Amino acid conjugated antimicrobial drugs: Synthesis, lipophilicity- activity relationship, antibacterial and urease inhibition activity

Article abstract of DOI:10.1016/j.ejmech.2017.12.089

Present work describes the in vitro antibacterial evaluation of some new amino acid conjugated antimicrobial drugs. Structural modification was attempted on the three existing antimicrobial pharmaceuticals namely trimethoprim, metronidazole, isoniazid. Twenty one compounds from seven series of conjugates of these drugs were synthesized by coupling with some selected Boc-protected amino acids. The effect of structural features and lipophilicity on the antibacterial activity was investigated. The synthesized compounds were evaluated against five standard American type culture collection (ATCC) i.e. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi strains of bacteria. Our results identified a close relationship between the lipophilicity and the activity. Triazine skeleton proved beneficial for the increase in hydrophobicity and potency. Compounds with greater hydrophobicity have shown excellent activities against Gram-negative strains of bacteria than Gram-positive. 4-amino unsubstituted trimethoprim-triazine derivative 7b have shown superior activity with MIC = 3.4 μM (2 μg/mL) for S. aureus and 1.1 μM (0.66 μg/mL) for E. coli. The synthesized compounds were also evaluated for their urease inhibition study. Microbial urease from Bacillus pasteurii was chosen for this study. Triazine derivative 7a showed excellent inhibition with IC₅₀ = 6.23 ± 0.09 μM. Docking studies on the crystal structure of B. pasteurii urease (PDB ID 4UBP) were carried out.

Full text of DOI:10.1016/j.ejmech.2017.12.089

Accepted Manuscript
Amino acid conjugated antimicrobial drugs: Synthesis, lipophilicity- activity
relationship, antibacterial and urease inhibition activity
Atta Ullah, Fatima Iftikhar, Muhammad Arfan, Syeda Tayyaba Batool Kazmi,
Muhammad Naveed Anjum, Ihsan-ul Haq, Muhammad Ayaz, Sadia Farooq, Umer
Rashid
PII:
S0223-5234(17)31120-0
10.1016/j.ejmech.2017.12.089
EJMECH 10067
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 September 2017
Revised Date: 14 December 2017
Accepted Date: 27 December 2017
Please cite this article as: A. Ullah, F. Iftikhar, M. Arfan, S.T. Batool Kazmi, M.N. Anjum, I.-u. Haq, M.
Ayaz, S. Farooq, U. Rashid, Amino acid conjugated antimicrobial drugs: Synthesis, lipophilicity- activity
relationship, antibacterial and urease inhibition activity, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2017.12.089.
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ACCEPTED MANUSCRIPT
Amino Acid Conjugated Antimicrobial Drugs: Synthesis, Lipophilicity-
Activity Relationship, Antibacterial and Urease Inhibition Activity
Atta Ullah, Fatima Iftikhar, Muhammad Arfan, Syeda Tayyaba Batool Kazmi, Muhammad
Naveed Anjum, Ihsan-ul Haq, Muhammad Ayaz, Sadia Farooq, Umer Rashid
Graphical Abstract

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Amino Acid Conjugated Antimicrobial Drugs: Synthesis, Lipophilicity-
Activity Relationship, Antibacterial and Urease Inhibition Activity
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Atta Ullah 1, Fatima Iftikhar 1, Muhammad Arfan 2, Syeda Tayyaba Batool Kazmi 3, Muhammad
Naveed Anjum 4, Ihsan-ul Haq 3, Muhammad Ayaz 1, Sadia Farooq 1, Umer Rashid 1,5,*
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1 Department of Chemistry, Hazara University, Mansehra 21120, Pakistan
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2 Department of Chemistry, School of Natural Sciences (SNS), National University of Sciences and
Technology (NUST), H-12, Islamabad 44000, Pakistan
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3 Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320,
Pakistan
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4 Department of Applied Chemistry, Government College University Faisalabad 38000, Pakistan.
5 Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
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Corresponding author. *E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com (Umer Rashid);
Tel. Off.: +92 (992) 383591-6
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23 Abstract
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Present work describes the in vitro antibacterial evaluation of some new amino acid conjugated
antimicrobial drugs. Structural modification was attempted on the three existing antimicrobial
pharmaceuticals namely trimethoprim, metronidazole, isoniazid. Twenty one compounds from
seven series of conjugates of these drugs were synthesized by coupling with some selected Boc-
protected amino acids. The effect of structural features and lipophilicity on the antibacterial
activity was investigated. The synthesized compounds were evaluated against five standard
American type culture collection (ATCC) i.e. Staphylococcus aureus, Bacillus subtilis,
Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi strains of bacteria. Our results
identified a close relationship between the lipophilicity and the activity. Triazine skeleton proved
beneficial for the increase in hydrophobicity and potency. Compounds with greater
hydrophobicity have shown excellent activities against Gram-negative strains of bacteria than
Gram-positive. 4-amino unsubstituted trimethoprim-triazine derivative 7b have shown superior
activity with MIC = 3.4 µM (2 µg/mL) for S. aureus and 1.1 µM (0.66 µg/mL) for E. coli. The
synthesized compounds were also evaluated for their urease inhibition study. Microbial urease
from Bacillus pasteurii was chosen for this study. Triazine derivative 7a showed excellent
inhibition with IC50= 6.23 + 0.09 µM. Docking studies on the crystal structure of B. pasteurii
urease (PDB ID 4UBP) were carried out.
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Key words: Structural modification; amino acid conjugates; trimethoprim; metronidazole;
isoniazid, Lipophilicity-Activity Relationship
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45 Introduction
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An antimicrobial drug is a substance that acts against all kinds of the microbial organisms
including bacteria, fungi, protozoa and viruses. In the history of chemotherapy, antimicrobials
are most successful forms that have contributed a lot to control the human ailment. The modern
antibiotic era is associated with the idea of Paul Ehrlich’s “magic bullets” concept and with the
serendipitous discovery of penicillin by Alexander Fleming in 1929. The antibiotic discoveries in
this era has propelled the drug research strategies and thousands of antibiotics were discovered
and used in the clinical practices. In fact, the antibiotics used today were originated in this era of
drug discovery between 1950 and 1970. Indeed after this golden era, there is a considerable gap
in the identification of new classes of the compounds. Unfortunately, the discovery efforts were
declined due to emergence of antibiotic resistance of pathogens over the last three decades [1-4].
