Exploring the ability of dihydropyrimidine-5-carboxamide and 5-benzyl-2,4-diaminopyrimidine-based analogues for the selective inhibition of L. major dihydrofolate reductase

Article abstract of DOI:10.1016/j.ejmech.2020.112986

To tackle leishmaniasis, search for efficient therapeutic drug targets should be pursued. Dihydrofolate reductase (DHFR) is considered as a key target for the treatment of leishmaniasis. In current study, we are interested in the design and synthesis of selective antifolates targeting DHFR from L. major. We focused on the development of new antifolates based on 3,4-dihydropyrimidine-2-one and 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine motif. Structure activity relationship (SAR) studies were performed on 4-phenyl ring of dihydropyrimidine (26–30) template. While for 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine, the impact of different amino acids (valine, tryptophan, phenylalanine, and glutamic acid) and two carbon linkers were explored (52–59). The synthesized compounds were assayed against LmDHFR. Compound 59 with the IC₅₀ value of 0.10 μM appeared as potent inhibitors of L. major. Selectivity for parasite DHFR over human DHFR was also determined. Derivatives 55–59 demonstrated excellent selectivity for LmDHFR. Highest selectivity for LmDHFR was shown by compounds 56 (SI = 84.5) and 58 (SI = 87.5). Compounds Antileishmanial activity against L. major and L. donovani promastigotes was also performed. To explore the interaction pattern of the synthesized compounds with biological macromolecules, the docking studies were carried out against homology modelled LmDHFR and hDHFR targets.

Full text of DOI:10.1016/j.ejmech.2020.112986

Journal Pre-proof
Exploring the ability of dihydropyrimidine-5-carboxamide and 5-benzyl-2,4-
diaminopyrimidine-based analogues for the selective inhibition of L. major
Dihydrofolate reductase
Maria Bibi, Naveeda Akhter Qureshi, Abdul Sadiq, Umar Farooq, Abbas Hassan,
Nargis Shaheen, Irfa Asghar, Duaa Umer, Azmat Ullah, Farhan Ahmad, Muhammad
Salman, Ahtaram Bibi, Umer Rashid
PII:
S0223-5234(20)30958-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112986
EJMECH 112986
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2020
Revised Date: 27 October 2020
Accepted Date: 28 October 2020
Please cite this article as: M. Bibi, N.A. Qureshi, A. Sadiq, U. Farooq, A. Hassan, N. Shaheen, I. Asghar,
D. Umer, A. Ullah, F. Ahmad, M. Salman, A. Bibi, U. Rashid, Exploring the ability of dihydropyrimidine-5-
carboxamide and 5-benzyl-2,4-diaminopyrimidine-based analogues for the selective inhibition of L.
major Dihydrofolate reductase, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112986.
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1
2
3
4
Exploring the ability of dihydropyrimidine-5-
carboxamide and 5-benzyl-2,4-diaminopyrimidine-
based analogues for the selective inhibition of L. major
Dihydrofolate reductase
5
6
7
8
Maria Bibi 1, Naveeda Akhter Qureshi 2, Abdul Sadiq 3, Umar Farooq 1, Abbas
Hassan 4, Nargis Shaheen 2, Irfa Asghar 5, Duaa Umer 5, Azmat Ullah 1, Farhan
Ahmad 1, Muhammad Salman 6, Ahtaram Bibi 7, Umer Rashid 1, *
9
1 Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus 22060,
Pakistan
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11
2 Department of Animal Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
12
13
3 Department of Pharmacy, Faculty of Biological Sciences, University of Malakand, Chakdara,
18000 Dir (L), KP, Pakistan.
14
4 Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
15
16
5 Department of Biotechnology, COMSATS University Islamabad, Abbottabad Campus 22060,
Pakistan
17
6 Department of Microbiology, National Institute of Health (NIH), Islamabad 45320, Pakistan
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19
7 Department of Chemistry, Faculty of Physical Sciences, Kohat University of Science and
Technology, 26000 Kohat, KP, Pakistan
20
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22
Corresponding author. *E-mail: umerrashid@cuiatd.edu.pk; umer.rashid73@gmail.com (Umer
Rashid);
1

23 Abstract
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25
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32
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34
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37
38
39
To tackle leishmaniasis, search for efficient therapeutic drug targets should be pursued.
Dihydrofolate reductase (DHFR) is considered as a key target for the treatment of leishmaniasis.
In current study, we are interested in the design and synthesis of selective antifolates targeting
DHFR from L. major. We focused on the development of new antifolates based on 3,4-
dihydropyrimidine-2-one and 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine motif. Structure
activity relationship (SAR) studies were performed on 4-phenyl ring of dihydropyrimidine (26-
30) template. While for 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine, the impact of different
amino acids (valine, tryptophan, phenylalanine, and glutamic acid) and two carbon linkers were
explored (52-59). The synthesized compounds were assayed against LmDHFR. Compound 59
with the IC50 value of 0.10 μM appeared as potent inhibitors of L. major. Selectivity for parasite
DHFR over human DHFR was also determined. Derivatives 55-59 demonstrated excellent
selectivity for LmDHFR. Highest selectivity for LmDHFR was shown by compounds 56 (SI =
84.5) and 58 (SI = 87.5). Compounds Antileishmanial activity against L. major and L. donovani
promastigotes was also performed. To explore the interaction pattern of the synthesized
compounds with biological macromolecules, the docking studies were carried out against
homology modelled LmDHFR and hDHFR targets.
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42
Key words: Leishmania major DHFR, Human dihydrofolate reductase, Methotrexate mimics;
antileishmanials; dihydropyrimidine-5-carboxamides; benzyl-2,4-diaminopyrimidines
43
44
2

45 1. Introduction
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47
48
49
50
51
52
53
The diseases caused by protozoan parasites are considered neglected tropical diseases (NTDs).
These are the major health issues and causes of mortality and economic hardships predominantly
in developing countries. Among these NTDs, leishmaniasis is the second-highest incidence of
parasitic disease in the world, second only to malaria, with high mortality and morbidity. The
NTDs primarily affect the people living in poverty with inadequate sanitary conditions and close
contact with the infectious vectors. There are 20 types of the parasite of Leishmania which cause
Leishmaniasis belongs to genus Leishmania and family trypanosomatidae. Among these species,
L. donovani, L. tropica, L. Mexicana and L. braziliensis are major causes of Leishmaniasis [1-7].
54
55
56
57
58
59
60
61
62
63
64
65
66
The available drugs to treat leishmaniasis have poor clinical efficacy, toxicity, and resistance
problems. Therefore, there is a dire need to find new drugs to treat leishmaniasis. To tackle
leishmaniasis, a search for efficient therapeutic drug targets should be pursued. Dihydrofolate
reductase (DHFR) is a ubiquitous enzyme and is involved in the folate reduction pathway. Since
it is involved in generating raw material for the replication of DNA, it is a key therapeutic target
for the treatment of many human diseases such as bacterial, mycobacterial, fungal, protozoal
infections, rheumatoid arthritis, and cancer [8-9]. Dihydrofolate reductase (DHFR) is considered
as a key target for the treatment of leishmaniasis. In human beings, the activities of DHFR and
thymidylate synthase (TS) are well separated. However, in contrast to human cell, leishmania
have a bifunctional dihydrofolate reductase-thymidylate (DHFR-TS) [10-12]. Among diverse
bioactive moieties, methotrexate (MTX, 1) and trimethoprim (TMP, 2) are clinically used DHFR
inhibitors. However, these two inhibitors have shown poor selectivity for leishmania DHFR [13-
17].
3

