 (d, J=7.6 Hz,
1H, NH), 7.90-7.92 (t, J=4.9 Hz, 1H, NH), 12.5 (br, 1H, OH); 13C NMR (125 MHz,
normal/DEPT-135): δ 18.69 (+ve, CH3Ala), 24.78 (-ve, CH2Pro), 29.11 (-ve, CH2Pro), 41.5 (-ve,
CH2Gly), 45.93 (-ve, CH2Pro), 50.48 (+ve, CHAla), 59.04 (+ve, CHPro), 65.83 (-ve, CH2), 128.18
(+ve, ArCH), 128.24 (+ve, ArCH), 128.81 (+ve, ArCH), 137.48 (ab, ArC), 156.11 (C=O),
167.14 (C=O), 173.05 (C=O), 173.72 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for
C18H23O6N3 ([M+H]+) 378.1659, found 378.1665.
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N-Boc Ala-OH (9). Compound 9 was synthesized according to general procedure B as thick oil;
yield 90%.
N-Boc-Ala-Gly-OMe (10). Compound 10 was synthesized according to general procedure C as
a thick oil; yield 50%,
= -20o (0.4, CHCl3); IR (ATR): 3379, 3309, 2981, 2506, 2359,
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2340, 1716, 1645, 1602 cm-1; H NMR (500 MHz, CDCl3, 25 C, TMS): δ 1.38 (d, J=6.84 Hz,
3H, CH3), 1.45 (s, 9H, CH3Boc), 3.76 (s, 3H, OCH3), 4.0 (t, J=4.84 Hz, 2H, CH2), 4.20-4.26 (m,
1H, CH), 5.1-5.14 (m, 1H, NH), 6.85 (br, 1H, NH); 13C NMR (125 MHz, normal/DEPT-135): δ
18.27 (+ve, CH3Ala), 28.3 (+ve, CH3Boc), 41.14 (-ve, CH2Gly ), 49.95 (+ve, CHAla), 52.35 (+ve,
OCH3), 80.23 (C(CH3)3), 155.55 (C=O), 170.19 (C=O), 173.04 (C=O); HRMS (microTOF-QII,
MS, ESI): Calcd for C11H20O5N2 ([M+Na]+) 283.1264, found 283.1258.
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Ala-Gly-OMe (11). Compound 11 was synthesized according to general procedure F as a thick
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oil; yield 80%,
= +10o (0.4, DMSO); IR (ATR): 3304, 3205, 3017, 2918, 2775, 2648,
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2512, 2361, 2094, 1770, 1720, 1645, 1576, 1418, 1395 cm-1; 1H NMR (500 MHz, DMSO-d6, 25
oC, TMS): δ 1.36-1.38 (m, 3H, CH3), 3.64 (s, 3H, OCH3), 3.82-3.86 (m, 1H, CHAla), 3.91-3.96
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(m, 2H, CH2Gly), 8.15-8.41 (br, 2H, NH2Ala), 8.90-8.93 (br, 1H, NHGly); C NMR (125 MHz,
normal/DEPT-135): δ 17.55 (+ve, CH3Ala), 41.02 (-ve CH2Gly ), 48.46 (+ve, CHAla), 52.29 (+ve,
OCH3), 80.23 (C(CH3)3), 170.32 (C=O), 170.61 (C=O); HRMS (microTOF-QII, MS, ESI):
Calcd for C6H11O3N2 ([M+H]+) 161.0920, found 161.0926.