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To combat the wave of resistance, novel drug discovery approaches has been emerged. These
approaches are strengthened due to advancement in the high-throughput pharmacological
screening and synthesis of diverse chemical libraries, rational drug design due to improvement in
protein structure determination and revolution in computing. Today, drug discovery scientists are
concerned with the discovery of new synthetic compounds as well as the chemical modification
of existing pharmaceuticals [4].
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Bacterial ureases are the key determinants of pathogenic symptoms in animals as well as in
human beings [5]. Urease is recognized by researchers as basic cause of pathologies stimulated
by a well-known bacteria, Helicobacter pylori (HP). It permits HP to uphold at low pH all
through colonization, thus playing pivotal role in producing peptic as well as gastric ulcers. For
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the eradication of H. pylori, a combination of a proton pump inhibitor (e.g. omeprazole) and two
antibiotics (Clarithromycin plus amoxicillin or metronidazole) are used as first-line therapy [6].
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A very important approach in today’s drug discovery process is the growing popularity of the
amino acids or peptide based drugs as therapeutic agents. Amino acids are critical to life, and
have many functions in metabolism. Structurally, amino acid are an important organic compound
self-possessed of amine (-NH2) and carboxylic (-COOH) functional group along with side chain
particular to each amino acid [7]. The essential elements of an amino acid are carbon, hydrogen,
oxygen and nitrogen, though other elements are found in the side-chains of several amino acids.
Due to structural diversity, they provide a balance between hydrophobicity and hydrophilicity
which is necessary for the cell membrane permeability and solubility. There are many drug
discovery initiatives where amino acids/peptides are successfully conjugated with the
biologically active cores and shown stability, enhanced potency, selectivity, permeability,
solubility and reduced toxicity [8-17].
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From all the above facts, we anticipated that amino acid conjugated antibacterial drugs may
result in new leads possessing good pharmacological activities. We have already reported a
successful example of structural modification of trimethoprim (1, TMP, Figure 1) that was
resulted in enhanced antibacterial potential [5]. This time, we tried to synthesize its conjugates
with amino acids. The other two drugs we chose were isoniazid (2) and metronidazole (3).
Isoniazid (INH) which is a lead antibacterial drug compound use to treat active tuberculosis
(T.B) infections and other skin problem. It is also used to stop the growth of bacteria.
Metronidazole (3, MTZ) belongs to nitroimidazole class of antibiotics and is used to treat a wide
variety of bacterial infections and diseases. Inspired by the structural diversity, activity profile of
these three clinically available drugs, we hypothesized that the structural modification of these
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drugs may result in a new leads possessing good pharmacological activities. The amino acids
chosen are valine (4), tryptophan (5) and phenylalanine (6) due to their structural diversity.
Tryptophan, a derivative of alanine, have a bicyclic indole ring linked at β-carbon atom of the
side chain. Due to this indole ring, the amino acid has both polar and lipophilic properties and
thus interacts with hydrophobic bilayer and with polar head groups via amine function. In
contrast to tryptophan, valine is hydrophobic and non-polar amino acid with aliphatic isopropyl
group. While, phenylalanine contains hydrophobic phenyl ring at β-position. Taking into
consideration the aforesaid, we envisaged that the coupling of these drugs with N-protected
amino acids may resulted in enhanced efficacy.
a)
O
OH
O
H2N
O
O
N
NH2
O2N
N
H
N
N
CH3
N
N
(3)
NH2
(1)
(2)
b)
NH2
OH
O
OH
NH2
(4)
O
O
OH
N
H
NH2
(5)
(6)
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Figure 1: Structures of the starting materials used in current study. (a) Commercially available drugs (1-
3); (b) Some selected amino acids (4-6)
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104 2. Results and Discussion
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2.1. Design strategy based on structure-property relationship
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Options for the treatment of Gram-negative microorganisms such as E. coli, P. aeruginosa are
limited due to multi-drug resistant or focus of the research for the development of drugs to
combat Gram-positive bacteria [18]. The study design of our current research is based on
influence of the lipophilicities on the uptake of target compounds by Gram-positive and negative
bacteria. The main goal of this study is to design the antibacterials that can; 1) penetrate to the
site of drug action, (2) interact with molecular targets within bacterium. The difference in the
composition of the outer cell envelop of the Gram-positive and negative bacteria hampers the
uptake of certain compounds [19-20]. The relative hydrophobicity/hydrophilicity and molecular
weight has a certain role in their uptake. We employed lipophilicity-activity and structure
activity relationship (SAR) strategies. The overall strategy is shown in Figure 2. Our
computational analysis have shown that the logP values have been increased to some extent by
the formation of amide or ester of the drugs (Figure 2a). Trimethoprim has four nitrogen atoms
but also contains three methoxy groups and a bridged methylene. The calculated LogP value for
trimethoprim is 1.28, whereas, for isoniazid and metronidazole -0.71 and -0.15, respectively. To
get our goal, we considered the introduction of triazine nucleus between drugs and amino acid.
Our computed logP values suggests that the introduction of triazine skeleton resulted in stronger
hydrophobicity (Figure 2b). Encouraged with these computational data, we proceed for the
synthesis and in vitro antibacterial study against some selected strains.
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Figure 2: Overall strategy for the design of target antibacterials
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2.2. Chemistry
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Boc-protected amino acids (8-10) were obtained by the reaction of amino acids (4-6) with
commercially available di-tert-butylcarbonate (7, t-BuOCO)2O) in 10% NaOH in dioxane
(Scheme 1). The 1H NMR spectrum of all the Boc-protected amino acids showed a nine protons
(three methyl groups) singlet in the range of 1.48-1.49 ppm.
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The coupling of N-protected Boc-amino acids with the already existing antimicrobial drugs was
carried out by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). 1-Hydroxy-
benzotriazole (HOBt) was used to avoid racemization and triethylamine (Et3N) was used to
maintain the pH. To obtain the final products, Boc group was de-protected by using
trifluoroacetic acid (TFA, CF3COOH) in dichloromethane (DCM, 10 mL / g of the compound.