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69
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71
72
73
74
75
76
77
78
Several studies have been reported on parasite DHFR with special emphasis on the selectivity
comparison with human enzyme. Cavazzuti et al., identified the inhibitors of folate-dependent
parasitic enzymes (PTR1 and DHFR-TS) via screening of folate-based library. Quinoxaline and
2,4-diaminopteridine derivatives showed selectivity and specificity against studied parasitic
enzymes compared with human enzymes. 2,4-diaminopteridine derivative (3, Figure 1) with Ki
= 4 μM against LmDHFR showed excellent selectivity (Ki for hDHFR = 10 μM). Identified
quinoxaline derivative (4) and amino quinoxaline (5) also showed selectivity towards LmDHFR.
However, derivative 6 showed selectivity toward human enzyme [18]. Hardy et al., tested a
library of 74 compounds against parasitic as well as hDHFR. Quinazoline derivative 7 showed
high activity and selectivity towards LmDHFR (IC50 for LmDHFR = 0.025 μM, for hDHFR =
0.040 μM). While pyrimidine derivative 8 showed selectivity towards hDHFR [19]. Chowdhury
et al., identified compound 9 as inhibitor of hDHFR [20].
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81
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83
84
85
86
87
Our research group have already reported C-5 and C-6 dihydropyrimidine (DHPM) derivatives
as potent antileishmanial agents [26]. Pyrazole derivative emerged as most potent antileishmanial
agent with IC50 value of 0.47 μg/mL (0.95 μM). The objective of the current research is to
design, synthesis, and evaluation of antifolates targeting Leishmania major dihydrofolate
reductase (LmDHFR). In current study, we synthesized 2,4-diaminopyrimidine and
dihydropyrimidine (DHPM) based methotrexate mimics and evaluate them for their potential
against L. major DHFR. Antileishmanial activity against promastigotes of L. major and L.
donovani was also performed. Interaction of the newly synthesized compounds with biological
targets was also explored.
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88
89
Figure 1. Potent anti-leishmanial agents reported recently
90 Results and discussion
91
2.1. Design strategy
92
93
94
95
96
97
98
Our design strategy of the current research is based on structural information of methotrexate.
The methotrexate structure consists of pteridine ring, p-aminobenzoic acid (PABA) and glutamic
acid (Figure 2b). A detailed SAR on 5-benzyl-2,4-diaminopyrimidines has already undertaken
by Chowdhury et al., and Gilbert et al., [11, 20]. We selected pyrimidine-2,4-diamine from
trimethoprim (5-benzyl-2,4-diaminopyrimidine derivative) and 3,4-dihydropyrimidine-2-one
scaffold for C-5 modifications. Apart from glutamate tail, 1-benzoylpiperidine-4-carboxylic acid,
and dimethyl 1-benzoylpiperidine-2,4-dicarboxylate mimics were also employed to explore their
5

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101
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103
104
105
106
effect on the selectivity between human and parasitic DHFR. In our current study, we designed
two strategies to explore the structure activity relationship (SAR) to synthesize more potent
antileishmanial drugs. For current study, we planned to synthesize variety of compounds
containing varying linker length. In strategy 1, SAR was carried out on 4-phenyl ring of the
DHPM-2-one core and glutamate core remains same. While in strategy 2, we planned SAR on 5-
(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine derivatives at amino acid point by using valine,
tryptophan, phenylalanine, and glutamic acid. A two-carbon linker was also attached to explore
the inhibition potential of these derivatives (Figure 3c).
107
108
Figure 2. Design strategy for current research
109
2.2. Chemistry
110
111
112
113
The general synthetic rout for the synthesis of compounds 26-30 is outlined in Scheme 1.
Compound 12 was synthesized in 51.3 % yield by refluxing a mixture of 4-aminobenzoic acid
(PABA, 11) and ethyl acetoacetate (10) in absolute ethanol. 2-oxo-3,4-dihydropyrimidine-5-
carboxylates (20-24) were synthesized by using our previously reported Biginelli protocol [21].
6

114
115
116
Acyl chloride derivatives of 20-24 were synthesized by the reaction with thionyl chloride in
dichloromethane (DCM). These acyl chloride derivatives were then reacted with glutamic acid
(25) to obtain target compounds (26-30).
O
O
O
O
O
O
OH
OH
Ethanol, Reflux
R
+
O
N
H2N
H
10
11
12
R
O
O
SnCl2.2H2O
ACN, Reflux
O
OH
+
12
H2N
NH2
+
HN
N
O
H
H
13-18
19
O
N
CH3
H
20-24
R
O
O
HO
O
O
HO
OH
NH2
i) SOCl2, DCM
ii)
N
O
25
H
HOOC
N
NH
25, NMM, DMF
H
R = H, 4-CH3, 4-OCH3, 4-NO2, 4-Cl
N
O
H
26-30
117
118
Scheme 1: Synthesis of dihydropyrimidine based methotrexate mimics
119
120
A multistep protocol for the synthesis of trimethoprim derivatives 56-63 is outlined in Schemes
2-4. Hydroxy trimethoprim derivative (31) was obtained by using our previously reported
method by using 48% HBr and trimethoprim (2) [22-24]. Compound 40 was then reacted with
1,2-dibromoethane (32) and bromoacetyl bromide (33) in DMF to afford 34-35 (Scheme 2).
Methyl esters (39-42) of amino acids (25, 36-38) were synthesized by reaction of amino acids by
drop-wise addition of thionyl chloride in methanol at 0 oC (Scheme 3).
121
122
123
124
125
7

NH2
N
NH2
N
OCH3
OCH3
OCH3
OH
N
48% HBr
N
Stir, 100 oC
3-4 Hr
H2N
H2N
OCH3
OCH3
2
31
X
Br
NH2
N
Br
OCH3
32-33
N
X = CH2 (32, 34)
X = C=O (33, 35)
X
O
Br
DMF,1 hr
H2N
OCH3
34, 35
126
127
Scheme 2: Synthesis of trimethoprim derivatives 34-35
128
O
O
R
R
SOCl2, CH3OH
Stirr,45 min
O
O
OH
OCH3
NH2
NH2
H3CO
OCH3
NH2
36-38
39-41
25
42
42
CH3
NH
R=
CH3
37, 40
38, 41
36, 39
129
130
Scheme 3: Esterification of amino acids
131
132
133
134
135
Boc-protection of 4-aminobenzoic acid (11) formed 43. Coupling of amino acid methyl esters
(39-42) yielded 44-47. Deprotection of Boc-amine using trifluoroacetic acid (TFA) and
subsequent reaction with 34-35 in DMF afforded 48-55. Free acids (56-63) were obtained by
alkaline hydrolysis of 48-55 (Scheme 2) [25-26].
136
8