N-Cbz-Ala-Gly-Pro-Ala-Gly-OMe (12). Compound 12 was synthesized according to general
o
procedure C as white solid; yield 40%, mp 102 C;
= -102.5o (0.4, CHCl3); IR (ATR):
3281, 3073, 2981, 2359, 2340, 2132, 1733, 1690, 1649, 1636, 1533, 1436 cm-1; H NMR (500
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MHz, CDCl3, 25 C, TMS): δ 1.38-1.39 (m, 3H, CH3Ala), 1.45-1.47 (m, 3H, CH3Ala), 2.03-2.09
(m, 2H, CH2Pro), 2.2-2.21 (m, 2H, CH2Pro), 3.59-3.58 (m, 1H,1H of CH2Pro), 3.63 (s, 3H, OCH3),
3.72-3.78 (m, 2H, 1H of CH2Gly + 1H of CH2Gly), 3.94 (br, 1H, 1H of CH2Pro), 4.16-4.27 (m, 2H,
1H of CH2Gly + 1H of CH2Gly), 4.39-4.41 (m, 1H, CHAla), 4.49-4.52 (m, 2H, 1Hof CHPro + 1H of
CHAla), 5.12 (s, 2H, CH2), 6.2 (d, J=7.46 Hz, 1H, NHAla), 7.06-7.07 (br, 1H, NHAla), 7.32-7.34
(br, 1H, NHGly), 7.35-7.37 (m, 5H, ArH), 7.38-7.40 (br, 1H, NHGly); 13C NMR (125 MHz,
normal/DEPT-135): δ 16.95 (+ve, CH3Ala), 17.16 (+ve, CH3Ala), 24.68 (-ve,CH2Pro), 29.30 (-
ve,CH2Pro), 41.01 (-ve, CH2Gly), 42.44 (-ve, CH2Gly), 47.63 (-ve,CH2Pro), 49.12 (+ve, CHAla),
49.87 (+ve, CHAla), 52.27 (+ve, OCH3), 61.28 (+ve,CHPro), 67.15 (-ve, CH2), 128.07 (+ve,
ArCH), 128.30 (+ve, ArCH), 128.58 (+ve, ArCH), 136.08 (ab, ArC), 156.69 (C=O), 169.69
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(C=O), 171.02 (C=O), 171.52 (C=O), 172.97 (C=O), 174.08 (C=O); HRMS (microTOF-QII,
MS, ESI): Calcd for C24H33O8N5 ([M+Na]+) 542.2221, found 542.2218.
Cbz-AF-OMe (13). Compound 13 was synthesized according to general procedure C as white
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solid, yield 80%, mp 98 C;
= +37.5o (0.4, CHCl3); IR (ATR): 3293, 3062, 3032, 2981,
2940, 2124, 1953, 1742, 1690, 1660, 1604, 1533, 1446 cm-1; 1H NMR (500 MHz, CDCl3, 25 oC,
TMS): δ 1.35 (d, J= 7.05 Hz, 3H, CH3Ala), 3.05 (dd, J= 14.29 Hz, J= 6.25 Hz, 1H, CH2Phe), 3.16
(dd, J = 13.69 Hz, J = 5.95 Hz, 1H, CH2Ph), 3.74 (s, 3H, OCH3), 4.24 (t, J= 6.32 Hz, 1H, CHPh),
4.86 (q, J= 7.59 Hz, 1H, CHala), 5.06-5.17 (m, 2H, CH2), 5.25- 5.34 (br, 1H, NH), 6.45- 6.51 (br,
1H, NH), 7.04- 7.15 (m, 2H, ArHPhe), 7.23- 7.28 (m, 3H, ArHPhe), 7.29-7.42 (m, 5H, ArHCbz);
13C NMR (125 MHz, normal/DEPT-135): δ 18.40 (+ve, CH3Ala), 37.81 (-ve, CH2Phe), 50.41 (+ve,
CHAla), 52.40 (+ve, OCH3), 53.18 (+ve, CHPhe), 67.07 (-ve, CH2), 128.24 (+ve, ArCH), 128.57
(+ve, ArCH), 128.59 (+ve, ArCH), 129.27 (+ve, ArCH), 135.64 (ab, ArCH), 136.16 (ab, ArC),
155.87 (C=O), 171.69 (C=O), 171.79 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for
C21H24O5N2 ([M+H]+) 385.1757 found 385.1666.
Cbz-AF-OH (14). Compound 14 was synthesized according to general procedure D as white
o
solid, yield 80%, mp 118 C;
= +10o (0.4, DMSO); IR (ATR): 3308, 3062, 3032, 2145,
1709, 1692, 1653, 1604, 1530, 1498, 1448, 1422, 1306, 1231 cm-1; 1H NMR (500 MHz, CDCl3,
o
25 C, TMS): δ 1.31 (d, J = 7.05 Hz, 3H, CH3Ala), 3.04 (dd, J = 14.48 Hz, J = 6.5 Hz, 1H,
CH2Phe), 3.21 (dd, J = 14.29 Hz, J = 5.49 Hz, 1H, CH2Phe), 4.28 (t, J = 5.92 Hz, 1H, CHPhe), 4.84
(q, J= 6.45 Hz, 1H, CHala), 5.07-5.14 (m, 2H, CH2), 5.47-5.64 (br, 1H, NH), 6.72-6.93 (br, 1H,
NH), 7.14-7.15 (m, 2H, ArHPhe), 7.21-7.26 (m, 3H, ArHPhe), 7.31-7.50 (m, 5H, ArHCbz); 13C
NMR (125 MHz, normal/DEPT-135) δ 18.22 (+ve, CH3Ala), 37.34 (-ve, CH2Phe), 50.36 (+ve,
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CHAla), 53.25 (+ve, CHPhe), 67.26 (-ve, CH2), 127.15 (+ve, ArCH), 128.10 (+ve, ArCH), 128.33
(+ve, ArCH), 129.57 (+ve, ArCH), 135.69 (ab, ArCH), 135.99 (ab, ArC), 155.15 (C=O), 172.53
(C=O), 174.20 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for C20H22O5N2 ([M+H]+)
371.1601, found 371.1572.