The coupling of trimethoprim (TMP, 1) and isoniazid (INH, 2) is shown in Schemes 1.
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The coupling of TMP (1) with Boc-protected amino acids (8-10) resulted in the formation of
amide at 4-position of the pyrimidine ring as major products (1a-c). The greater susceptibility of
4-amino group in 2,4-di amino pyrimidines is due to the fact that carbon bearing amino group at
2-position is directly adjacent to two N-atoms of the pyrimidine ring, i.e. under the effect of two
electron-withdrawing groups. While, in case of C-4, it experiences this effect from only one side.
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This is an evident from the shielded protons of 4-NH2 group in the H NMR spectrum of
trimethoprim as compared to the more de-shielded protons of amino group at 2-psoition. In the
1H NMR spectra of compounds 1a-c and 4a-c, broad singlets in the range of 7.97-8.03 were
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assigned to 4-NH2 protons. While, in the H NMR spectra of 4-amino unsubstituted compounds
7a-c, peaks at 7.42-7.46 were assigned to 2-NH2 protons. Furthermore, in trimethoprim, there is
an additional electron-donating group at C-5, which further enhances the nucleophilicity at 4-
position. Therefore, as per our expectations the 4-amino substituted products were found to be
the major product in every case. These results are also consistent with our previously reported
results [5].
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O
O
O
N
NH2
N
O
NH
b, d
R
NH2
O
O
R
a
R
1a-c
OH
Boc2O
+
OH
NH2
NHBoc
c, d
O
N
4-6
7
H
N
8-10
R
N
H
NH2
O
2a-c
R=
N
H
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Scheme 1: Synthesis of target compounds 1a-c and 2a-c. Reagents and conditions: a) Dioxane, NaOH;
b) TMP (1), HOBt, EDC, Et3N, CHCl3; c) INH (2), HOBt, EDC, Et3N, CHCl3; d) CF3COOH, DCM
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Metronidazole is an important 5-nitroimidazole derivative. Mao et al reported the salicylic acid
derivatives by coupling with metronidazole showed promising inhibition of H. pylori urease
[21]. Keeping in view this activity, we decided to synthesize some derivatives by the reaction of
carboxylic hydroxyl group of N-protected amino acid and pendant hydroxyl group of
metronidazole. A two-step reaction involves first the formation of metronidazole tosylate (12) by
the reaction of metronidazole (3) with p-toluenesulfonyl (11). Treatment of 12 with N-protected
amino acids (8-10) in DMF using K2CO3 as base followed by deprotection of N-Boc using
trifluoroacetic acid in DCM yield our desired products (3a-c) (Scheme 2).
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O
O S
OH
O
Cl
O
CH3
a
O2N
S
O2N
N
N
+
O
CH3
CH3
CH3
N
N
12
11
3
O
N
CH3
O S
O
R
OH
b, c
O
N
O
CH3
O2N
N
+
O2N
Boc
O
N
H
R
CH3
N
H2N
12
8-10
3a-c
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Scheme 2: Synthesis of target compounds 3a-c. Reagents and conditions: a) DCM, TEA; b) K2CO3,
DMF; c) CF3COOH, DCM
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Our computed logP values indicate the introduction of triazine nucleus enhances the
hydrophobicity. Hence in another attempt, we synthesized di-substituted triazine hybrids of all
the three studied drugs and amino acid. Triazine scaffolds play important role in exerting specific
biological effects due to favorable drug-target interactions [22]. Here, we tried commercially
available 2,4-dichloro-6-methoxy-1,3,5-triazine (13) to react with the drugs and N-protected
amino acids via two-step protocol followed by deprotection to produce hybrids (4a-c, 5a-c and
6a-c Scheme 3). In first substitution step, nucleophilic attack of the drugs was carried out by
o
stirring the reactants at 0 C to yield products 14-16 (Scheme 3). In second step same mixture
was refluxed with Boc-protected amino acids and finally deprotection step with TFA in DCM
yielded required products (Scheme 3).
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H2N
O
R
O
Cl
H
N
N
H
N
N
HN
HN
N
d,e
N
N
N
O
O
O
N
O
N
15
5a-c
b
NH2
N
N
NH2
O
O
N
O
N
O
N
N
N
O
O
O
N
O
d,e
O
HN
a
N
N
N
N
O
HN
N
O
Cl
Cl
Cl
13
14
NH2
4a-c
R
c
O
NO2
N
H3C
O
N
N
N
d,e
O
R
N
Cl
N
N
O
N
O
N
CH3
H2N
N
NO2
6a-c
O
16
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Scheme 3: Synthesis of target compounds 4a-c, 5a-c and 6a-c. Reagents and conditions: a) Compound 1,
K2CO3, THF, 0 oC, b) Compound 2, K2CO3, THF, 0 oC, c) Compound 3, K2CO3, THF, 0 oC, d)
Compounds 8-10, K2CO3, THF, reflux, e) CF3COOH, DCM
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As discussed in our previous report, unsubstituted 4-NH2 is necessary for the target enzyme
inhibition [5]. In current study, we have observed some inhibition and moderate activity from our
synthesized products in Scheme 1 (1a-c) and Scheme 3 (4a-c). We again here tried the
substitution at trimethoxy ring. Compounds 7a-c were synthesized by hydrolyzing the methoxy
group using 48% HBr to obtain 17 (Scheme 4). Further two step protocol was employed as in
schemes 3 to obtain triazine derivatives 7a-c (Scheme 4).
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NH2
N
NH2
O
O
O
N
a
N
H2N
H2N
N
OH
O
N
O
17
1
NH2
N
NH2
O
N
O
O
O
O
N
b, c
N
N
O
N
N
+
H2N
OH
H2N
N
O
Cl
N
Cl
O
O
NH2
7a-c
17
12
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Scheme 4: Synthesis of target compounds 7a-c. Reagents and conditions: a) 48% HBr; b) Compounds
8-10, K2CO3, THF, reflux, e) CF3COOH, DCM
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2.3. In vitro antibacterial studies
Targeted conjugates were evaluated for their antibacterial activity against two Gram-positive i.e.
