Modification of the lead molecule: Tryptophan and piperidine appended triazines reversing inflammation and hyeperalgesia in rats

Article abstract of DOI:10.1016/j.bmc.2019.115246

The structural optimization of the molecules making them to fit into the active site pocket of COX-2 occupying the same space as covered by the natural substrate arachidonic acid helped in the emergence of compound 10 as an efficacious anti-inflammatory a

Full text of DOI:10.1016/j.bmc.2019.115246

Journal Pre-proofs
Modification of the Lead Molecule: Tryptophan and Piperidine Appended
Triazines Reversing Inflammation and Hyeperalgesia in Rats
Priya Kumari, Sukhmeet Kaur, Jashanpreet Kaur, Rajbir Bhatti, Palwinder
Singh
PII:
S0968-0896(19)31598-6
https://doi.org/10.1016/j.bmc.2019.115246
BMC 115246
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
15 September 2019
23 November 2019
29 November 2019
Please cite this article as: P. Kumari, S. Kaur, J. Kaur, R. Bhatti, P. Singh, Modification of the Lead Molecule:
Tryptophan and Piperidine Appended Triazines Reversing Inflammation and Hyeperalgesia in Rats, Bioorganic
&
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115246
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Modification of the Lead Molecule: Tryptophan and Piperidine Appended Triazines
Reversing Inflammation and Hyeperalgesia in Rats
ψ
ψ
€
€
ψ
Priya Kumari, Sukhmeet Kaur, Jashanpreet Kaur, Rajbir Bhatti, Palwinder Singh
ψDepartment of Chemistry, Centre for Advanced Studies, Guru Nanak Dev University, Amritsar-
1
43005. India
€Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005. India
Ph. 91-183-2258802-09 x 3278, FAX: 91-183-2258820,
e-mail: palwinder_singh_2000@yahoo.com
Abstract. The structural optimization of the molecules making them to fit into the active site
pocket of COX-2 occupying the same space as covered by the natural substrate arachidonic acid
helped in the emergence of compound 10 as an efficacious anti-inflammatory agent. Selective for
COX-2 over COX-1, compound 10 exhibited IC 0.02 µM for COX-2 and reversed acetic acid
5
0
-1
induced inflammation in rats by 73% when used at 10 mg kg dose and the same dose of the
compound also rescued the animals from inflammatory phase of formalin induced hyperalgesia.
As evidenced by the results of molecular modeling studies supported by the nuclear Overhauser
enhancement data, the appropriate geometry of the molecule in the active site pocket of COX-2
contributing to its H-bond/hydrophobic interactions with Ser530, Trp387 and Tyr385 seems
responsible for the enzyme inhibitory activity of the compound.
1

Introduction
Inflammation is the first line defense of the body against the invasion of pathogens but its
prevalence for a long time leads to the emergence of various diseases such as diabetes, Alzheimer’s
disease and rheumatoid arthritis.1-5 Under the influence of inflammatory stimulants; starting with
the phospholipase triggered release of membrane bound arachidonic acid (AA) followed by
cytokines/ TNF-α/ NO synthetase induced activation of cyclooxygenases (COX), it is the COX-2
metabolized arachidonic acid that is responsible for the production of inflammatory prostaglandins
PGE2.6-11 Therefore, irrespective of the constitutive role of COX-1 and COX-2 in the production
of desirable metabolites for the aggregation of platelets, maintaining renal blood flow and lining
of gastric mucosa, homeostasis, working of cardiac muscles and smoothing of labor pain,12,13 the
induced COX-2 is responsible for the culmination of inflammation.14,15 In this context, overcoming
the obvious limitations of COX-1/COX-2 non-selective anti-inflammatory drugs such as aspirin,
indomethacin, ibuprofen,16,17 the launch of COX-2 selective drugs (COXIBS)18-21 was a cutting-
edge achievement in the treatment of inflammatory diseases. However, the inhibition of COX-2
leading imbalanced production of prostacyclins and thromboxane results in the emergence of
cardiovascular problems such as stroke and cardiac death22-27 affecting the clinical scope of
COXIBS, especially after the withdrawal of rofecoxib and valdecoxib.28,29 Hence, a major
challenge that remains for anti-inflammatory agents to reach their full potential is to search for
new chemical entities with desirable selectivity for COX-2 and consequently better biological
profile.
The structural analysis of COX-2 in association with its natural substrate shows that AA is
present in the extended L/U-shaped geometry in the active site pocket of the enzyme.30,31 Since
the active conformation (acquired in the active site pocket of the enzyme) of the enzyme inhibitor
2

affects its efficacy, the electronic and stereochemical parameters of the inhibitor should desirably
match to the active site of the enzyme. In order to develop such ligands, it was planned to modify
3
2
the lead molecule 1 (Figure 1) by introducing amino acid in place of either the pyrrolidine moiety
or the 4-(4-aminophenyl)morpholin-3-one making the resulting molecule to acquire a
conformation overlapping with AA in the active site of the enzyme. The series of compounds 2 –
1
5 (Figure 1) was synthesized and evaluated for the biological activity.
Figure 1. Previous molecule 1 and the library of compounds 2–15 for the present studies.
Results and Discussion
The syntheses of compounds 2–6 were accomplished through the key intermediate 16 that was
o
obtained by the reaction of cyanuric chloride with 4-(4-aminophenyl)morpholin-3-one at 0-5 C.
The reaction of compound 16 with L-phenylalanine methyl ester hydrochloride/ L-proline methyl
ester hydrochloride/ anthranilic acid/ L-isoleucine methyl ester hydrochloride/ N-Boc-L-
o
tryptophan methyl ester hydrochloride at 40 C in the presence of Et N and NaH in dry ACN
3
provided respective compounds 2–6. In order to provide rigidity to the substituents at the triazine
ring, the tryptophan was made to react through its side chain by using N-Boc tryptophan methyl
ester. Compound 7 was procured by the deprotection of Boc group of compound 6 in the presence
of trifluoroacetic acid (TFA) (Scheme 1).
3

Cl
N
Cl
Cl
N
N
N
N
N
N
N
HN
O
N
HN N (S)NH
Cl
Cl
COOCH3
O
(
S)
O
N
N
NH2
(i)
N
H CO
3
O
(ii)
O
2
7
Cl
(vii)
Cl
N
N
N
Cl
N
HN
O
N
Cl
(
vi)
HN
O
N
N
(
iii)
N
N
(S)
HN
O
N
N
H CO
3
O
N
16
(S)
O
HN
Boc
N
O
3
COOCH3
(v)
(iv)
O
6
Cl
Cl
N
N
COOCH
N
N
3
(
S)
HN
N
N(S)
HN
N
NH
H
COOH
N
N
O
O
O
4
O
5
Scheme 1. Reaction conditions:
o
(i) Acetone, 4-(4-aminophenyl)-3-morpholinone, 0-5 C
o
(
ii) ACN, Et N, L-phenyl alanine methyl ester hydrochloride, 40 C
3
o
(
(
(
(
(
iii) ACN, Et N, L-proline methyl ester hydrochloride, 40 C
3
o
iv) ACN, Et N, anthranilic acid, 40 C
3
o
v) ACN, Et N, L-isoleucine methyl ester hydrochloride, 40 C
3
o
vi) ACN, NaH, N-Boc-L-tryptophan methyl ester hydrochloride, 40 C
o
vii) DCM, TFA, 25 C
Treatment of cyanuric chloride with 1 equivalent morpholine / piperidine in the presence of
o
33
Et N at 0-5 C resulted into the formation of compounds 17 and 18. Compounds 17 and 18 were
3
then treated with N-protected amino acid esters in the presence of NaH and NaOH, TBABr in dry
ACN and DCM at room temperature to procure compounds 19-22. In case of compound 22, the
ester group of tryptophan was hydrolyzed in situ to the corresponding carboxyl group. Removal
of Boc group of compounds 19-22 gave compounds 8-11 (Scheme 2).
4