COOH
COOH
i) SOCl2, DMF (1 drop),
Boc2O, 1M NaOH
1,4-dioxane
DCM, Reflux, 18 hr
Boc
H2N
N
ii) 39-41, Et3N, DCM
Stirr, 3-4 hr
H
12
43
NH2
O
O
OCH3
OCH3
N
N
H
i) TFA, DCM
N
H
R
N
H2N
O
Boc
H
N
ii) 34, Reflux, 16 hr
N
OCH3
R
H
44-47
48-51
O
O
OCH3
NH2
N
O
O
i) 1M NaOH / CH3OH
stirr, rt 8-14 h
H
N
H2N
N
H
O
N
R
ii) 1N HCl
52-55
O
O
OH
137
138
Scheme 4: Synthesis of trimethoprim-based methotrexate mimics with ethylene linker (52-55).
139
-
NH2
H3CO
NH2
N
R
NH2
N
OCH3
O
O
OCH3
O
N
H
N
N
39-42
R
Br
H2N
O
H2N
O
OCH3
i)DMF, K2CO3
Reflux, 3h
OCH3
O
OH
56-59
35
ii) 1M NaOH stirr
140
141
Scheme 5: Synthesis of trimethoprim-based methotrexate mimics with oxoethyl linker (56-59).
2.3. In vitro L. major dihydrofolate reductase (LmDHFR) inhibition
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The synthesized compounds were evaluated for enzyme inhibition assays against leishmania
major DHFR isolated at promastigote stage. The inhibitory potential of both series given as IC50
value is presented in Tables 1. From dihydropyrimidine series (26-30), the tested compounds 26,
28 and 29 showed excellent inhibition in submicromolar range with IC50 value of 0.38, 0.25 and
9

148
149
150
151
0.19
value in low micromolar range (0.21-0.81
excellent potency for LmDHFR. These determined IC50 values of these derivatives are from 0.10
M to 0.18 M.
μ
M, respectively. While the compounds 52-55 showed good LmDHFR inhibition with IC50
μM). Tested oxoethyl derivatives 56-59 showed
μ
μ
152
153
154
155
156
157
158
159
Subsequently, to demonstrate the selectivity for parasite enzyme over human enzyme, we
performed in-vitro inhibition studies on recombinant human DHFR (rhDHFR).
Dihydropyrimidine derivatives 26-30 showed good selectivity with the selectivity index in the
range of 2.2-3.2. PABA containing trimethoprim derivatives 52-55 showed poor selectivity for
LmDHFR. However, acetyl spacer containing trimethoprim derivatives 56-59 displayed
comparatively low affinity with rhDHFR and high selectivity for LmDHFR. Highest selectivity
was shown by compound 56 (SI = 84.5) and 58 (SI = 87.5). The selectivity index of compounds
57 and 59 is 10.2 and 16.3 respectively.
160
Table 1: Enzyme inhibition activities of the synthesized compounds
R
HO
O
O
N
H
O
HOOC
N
NH
H
N
O
H
LmDHFR IC50
M)
No.
R
h
DHFR IC50 (
μ
M)
SI
(
μ
H
0.84 + 0.55
ND
2.2
-
26
27
0.38 + 0.03
4-CH3
1.91 + 0.07
4-OCH3
1.63 + 0.11
ND
-
28
4-NO2
4-Cl
0.81 + 0.03
0.53 + 0.01
3.2
2.8
29
30
0.25 + 0.01
0.19 + 0.01
10

NH2
N
NH2
N
O
O
O
O
N
H
N
O
H
N
N
R
H2N
H
N
H2N
O
R
O
O
OH
O
56-59
52-55
O
OH
CH3
CH3
0.81 + 0.10
ND
0.04 + 0.00
0.05
52
0.03 + 0.00
-
NH
53
54
0.53 + 0.13
0.21 + 0.10
0.13 + 0.04
0.09 + 0.01
0.01 + 0.00
10.98 + 0.05
0.2
O
0.05
84.5
55
56
HO
CH3
CH3
NH
0.18 + 0.03
1.83 + 0.03
57
10.2
0.14 + 0.01
0.10 + 0.01
12.26 + 0.13
1.63 + 0.11
87.5
16.3
58
59
O
HO
Methotrexate
TMP
-
0.005
1.03
20.5
161
162
Selectivity index = IC50 ratio (hDHFR/lmDHFR)
11

163
2.4. In vitro antileishmanial assay against promastigotes of L. major and L. donovani
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169
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In-vitro antileishmanial activity was carried out against promastigotes of L. major and L.
donovani. The IC50 values of the antileishmanial activity of series 26-30 and 52-59 are enlisted in
Tables 2. The synthesized compounds (26-30) showed good to excellent antiparasitic activities.
The compounds were efficient against the two species of the parasite L. major and L. donovani
and except compound 28, all other showed inhibition in low micro-molar concertation. The
compound 27 showed IC50 values against L. major in 2.21+ 0.01 µM. This compound showed
IC50 values of 2.56 + 0.01 µM against L. donovani. Nitrophenyl derivative 29 showed excellent
antileishmanial activities with low IC50 values of 1.23 + 0.66 µM against L. major and 0.94 +
0.11 µM against L. donovani. Compound 30 also showed good IC50 values of 2.68 + 0.15 µM
against L. major and 3.01 + 0.09 µM against L. donovani.
174
175
176
177
178
179
180
181
The compounds having diaminopyrimidine (52-59) showed excellent activities against both the
tested parasitic species L. major and L. donovani. The compound 52 showed with low IC50
values of 0.67 + 0.01 µM against L. major and IC50 values of 0.73 + 0.01 µM against L.
donovani. The compound 53 showed with low IC50 values of 0.83 + 0.01 µM against L. major
and IC50 values of 1.36 + 0.06 µM against L. donovani. The compound 54 showed with low IC50
values of 0.98 + 0.02 µM against L. major and IC50 values of 0.95 + 0.04 µM against L.
donovani. The compound 55 showed with low IC50 values of 0.19+0.01 µM against L. major and
IC50 values of 0.11 + 0.01 µM against L. donovani.
182
183
184
However, compounds without PABA linker (56-59) were not able to show high potency
compared with X=CH2. Compound 59 from third series showed the activities against with IC50
values of 12.34 + 1.01 µM against the L. major and 8.94 + 0.83 µM against the L. donovani.
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186
Other compounds of the scheme showed larger inhibitory concentrations as compared to
compound 59.
187
Table 2: Results (μM ) in of in-vitro anti-leishmanial of compounds 26-29, 52-59.
Antileishmanial activity (Promastigotes)
IC50 ( M)
μ
No.
L. major
L. donovani
5.48 + 0.23·
2.21 + 0.12
15. 1 + 1.11
5.82 + 0.19
2.56 + 0.01
17.4 + 0.79
26
27
28
1.23 + 0.06
2.68 + 0.15
0.67 + 0.01
0.83 + 0.01
0.98 + 0.02
0.19 + 0.01
0.94 + 0.11
3.01 + 0.09
0.73 + 0.01
1.36 + 0.06
0.95 + 0.04
0.11 + 0.01
29
30
52
53
54
55
24.49 + 1.08
26.67 + 0.97
22.29 + 0.85
28.51 + 1.23
39.10 + 1.41
42.88 + 1.71
56
57
58
12.34 + 1.01
8.94 + 0.83
59
0.60 + 0.05
-
-
Amphotericin B
3.27 + 0.23
Sodium stibogluconate
188
189
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190
191
192
2.5. Docking study
193
194
195
196
197
198
199
200
201
202
203
Docking studies of the synthesized compounds were performed using Molecular Operating
Environment (MOE 2016.0802). We docked the synthesized compounds against leishmania
major (LmDHFR). As, the crystal structure of LmDHFR is not available, a three-dimensional
structure of bifunctional dihydrofolate reductase-thymidylate synthase (LmDHFR-TS) was
constructed by using Swiss-Model webserver (https://swissmodel.expasy.org/). Best model was
selected from five models and refinement of the constructed model (94.6 % amino acids were
positioned in favored regions, 4.1 % in allowed regions and 1.4 % in outlier region) was
validated by Ramachandran plot (Figure S-1). Moreover, we docked methotrexate into the
DHFR region. The amino acid residues present in the modelled LmDHFR active site were
compared with the literature [10-11, 27]. The ribbon diagram of homology modelled LmDHFR
with MTX is shown in Figure 3.
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205
Figure 3: MTX bound to homology modelled L. major DHFR.
206
207
208
209
210
211
212
213
214
215
After validation of docking accuracy (details in Supplementary material), we docked the tested
compounds into the active site of modeled LmDHFR. The 3-D interaction plot of compound 55,
59 and 29 are shown in Figure 5a-c. Compound 55 forms hydrogen bond interactions with
Ala32 and Thr83. Phe91 forms π-π stacking interactions with phenyl ring of trimethoprim. Ile45
also forms π-π T-shaped interactions. While Met53 involved in π-sulfur type of interactions to
stabilize the ligand-enzyme complex (Figure 5a). Ala32, Gly42 and Ser86 forms hydrogen bond
interactions with compound 59. Phe91 forms π-π stacking interaction with pyrimidine ring.
Met53 forms π-sulfur type of interactions. Val87 also forms π-σ type of interactions (Figure 5b).
Dihydropyrimidine analogue 29 forms hydrogen bond interactions with Val49, Thr83, Gln160
and Arg191. Ile45 forms π-alkyl interactions (Figure 5c).
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217
218
Figure 5. The 3-D interaction plot of compound (
LmDHFR
a) 55; (b) 59 and (c) 29 into the homology modelled
219 3. Conclusions
220
221
222
223
224
225
226
227
228
A series of new 13 compounds were designed and synthesized having dihydropyrimidine and 5-
(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine core. Our current research describes the synthesis
and biological evaluation of some structural mimics of methotrexate. The focus of the current
research was on the structural replacement of pteridine core of methotrexate with 5-benzyl-2,4-
diaminopyrimidine core (from trimethoprim) and dihydropyrimidine core and 5-
carboxamido)benzamido)pentanedioic
dihydropyrimidine derivatives (26-30), the structure activity relationship was performed on 4-
phenyl ring of the DHPM-2-one core. These derivatives (26-30) showed good selectivity SI =
acid
glutamate
core
remains
same..
For
(
2.2-3.2) for parasite enzyme (LmDHFR) over human enzyme. While, for 5-benzylpyrimidine-
16