S. aureus (ATCC- 6538), B. subtilis (ATCC-6633) and three Gram-negative i.e. E. coli (ATCC-
25922), S. typhi (ATCC-14028) and P. aeruginosa (ATCC-15442) strains of bacteria. Minimum
inhibitory concentration (MIC) values of these compounds were calculated using standard
methods. Antibacterial activity of the synthesized compounds was compared with trimethoprim,
ciprofloxacin and roxithromycin.
2.3.1. Antibacterial potency of trimethoprim derivatives
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Bioassay data of synthesized trimethoprim derivatives 1a-c, 4a-c and 7a-c is shown in Table 1.
It revealed that among the direct conjugates, conjugates of the trimethoprim 1a-c and triazine
derivatives (4a-c) where 4-NH2 is blocked showed moderate to poor activity. These results are
contrary to our previously reported results where the substitution at 4-NH2 group resulted in the
complete loss in antibacterial activity [5]. Compound 1a has shown some activity against Gram-
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positive bacteria (MIC= 82 µM for S. aureus and 68.3 µM for B. subtilis). Conjugation with
tryptophan and phenylalanine increased the inhibitory potency against Gram-negative bacteria
and decrease the anti-Gram-positive activity. On the other hand, triazine derivatives 4a-c anti-
Gram-negative inhibition is dominant. 4c showed good inhibition against P. aeruginosa with
MIC value of 28.4 µM (16 µg/mL). This results showed that there is no permeability problem for
the compounds. The moderate activity may be due to blocked 4-NH2.
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To further study of SAR of trimethoprim derivatives, we proceed for our previously reported
strategy and trimethoxyphenyl ring was further used for investigations. Here, the effect of
unsubstituted 4-NH2 on antibacterial potency was explored. Excellent inhibition was observed
for all types of bacterial strains under study. Superior activity was shown by compound 7b not
only against Gram-negative bacteria but also for Gram-positive strains. The MIC for E. coli = 1.1
µM (0.66 µg/mL); for S. typhi 2.3 µM (1.33 µg/ mL) and for P. aeruginosa 1.4 µM (0.83
µg/mL). The MIC value for Gram-positive S. aureus was found 3.4 µM (2 µg/ml) and for B.
subtilis 6.8 µM (4 µg/mL). In the tested concentrations, MTZ and INH did not show any
inhibition. The results are shown in Table 1.
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Table 1: SAR and antibacterial activity of trimethoprim derivatives
NH2
O
N
O
O
N
NH2
NH2
N
O
N
O
O
N
O
N
O
O
O
N
N
O
N
N
N
N
O
O
HN
R
HN
O
R
O
H2N
O
NH2
H2N
NH2
4a-c
1a-c
7a-c
R
Antibacterial activity
MIC in µM (µg/mL) b
Compound
No.
R
LogP a
Gram Positive
Sa
Gram Negative
Bs
Ec
St
Pa
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82.2
(32)
68.3
(26.6)
218.9
(85.3)
136.9
(53.3)
1.30
2.23
164.4 (64)
67.1 (32)
1a
1b
134.3
(64)
223.7
(106.6)
55.8
(26.6)
67.1
(32)
N
H
243.8
(106.6)
243.8
(106.6)
97.4
(42.6)
73.1
(32)
2.14
2.68
73.1 (32)
62.2 (32)
1c
4a
165.7
(85.3)
124.4
(64)
82.8
(42.6)
51.7
(26.6)
212.8
(128)
212.8
(128)
53.2
(32)
3.23
53.2 (32) 53.2 (32)
4b
N
H
303.2
(170.6)
227.5
(128)
47.3
(26.6)
3.20
2.19
56.8 (32) 28.4 (16)
4c
7a
10.6
(5.3)
7.9
(4)
6.6
(3.3)
6.6 (3.3) 15.9 (8)
7b
1.98
2.64
3.4 (2)
6.8 (4)
1.1 (0.66) 2.3 (1.33) 1.4 (0.83)
N
H
19.3
(10.6)
7c
12 (6.6)
3.6 (2)
55.1 (16) NP d
30.2 (10) NP
0.6 (0.5) NP
7.3 (4)
6 (3.3)
NP
22.7
(6.6)
Trimethoprim
NP
30.2
(10)
30.2
(10)
Ciprofloxacin
Roxithromycin
30.2 (10)
0.6
(0.5)
0.3
(0.25)
0.6 (0.5)
Metronidazole
Isoniazid
NI e
NI
NI
NI
NI
NI
NI
NI
NI
NI
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a LogP values were calculated via VCCL online software; b The reported MIC values in µM. MIC value in µg/ml are shown in parenthesis.
These MIC values are an average of at least three individual measurements; c The values shown in bold are the better inhibitors than the standard
drugs used; d NP= Not performed; e NI= No inhibition shown in tested concentration; S. aureus (ATCC- 6538), B. subtilis (ATCC-6633), E. coli
(ATCC-25922), S. typhi (ATCC-14028) and P. aeruginosa (ATCC-15442).
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2.3.2. Antibacterial potency of isoniazid derivatives
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Except compound 2b, the SAR analogues having isoniazid (2a and 2c) were found inactive and
showed no inhibitory efficacy in the tested concentration domain. This inactivity is probably due
to cell impermeability. Tryptophan conjugate (2b) demonstrated some weak inhibition against
Gram-positive strains. However, addition of triazine skeleton (5a-c) was helpful for improving
activity. Compound 5a showed low MIC values for tested Gram-negative strains with MIC value
of 59 µM (21.3 µg/mL) against E. coli. Compound 5b exhibited low MIC values for both types
of strains. It showed MIC value of 47.5 µM (21.3 µg/mL) and 71 µM (32 µg/mL) against S.
aureus and B. subtilis. While, MIC values for E. coli
µM and 60 µM respectively (Table 2).
, S. typhi and P. aeruginosa are 36 µM, 36
234
Table 2: SAR and antibacterial activity of isoniazid derivatives
O
NH2
R
H
N
N
O
O
O
N
N
H
H
R
N
N
N
N
O
N
H
NH2
O
2a-c
5a-c
Antibacterial activity
MIC in µM (µg/mL) a
Compound
No.