Cl
N
Cl
N
N
N
N
N
(ii)
N
O
N
O
(iv)
X
X
(
S) COOCH3
(
S) COOCH3
Cl
N
Cl
N
NH2
NHBoc
(i)
N
N
N
N
19, 20
8, 9
Cl
Cl
Cl
N
X
Cl
Cl
17,18
N
N
N
N
N
N
N
(iii)
N
N
N
O
(
S)
(
S)
HOOC
H COOC
3
NHBoc
NHBoc
22
8, 17, 19: X = CH2
21
9, 18, 20: X = O
(iv)
(iv)
Cl
N
Cl
8.41
H
Cl
N
H
N
N
N
N
7
H
.98
N
N
H
H
H
NH2
H
N
N
N
H
O
H
N
N
H
N
N
2' H
7
.98
H 2
O
2
' H
O
H
NOE
H
(Z)
8
.41
H
H
H
NOE
3.68-3.75
H
3.68-3.75
10
(S)
H
H
H
H
H
H
H
H
4
H
HOOC
H
H
H
11
7
H
7.40
NH2
H
NH2
O
H
H
H
H
H
H
O
3
.58
H
H
o
11
H
B
A
10
Scheme 2. Reaction conditions
(
(
(
(
i) Acetone, Et N, morpholine/piperidine, 0-5 C
3
o
ii) DCM, NaOH, TBABr (cat.), N-Boc-L-tyrosine methyl ester hydrochloride, 25 C
iii) Dry ACN, NaH, N-Boc-L-tryptophan methyl ester hydrochloride, 25 C
o
o
iv) DCM, TFA, 25 C
Making further modification in the compounds in extending the space between the amino acid
residue and the triazine ring of compounds 8–11 and also keeping in view the medicinal
significance of azole moiety, compounds 12–15 were synthesized. The presence of azole moiety
was also considered to provide some rigidity to this arm of the molecule. The key intermediates
2
4 were obtained from cyanuric chloride through compound 23 (Scheme 3). Reactions of
compounds 24 with benzyl azide, (S)-phenylalanine azide, (S)-tyrosine azide and (S)-tryptophenyl
azide provided respective compounds 12(a-d), 13(a-d), 14(a-c) and 15(b,e).
5

Cl
N
N3
Cl
N
N
N
N
N
(iii)
R
O
N
N
Cl
Cl
N
(i)
Cl
N
1
2(a-d)
N3 (S)
O
N
N
Cl
N
O
Cl
O
N
N
O
23
(iii)
R
O
N (S)
N
N
Cl
O
(ii)
N
N
13(a-d)
R
N
O
N3 (S)
O
Cl
24
O
OH
N
N
O
R
(iii)
R
N
O
N (S)
N
N
O
a
N
N
OH
1
4(a-c)
b
O
N3
O
Cl
(
S)
O
c
HN
N
O
N
N
N
O
O
H
O
(S)
R
N
N
N
O
d
e
HN
N
Cl
(iii)
N
NH
15(b,e)
o
o
Scheme 3. (i) propargyl alcohol, TBABr, NaOH, DCM, 0 C, 2h (ii) RH, Et N, acetone, 0 C
3
o
(
iii) sodium ascorbate, CuSO .5H O, EtOH:water (9:1), 60 C.
4
2
While all the compounds were characterized with the help of NMR, mass, IR spectral data,
1
1
their conformation was checked by recording H- H NOESY spectra. For compound 10 (Scheme
), all the H and C signals were assigned (Figure S26-S36) and also confirmed with the NMR
2
spectra of its precursor 21 (Figure S98-S104). Since the conformation acquired by the molecule in
the active site pocket of the enzyme is crucial for its activity, the orientation of the piperidine and
tryptophan substituents on the triazine template in compound 10 was ascertained from the nuclear
Overhauser effect (nOe) cross peaks. The nOe between 2-H (δ 7.98) and 2´-H (δ 3.68) was
observed (H -H , Figure 2A) indicating that compound 10 exists in form ‘A’ (Scheme 2).
2
2´
Interestingly, the observation of nOe between 2´-H and the aromaticH at δ 8.41 (H -H , Figure 2)
7
2´
6

pointed also towards the presence of form ‘B’ of compound 10 (Scheme 2). Similar population of
o
o
the forms ‘A’ and ‘B’ of 10 was observed at 13 C (Figure 2B) and 35 C (Figure 2C) and even by
changing the solvent from CDCl to DMSO-d (Figure 2D). The placement of the side chain of
3
6
tryptophan was also confirmed by the visibility of nOe signals between H – OCH /H / H and
2
3
10
11
1
13
H – OCH / H /H . Therefore, not showing two sets of signals in the H and C NMR spectra, it
4
3
10
11
seems that there is rapid inter-conversion between the two forms of compound 10 in the solution
phase. However, during the molecular docking of compound 10 in the active site pocket of COX-2,
the compound has preferably acquired conformation ‘A’ that may be attributed to its local polar
and non-polar interactions with Tyr385 and Trp387,
Figure 2. NOESY spectra of compound 10 (NS 32, NE 256, mixing time 500 ms, CDCl ) recorded
3
o
o
o
at: (A) 25 C (400 MHz), (B) 13 C (500 MHz), (C) 35 C indicating the existence of 10 in both
o
forms ‘A’ and ‘B’ (Scheme 2), (D) 35 C (DMSO-d ) also showing forms ‘A’ and ‘B’.
6
7

respectively. Among compounds 12-15, no nOe was observed between any of the Hs’ of the two
arms of the compounds (Figure 3A 3C, Figure S56,S69,S86) except in case of compound 12b and
1
3b that showed nOe between 2-H and 3′-H (Figure 3B, 3D, Figure S61). Most probably, the ether
linkage between the triazine and the azole moiety and the nature of substituent at N-6 of azole
provided flexibility and flattened the azole arm of the compounds.
Cl
C
D
Cl
N
N
O
2
N
N
N
3
H
N
O
X
N
8
H
7
COO
H
CH
N
H
7
N
H
5
H
H
a
2
N
N
H
16 H
H
O
H
8
1
b H
2
'
9
H
H
N
O
H
N
2'
3
'
N
H
17H
H
H
11
H
H H
X
a
H
X
H
NOE
H
3
'
NOE
N
18H
1
2
bH
H
H
H
NOE
X
1
1
Figure 3. H- H NOESY spectrum of compound (A) 15b and (B) 12b and their respective
conformations (C) and (D).
Biological studies
In Vitro COX inhibitory assay. Enzyme inhibitory assays of the compounds were performed
3
4
35
using the protocol provided with the kit as well as the one described in the previous report.
-4
-8
Compounds were screened for their COX-1 and COX-2 inhibitory activity at 10 to 10 M
8