229
230
231
232
233
234
235
236
237
238
2,4-diamine derivatives, the impact of different amino acids (valine, tryptophan, phenylalanine,
and glutamic acid) and two carbon linkers between 4-aminobenzoic acid and 5-(3,5-
dimethoxybenzyl)pyrimidine-2,4-diamine core were explored. Compounds 52-55 showed poor
selectivity for LmDHFR While compounds 56-59 have shown excellent inhibition potential and
selectivity for LmDHFR. The interaction pattern of the synthesized compounds homology
modelled LmDHFR and hDHFR was explored by using docking simulations. the interaction
pattern of the synthesized compounds against targets. In conclusion, dihydropyrimidine5-
carboxamides and 5-benzylpyrimidine-2,4-diamine are important scaffolds parasitic
dihydrofolate reductase inhibition and their further structural modification may lead to the
synthesis of more potent antileishmanials.
239
240
241
242 4. Material and methods
243
244
245
246
247
248
249
250
All the reagents and solvents were purchased from standard commercial vendors and were used
without any further purification. All the substituted aromatic aldehydes, ethyl acetoacetate, urea,
L-glutamic acid, PABA, 1,2-dibromoethane and bromoacetyl bromide were purchased from
Sigma Aldrich. Amino acids were purchased from Scharlau Labs. 1H and 13C-NMR spectra were
recorded in deuterated solvents on a Bruker spectrometer at 300 and 75 MHz respectively using
tetramethyl silane (TMS) as internal reference. Chemical shifts are given in δ scale (ppm). The
progress of all the reactions was monitored by TLC on 2.0 x 5.0 cm aluminum sheets pre-coated
with silica gel 60F254 with a layer thickness of 0.25 mm (Merck). LC-MS spectra were obtained
17

251
252
253
254
255
using Agilent technologies 1200 series high performance liquid chromatography comprising of
G1315 DAD (diode array detector) and ion trap LCMS G2445D SL. Elemental analyses were
conducted using Elemental Vario EI III CHN analyzer. Final products were checked for their
purity on Schimadzu HPLC system using C18 RP-column and isocratic solvent system
(mentioned in experimental) part at room temperature.
256
4.1. General method for the synthesis of 4-(3-oxobutanamido)benzoic acid (12)
257
258
259
260
261
262
263
Compound 12 was synthesized by refluxing a mixture of 4-aminobenzoic acid (20 mmol) and
ethyl acetoacetate (20 mmol) in absolute ethanol. After completion of reaction (TLC), the
reaction mixture was cooled and solid formed was filtered. The crude product was purified by
silica gel column chromatography using n-hexane : EtOAc (7:1). Yield = 51 %; 1H NMR = (300
MHz, DMSO - d
6
)
δH
(ppm): 12.33 (s, 1H, OH), 9.08 (s, 1H. NH), 8.11 (d, 2H, J = 8.1 Hz, ArH),
), 2.21 (s, 3H, CH3).
7.80 (d, 2H, J = 8.1 Hz, ArH), 3.32 (s, 2H, CH
2
264
4.2. General method for the synthesis of compounds (20-24)
265
266
267
268
269
A mixture of aromatic aldehydes (13-18, 10 mmol) and 4-(3-oxobutanamido)benzoic acid (12,
10 mmol) and urea (19, 10.2 mmol) stirred under reflux in acetonitrile using tin(II) chloride
dihydrate (SnCl2.2H2O). After the completion of the reaction (TLC), the mixture was poured into
ice. The resulting solid was washed with water and recrystallize with appropriate solvent to
furnish 20-24.
270
271
4.2.1. 4-(6-Methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamido)benzoic
acid (20)
18