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Cbz-AFP-OMe (15). Compound 15 was synthesized according to general procedure C as white
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solid, yield 40%, mp 94 C;
= -42.5o (0.4, CHCl3); IR (ATR): 3289, 3062, 2974, 2124,
1938, 1744, 1686, 1632, 1528, 1438, 1364, 1321 cm-1; 1H NMR (500 MHz, CDCl3, 25 oC, TMS)
(two inseparable isomers, 3:1): δ 1.31 (d, J=7.01 Hz, 3H, CH3Ala), 1.38 (d, J=7.01 Hz, 3H,
CH3Ala), 1.48-1.60 (m, 1H, CH2Pro), 1.63-1.75 (m, 2H, CH2Pro), 1.85-2.00 (m, 4H, CH2Pro), 2.05-
2.13 (m, 1H, CH2Pro), 2.88-2.93 (m, 1H, CH2Pro), 2.96-2.99 (m, 2H, CH2Phe), 3.04-3.07 (m, 1H,
CH2Pro), 3.12-3.21 (m, 2H, CH2Phe), 3.31-3.35 (m, 1H, CHPro), 3.50-3.68 (m, 2H, CH2Pro), 3.68 (s,
3H, OCH3), 3.76 (s, 3H, OCH3), 4.21-4.35 (m, 1H, CHPhe), 4.49-4.50 (m, 1H, CHPro), 4.68-4.73
(m, 1H, CHPhe), 4.94 (q, J= 7.05 Hz, 1H, CHala), 5.06-5.16 (m, 4H, CH2), 5.27 (br, 1H, NH), 5.42
(br, 1H, NH), 6.86 (br, 2H, NH), 7.21-7.30 (m, 10H, ArH), 7.32-7.37 (m, 10H, ArCH); 13C NMR
(125 MHz, normal/DEPT-135): δ 18.72 (+ve, CH3Ala), 19.11 (+ve, CH3Ala), 22.23 (-ve, CH2Pro),
24.5 (-ve,CH2Pro), 29.0 (-ve, CH2Pro), 30.50 (-ve, CH2Pro), 38.58 (-ve, CH2Phe), 40.58 (-ve,
CH2Phe), 46.07 (-ve, CH2Pro), 46.85 (-ve, CH2Pro), 50.46 (+ve, CHAla), 51.98 (+ve, OCH3), 52.24
(+ve, OCH3), 52.67 (-ve, CHPhe), 52.81 (-ve, CHPhe), 58.92 (+ve, CHPro), 59.02 (+ve, CHPro),
67.01 (-ve, CH2), 126.96 (+ve, ArCH), 127.16 (+ve, ArCH), 128.15 (+ve, ArCH), 128.18 (+ve,
ArCH), 128.38 (+ve, ArCH), 128.54 (+ve, ArCH), 128.59 (+ve, ArCH), 129.36 (+ve, ArCH),
129.79 (+ve, ArCH), 135.86 (ab, ArC), 136.24 (ab, ArC), 155.89 (C=O), 169.94 (C=O), 171.59
(C=O), 172.36 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for C26H31O6N3 ([M+Na]+)
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504.2105, found 504.2131. Two isomers are most probably coming due to the different
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Cbz-AFP-OH (16). Compound 16 was synthesized according to general procedure C and D as
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white solid, yield 90%, mp 82 C;
2976, 2879, 2145, 1718, 1619, 1522, 1498, 1449, 1328 cm-1; 1H NMR (500 MHz, CDCl3, 25 oC,
TMS) (two inseparable isomers, 3:1): δ 1.34 (d, J= 6.62 Hz, 3H, CH3Ala), 1.41 (d, J= 6.82 Hz,
3H, CH3Ala), 1.53-1.62 (m, 2H, CH2Pro), 1.83-1.87 (m, 3H, CH2Pro), 2.06-2.15 (m, 3H, CH2Pro),
2.90-2.95 (m, 1H, CH2Pro), 2.98-3.02 (m, 2H, 1H of CH2Phe + 1H of CH2Pro), 3.08-3.12 (m, 1H,
CH2Phe), 3.26-3.30 (m, 1H, CH2Phe), 3.50-3.56 (m, 1H, CHPro), 3.60-3.61 (m, 1H, CHPro), 4.35-
4.37 (m, 2H, CHAla), 4.46-4.48 (m, 2H, CHPro), 5.00-5.02 (m, 2H, CHPhe), 5.07-5.10 (m, 2H,