concentrations in triplicate. The COX-1, COX-2 inhibitory activities were quantified in terms of
0% inhibitory concentration (IC ) of the compounds by measuring the production of
5
5
0
prostaglandins by both the enzymes in the presence of test compounds. Compounds 2 and 5
showed lower efficacy for COX-2 inhibition with IC (COX-2) 8 μM and 6 μM, respectively in
5
0
comparison to compound 1 and it may be attributed to the presence of one flexible substituent in
the form of Phe and Ile in these compounds. Supporting the contributions of the molecular structure
to the efficacy of the compound, the COX-2 inhibitory activity of compound 4 was improved in
comparison to that of compounds 2 and 5 and it was probably accredited to the less flexibility of
the two substituents and acquiring rigid geometry by the molecule. Compounds 7 and 10 showed
similar efficacies for COX-2 inhibition but better than compound 1 and comparable to that of
celecoxib and diclofenac (Table 1). It seems that the presence of indole moiety (of Trp) near to the
triazine ring induced more rigidity in the molecule and confined it to the active site pocket of the
enzyme. In case of compound 11, the efficacy for COX-2 inhibition was appreciable, though less
than that of compound 10 but its selectivity index was poor. For compounds 12–15, no significant
activity was observed except in case of compounds 12b and 13b. The lower activity of these
compounds may be due to their flexible conformation in comparison to compounds 7 and 10 (also
discussed in the later section). Therefore, the results of enzyme immunoassays favored that
compounds 7 and 10, with selectivity index for COX-2 higher than that of indomethacin and
diclofenac but comparable to that of celecoxib, are the suitable candidates for their further
investigations for analgesic and anti-inflammatory activity by using the animal models.
Table 1. IC (μM) and the selectivity index of the compounds against COX-1 and COX-2.
5
0
9


Compound
IC (μM)
Selectivity index*
5
0
COX-1
COX-2
2
4
5
7
8
>10
>10
>10
>10
>10
>10
1.0
8.0
>1.2
>10
>1.6
>333
>2.0
>500
10
1.0
6.0
0.03
5.0
1
1
0
1
0.02
0.10
5.0
1
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
5b
5e
8.0
1.6
1
>10
6.0
0.10
-
>5
1
1
4.0
7.0
0.6
>1.3
>2
1
>10
>10
8.0
6.0
1
0.20
9.0
1
0.9
1
5.0
8.0
0.6
1
>10
>10
>10
>10
>10
>10
0.08
0.07
15
4.0
>0.25
>0.3
>0.3
1
1
3.0
1
3.0
1
>10
>10
0.06
0.96
0.02
0.04
1
1
1
>160
0.08
3.5
Indomethacin
Diclofenac
Celecoxib
375
ψAverage of the three values with deviation <2%. *IC50 (COX-1)/(COX-2)
Human whole blood assay.34,36 The selectivity of compounds 7 and 10 for COX-2 over COX-1
was also ascertained with human whole blood assay in which these compounds did not lower TXB2
1
0

level of calcium ionophore activated COX-1 sample in comparison to the control whereas the
formation of PGE in the LPS activated COX-2 sample was considerably decreased when
2
compared to the control (Table 2, Figure 4).
Table 2. Human whole blood assay data showing selectivity of compounds for COX-2.
TXB (ng/mL)
PGE (ng/mL)
2
2
-
calcium
+ calcium
ionophore
1.20
-LPS
0.33
+LPS
ionophore
0.35
Control
1.40
0.98
0.53
0.45
Indomethacin (1 μM)
Compound 7 (1 μM)
Compound 10 (1 μM)
0.59
0.90
0.84
Figure 4. Inhibition of (A) TxB in calcium-ionophore-stimulated whole blood and (B) PGE in
2
2
LPS-stimulated whole blood by compounds 7 and 10.
Screening for anti-inflammatory and analgesic activities
The acetic acid induced writhing has been considered as one of the most sensitive tools for the
screening of anti-inflammatory and analgesic activities of various test compounds. The abdominal
constrictions after i.p. administration of acetic acid are postulated to be induced by the activation
3
7
of local peritoneal receptors. Treatment with indomethacin, compounds 7 and 10 at a dose of 10
−
1
mg kg significantly decreased writhes by 70%, 73% and 65%, respectively as compared to acetic
acid treated rats. Treatment of animals with compounds 2, 3, 5, 6 and 9 also decreased the number
1
1

of writhings but the effect of these compounds was less as compared to compounds 7 and 10
(Figure 5). Compounds 7 and 10 were taken up for further in-vivo studies.
3
Cmpd 7 Cmpd 9 Cmpd
Cmpd 2 Cmpd 5 Cmpd 6
Cmpd 10
Indomethacin
Figure 5. Effect of compounds 2, 3, 5, 6, 7, 9 and 10 on acetic acid induced writhing in rats. All data
a
was expressed as mean ± SEM (n=5). p<0.05 vs vehicle control. Nine groups, with 5 animals in each
group, of Wistar rats with body weight ranging from 200-250 g were used.
Effect of compound treatment on formalin induced pain
Formalin induced paw-licking test was used to validate the peripheral and central
3
8
antinociceptive effects of the compounds. Intraplantar injection of formalin was given to produce
biphasic hyperalgesia in rats. The first phase was the neurogenic phase (1–5 min) and the second
phase was the inflammatory hyperalgesia (20–30 min). Formalin injection resulted into the
increased number of flinchings in both phases (Figure 6). Treatments with indomethacin,
compounds 7 and 10 attenuated hyperalgesia in the neurogenic phase as well as the inflammatory
phase. However, statistically significant reduction in hyperalgesia was observed only in the
inflammatory phase as compared to the vehicle treated control group (Figure 6). Compound 10
1
2

exhibited better anti-inflammatory effect than that of indomethacin whereas the effect of
compound 7 was comparable to indomethacin.
Figure 6. Effect of compounds 7 and 10 on formalin induced hyperalgesia. All the values are expressed as
a
Mean ± SEM (n=5). All data was expressed as mean ± SEM (n=5). p<0.05 vs vehicle control. Four groups,
with 5 animals in each group, of Wistar rats with body weight ranging from 200-250 g were used.
Immunohistochemistry
Immunohistological staining using COX-2 antibody revealed an enhanced expression of COX-
2
as indicated by the increased staining intensity in the spinal sections of formalin treated rats in
comparison to the normal and compound 7 and 10 treated rats (Figure 7). It was apparent from
figure 7c that the range of fluorescence intensity was significantly reduced in the case of compound
treated animals indicating that the expression of COX-2 is suppressed in the presence of
compounds 7 and 10. Hence, supporting the design of the molecules and the results of enzyme
immunoassays, the analgesic and anti-inflammatory effects of the compounds through targeting
COX-2 were confirmed.
1
3