1
272
273
274
275
276
White solid; R
f
= 0.41 (CH2Cl2 /CH3OH 5:1); Yield = 53 %; m.p. 212-214 ºC; H NMR = (300
MHz, DMSO - d
6
)
δH
(ppm): 12.35 (s, 1H, OH), 9.16 (s, 1H. NH), 8.40 (s, 1H, NH), 8.11 (d, 2H,
J = 7.8 Hz, Ar-H), 7.86 (d, 2H, J = 7.8 Hz, Ar-H), 7.69 (s, 1H, NH), 7.18-7.11 (m, 5H, ArH),
5.07 (d, 1H, J = 3.3, CH), 2.24 (s, 3H, CH3); LC-MS: m/z = 351.2 [M+H]+; Analysis calculated
for C19H17N3O4: C, 64.95; H, 4.88; N, 11.96; Observed;; C, 64.99; H, 4.86; N, 11.94.
277
278
4.2.2.
4-(6-methyl-2-oxo-4-p-tolyl-1,2,3,4-tetrahydropyrimidine-5-carboxamido)benzoic
acid (21)
1
279
280
281
282
283
284
White solid; Rf = 0.50 (CH2Cl2/ CH3OH 5:1); Yield = 55 %; m.p. 219-221ºC; H NMR= (300
MHz, DMSO- d6) δH (ppm): 12.37 (s, 1H, OH), 9.13 (s, 1H. NH), 8.42 (s, 1H, NH), 8.13 (d, 2H,
J = 8.1 Hz, Ar-H), 7.88 (d, 2H, J = 8.1 Hz, Ar-H), 7.65 (s, 1H, NH), 7.06. (d, 2H, J = 7.5 Hz, Ar-
H), 6.84 (d, 2H, J = 7.5 Hz, Ar-H), 5.05 (d, 1H, J = 3.3 Hz, CH), 2.43 ( s, 3H, CH3), 2.24 ( s, 3H,
CH3); LC-MS: m/z = 365.2 [M+H]+; Analysis calculated for C20H19N3O4: C, 65.74; H, 5.24; N,
11.50; Observed;; C, 64.71; H, 5.26; N, 11.51.
285
286
287
4.2.3. 4-(4-(4-Methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamido) benzoic acid (22)
1
288
289
290
291
292
293
Light yellow solid; R
(300 MHz, DMSO- d
f
= 0.51 (CH
2
Cl2
/CH
3
OH 5:1); Yield = 61 %; m.p. 231-233 ºC; H NMR=
6
)
δ (ppm): 12.39 (s, 1H, OH), 9.14 (s, 1H. NH), 8.44 (s, 1H, NH), 8.13 (d,
H
2H, J = 8.1 Hz, Ar-H), 7.87 (d, 2H, J = 8.1 Hz, Ar-H), 7.65 (s, 1H, NH), 7.14. (d, 2H, J = 7.8 Hz,
Ar-H), 6.80 (d, 2H, J = 7.8 Hz, Ar-H), 5.03 (d, 1H, J = 3.0 Hz, CH), 3.83 ( s, 3H, OCH3), 2.25 (
s, 3H, CH3); LC-MS: m/z = 381.13 [M+H]+; Analysis calculated for C20H19N3O5: C, 62.99; H,
5.02; N, 11.02; Observed;; C, 62.95; H, 5.04; N, 11.05.
19

294
295
4.2.4.
4-(6-Methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido)
benzoic acid (23)
1
296
297
298
299
300
301
Light yellow solid; R
f
= 0.54 (CH
2
Cl2
/CH OH 5:1); Yield = 48 %; m.p. 222-224 ºC; H NMR=
3
(300 MHz, DMSO- d
6
)
δH
(ppm): 12.38 (s, 1H, OH), 9.14 (s, 1H. NH), 8.41 (s, 1H, NH), 8.21 (d,
2H, J = 8.7 Hz, Ar-H), 8.12 (d, 2H, J = 8.4 Hz, Ar-H), 7.86 (d, 2H, J = 8.4 Hz, Ar-H), 7.64 (s,
1H, NH), 7.53 (d, 2H, J = 8.7 Hz, Ar-H), 5.07 (d, 1H, J = 3.0 Hz, CH), 2.25 (s, 3H, CH3); LC-
MS: m/z = 397.11 [M+H]+; Analysis calculated for C19H16N4O6: C, 57.58; H, 4.07; N, 14.14;
Observed;; C, 57.52; H, 4.09; N, 14.16.
302
303
4.2.5. 4-(4-(4-Chlorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido)
benzoic acid (24)
1
304
305
306
307
308
309
Lemon colored solid; R
f
= 0.56 (CH
2Cl2
/CH
3
OH 5:1); Yield = 60 %; m.p. 206-208 ºC ; H
NMR= (300 MHz, DMSO-d
6
)
δ (ppm): 12.35 (s, 1H, OH), 9.15 (s, 1H. NH), 8.41 (s, 1H, NH),
H
8.12 (d, 2H, J = 8.1 Hz, Ar-H), 7.94 (d, 2H, J = 8.1 Hz, Ar-H), 7.66 (s, 1H, NH), 7.16 (d, 2H, J =
8.4 Hz, Ar-H), 6.91 (d, 2H, J = 8.4 Hz, Ar-H), 5.06 (d, 1H, J = 2.7 Hz, CH), 2.23 ( s, 3H, CH3);
LC-MS: m/z = 385.2 [M+H]+ ; Analysis calculated for C19H16ClN3O4: C, 59.15; H, 4.18; N,
10.89; Observed; : C, 59.19; H, 4.15; N, 10.87;
310
4.3. General method for the synthesis of target compounds 26-30
311
312
313
314
To a solution of compounds 20-24 (5 mmol) in DCM, 1 drop of DMF was added followed by
freshly distilled thionyl chloride (5 mmol) was added dropwise. The solution was heated to
reflux. After the completion of reaction, solvent and excess thionyl chloride were removed by
distillation. The crude solid residues were used for further reactions.
20

315
316
317
318
The obtained acid chloride solid residues and L-glutamic acid (25) were dissolved in DMF in the
presence of N-methylmorpholine (NMM). The resulting solutions were heating to reflux. After
completion of reaction (consumption of starting material), the mixture was poured into ice. The
resulting solid was washed with water.
319
320
4.3.1. (4-(4-Methyl-2-oxo-6-phenylhexahydropyrimidine-5-carboxamido) benzoyl) glutamic
acid (26)
321
322
323
324
325
326
327
328
329
330
Yellow solid; R
f
= 0.41 (CH
2
Cl
2
/CH OH/GAA; 7 : 3 : 0.2); Yield = 47 %; m.p. 151-153 ºC;
3
HPLC purity = 98.8 % (C18 RP, 1% TFA in MeOH / H2O-92:8), TR = 8.2 min. 1H NMR = (300
MHz, DMSO - d
6
)
δH (ppm): 12.27 (s, 2H, 2 × COOH), 9.16 (s, 1H. NH), 8.43-8.37 (m, 2H,
CONH and CONHCH), 8.12 (d, 2H, J = 8.1 Hz), 7.74 (d, 2H, J = 8.1 Hz), 7.54 (s, 1H, NH),
7.13-7.01 (m, 5H, ArH), 5.11 (s, 1H, J = 3.0 Hz, CH ), 4.53-4.46 (m, 1H, CH), 2.40 (t, 2H, J =
13
6.9 Hz, CH
2
), 2.26 ( s, 3H, CH3), 2.16-1.99 (m, 2H, CH2); C-NMR, (75 MHz, DMSO-d6):
δC
(ppm); 17.4, 26.36 (2C), 30.44, 47.2, 54.2, 56.5, 57.1, 117.5 (2C), 121.6, 125.7, 128.3 (2C),
128.2, 129.9, 137.2, 141.4, 150.8 (2C), 167.1, 177.3 (2C), 178.3; LC-MS: m/z = 480.2 [M+H]+;
Analysis calculated for C24H24N4O7: C, 60.00; H, 5.03; N, 11.66; Observed; C, 59.77; H, 5.41;
N, 11.58
331
332
4.3.2. (4-(4-Methyl-2-oxo-6-(p-tolyl) hexahydropyrimidine-5-carboxamido) benzoyl)
glutamic acid (27)
333
334
335
336
337
338
Off-white solid; R
f
= 0.48 (CH
2
Cl
2
/CH OH/GAA; 7 : 3 : 0.2); Yield = 42 %; m.p. 163-165 ºC;
3
HPLC purity = 97.7 % (C18 RP, 1% TFA in MeOH / H2O-92:8),. TR = 10.6 min. 1H NMR=(300
MHz, DMSO- d
6
)
δ (ppm): 12.29 (s, 2H, 2 × COOH), 9.15 (s, 1H. NH), 8.42 (br s, 2H, CONH
H
and CONHCH), 8.10 (d, 2H, J = 8.1 Hz), 7.73 (d, 2H, J = 8.1 Hz), 7.56 (s, 1H, NH), 7.10 (d, 2H,
J = 7.5 Hz), 6.80 (d, 2H, J = 7.5 Hz), 5.13 (s, 1H, J = 3.3 Hz, CH ), 4.53-4.46 (m, 1H, CH), 2.39
(t, 2H, J = 6.9 Hz, CH
2
), 2.45 ( s, 3H, CH3), 2.25 ( s, 3H, CH3), 2.16-1.99 (m, 2H, CH2);
21