R
LogP b
Gram Positive
Gram Negative
Sa
Bs
Ec
St
Pa
-0.41
0.64
0.05
NI
NI
NI
NI
NI
2a
2b
2c
98.9
(32)
98.9
(32)
527.7
(170.6)
792
(256)
660
(213.3)
N
H
NI
NI
NI
NI
NI
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117.8
(42.6)
177
(64)
59
(21.3)
74
(32)
74
(26.6)
1.58
2.23
2.50
5a
5b
47.5
(21.3)
71
(32)
36
(16)
36
(16)
60
(26.6)
N
H
104
(42.6)
156
(64)
52
(21.3)
78
(32)
65
(26.6)
5c
a
235
236
The reported MIC values in µM. MIC value in µg/ml are shown in parenthesis. These MIC values are an average of at least three individual
measurements;
237
2.3.3. Antibacterial potency of metronidazole derivatives
238
239
240
241
242
243
244
245
246
247
248
249
Metronidazole derivatives 3a-c demonstrated variable inhibitory efficacy against all the bacterial
strains. Compound 3a exhibited poor inhibition against Gram-negative strains, while compound
3b exhibited moderate anti-E. coli with MIC value of 74.4 µM (26.6 µg/mL). Compound 3c
showed good anti-S. aureus inhibitory potency with MIC value of 50.3 µM (16 µg/mL). Here,
again the addition of triazine skeleton proved beneficial for the increase in potency. Depending
upon the structural features, the target compounds 6a-c demonstrated weak to excellent
inhibitory potential. Compound 6a showed weak inhibition for both types of the tested strains.
Low MIC value was shown by compound 6b and 6c against both the tested strains. Compound
6b with MIC = 2 µM (1 µg/mL) against E. coli also inhibited S. typhi and P. aeruginosa with
MIC of 6.8 µM (3.3 µg/mL) and 4 µM (2 µg/mL) respectively. Excitingly, it also exhibited
remarkable activity against S. aureus and B. subtilis (MIC = 16.5 µM and 13 µM respectively)
superior to trimethoprim and ciprofloxacin.
250
251
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Table 3: SAR and antibacterial activity of metronidazole derivatives
NO2
N
CH3
N
O
NH2
R
N
O
N
O
N
N
O2N
CH3
O
N
R
O
H2N
O
3a-c
6a-c
Antibacterial activity
MIC in µM (µg/mL)a
Compound
No.
LogP
R
Gram Positive
Gram Negative
b
Sa
Bs
Ec
St
Pa
118.5
(32)
158
(42.6)
631.6
(170.6)
474
(128)
0.45
1.74
473 (128)
3a
3b
179
(64)
239
(85.3)
74.4
(26.6)
89.5
(32)
119.2
(42.6)
N
H
50.3
(16)
83.6
(26.6)
201
(64)
201
(64)
268
(85.3)
1.04
1.46
3c
6a
81
(32)
81
(32)
67.3
(26.6)
81
(32)
54
(21.3)
16.5
(8)
6.8
(3.3)
4.0
(2.0)
1.93
6b
13 (6.3) 2.0 (1.0)
N
H
24
(10.6)
18
(8)
9
(4)
2.13
18 (8) 7.4 (3.3)
6c
a
253
254
The reported MIC values in µM. MIC value in µg/ml are shown in parenthesis. These MIC values are an average of at least three individual
measurements;
255
256
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257
2.4. Lipophilicity-activity relationship
258
259
260
261
262
263
264
265
266
267
It has been reported that the main cause of the failure of the antibiotic drugs is lack of their
penetration due to a formidable barrier i.e. cell wall. As discussed the difference in the
composition of the outer cell envelop of the Gram-positive and negative bacteria hampers the
uptake of certain compounds. There are a number of examples in which the tested drug is
effective against Gram-negative bacteria but at the same time ineffective against Gram-positive
strain. A number of cheminformatics investigations have been performed on small to large data
bases to analyze the effect of physiochemical properties on the antibacterial activity [19, 23-24].
Amongst the physiochemical properties that determine the pharmacokinetic properties of the
drugs, hydrophobicity/hydrophilicity is important characteristic to improve the pharmacokinetic
properties and thus in turn penetration into the site of action.
268
269
270
271
272
273
274
275
276
277
278
In present research, we tried to investigate the influence of the lipophilicities on the uptake of
target compounds by Gram-positive and negative bacteria. The computed logP values are
presented in Tables 1-3. The logP values of all the synthesized compounds lies between -0.41 to
3.23. In general, it can be concluded that the antibacterial activities increases with increase in the
hydrophobicity. The logP values of trimethoprim derivatives 1a-c showed greater lipophilicity
and the logP values of 1b and 1c is 2.23 and 2.14, respectively. However, valine conjugate 1a
showed comparatively low logP value (1.30) and effective against Gram-positive strains. The
logP values of the triazine derivatives (4a-c and 7a-c) of trimethoprim followed the order of 4b >
4c > 4a > 7c > 7a > 7b. Among all the target compounds 4-NH2 substituted derivatives 4b and
4c showed highest values of logP. Compounds 7a-c with logP values in the range of 1.98-2.64
showed high inhibition potency for both tested strains.
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279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
Isoniazid derivatives (2a-c) possessed the lowest logP values and are strongly hydrophilic. This
hydrophilicity is likely to the reason of their inactivity due to cell impermeability. However, with
introduction of triazine nucleus (5a-c) their logP values increases in the range of 1.58-2.5. The
logP values of the metronidazole derivatives (3a-c and 6a-b) followed the order 6c > 6b > 3b >
6a > 3c > 3a. In comparison to first series of metronidazole derivatives (3a-c), triazine
derivatives (6a-c) showed better hydrophobicity and also bioactivity. Compound 6b and 6c
(logP= 1.93 and 2.13) inhibit both types of strains equally. In conclusion, triazine derivatives
have highest logP values. It can be seen from Figure 3 that for most active compounds, our
studied cell permeability parameter logP lies in the range of 1.98-2.68. The MIC values in this
range of LogP values lies between 0.6 – 10 µg/mL. These compounds have shown excellent
activities against Gram-negative strains of bacteria than Gram-positive. However in few cases,
compounds falling under the same range of logP have also shown excellent activity against
Gram-positive species (e.g. S. aureus). This high activity or in other words high cell permeability
may be due to other physiochemical parameters such as polar surface area or high molecular
weight.