Figure 7. (a) Expression of COX-2 in the lumbar spinal region, (b) graph showing fluorescent
intensity in the spinal sections of control and treated rats, (c) range of intensity (ROI) plot of images
showing considerable reduction in fluorescence intensity in the case of compound 7 and compound
1
0 treated animals.
Acute toxicity studies. Although slight restless behavior was observed in the first four hours,
-1
animals treated with an oral dose of 2000 mg kg of compound 10 did not show any marked
1
4

behavioral alterations during the 14 days period of observation. Histological sections taken at the
end of study indicated normal liver and kidney (Figure 8) indicating that the compound was non-
-1
toxic even at 2000 mg kg dose.
A
B
C
D
Figure 8. Photomicrographs showing sections of (A) normal liver, (B) compound 10 treated liver,
C) normal kidney and (D) compound 10 treated kidney at 10X magnification.
(
Correlation of molecular docking conformations, nOe data and the biological activity
Since the conformation of the ligand in the active site pocket of the enzyme affects its
interactions with the amino acid residues and hence the efficacy of the compound, the trends of
the compounds’ COX-2 inhibitory activities were rationalized with the help of molecular docking
studies and further confirming the docked conformation of the molecule with its nOe data.
Although the NMR data indicated the existence of both forms ‘A’ and ‘B’ for compound 10 in the
solution phase (Scheme 2, Figure 2), its molecular docking in the active site pocket of COX-2
1
5

preferred form ‘A’ occupying the same pocket where AA sits. The indole fragment of 10 was
locked with Trp387 through non-polar interactions (arrow 1, Figure 9A). Similar to the carboxyl
group of AA, the ester part of the compound interacted through H-bonding with Tyr385 and Ser530
and the piperidine moiety was placed in the hydrophobic sub-pocket of Val116, Leu117 and
Leu531 (arrow 2, Figure 9A). It is pertinent to mention that the geometry of the molecule (as ‘A’,
‘B’ of 10) in the active site pocket of the enzyme is affected by the local interactions with the
amino acid residues. In comparison to the placement of compound 1 in the active site pocket of
the enzyme (Figure 9D) where the small pyrrolidine (relative to piperidine) is pulled in the
hydrophobic sub-pocket (Val116, Leu117, Leu531) dragging rest of the molecule; the large
piperidine moiety and the hooking of indole fragment with Trp387 (arrow marks, Figure 9A)
pushed the carboxyl part of Trp residue of compound 10 near to Ser530 and Tyr385 and these
interactions probably enhanced the efficacy of 10. However, none of the compounds 2, 4 and 5
exhibited such type of interactions (Figure 9E, F, G). The conformations of compounds 11 and 7
in the active site pocket of COX-2 were also similar to compound 10 (Figure 9H, 9K) but probably
the more hydrophilicity of 11 (Table S1) in comparison to 10 made it less potent. Moreover,
compare to the piperidine moiety of 10 occupying the hydrophobic sub-pocket of the enzyme,
relatively less hydrophobic morpholine unit of compound 11 was placed in the same sub-pocket
creating a bit of unfavorable environment. A nice structure-activity correlation was also visible for
the series of compounds 12-15. Compounds 12-15 were hard to acquire close conformation like
that of compound 10 and were also found less effective against COX-2. Among compounds 12
and 13, only in case of 12b and 13b, nOe was observed between the morpholine unit and
etheralCH and their molecular docking structures also acquired quite closed conformations
2
(Figure 9I, Figure S62). Correspondingly, the COX-2 inhibitory activity of
1
6

1
7

3
9
Figure 9. (A) Compound 10 docked in the active site of COX-2 (PDB ID 1CVU) occupying the same
pocket where AA sits (AA shown in green). Non-polar hydrogens are omitted for clarity. (B) Another view
of compound 10 bound to COX-2 active site. (C) 2D view of compound 10 showing H-bonding and π-π
interactions with the enzyme. Molecular docking of (D) compound 1, (E) compound 2, (F) compound 5,
(G) compound 4, (H) compound 11, (I) compound 12b, (J) compound 15b and (K) compound 7 in the
active site pocket of COX-2.
compounds 12b and 13b was better than that of other compounds in the series though these were
less potent than compound 10. None of the compounds 14 and 15 exhibited nOe between the two
substituents of the triazine moiety and their molecular docking conformations were also seen
flattened where a part of the molecule was placed outside the active site pocket of the enzyme
(Figure 9J, Figure S87). Therefore, besides the justification for the structure activity relationship,
the scrutining of the molecular architect of the compounds, the molecular interactions with the
target enzyme and the enzyme inhibitory activity data may help in the design of more such
molecules.
3
D-Quantitative Structure−Activity Relationship Study Model
Using the partial least square (PLS) factor of three, the field-based 3D-quantitative
structure−activity relationship (QSAR) model was generated by correlating the activity with the
steric, electrostatic, hydrophobic, hydrogen bond donor (HBD) and hydrogen bond acceptor
(HBA) factors (Figure S174). Leave-one-out cross-validation and non-cross- validation analysis
2
2
gave R CV and R 0.2117 and 0.8785, respectively and the standard error of estimate was 0.3803
1
8

with the F ratio 28.9 (Tables 3). The steric, electrostatic, hydrophobic, HBA, and HBD fields were
found to contribute by 0.44, 0.13, 0.21, 0.16, and 0.04, respectively towards the activity of the
compounds (Table 4, Figure S174). The QSAR model also showed nice correlation between the
experimental and the predicted activity for both training and test set of compounds (Figure 10).
Keeping in view the constrained and hydrophobic active site pocket of the enzyme, expectedly, it
was observed that the steric factor along with the hydrophobic parameters were the major
contributors to the biological activity of the molecules under the present investigation.
Table 3. PLS-Based Statistical Parameters for the Selected 3D-QSAR Modela
R2
R CV
2
R2
Stability
F
P
RMSE Q2
Pearson-r
Factors SD
Scramble
1
2
3
0.4942 0.7606 0.2228
0.4015 0.8533 0.3417
0.3803 0.8785 0.2117
0.5455
0.7288
0.8200
0.559
0.507
0.337
44.5 1.06e-05 0.76
37.8 3.82e-06 0.75
28.9 8.94e-06 0.79
0.1963 0.7511
0.2033 0.7398
0.1225 0.6985
a
2
SD, standard deviation of regression; R, squared value of R for the regression; F, variance ratio. Large value of F
indicates more statistical regression; P, significance level of variance ratio. Smaller value indicates a greater degree
2
of confidence; rmse, root-mean-square error; Q, squared value of Q for the predicted activities; Pearson-R, Pearson-
R value for the correlation between the predicted and observed activity for the test set.
Table 4. Contribution of various factors towards the biological activity of the molecules.
Factors
Guassian Steric
Guassian
electrostatic
0.112438
0.128202
0.132070
Guassian
hydrophobic
0.180919
0.204815
0.211862
Guassian Hbond Guassian Hbond
acceptor
0.158270
0.158987
0.163523
donor
1
2
3
0.522378
0.472994
0.447929
0.025996
0.035002
0.044616
1
9