339
340
341
342
343
13CNMR, (75 MHz, DMSO-d
6
):
δ δC (ppm); 17.6,
(ppm); 13C-NMR, (75 MHz, DMSO-d6):
C
27.35 (3C), 31.41, 47.8, 54.6, 56.8, 58.1, 117.9 (2C), 122.4, 125.5, 124.5 (2C), 128.7, 129.8,
137.7, 142.3, 151.7 (2C), 167.9, 177.6 (2C), 179.6. LC-MS analysis: m/z = 494.180[M+H]+;
Analysis calculated for C25H26N4O7: C, 60.72; H, 5.30; N, 11.33 Observed; C, 60.52; H, 5.66; N,
11.32
344
345
4.3.3. (4-(4-(4-Methoxyphenyl)-6-methyl-2-oxohexahydropyrimidine-5-carboxamido)
benzoyl) glutamic acid (28)
346
347
348
349
350
351
352
353
354
355
Light yellow solid; R
f
= 0.43 (CH
2
Cl
2
/CH
3
OH/GAA; 7 : 3 : 0.2); Yield = 47 %; m.p. 184-186
1
ºC; HPLC purity = 98.0 % (C18 RP, 1% TFA in MeOH / H2O-92:8). TR = 9.5 min. H NMR=
(300 MHz, DMSO- d
6
)
δH (ppm): 12.28 (s, 2H, 2 × COOH), 9.15 (s, 1H. NH), 8.40 (br s, 2H,
CONH and CONHCH), 8.11 (d, 2H, J = 8.1 Hz), 7.74 (d, 2H, J = 8.1 Hz), 7.55 (s, 1H, NH), 7.16
(d, 2H, J = 7.8 Hz), 6.78 (d, 2H, J = 7.5 Hz), 5.11 (s, 1H, J = 3.0 Hz, CH ), 4.52-4.45 (m, 1H,
CH), 2.40 (t, 2H, J = 6.6 Hz, CH
2
), 2.84 ( s, 3H, OCH3), 2.24 ( s, 3H, CH3), 2.15-1.99 (m, 2H,
C (ppm): 18.2, 28.3 (3C), 3.4, 48.3, 54.9, 56.9, 58.7,
13
CH2); C-NMR, (75 MHz, DMSO-d6):
δ
117.9 (2C), 123.3, 125.3, 124.6(2C), 128.9, 129.6, 137.3, 142.3, 151.9 (2C), 168.1, 178.3 (2C),
179.9; LC-MS: m/z = 510.175 [M+H]+; Analysis calculated for C25H26N4O8 ;C, 58.82: H, 5.13;
N, 10.98; Observed; C, 58.63: H, 5.53; N, 10.96
356
357
4.3.4. (4-(4-Methyl-6-(4-nitrophenyl)-2-oxo-hexahydropyrimidine-5-carboxamido) benzoyl)
glutamic acid (29)
358
359
360
Light brown solid; R
f
= 0.40 (CH
2
Cl
2
/CH
3
OH/GAA; 7 : 3 : 0.2); Yield = 41 %; m.p. 144-146 ºC;
1
HPLC purity = 97.3% (C18 RP, 1% TFA in MeOH / H2O-92:8), TR = 8.2 min. H NMR= (300
MHz, DMSO- d
6
)
δH
(ppm): 12.30 (s, 2H, 2 × COOH), 9.14 (s, 1H. NH), 8.49 (d, 1H, J = 7.5 Hz.
22

361
362
363
364
365
366
367
CONH), 8.36 (br s, 1H, CONHCH), 8.23 (d, 2H, J = 8.7 Hz), 8.15 (d, 2H, J = 8.1 Hz), 7.76 (d,
2H, J = 8.1 Hz), 7.59 (s, 1H, NH), 7.48 (d, 2H, J = 8.7 Hz), 5.15 (s, 1H, J = 3.3 Hz, CH ), 4.52-
4.45 (m, 1H, CH), 2.40 (t, 2H, J = 6.6 Hz, CH
2
), 2.25 ( s, 3H, CH3), 2.15-1.99 (m, 2H, CH2);
13C-NMR, (75MHz, DMSO-d
6
):
δC (ppm): 18.5, 28.5 (3C), 30.5, 48.5, 54.6, 57.3, 59.5, 118.5
(2C), 123.5, 125.6, 124.7(2C), 127.9, 130.6, 137.5, 142.5, 152.5 (2C), 168.5, 178.7 (2C), 180.9;
LC-MS: m/z = 525.150 [M+H] ; Analysis calculated for C24H23N5O9 ;C, 54.86: H, 4.41; N,
+
13.33; Observed; C, 58.64: H, 5.53; N, 10.96.
368
369
4.3.5. (4-(4-(4-Chlorophenyl)-6-methyl-2-oxohexahydropyrimidine-5-carboxamido)
benzoyl) glutamic acid (30)
370
371
372
373
374
375
376
377
378
379
Lemon colored solid; R
f
= 0.48 (CH
2
Cl
2
/CH
3
OH/GAA; 7 : 3 : 0.2); Yield = 50 %; m.p. 156-158
ºC ; HPLC purity = 100.0 %. (C18 RP, 1% TFA in MeOH / H2O-92:8), TR = 11.9 min. 1H NMR=
(300 MHz, DMSO-d
6
)
δ (ppm): 12.27 (s, 2H, 2 × COOH), 9.16 (s, 1H. NH), 8.43 (br s, 2H,
H
CONH and CONHCH), 8.12 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.4 Hz), 7.54 (s, 1H, NH), 7.20
(d, 2H, J = 8.1 Hz), 6.99 (d, 2H, J = 8.1 Hz), 5.10 (s, 1H, J = 3.3 Hz, CH ), 4.51-4.46 (m, 1H,
CH), 2.41 (t, 2H, J = 7.2 Hz, CH
2
), 2.25 ( s, 3H, CH3), 2.17-1.99 (m, 2H, CH2); 13C-NMR, (75
MHz, DMSO-d6): C (ppm): 18.7, 28.5 (3C), 30.7, 48.8, 54.9, 57.8, 59.7, 118.9 (2C), 123.9,
δ
125.8, 124.3(2C), 128.2, 130.8, 137.7, 142., 152.9 (2C), 169.2, 178.9 (2C), 180.1 LC-MS
analysis: m/z= 515.14 [M+H]+; Theoretical perusal Calculated for : C24H23ClN4O7 ; C, 55.98 ; H,
4.50 ; N, 10.88 ; Observed; ; C, 55.72 ; H, 4.89 ; N, 10.90
380 4.4. General method for the synthesis of hydroxy-trimethoprim (31)
23