CH2), 5.15 (s, 2H, CH2), 5.80 (d, J= 6.84 Hz, 1H, NHAlab), 6.11 (d, J= 6.82 Hz, 1H, NH), 7.20-
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7.38 (m, 20H, ArH), 7.53 (br, 1H, NH), 8.03-8.04 (d, J = 7.31 Hz, 1H, NH); C NMR (125
MHz, normal/DEPT-135): δ 19.76 (+ve, CH3Ala), 20.63 (+ve, CH3Ala), 22.65 (-ve, CH2Pro), 24.83
(-ve, CH2Pro), 28.37 (-ve, CH2Pro), 39.76 (-ve, CH2Phe), 40.29 (-ve, CH2Phe), 46.21 (-ve, CH2Pro),
47.57 (-ve, CH2Pro), 50.24 (+ve, CHAla), 52.71 (-ve, CHPhe), 59.04 (+ve, CHPro), 59.49 (+ve,
CHPro), 67.21 (-ve, CH2), 127.12 (+ve, ArCH), 128.10 (+ve, ArCH), 128.24 (+ve, ArCH), 128.48
(+ve, ArCH), 128.33 (+ve, ArCH), 129.64 (+ve, ArCH), 136.09 (ab, ArC), 156.18 (C=O);
HRMS (microTOF-QII, MS, ESI): Calcd for C25H29O6N3 ([M+H]+) 468.2129, found 468.1710.
N-Boc-Phe-OH (17). Compound 17 was synthesized according to general procedure B as a thick
oil; yield 85%.
Boc-FG-OMe (18). Compound 18 was synthesized according to general procedure C as white
solid, yield 50%, mp 84 oC;
= -5o (0.4, CHCl3); IR (ATR): 3386, 3302, 2978, 2931, 2104,
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2001, 1753, 1686, 1656, 1530, 1511, 1436 cm-1; H NMR (500 MHz, CDCl3, 25 C, TMS): δ
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1.41 (s, 9H, CH3Boc), 3.07-3.15 (m, 2H, CH2Phe), 3.74 (s, 3H, OCH3), 3.95 (dd, J = 18.07, 4.23
Hz, 1H, CH2Gly), 4.03 (dd, J = 18.45, 5.76 Hz, 1H, CH2gly), 4.38- 4.50 (m, 1H, CHPhe), 5.08-5.14
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(m, 1H, NH), 6.49-6.60 (br, 1H, NH); C NMR (125 MHz, normal/DEPT-135): δ 28.24 (+ve,
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CH3Boc), 38.34 (-ve, CH2Phe), 41.17 (-ve CH2Gly ), 52.35 (+ve, OCH3), 55.62 (+ve, CHPhe), 80.31
(C(CH3)3), 126.95 (+ve, ArCH), 128.65 (+ve, ArCH) , 129.31 (+ve, ArCH), 136.58 (ab, ArCH),
169.89 (C=O), 170.60 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for C17H24O5N2
([M+H]+) 337.1757, found 337.1754.
FG-OMe (19). Compound 19 was synthesized according to general procedure C and F as thick
yellowish oil, yield 82%,
= +15o (0.4, DMSO); IR (ATR): 3274, 3090, 2689, 1986, 1886,
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1735, 1559, 1442, 1407, 1271, 1304, 1133 cm-1; H NMR (500 MHz, CDCl3, 25 C, TMS): δ
3.07- 3.28 (m, 2H, CH2Phe), 3.59 (s, 3H, OCH3), 3.74-3.79 (m, 1H, CH2Gly), 3.95-3.99 (m, 1H,
CH2Gly), 4.39-4.54 (m, 1H, CHPhe), 7.15-7.27 (m, 5H, ArCH), 7.30-7.35 (br, 1H, NH), 8.18-8.48
(m, 2H, NH2Phe); 13C NMR (125 MHz, normal/DEPT-135) δ 37.32 (-ve, CH2Phe), 41.06 (-ve
CH2Gly), 52.40 (+ve, OCH3), 54.72 (+ve, CHPhe), 127.75 (+ve, ArCH), 128.91 (+ve, ArCH),
129.52 (+ve, ArCH), 133.91 (ab, ArCH), 169.24 (C=O), 170.05 (C=O); HRMS (microTOF-QII,
MS, ESI): Calcd for C12H16O3N2 ([M+H]+) 237.1233, found 237.1301.