Figure 10. Scatter plot of observed versus predicted activity for training and test set compounds.
A comparison of the ADME profile of compounds 1, 7, 10 and 11 was indicative of the fact
that the blood brain barrier effect of compounds 7, 10 and 11 was improved over that of compound
1
(Table 3) whereas the solubility factor of 7 and 10 was expectedly but desirably reverse to that
of compounds 1 and 11. Cytochrome P4502D6 (CYP2D6) and cytochrome P4503A4 (CYP3A4)
catalyze the oxidation of organic compounds and are respectively responsible for the metabolism
4
0
and elimination of 20% and 40% of all pharmaceutical drugs from the body. In addition to the
weak substrate to CYP3A4, compound 10 is more likely to get metabolized by CYP2D6 whereas
compounds 1 and 11 are not the substrates of this enzyme. As per the Marine-Darby Canine kidney
(MDCK) cell model, the permeability of compounds 7 and 10 was quite different from that of
compound 1. However, compounds 1, 7, 10 and 11 exhibited similar plasma protein binding and
human intestinal absorption. Therefore, as per the predicted model, superior pharmacological
parameters of compound 7 and 10 over those of compound 1 are expected.
Table 3. ADME profile of compound 1, 7, 10, 11.41
2
0

Property
Effect
10
1
7
11
Plasma Protein Binding (%)
85.69
0.01
4.91
96.83
Non
87.62
0.09
0.03
97.19
85.08
67.57
BBB (Cbrain/Cblood
)
0.66
0.74
0.19
5.81
Pure water solubility (mg/mL)
Human intestinal absorption (HIA) (%)
CYP2D6 substrate
98.25
96.86
Weakly Substrate
Non
CYP3A4 substrate
Weakly Weakly
0.12 0.06
Weakly Weakly
Marine-Darby Canine kidney (MDCK) (nm/sec)
0.08
0.64
Conclusions
Keeping in view the essentiality of geometrical and electronic compatibility between the enzyme and its
inhibitor, the modification of compound 1 was made for developing new anti-inflammatory agent. The
replacement of pyrrolidine and morpholinone moiety of compound 1 with piperidine and tryptophan
provided compound 10 that was placed in the COX-2 active site more elegantly than compound 1 and its
anti-inflammatory profile was improved over that of compound 1. Compound 10 did not show toxicity even
-1
-1
at dose 2000 mg kg and it was capable to reduce acetic acid induced inflammation by 73% at 10 mg kg
dose and also rescued the animal from formalin induced inflammatory phase of hyperalgesia. As depicted
from the results of molecular modeling studies supported with nOe data, it seems that the geometry and
appropriate placement of polar, non-polar units of the molecule in the corresponding region of the enzyme
are the pre-requisites for an effective COX-2 inhibitor. Overall, the medicinal and pharmacological features
of compound 10 favor its candidature for pursuing further studies.
Experimental section
Chemistry
All the reagents purchased from suppliers were used as such without any further purification.
Acetonitrile was dried by refluxing over phosphorus pentoxide (P O ) and stored over activated 4
2
5
Ǻ molecular sieves. Melting points were determined in open capillaries and were uncorrected.
2
1

Reactions were monitored by thin layer chromatography (TLC) performed on glass plates coated
with silica gel (GF254) and further visualized under UV light and iodine atmosphere. Column
chromatography was performed on silica gel 60-120 mesh size and hexane - ethyl acetate were
1
13
used as eluents. H, C NMR spectra were recorded on Bruker 500 MHz and 125 MHz NMR
spectrometer, using CDCl and DMSO-d as solvents and TMS as internal standard. Chemical
3
6
shifts are reported in ppm relative to trimethyl silane (TMS), multiplicity (s = singlet, d = doublet,
t = triplet, m = multiplet, dd = double doublet, br = broad) and coupling constant (J in Hz). For
1
3
C and DEPT-135 NMR spectra; positive signals correspond to CH and CH carbons, negative
3
signals correspond to CH carbons. Mass spectra were recorded on Bruker Micro TOF QII mass
2
spectrometer using ESI (electrospray ionization) ion source in both +ve and –ve mode for high
resolution mass spectra (HRMS) and Shimadzu MS-2020 for MS. Using KdScientific automated
pump, with flow rate 180 μL/h, 50 μM solution in ACN-water formic acid (7:2.9:0.1) was injected
o
to electrospray ionization source. Desolvation was performed with dry N gas heated at 180 C.
2
Various parameters of mass spectrometer were optimized for maximum ion abundance. Typically
-7
the Capillary voltage was 4500 V and vacuum was maintained at 3-4×10 mbar. Sodium formate
was used as internal calibrant. The percentage purity >98% of the compounds was checked with
q1H NMR spectrum (Figure S173) and their HRMS were recorded.
General procedure for the synthesis of compounds
Procedure A. To a cooled solution of cyanuric chloride/compound 23 (1 mmol) in acetone (20
o
mL) at 0 C, a solution of amine/substituted aniline (0.9 mmol) in acetone was added dropwise.
o
The reaction mixture was stirred for 2h at 0 C. After the completion of reaction (monitored by
TLC), water (50 mL) was added to the reaction mixture and it was extracted with ethyl acetate.
The organic layer was separated, dried over anhydrous Na SO , and crude product was obtained
2
4
2
2

after concentrating it under vacuum. Compounds 16–18 and 24 were procured after purifying this
crude product by column chromatography using hexane/ethyl acetate as eluent. Triethyl amine was
used for the synthesis of 17, 18 and 24.
Procedure B. N-Protection of L-amino acid methyl ester hydrochloride.
To the solution of L-amino acid methyl ester hydrochloride (1 mmol) in acetone-water (30 mL,
1
:1 v/v), Na CO (1.5 mmol) was added followed by the addition of di-tert-butylpyrocarbonate (1
2 3
o
o
o
mmol) in portions at 0 C. The reaction mixture was stirred for 1 h at 0 C and then at 25 C for
1
.5 h. The reaction mixture was then extracted with ethyl acetate (2×50 mL). The aqueous part
was acidified with dil HCl, and the separated oil was extracted with ethyl acetate and dried over
anhydrous Na SO . The ethyl acetate was evaporated under vacuum to obtain desired N-Boc-L-
2
4
amino acid methyl ester.
o
Procedure C. To the solution of compound 16 (1 mmol) in dry ACN (50 mL) at 40 C, solution of
amino acid methyl ester hydrochloride (1 mmol) neutralized with Et N (1 mmol ) in 5 mL dry
3
ACN was added. After completion of the reaction (TLC), water (50 mL) was added to the reaction
mixture and it was extracted with ethyl acetate (4×50 mL). The combined organic layers were
passed through anhydrous Na SO . The crude product was obtained by evaporating ethyl acetate
2
4
under vacuum and it was purified by column chromatography using ethyl acetate – hexane as the
eluent.
Procedure D. To the solution of compounds 17 / 18 (1 mmol) and N-Boc-L-tyrosine methyl ester
o
(
1 mmol) in DCM at 25 C tetrabutyl ammonium bromide (catalytic amount) was added. Then,
5
0% NaOH (2.5 mmol) was added drop-wise to the reaction mixture and stirred till the completion
of the reaction (TLC). The reaction was quenched with water (50 mL) and it was extracted with
chloroform. The organic layer was separated and dried over anhydrous Na SO and concentrated
2
4
2
3