381
382
383
Hydroxy trimethoprim derivative 31 was obtained by using our previously reported procedure.
Yield = 52 %; m.p. 282-285ºC ; 1H NMR= (300 MHz, DMSO-d
): δ (ppm): 8.53 (s, 1H), 7.87 (s,
6
2H), 7.66 (s, 1H), 7.38 (s, 2H), 6.69 (s, 2H), 3.73 (s, 6H), 3.53 (s, 2H).
384
385 4.5. General method for the synthesis of trimethoprim derivatives 34-35
386
387
388
389
390
391
392
To a stirred solution of hydroxy-TMP (10 mmol) and potassium carbonate in DMF, 1,2-
dibromoethane (32) / bromoacetyl bromide (33) were added dropwise at room temperature. The
o
solution was stirred at 70-80 C. After completion of the reaction, the solvent was evaporated
under vacuum and the residue was dissolved in ethyl acetate. The organic layer was washed with
sodium bicarbonate and brine. The organic layer was dried over anhydrous magnesium
hydroxide. Finally, the solvent was evaporated under vacuum. The crude solid was purified using
silica gel column chromatography using n-hexane / EtOAc (7:1).
393
4.5.1. 5-(4-(2-Bromoethoxy)-3,5-dimethoxybenzyl)pyrimidine-2,4-diamine (34)
394
395
396
397
Yield = 56 %; 1H NMR= (300 MHz, DMSO- d6) δH (ppm): 7.79 (s, 1H), 7.67 (s, 2H, NH2), 7.35
(s, 2H, NH2), 6.70 (s, 2H), 4.82 (t, 2H, J = 6.9 Hz), 3.92 (t, 2H, J = 6.9 Hz), 3.73 (s, 6H, OCH3),
3.53 (s, 2H). LC-MS: m/z = 383.1 [M+H]+; Analysis calculated for C15H19BrN4O3: C, 47.01; H,
5.00; N, 14.62; Observed; C, 47.05; H, 4.09; N, 14.61.
398
4.5.2. 4-((2,4-Diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenyl 2-bromoacetate (35)
399
400
Yield = 62 %; 1H NMR= (300 MHz, DMSO-d6) δH (ppm): 7.78 (s, 1H), 7.66 (s, 2H, NH2), 7.34
(s, 2H, NH2), 6.72 (s, 2H), 4.55 (s, 2H), 3.76 (s, 6H, OCH3), 3.54 (s, 2H). LC-MS: m/z =
24

401
402
396.043 [M+H]+ ; Analysis calculated for C15H17BrN4O4: C, 45.36; H, 4.31; N, 14.10; Observed;
C, 45.39; H, 4.29; N, 14.08.
403 4.6. General method for the synthesis of amino acid esters (39-42)
404
405
406
407
408
The amino acids (25, 36-38) (20.0 mmol) were taken in a round bottom flask in methanol under
N2, thionyl chloride (22 mmol), 44 mmol in the case of glutamic acid (25), was added dropwise
to the mixture. The mixture was stirred for 2 hours. The reaction mixture was concentrated under
vacuum. To the concentrated mixture concentrated NaHCO3 solution was added until pH is 8-9.
The organic layer was extracted, and amino esters (39-42) were obtained.
409 4.7. General method for the synthesis of NH-Boc protected compounds 44-47
410
411
412
413
In a round bottom flask 4-amino benzoic acid (12, 20 mmol), was taken in 1, 4-dioxane (25 mL)
o
and 1M NaOH (1 mL) was added. The mixture was stirred at 0 C. To the solution, Boc2O (1.2
eq, 24 mmol) was added and mixture was stirred at low temperature than at room temperature for
4 hours. The precipitates of compound 43 was obtained.
414
415
416
417
In a next step, thionyl chloride (2.5 eq.) was added dropwise to the solution of compound 43 (10
mmol) in DCM (25 mL) and 1 drop of DMF and refluxed for 18 hours under nitrogen. After
completion of reaction, the solvent was evaporated under vacuum and acyl chloride derivative
was obtained. The crude product was used without further purification in the next reactions.
418
419
420
421
422
The synthesized acyl chloride derivative and amino acid esters 39-42 (5.0 mmol) were added in
DCM (10 mL) as solvent. To the mixture triethylamine base (3 eq) was added. The mixture was
stirred for 3-4 hours. After the completion of the reaction the mixture diluted with DCM and
added 1M HCl. The mixture was extracted. The organic layer was proceeded under vacuum
evaporation. The crystals of compounds (44-47) were obtained.
25

423
424
Methyl 2-(4-(tert-butoxycarbonyl) benzamido)-3-methylbutanoate (44)
1H NMR= (300 MHz, DMSO- d6) δH (ppm): 8.30 (s, 1H, CONH), 8.14 (s, 1H, NH-Boc), 8.01 (d,
425
426
427
2H, J = 8.4 Hz), 7.76 (d, 2H, J = 8.4 Hz), 4.88 (d, 1H, J = 7.2 Hz), 3.65 (s, 3H, OCH3), 2.31 (m,
1H), 1.42 (s, 9H), 0.89 (s, 6H, 2CH3). LC-MS: m/z = 351.2 [M+H]+ ; Analysis calculated for
C18H26N2O5: C, 61.70; H, 7.48; N, 7.91; Observed; C, 47.05; H, 4.09; N, 14.61.
428
429
430
Methyl 2-(4-(tert-butoxycarbonyl)benzamido)-3-(3a,7a-dihydro-1H-indol-3-yl)propanoate
(45)
431
432
433
434
435
436
1H NMR= (300 MHz, DMSO- d6) δH (ppm): 10.56 (s, 1H, NH-indole), 8.43 (d, 1H, J = 7.2 Hz,
CONHCH), 8.16 (s, 1H, NH-Boc), 8.03 (d, 2H, J = 8.4 Hz), 7.75 (d, 2H, J = 8.4 Hz), 7.41 (m,
2H, Ar-H), 7.26 (s, 1H, Ar-H), 7.14 (m, 2H, Ar-H), 5.01 (dd, 1H, J = 10.2, 9.9 Hz, CH), 3.67 (s,
3H, OCH3), 3.41 (dd, 1H, J = 16.5, 10.5 Hz, CHH), 3.08 (dd, 1H, J = 16.8, 9.6 Hz, CHH), 1.44
(s, 9H). LC-MS: m/z = 439.21 [M+H]+ ; Analysis calculated for C24H29N3O5: C, 65.54; H, 6.65;
N, 9.56; Observed; : C, 6.52; H, 6.67; N, 9.52;
437
438
Methyl 2-(4-(tert-butoxycarbonyl)benzamido)-3-phenylpropanoate (46)
1H NMR= (300 MHz, DMSO- d6) δH (ppm): 8.33 (br s, 1H, CONHCH), 8.14 (s, 1H, NH-Boc),
439
440
441
442
8.01 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.4 Hz), 7.11 (m, 5H, Ar-H), 4.99 (dd, 1H, J = 12.3, 7.8
Hz, CH), 3.62 (s, 3H, OCH3), 4.77 (dd, 1H, J = 17.1, 12.3 Hz, CHH), 2.75 (dd, 1H, J = 17.1, 7.5
Hz, CHH), 1.42 (s, 9H, 3 × CH3). LC-MS: m/z = 398.4 [M+H]+; Analysis calculated for
C22H26N2O5: C, 66.32; H, 6.58; N, 7.03; Observed: C, 66.36; H, 6.56; N, 7.01.
443
Dimethyl 2-(4-(tert-butoxycarbonyl)benzamido)pentanedioate (47)
26