294
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128
120
112
104
96
88
80
72
64
56
48
40
32
24
16
8
0
-1
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
LogP
295
296
297
298
Figure 3: Distribution of logP values in relationship with antibacterial activity. For most active
compounds, logP lies in the range of 1.98-2.68. Excellent activity= 0.6 – 10 g / mL; moderate activity=
11-32 g / mL; > 32 g / mL poor activity (Blue dot for S. aureus and red for E. coli.)
µ
µ
µ
299
2.4. In vitro urease inhibition activity against B. pasteurii
300
301
302
303
304
305
306
307
308
309
The structures of all ureases exhibit common similarities regardless of their source. Due to these
similarities, inhibition of one urease isozyme will likely consider as inhibition of other isozyme
[25]. Therefore, we carried out our studies on Bacillus pasteurii. The synthesized conjugates of
drugs were screened for their urease inhibition according to Weatherburn indophenols method by
the production of ammonia [26]. Trimethoprim derivatives (1a-c) displayed the IC50 value in the
range of 31.34-52.41 µM. While, isoniazid derivatives (2a-c) showed potency in the range of
21.11-48.6 µM. Metronidazole derivatives (3a-c) showed better inhibitor activity in the range of
13.61-29.53 µM. Metronidazole conjugate with tryptophan (3b) was emerged as the most potent
compound of this series with IC50 value of 13.61 µM. Derivatives 4a-c showed good urease
inhibition with IC50 value in the range of 11.97-19.29 µM. Isoniazid derivatives 5a-5c showed
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310
311
312
313
moderate inhibition of B. pasteurii urease. The metronidazole derivatives 6a-c exhibited good
inhibition wit IC50 value in the range of 11.43-19.51 µM. Excellent urease inhibition was
demonstrated by trimethoprim derivatives 7a-c. The IC50 value lies between 6.23-11.51 µM.
Compound 7a with IC50 of 6.23 + 0.09 µM emerged as the most potent compound.
314
Table 4: In vitro urease inhibition activity against B. pasteurii
Compound
No.
IC50 (µM± S.E.M) or %age
inhibition
Compound
No.
IC50 (µM± S.E.M) or %age
inhibition
52.41 + 0.42
35.10 + 0.39
31.34 + 0.88
48.6 + 1.02
29.31 + 0.89
37.29 + 0.28
30.98 + 1.09
19.51 + 0.65
16.32 + 0.69
11.43 + 0.33
6.23 + 0.09
11.51 + 0.17
8.33 + 0.11
13%
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
37.01 + 0.97
21.11 + 0.18
29.53 + 0.33
13.61 + 0.14 c
27.44 + 0.56
19.29 + 0.35
11.97 + 0.11
13.75 + 0.42
6b
6c
7a
7b
7c
Metronidazole
Thiourea
21+ 0.10
315
316
2.7. Docking studies on Bacillus pasteurii urease (BPU)
317
318
319
320
Docking study of the synthesized compounds was carried out by using AutoDock 4.2. Crystal
structure of B. pasteurii urease was retrieved from protein data bank (PDB ID 4UBP, 1.55 Å
resolution) with acetohydroxamic acid (HEA) as co-crystalized ligand [27]. The reliability of the
docking was checked by performing the re-docking of co-crystalized ligand acetohydroxamic
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321
322
acid (HEA). The root mean square deviation (RMSD) between co-crystallized and re-docked
conformation is 1.14 Å.
323
324
325
326
327
328
329
330
331
332
333
The bioactivity data presented in Table 4 indicated that among all the synthesized compounds
TMP-triazine derivatives (4a-c and 7a-c) showed good to excellent activities. The visual
inspection of the lowest energy docked pose of the TMP derivative compound 4b (IC50 = 11.97
+ 0.11 µM) showed that it coordinates with bi-nickel center (Ni798 and Ni799) via its 2,4-
diaminopyrimidine ring of TMP (Figure 4a). Carbamylated Lys490 (KCX490, a non-standard
bridging residue) forms hydrogen bond interaction with 2-NH2 group of pyrimidine ring. The
guanidinium group of Arg339 forms hydrogen bond donor interactions with one of the methoxy
oxygen of the TMP and also with nitrogen atom of triazine ring. Gly280 forms bifurcated
hydrogen bond interactions with both the amino groups of TMP pyrimidine ring. Cyst322 forms
a
π-sulphur interaction with its sulphur atom and
π-system of triazine ring. Imidazole ring of
His249 is observed to form a T-shaped interactions with pyrimidine ring of TMP (Figure 4b).
π-π
334
335
336
337
338
339
Figure 4: a) Depiction of the lowest energy docking-poses of the compound 4b into the binding site of
B. pasteurii urease (PDB ID 4UBP). A ribbon model of the enzyme is presented; (b) Close-up depiction
of the docking pose of compound 4b showing different types of ligand-enzyme interactions in the
binding site of 4UBP. The key residues are represented as green stick model. Ni atoms (798 and 799) are
shown in blue ball model.
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340
341
Compound 7a emerged as the most potent inhibitor of the BPU with IC50 value of 6.23 + 0.09
µM. The visual inspection of the lowest energy docked pose of the most active compound 7a
342
343
344
345
346
347
348
showed that it coordinates with bi-nickel center (Ni798 and Ni799) via its 2,4-diaminopyrimidine ring
of TMP (Figure 5a). A difference in the activity of 4b and 7a is the unsubstituted 4-NH2 on pyrimidine
ring. In 7a, stronger ligation with Ni bi-center and surrounding residues was observed than substituted on
(4b). 4-NH2 group is involved in the hydrogen bonding (HB) with Gly280 and Asp363. While, 2-NH2
group establishes HB interactions with His137 and His139. Ring nitrogen (N-1) forms HB with His249
and His275. His324 forms a HB interactions with carbonyl oxygen of valine. Apart from HB interactions,
many π-CH interactions also stabilize the ligand-enzyme complex (Figure 5b)
.