under vacuum to obtain crude product which was then purified by column chromatography using
ethyl acetate – hexane as eluent. Thus compound 19 and 20 were procured.
Procedure E. NaH (1.5 mmol) was washed 3-4 times with dry hexane and then dry ACN was
added. N-Boc-L-tryptophan methyl ester (1 mmol) was added to the suspension of NaH in ACN
o
and the reaction mixture was stirred for 5 min at 25 C until the reactant gets dissolved. Then,
compound 16/17/18 was added and the reaction mixture was stirred and monitored with TLC. The
reaction was quenched by the addition of ice cold water and extracted with ethyl acetate (2×50
mL). The organic layers were separated, and dried over anhydrous Na SO . The crude product
2
4
obtained after concentrating ethyl acetate under vacuum was purified by column chromatography
using hexane-ethyl acetate as eluent to procure compounds 6, 21, 22.
Procedure F. Removal of protecting group.
For deprotection of Boc group of compounds 6 and 19-22, solution of the compound (1 mmol) in
dichloromethane (DCM) was stirred with 10 mmol TFA. The reaction was monitored by TLC and
after its completion, DCM was evaporated under vacuum and compounds 7 and 8-11 were
procured after washing of the crude product with diethyl ether.
Procedure G. To a solution of compound 24 (1 mmol) in 18 mL ethanol; sodium ascorbate (5 mol
%
) in 1 mL water, CuSO .5H O (2 mol %) in 1 mL water and benzyl azide / L-phenylalanine
4 2
methyl ester azide / L-tyrosine methyl ester azide / L-tryptophan methyl ester azide were added in
o
sequence and reaction mixture was stirred for 14 h at 60 C. After the completion of the reaction
(TLC), the reaction mixture was diluted with water and extracted with ethyl acetate (2×50 mL).
The combined organic layers were passed through anhydrous Na SO and concentrated under
2
4
vacuum to obtain crude product which was then purified by column chromatography using ethyl
acetate – hexane as eluent. Thus compounds 12(a-d), 13(a-d), 14(a-c) and 15(b,e) were procured.
2
4

Procedure H. To the solution of cyanuric chloride (1 mmol) and propargyl alcohol (1 mmol) in
o
DCM at 0 C tetrabutyl ammonium bromide (catalytic amount) was added. Then, 50% NaOH (2.5
mmol) was added dropwise to the reaction mixture and stirred till the completion of the reaction
(TLC). The reaction was quenched with water (50 mL) and it was extracted with chloroform. The
organic layer was separated and dried over anhydrous Na SO and concentrated under vacuum to
2
4
obtain crude product which was then purified by column chromatography using ethyl acetate –
hexane as eluent. Thus compound 23 was procured.
Methyl
(4-chloro-6-((4-(3-oxomorpholino)phenyl)amino)-1,3,5-triazin-2yl)-L-phenyl
alaninate (2). Compound 2 was synthesized according to general procedure C using compound
o
1
3
=
2 and L-phenylalanine methyl ester hydrochloride. White solid, yield 67%, mp 139 C. IR (ATR):
-1
25
265, 2363, 2117, 1878, 1744, 1572, 1505, 1416, 1237, 1125, 984, 924, 805, 745, 499 cm . [α]
D
o
1
+37.5 (0.4, CHCl ). H NMR (500 MHz, CDCl ) δ 3.12 (dd, J = 13.5, 7.0 Hz, 1H of PhCH ),
3
3
2
3
.23 (dd, J = 13.5, 4.6 Hz, 1H of PhCH ), 3.71-3.76 (m, 5H, OCH + CH ), 4.03-4.04 (m, 2H,
2 3 2
CH ), 4.35 (s, 2H, CH ), 4.88-4.89 (m, 1H, CH), 5.88 (br, 1H, NH), 7.12-7.14 (m, 2H, ArH ), 7.26-
2
2
1
3
7
4
.33 (m, 6H, ArH+ NH), 7.53-7.54 (m, 2H, ArH); C NMR (125 MHz, CDCl ) δ 37.9 (-ve, CH ),
3 2
9.7 (-ve, CH ), 52.5 (+ve, OCH ), 55.1 (+ve, CH), 64.1 (-ve, CH ), 68.6 (-ve, CH ), 121.3 (+ve,
2
3
2
2
ArCH), 126.0 (+ve, ArCH), 127.4 (+ve, ArCH), 128.8 (+ve, ArCH), 129.1 (+ve, ArCH), 135.2
(ab, ArC), 136.2 (ab, ArC), 137.2 (ab, ArC), 163.8 (ab, C), 163.9 (ab, C), 165.4 (ab, C), 166.8
+
(
C=O), 171.4 (C=O). HRMS (microTOF-QII, MS, ESI): Calcd for C H N O Cl ([M+Na] )
2
3
23
6
4
5
05.1362, found 505.1359.
Methyl (4-chloro-6-((4-(3-oxomorpholino)phenyl)amino)-1,3,5-triazin-2yl)-L-prolinate (3).
Compound 3 was synthesized according to general procedure C using compound 12 and L-proline
o
methyl ester hydrochloride. White solid, yield 72%, mp 225 C. IR (ATR): 3272, 3175, 3123, 3004,
2
5