Cbz-AFPFG-OMe (20). Compound 20 was synthesized according to general procedure C as
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white solid, yield 40%, mp 78 C;
= -67.5o (0.4, CHCl3); IR (ATR): 3293, 3030, 2952,
2124, 1718, 1630, 1507, 1438, 1373, 1340 cm-1; 1H NMR (500 MHz, CDCl3, 25 oC, TMS) (two
inseparable isomers, 1:1): δ 0.66-0.68 (m, 1H, CH2Pro), 0.94-1.00 (m, 1H, CH2Pro), 1.30 (d, J=
6.88 Hz, 3H, CH3Ala), 1.31-1.36 (m, 1H, CH2Pro), 1.38 (d, J= 7.01 Hz, 3H, CH3Ala), 1.76-1.79 (m,
1H, CH2Pro), 1.80-1.89 (m, 2H, CH2Pro), 1.90- 1.96 (m, 1H, CH2Pro), 2.00- 2.10 (m, 1H, CH2Pro),
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2.76- 2.84 (m, 2H, CH2Phe), 2.98- 3.06 (m, 4H, 1H CH2Pro + 2H CH2Phe + 1H CH2Phe), 3.11- 3.17
(m, 1H, CH2Pro), 3.19- 3.24 (m, 2H, CH2Phe), 3.24- 3.28 (m, 1H, CH2Pro), 3.30-3.34 (m, 1H,
CH2Phe), 3.48-3.51 (m, 1H, CHPro), 3.61-3.65 (m, 1H, CH2Pro), 3.66 (s, 3H, OCH3), 3.74 (s, 3H,
OCH3), 3.79-3.91 (m, 2H, CH2Gly), 4.17- 4.23 (m, 3H, CHAla + 1H of CH2Gly + 1H of CH2Gly),
4.32-4.35 (m, 1H, CHAla), 4.40 (m, 1H, CHPro), 4.48-4.50 (m, 1H, CHPhe), 4.53-4.57 (m, 1H,
CHPhe), 4.72 (q, J = 7.46 Hz, 1H, CHPhe), 4.88 (q, J = 7.96 Hz, 1H, CHPhe), 5.08-5.17 (m, 4H,
CH2cbz), 5.61-5.62 (m, 1H, NH), 6.41 (d, J= 8.29 Hz, 1H, NH), 6.76 (br, 1H, NH), 6.81-6.83 (m,
2H, NH), 7.14-7.15 (m, 3H, ArCH), 7.20-7.21 (m, 7H, NH+ ArCH), 7.25-7.26 (m, 6H, ArCH),
7.30-7.32 (m, 15H, ArCH), 7.34-7.39 (m, 10H, ArCH), 7.68 (d, J = 7.32 Hz, 1H, NH); 13C NMR
(125 MHz, normal/DEPT-135): δ 17.51 (+ve, CH3Ala), 18.42 (+ve, CH3Ala), 21.20 (-ve,CH2Pro),
24.97 (-ve,CH2Pro), 28.86 (-ve,CH2Pro), 30.86 (-ve,CH2Pro), 36.50 (-ve,CHPhe), 37.04 (-ve,CHPhe),
38.14 (-ve,CHPhe), 38.62 (-ve,CHPhe), 40.98 (-ve,CH2Gly), 41.30 (-ve,CH2Gly), 46.51 (-ve,CH2Pro),
47.59 (-ve,CH2Pro), 50.10 (+ve, CHAla), 50.43 (+ve, CHAla), 50.89 (+ve, OCH3), 52.19 (+ve,
OCH3), 52.31 (+ve, CHPro), 53.70 (+ve, CHPro), 53.91 (+ve, CHPro), 55.60 (+ve, CHPhe), 60.93
(+ve, CHPro), 61.13 (+ve, CHPro), 67.09 (-ve, CH2), 67.21 (-ve, CH2), 126.82 (+ve, ArCH),
127.11 (+ve, ArCH), 127.23 (+ve, ArCH), 127.65 (+ve, ArCH), 128.81 (+ve, ArCH), 128.29
(+ve, ArCH), 128.36 (+ve, ArCH), 128.59 (+ve, ArCH), 128.67 (+ve, ArCH), 128.70 (+ve,
ArCH), 129.22 (+ve, ArCH), 129.36 (+ve, ArCH), 129.42 (+ve, ArCH), 134.93 (ab, ArC),
136.05 (ab, ArC), 136.11 (ab, ArC), 136.69 (ab, ArC), 137.38 (ab, ArC), 170.12 (C=O), 170.30
(C=O), 170.74 (C=O), 170.84 (C=O), 170.87 (C=O), 171.28 (C=O), 171.28 (C=O), 171.55
(C=O), 171.67 (C=O); HRMS (microTOF-QII, MS, ESI): Calcd for C37H43O8N5 ([M+H]+)
686.3184, found 686.3113.