444
445
446
447
448
1H NMR= (300 MHz, DMSO- d6) δH (ppm): 8.32 (d, 1H, J = 7.5 Hz CONHCH), 8.12 (s, 1H,
NH-Boc), 7.97 (d, 2H, J = 8.1 Hz), 7.79 (d, 2H, J = 8.1 Hz), 4.52-4.42 (m, 1H, CH), 3.63 (s, 3H,
OCH3), 3.53 (s, 3H, OCH3), 2.41 (t, 2H, J = 6.6 Hz, CH2), 2.16-1.99 (m, 2H, CH), 1.45 (s, 9H, 3
× CH3). LC-MS: m/z = 394.17 [M+H]+ ; Analysis calculated for C19H26N2O7: C, 57.86; H, 6.64;
N, 7.10; Observed; C, 57.69; H, 4.07; N, 14.61;
449 4.8. General method for the synthesis of target compounds 52-55
450
451
452
453
For deprotection of amino group, compounds 44-47 were taken in a round bottom flask and 25
mL of DCM solvent (7 ml) was added. Then added trifluoroacetic acid (6 eq; 20.0 mmol) and
o
water in 1:1 ratio at 0 C. The reaction was stirred for 2 hours and after completion of the
reaction deprotected amino acid product was obtained under vacuum evaporation.
454
455
456
457
458
To a solution of compounds 34 in DCM added compounds 44-47 and mixture was refluxed for
16 hours. After the completion of the reaction the mixture was washed with water and ethyl
acetate. The mixture was washed and filtered, and precipitate was obtained under vacuum
evaporation to crude products. The crude mixture was purified using silica gel column
chromatography using n-hexane / EtOAc (1:1) to obtain compounds 48-51.
459
460
461
462
463
464
465
To a stirred solution of esters 48-51 (5 mmol) in methanol (20 mL), 4 mL of sodium hydroxide
(1M) was added and the reaction mixture was stirred over night at room temperature. Finally, the
mixture was neutralized using 1 M HCl solution and concentrated in vacuum. The solution was
further acidified to a pH of 2-3 with 1M HCl and resulting product was extracted with 3 x 15 mL
of ethyl acetate. Combined organic layer was washed with brine washed with brine (2 x 10 mL)
and dried over anhydrous MgSO4. The resulting precipitated carboxylic acids were collected by
filtration.
27

466
467
(4-((2-(4-((2,4-Diaminopyrimidine-5-yl)methyl)-2,6-dimethoxyphenoxy)ethyl)amino)
benzoyl)valine (52)
o
468
469
470
471
472
473
474
475
476
477
White solid; R
HPLC purity = 99.1 %. (C18 RP, 1% TFA in MeOH / H2O-90:10), TR = 16.5 min. H NMR =
(300 MHz, DMSO-d (ppm): 12.26 (s, 1H, OH-valine), 8.29 (s, 1H, -NH), 7.98 (s, 2H, NH2),
f
= 0.48 (CH
2
Cl2
/CH OH/GAA; 8 : 1 : 0.2); Yield = 45 %; m.p. 131-133 C;
3
1
6
)
δ
H
7.81 (d, 2H, J = 7.8 Hz), 7.68 (s, 1H), 7.42 (s, 2H, NH2), 6.87 (d, 2H, J = 7.8 Hz), 6.69 (s, 2H),
5.10 (d, 1H, J = 6.9 Hz), 4.88 (s, 1H, NH), 4.44 (t, 2H, J = 6.9 Hz), 3.80 (s, 6H), 3.63 (s, 2H),
13
3.42 (t, 2H, J = 6.9 Hz), 2.41-2.32 (m, 1H, CH), 0.99 (t, 6H, J = 6.9 Hz). C-NMR, (75 MHz,
DMSO-d6): δC (ppm): 18.5 (2c), 30.5, 36.6, 42.4, 56.3(2c), 62.86, 6., 101.8 (C), 105.3, 112.5
(2C), 122.6, 128.9, 130.3 (2C), 134.6, 150.9, 153.5 (2C), 161.3, 162.5, 167.7, 172.9. LC-MS
analysis: m/z = 538.25 [M+H] +; Analysis calculated for C27H34N6O6: C, 60.21; H, 6.36; N,
15.60; Observed; C, 60.29; H, 6.35; N, 15.57.
478
479
(4-((2-(4-((2, 4-Diaminopyrimidine-5-yl)methyl)-2,6dimethoxyphenoxy)ethyl) amino)
benzoyl) tryptophan (53)
o
480
481
482
483
484
485
486
487
488
Pale white solid; R
f
= 0.53 (CH
2
Cl2
/CH
3
OH/GAA; 8 : 1 : 0.2); Yield = 41 %; m.p. 163-165 C ;
1
HPLC purity = 99.4 % (C18 RP, 1% TFA in MeOH / H2O-90:10), TR = 17.3 min. H NMR =
(300 MHz, DMSO-d
6
)
δ (ppm): 12.54 (s, 1H, OH-tryp), 10.59 (s, 1H, NH-imd), 8.33 (d, 1H, J =
H
7.5, CONHCH), 7.96 (d, 2H, J = 8.4 Hz, Ar-H), 7.89 (br s, 2H, NH2), 7.68 (s, 1H, Ar-H), 7.41
(m, 2H, Ar-H), 7.34 (br s, 2H, NH2), 7.26 (s, 1H, Ar-H), 7.14 (s, 2H, Ar-H), 6.93 (d, 2H, J = 8.4
Hz, Ar-H), 6.67 (s, 2H, Ar-H), 5.00 (dd, 1H, J = 10.2, 9.9 Hz, CH), 4.87 (s, 1H, NH), 4.47 (t,
2H, J = 6.9 Hz), 3.83 (s, 6H, OCH3), 3.63 (s, 2H, CH2), 3.51 (t, 2H, J = 6.9 Hz), 3.36 (dd, 1H, J
= 16.5, 10.5 Hz, CHH), 3.08 (dd, 1H, J = 16.8, 9.6 Hz, CHH). 13C-NMR, (75 MHz, DMSO-d6):
δ
C (ppm): 25.8, 35.8, 42.7, 56.5 (2c), 59.3, 69.5, 98.4, 102.6 (2C), 105.4, 111., 112.69 (2C),
28