349
350
351
352
353
Figure 5: a) Depiction of the lowest energy docking-poses of the most active compound 7a into the
binding site of B. pasteurii urease (PDB ID 4UBP). A ribbon model of the enzyme is presented; (b)
Close-up depiction of the docking pose of compound 7a showing different types of ligand-enzyme
interactions
354 3. Conclusion
355
356
357
358
359
In summary, a series of some novel derivatives of three existing antimicrobial pharmaceuticals
namely trimethoprim, metronidazole and isoniazid were synthesized by coupling with some
selected Boc-protected amino acids. These derivatives were assayed for their antibacterial
activity against S. aureus, B. subtilis, E. coli, S. typhi and P. aeruginosa. The results of the study
revealed that the lipophilicity is an important physio-chemical characteristic for antibacterial
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360
361
362
363
364
365
activity. In general high inhibition was demonstrated by the compounds having logP lies in the
range of 1.98-2.68. With few exceptions, compounds with greater hydrophobicity are more
active against Gram-negative strains. Compounds 7a-c and 6b showed good activity against all
the tested strains. Urease inhibition activity against Bacillus pasteurii was also evaluated. These
findings suggest that these compounds are a good starting point for the rational development of
new antibacterials.
366 4. Material and methods
367
368
369
370
371
372
373
374
375
376
377
378
All the reagents and solvents were purchased from standard commercial vendors and were used
without any further purification. Amino acids, (BOC)2O and EDC were purchased from Merck.
Trimethoprim, metronidazole and isoniazid was taken from Islamabad Pharmaceutical Products,
1
Islamabad. H and 13C-NMR spectra were recorded in deuterated solvents on a Bruker
spectrometer at 300 and 75 MHz respectively using tetramethylsilane (TMS) as internal
reference. Chemical shifts are given in
δ scale (ppm). Melting points were determined in open
capillaries using Gallenkamp melting point apparatus (MP-D). 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 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 a LECO-
932 CHNS Analyzer (LECO Corporation, USA).
379
380
4.1. Synthesis of Boc-N-Protected Amino acids (8-10)
Amino group of amino acids (4-6) was protected with Boc group by following procedure:
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381
382
383
384
385
386
387
388
The solution of amino acid (4-6) in 75 mL of dioxane, 40 mL of H2O and 408 mL solution of IM
NaOH was stirred and cooled in an ice bath. (BOC)2O (9g, 41.14 mmol) was added to solution
and stirred for one hour at room temperature. The solution was concentrated under vacuum to
about 30-40 mL. Cooled in an ice water bath, covered with layer of ethyl acetate (25mL) and
acidified with dilute solution of KHSO4 to pH 2-3. The aqueous layer was extracted with ethyl
acetate and washed with distilled H2O, brine and dried over MgSO4. The solvent was removed
under reduced pressure and residue was chromatographed on a silica gel column using methanol
2 % solution in chloroform as an eluent to afford pure product in 82 % yield.
389
4.1.1. 2-(tert-butoxycarbonyl)-3-methylbutanoic acid (8)
1
390
391
White powder, H NMR (300 MHz, CDCl3):
δ
0.98 (d, 6H, J=6.9 Hz, CH3), 1.48 (s, 9H, 3 x
CH3), 2.41 (m, 1H, CH), 5.09 (d, 1H, CH), 7.95 (brs, 1H, NHBoc), 11.28 (br s, 1H, OH).
392
4.1.2. 2-(tert-butoxycarbonyl)-3-(1H-indol-3-yl)propanoic acid (9)
393
394
395
White powder, 1H NMR (300 MHz, CDCl3):
δ 1.49 (s, 9H, 3 x CH3), 3.43(m, 2H, CH2), 4.96 (t,
1H, =8.4 Hz,CH), 7.20 (m, 2H, Ar-H), 7.33 (s, 1H, Ar-H), 7.45 (m, 2H, Ar-H), 7.98 (brs, 1H,
J
NHBoc), 10.60 (brs, 1H, NH-indole), 11.30 (brs, 1H, OH).
396
4.1.3. 2-(tert-butoxycarbonyl)-3-phenylpropanoic acid (10)
1
397
398
White powder, H NMR (300 MHz, CDCl3):
δ
1.43 (s, 9H, 3 x CH3), 3.24 (dd, 1H, CH2), 3.73
(dd, 1H, CH2), 4.93 (dd, 1H, CH), 7.14 (m, 5H, Ar-H), 7.97 (br s, 1H, NH), 11.29 (brs, 1H, OH).
4.2. Procedure for the deprotection of Boc
399
400
401
A fully protected amino acid (0.86 g, 3.32 mmol) was dissolved by stirring in CH2C12 (7 mL)
then at 0o under N2, CF3COOH (8 equiv. 2.05mL, 26.56 mmol) was added drop wise by
25

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o
402
403
maintaining 0 C temp of ice bath. The mixture was allowed to stir at room temperature for 6
hours. The organic solvent was evaporated and the residue was dried under vacuum.
404
4.3. General procedure for the coupling of N-Boc amino acids and antimicrobial drugs
405
406
407
408
409
410
411
A mixture of Boc-protected amino acid (4 mmol) and HOBt (0.62 g, 4 mmol) in CHCl3 was
o
stirred and cooled to 0 C (ice bath). Then, antibacterial drugs (1-3) (4 mmol) triethylamine (4
mmol) and EDC (4 mmol) were added. The mixture was allowed to warm to room temperature
and stirring continued overnight. The mixture was diluted with CHCI3 and washed with 1 N HC1
(thoroughly), aq. sat. NaHCO3 and NaCl solution. The organic phase was dried MgSO4 and
evaporated. The crude residues were purified by column chromatography using n-hexane/ ethyl
acetate resulted in 71-80 % yield.