-1
25
o
2
363, 2117, 1908, 1751, 1640, 1565, 1490, 1394, 1162, 991, 842, 559 cm . [α] = -120.0 (0.4,
D
1
CHCl ). H NMR (500 MHz, CDCl ) δ 2.00-2.10 (m, 3H, CH ), 2.32-2.33 (m, 1H, CH ), 3.67 (s,
3
3
2
2
2
H, CH ), 3.75-3.76 (m, 4H, OCH + 1H CH ), 3.82-3.84 (m, 1H, CH ), 4.03 (m, 2H, CH ), 4.34
2 3 2 2 2
(s, 2H, CH ), 4.60-4.66 (m, 1H, CH), 7.15-7.17 (m, 1H, NH), 7.28-7.30 (m, 2H, ArH), 7.52-7.54
2
1
3
(
m, 1H, ArH), 7.63-7.64 (m, 1H, ArH); C NMR (125 MHz, CDCl ) δ 23.8 (-ve, CH ), 30.2 (-ve,
3
2
CH ), 47.3 (-ve, CH ), 49.7 (-ve, CH ), 52.3 (+ve, OCH ), 59.6 (+ve, CH), 64.1 (-ve, CH ), 68.6
2
2
2
3
2
(
-ve, CH ), 120.6 (+ve, ArCH), 125.9 (+ve, ArCH), 136.6 (ab, ArC), 136.8 (ab, ArC), 163.4 (ab,
2
C), 163.5 (ab, C), 166.7 (ab, C), 172.6 (C=O), 172.7 (C=O). HRMS (microTOF-QII, MS, ESI):
+
Calcd for C H N O Cl ([M+Na] ) 455.1205, found 455.1256.
19
21
6
4
2
-((4-Chloro-6-((4-(3-oxomorpholino)phenyl)amino)-1,3,5-triazin-2-yl)amino)benzoic acid
(4). Compound 4 was synthesized according to general procedure C using compound 12 and
o
anthranilic acid. Creamish solid, yield 67%, mp 240 C (dec), IR (ATR): 3295, 3101, 3026, 2363,
-
1 1
2
3
1
117, 1878, 1662, 1565, 1498, 1118, 998, 916, 760, 663 cm . H NMR (500 MHz, DMSO-d ) δ
6
.73 (t, J = 4.7 Hz, 2H, CH ), 3.98 (t, J = 4.5 Hz, 2H, CH ), 4.20 (s, 2H, CH ), 7.18 (t, J = 7.3 Hz,
2
2
2
H, ArCH), 7.37 (d, J= 8.4 Hz, 2H, ArCH), 7.63-7.65 (m, 3H, ArCH), 8.01 (d, J = 6.2 Hz, 1H,
ArCH), 8.41 (m, 1H, ArH), 10.40-10.45 (br, 1H, NH), 11.08 (br, 1H, NH), 11.15 (s, 1H, COOH).
1
3
C NMR (125 MHz, DMSO-d ) δ: 49.5 (-ve, CH ), 63.9 (-ve, CH ), 68.23 (-ve, CH ), 122.1 (+ve,
6
2
2
2
ArCH), 123.3 (+ve, ArCH), 126.1 (+ve, ArCH), 131.7 (+ve, ArCH), 134.1(+ve, ArCH), 136.6 (ab,
ArC), 137.8 (ab, ArC), 140.4 (ab, ArC), 150.3 (ab, ArC), 164.3 (ab, C), 166.4 (C=O), 169.8 (ab,
C), 169.9 (ab, C), 171.5 (C=O). HRMS (microTOF-QII, MS, ESI): Calcd for C H N O Cl ([M-
2
0
17
6
4
-
H] ) 439.0916, found 439.0941.
Methyl (2S)-2-((4-chloro-6-((4-(3-oxomorpholino)phenyl)amino)-1,3,5-triazin-2-yl)amino)-
-methylpentanoate (5). Compound 5 was synthesized according to general procedure C using
3
2
6

o
compound 12 and L-isoleucine methyl ester hydrochloride. White solid, yield 60%, mp 192 C. IR
(ATR): 3399, 3272, 3093, 2967, 2363, 1878, 1736, 1640, 1498, 1416, 1237, 1155, 984, 924, 834,
-
1
25
o
1
6
1
85 cm . [α] = -25.0 (0.4, CHCl ). H NMR (500 MHz, CDCl ) δ 0.95-0.96 (m, 6H, 2×CH ),
D 3 3 3
.25-1.28 (m, 1H, CH ), 1.50-1.55 (m, 1H, CH ), 1.97 (m, 1H, CH), 3.73-3.77 (m, 5H,
2
2
OCH +CH ), 4.04 (s, 2H, CH ), 4.35 (s, 2H, CH ), 4.58-4.60 ( m, 1H, CH), 5.98 (br, 1H, NH),
3
2
2
2
1
3
7
.29-7.30 (m, 3H, ArCH + NH), 7.55-7.57 (m, 2H, ArCH). C NMR (125 MHz, CDCl ) δ 11.6
3
(
+ve, CH ), 15.4 (+ve, CH ), 25.2 (-ve, CH ), 37.7 (+ve, CH), 49.7 (-ve, CH ), 52.2 (+ve, OCH ),
3 3 2 2 3
5
7.9 (+ve, CH), 64.1 (-ve, CH ), 68.6 (-ve, CH ), 121.5 (+ve, ArCH), 126.0 (+ve, ArCH), 136.3
2 2
(ab, ArC), 137.1 (ab, ArC), 163.9 (ab, C), 165.7 (ab, C), 165.9 (ab, C),166.8 (C=O), 171.6 (C=O).
+
HRMS (microTOF-QII, MS, ESI): Calcd for C H N O Cl ([M+Na] ) 471.1518, found 471.1495.
2
0
25
6
4
α
Methyl N -(tert-butoxycarbonyl)-1-(4-chloro-6-((4-(3-oxomorpholino)phenyl)amino)-1,3,5-
triazin-2-yl)-L-tryptophanate (6). Compound 6 was synthesized according to general procedure
E using compound 12 and N-Boc-L-tryptophan methyl ester. Creamish solid, yield 66%, mp 193
oC. IR (ATR): 3347, 3272, 3145, 2981, 2363, 1878, 1744, 1654, 1550, 1461, 1341, 1237, 1133,
-1 1
9
24, 797, 678, 611, 551 cm . H NMR (500 MHz, CDCl ) δ 1.42 (s, 9H, OC(CH ) ), 3.19-3.31
3 3 3
(m, 2H, CH ), 3.70 (s, 3H, OCH ), 3.81 (m, 2H, CH ), 4.07 (m, 2H, CH ), 4.38 (s, 2H, CH ), 4.70
2 3 2 2 2
(m, 1H, CH), 5.18 (s, 1H, NH), 7.28-7.41 (m, 4H, ArCH + NH), 7.51-7.52 (d, J = 7.01 Hz, 1H,
1
3
ArCH), 7.61 (m, 3H, ArCH ), 7.92 (s, 1H, ArCH), 8.55-8.66 (m, 1H, ArCH); C NMR (125 MHz,
CDCl ) δ 28.0 (-ve, CH ), 28.3 (+ve, CH ), 49.7 (-ve, CH ), 52.4 (+ve, OCH ), 53.6 (+ve, CH),
3
2
3
2
3
6
4.1 (-ve, CH ), 68.6 (-ve, CH ), 80.1 (C(CH ) ). 115.2 (ab, ArC), 117.2 (+ve, ArCH), 119.1 (+ve,
2 2 3 3
ArCH), 123.4 (+ve, ArCH) , 124.80 (+ve, ArCH), 126.1 (+ve, ArCH), 131.7(ab, ArC), 132.17 (ab,
ArC), 135.4 (ab, ArC), 155 .0 (C=O), 164.5 (ab, C), 166.9 (C=O), 170.4 (ab, C), 170.6 (ab, C),
2
7