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Cbz-MG-OH (21). Compound 21 was synthesized according to general procedure C and D as
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white solid, yield 86.95%, mp 74 C; IR (ATR): 3313, 3032, 2972, 2920, 2510, 2361, 2130,
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1751, 1669, 1634, 1606, 1526 cm-1; H NMR (500 MHz, DMSO-d6, 25 C, TMS): δ 1.78-1.83
(m, 1H, CH2Met), 1.89-1.91 (m, 1H, CH2Met), 2.03 (s, 3H, CH3Met), 2.47-2.48 (m, 2H, CH2Met),
3.69 (dd, J = 17.71 Hz, J= 5.53 Hz, 1H, CH2Gly), 3.81 (m, dd, J = 17.52 Hz, J= 5.80 Hz, 2H,
CHGly), 4.10-4.14 (m, 1H, CHMet), 4.99-5.05 (m, 2H, CH2), 7.31-7.36 (m, 5H, ArCH), 7.51 (d, J=
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7.98 Hz, 1H, NH), 8.21 (t, J= 5.53 Hz, 1H, NH); C NMR (125 MHz, normal/DEPT-135): δ
15.03 (+ve, CH3Met), 30.01 (-ve, CH2Met), 32.15 (-ve, CH2Met), 41.5 (-ve, CH2Gly ), 54.20 (+ve,
CHMet), 65.92 (-ve, CH2), 128.17 (+ve, ArCH), 128.26 (+ve, ArCH), 128.80 (+ve, ArCH),
137.45 (ab, ArC), 156.42 (C=O), 171.57 (C=O), 172.30 (C=O); HRMS (microTOF-QII, MS,
ESI): Calcd for C15H20O5N2S ([M+Na]+) 363.0985, found 363.1033.
PF-OMe (22). Compound 22 was synthesized according to general procedure C and F as thick
oil, yield 68.28%,
= -20o (0.4, DMSO); IR (ATR): 3321, 3136, 3028, 3002, 2340, 2357,
2133, 1735, 1653, 1533, 1448, 1375 cm-1; HRMS (microTOF-QII, MS, ESI): Calcd for
C16H20O4N2 ([M+H]+) 221.0954, found 221.0937.
Cbz-MGPF-OMe (23). Compound 23 was synthesized according to general procedure C as
creamish solid, yield 85%, mp 150 oC;
= -37.5o (0.4, CHCl3); IR (ATR): 3092, 2958, 3309,
3285, 2920, 2361, 2340, 2128, 1716, 1753, 1694, 1643, 1530, 1431, 1455 cm-1; H NMR (500
MHz, CDCl3, 25 oC, TMS): δ 1.81-1.87 (m, 1H, CH2Pro), 1.94-1.99 (m, 3H, CH2Pro+1H CH2Met),
2.11-2.15 (m, 4H, CH3Met+1H CH2Met), 2.31-2.32 (m, 1H, CH2Pro), 2.57-2.59 (m, 2H, CH2Met),
3.00 (dd, J = 14.41, 7.10 Hz, 1H, CH2Phe), 3.21 (dd, J= 13.91 Hz, J= 5.14 Hz, 1H, CH2Phe), 3.30-
3.38 (m, 2H, CH2Pro), 3.74 (s, 3H, OCH3), 3.85 (dd, J= 17.8, 3.42 Hz, 1H, CH2Gly), 4.02 (dd, J=
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