+
1
72.4 (C=O). HRMS (microTOF-QII, MS, ESI): Calcd for C H N O Cl ([M+Na] ) 644.1995,
30 32 7 6
found 644.1979.
Methyl
1-(4-chloro-6-((4-(3-oxomorpholino)phenyl)amino)-1,3,5-triazin-2-yl)-L-
tryptophanate (7). Compound 7 was synthesized according to general procedure F using
o
compound 6. White solid, yield 80%, mp 154 C (dec). IR (ATR): 3265, 2959, 2363, 2109, 1886,
-1
25
o
1
1
744, 1669, 1550, 1446, 1341, 1237, 1118, 991, 797 cm . [α] = +2.5 (0.4, CH OH). H NMR
D 3
(
500 MHz, DMSO-d ) δ 3.34-3.46 (2H, CH , buried under the DMSO signal at 3.46, detected with
6 2
HSQC), 3.71-3.78 (m, 5H, CH + OCH ), 4.01 (m, 2H, CH ), 4.23 (s, 2H, CH ), 4.43 (m, 1H, CH),
2
3
2
2
7
8
.33 (m, 1H, ArCH), 7.45-7.49 (m, 2H, ArCH), 7.59 (m, 1H, ArCH), 7.69 (m, 2H, ArCH), 8.07-
1
3
.12 (m, 1H, ArCH), 8.43-8.49 (m, 2H, ArCH), 10.88 (br, 1H, NH); C NMR (125 MHz, DMSO-
d ) δ 26.0 (-ve, CH ), 49.5 (-ve, CH ), 52.2 (+ve, OCH ), 53.3 (+ve, CH), 64.0 (-ve, CH ), 68.2 (-
6
2
2
3
2
ve, CH ), 115.1 (ab, ArC), 117.2 (+ve, ArCH), 119.4 (+ve, ArCH), 122,2 (+ve, ArCH), 123.6 (+ve,
2
ArCH), 125.0 (+ve, ArCH), 125.4 (+ve, ArCH), 126.3 (+ve, ArCH), 131.1 (ab, ArC), 135.4 (ab,
ArC), 136.3 (ab, ArC), 164.2 (ab, C), 164.6 (ab, C), 165.2 (ab, C), 166.5 (C=O), 170.0 (C=O).
+
HRMS (microTOF-QII, MS, ESI): Calcd for C H N O Cl ([M+Na] ) 544.1471, found 544.1461.
2
5
24
7
4
Methyl
(S)-2-amino-3-(4-((4-chloro-6-(piperdin-1-yl)-1,3,5-triazin-2-yl)oxy)phenyl)
propanoate (8). Compound 8 was synthesized according to general procedure F using compound
1
7
3
5. Thick oil, yield 80%. IR (ATR): 3347, 3026, 2929, 2087, 1729, 1669, 1580, 1446, 1252, 1200,
-1
25
o
1
90, 536 cm . [α]
= +10.0 (0.4, CHCl ). H NMR (500 MHz, CDCl ) δ 1.56-1.66 (m, 6H,
3 3
D
×CH ), 3.35 (m, 2H, CH ), 3.65-3.79 (m, 7H, 2×CH + OCH ), 4.31 (m, 1H, CH), 7.10 (m, 2H,
2
2
2
3
1
3
ArCH), 7.30 (m, 2H, ArCH); C NMR (125 MHz, CDCl ) δ 24.3 (-ve, CH ), 25.6 (-ve, CH ),
3
2
2
2
5.7 (-ve, CH ), 35.6 (-ve, CH ), 45.0 (-ve, CH ), 45.2 (-ve, CH ), 53.3 (+ve, OCH ), 54.3 (+ve,
2 2 2 2 3
CH), 122.2 (+ve, ArCH), 130.7 (+ve, ArCH), 131.4 (ab, ArC), 151.4 (ab, ArC), 164.8 (ab, C),
2
8

1
69.2 (ab, C), 170.4 (ab, C), 170.9 (C=O). HRMS (microTOF-QII, MS, ESI): Calcd for
+
C H N O Cl ([M+H] ) 394.1484, found 392.1337.
1
8
22
5
3
Methyl (S)-2-amino-3-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)oxy)phenyl)propanoate
(9). Compound 9 was synthesized according to general procedure F using compound 16. White
o
solid, yield 78%, mp 142 C. IR (ATR): 3362, 3056, 2967, 2087, 1997, 1744, 1677, 1580, 1498,
-1
25
o
1
1
394, 1267, 1177, 1103, 1021, 790, 536, 484 cm . [α]
= -5.0 (0.4, CHCl ). H NMR (500
3
D
MHz, CDCl ) δ 3.30 (m, 2H, CH ), 3.68-3.72 (m, 9H, 3×CH + OCH ), 3.86 (m, 2H, CH ), 4.25
3
2
2
3
2
1
3
(
m, 1H, CH), 7.09 (m, 2H, ArCH), 7.26 (m, 2H, 2×ArCH); C NMR (125 MHz, CDCl ) δ 35.7 (-
3
ve, CH ), 44.1 (-ve, CH ), 44.3 (-ve, CH ), 53.1 (+ve, OCH ), 54.1 (+ve, CH), 66.3 (-ve, CH ),
2
2
2
3
2
6
6.4 (-ve, CH ), 122.1 (+ve, ArCH), 130.6 (+ve, ArCH), 131.5 (ab, ArC), 151.3 ( ab, ArC), 165.4
2
(ab, C), 169.3 (ab, C), 170.5 (ab, C), 171.2 (C=O). HRMS (microTOF-QII, MS, ESI): Calcd for
+
C H N O Cl ([M+H] ) 394.1277, found 394.1077.
1
7
20
5
4
Methyl 1-(4-chloro-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)-L-tryptophanate (10). Compound 10
was synthesized according to general procedure F using compound 17. Creamish solid, yield 77%,
o
mp 160 C (dec). IR (ATR) 2937, 2862, 2363, 2102, 1751, 1669, 1565, 1237, 1125, 984, 797, 514
-1 1
cm . H NMR (500 MHz, CDCl ) δ: 1.50-1.55 (m, 6H, 3CH ), 3.29 (m, 2H, CH ), 3.58 (s, 3H,
3
2
2
OCH ), 3.68-3.75 (m, 4H, 2CH ), 4.15 (m, 1H, CH), 7.10 (m, 1H, ArCH), 7.18 (m, 1H, ArCH),
3
2
1
3
7
2
5
.40 (m, 1H, ArCH), 7.98 (s, 1H, ArCH), 8.41 (m, 1H, ArCH). C NMR (125 MHz, CDCl ) δ:
3
4.3 (-ve, CH ), 25.5 (-ve, CH ), 25.6 (-ve, CH ), 26.1 (-ve, CH ), 44.8 (-ve, CH ), 45.0 (-ve, CH ),
2
2
2
2
2
2
2.8 (+ve, CH), 53.2 (+ve, OCH ), 113.5 (ab, ArC), 116.5 (+ve, ArCH), 118.7 (+ve, ArCH), 123.0
3
(+ve, ArCH), 124.4 (+ve, ArCH), 125.0 (+ve, ArCH), 130.5 (ab, ArC), 135.3 (ab, ArC), 161.8 (ab,
C), 162.4 (ab, C), 163.5 (ab, C), 169.6 (C=O). HRMS (microTOF-QII, MS, ESI): Calcd for
+
C H N O Cl ([M+H] ) 415.1644, found 415.1623.
2
0
23
6
2
2
9