412
413
4.3.1. N-(5-(3,4,5-trimethoxybenzyl)-2-aminopyrimidin-4-yl)-2-amino-3-methylbutanamide
(1a)
414
415
416
417
418
419
420
421
422
1a was synthesized according to the general procedure by using N-Boc-valine (4 mmol) and
trimethoprim (4 mmol). Deprotection of N-Boc according to procedure in section 4.2 resulted in
the pure product 1a. White solid; Rf = 0.51 (CHCl3/MeOH 6:1); Yield 74%; mp. 183-185 ºC; 1H
NMR (300 MHz, DMSO-d6):
δ 0.98 (d, 6H, J=6.6 Hz, CH3), 2.42 (m, 1H, CH), 3.63 (s, 2H,
CH2), 3.81 (s, 9H, O-CH3), 5.09 (d, 1H,
J=6.6 Hz, CH), 6.59 (brs, 2H, NH2), 6.76 (s, 2H, Ar-H),
13
7.66 (s, 1H, Ar-H), 8.01 (brs, 2H, NH2), 8.73 (br s, 1H, NH); C-NMR (75 MHz, DMSO-d6):
18.3 (2C), 34.3, 36.4, 57.3 (3C), 63.1, 109.3 (2C), 111.4, 128.9, 140.2, 151.6 (2C), 156.0, 160.4,
162.6, 171.8.; LC-MS: m/z=390.2 [M+H]+; Anal. Calcd for C19H27N5O4: C, 58.60; H, 6.99; N,
17.98; Found: C, 58.63; H, 6.97; N, 17.95.
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424
4.3.2.
N-(5-(3,4,5-trimethoxybenzyl)-2-aminopyrimidin-4-yl)-2-amino-3-(1H-indol-3-
yl)propanamide (1b)
425
426
427
428
429
430
431
432
433
434
1b was synthesized according to the general procedure by using N-Boc tryptophan (4 mmol) and
trimethoprim (4 mmol). After deprotection pure product was obtained in 71% yield. White solid;
Rf = 0.54 (CHCl3/MeOH 4:1) ; mp. 216-218 ºC; 1H NMR (300 MHz, DMSO-d6):
CH2), 3.69 (s, 2H, CH2), 3.85 (s, 9H, 3 x OCH3), 4.96 (t, 1H, =8.4 Hz,CH), 6.59 (brs, 2H, NH2),
δ 3.43 (m, 2H,
J
6.77 (s, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 7.31 (s, 1H, Ar-H), 7.43 (m, 2H, Ar-H), 7.65 (s, 1H, Ar-
13
H
), 8.03 (brs, 2H, NH2), 8.75 (br s, 1H, NH), 10.63 (br s, 1H, NH-indole); C-NMR (75 MHz,
DMSO-d6): 32.4, 36.4, 56.8, 57.4 (3C), 109.1 (2C), 109.8, 111.0, 112.4, 117.7, 121.4 (2C),
122.0, 124.1, 126.6, 129.1, 134.2, 138.1, 152.5, 155.8, 160.5, 162.6, 172.0.; LC-MS: m/z= 477.2
[M+H]+; Anal. Calcd for C25H28N6O4: C, 63.01; H, 5.92; N, 17.64; Found: C, 63.03; H, 5.90; N,
17.62.
435
436
4.3.3.
N-(5-(3,4,5-trimethoxybenzyl)-2-aminopyrimidin-4-yl)-2-amino-3-phenyl
propanamide (1c)
1
437
438
439
440
441
442
443
White solid; Rf = 0.53 (CHCl3/MeOH 4:1); Yield 74%; mp. 203-205 ºC; H NMR (300 MHz,
DMSO-d6):
δ 3.42 (m, 2H, CH2), 3.68 (s, 2H, CH2), 3.81 (s, 9H, O-CH3), 4.97 (t, 1H, J=8.4 Hz,
CH), 6.69 (brs, 2H, NH2), 6.78 (s, 2H, Ar-H), 7.14 (m, 5H, Ar-H), 7.65 (s, 1H, Ar-H), 8.03 (brs,
2H, NH2), 8.76 (br s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6): 36.3, 38.4, 55.9 (3C), 57.3,
107.7, 109.2, 111.3, 124.9, 126.4, 129.1(2C), 130.7 137.4, 142.5, 151.5 (2C), 155.9 (2C), 160.4,
162.4, 172.0.; LC-MS: m/z= 438.2 [M+H]+; Anal. Calcd for C23H27N5O4: C, 63.14; H, 6.22; N,
16.01; Found: C, 63.12; H, 6.20; N, 16.03.
444
4.3.4. N'-(2-amino-3-methylbutanoyl)isonicotinohydrazide (2a)
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1
445
446
447
448
449
450
White solid; Rf = 0.57 (CHCl3/MeOH 4:1); Yield 79%; mp. 146-148 ºC; H NMR (300 MHz,
DMSO-d6): 0.98 (d, 6H, = 6.6 Hz, CH3), 2.40 (m, 1H, CH), 5.07 (d, 1H, = 6.6 Hz, CH),
6.61 (brs, 2H, NH2), 7.68 (d, 2H, = 6.0 Hz, Ar-H), 8.73 (d, 2H, = 6.0 Hz, Ar-H), 9.22 (br s,
δ
J
J
J
J
1H, NH), 9.64 (br s, 1H, NH); 13C-NMR (75 MHz, DMSO-d6): 18.4 (2C), 34.2, 62.9, 121.3,
122.5, 141.4, 147.6, 148.1, 162.4, 171.0.; LC-MS: m/z= 237.1 [M+H]+; Anal. Calcd for
C11H16N4O2: C, 55.92; H, 6.83; N, 23.71; Found: C, 55.98; H, 6.81; N, 23.69.
451
4.3.5. N'-(2-amino-3-(1H-indol-3-yl)propanoyl)isonicotinohydrazide (2b)
1
452
453
454
455
456
457
458
459
White solid; Rf = 0.51 (CHCl3/MeOH 4:1); Yield 73%; mp. 189-191 ºC; H NMR (300 MHz,
CDCl3):
1H, =10.2, 9.9 Hz, CH), 6.58 (brs, 2H, NH2), 7.19 (m, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 7.40 (m,
2H, Ar-H), 7.67 (d, 2H, = 6.0 Hz, Ar-H), 8.74 (d, 2H, = 6.0 Hz, Ar-H), 9.22 (br s, 1H, NH),
δ 3.04 (dd, 1H, J=16.8, 9.6 Hz, CHH), 3.48 (dd, 1H, J=16.5, 10.5 Hz, CHH), 4.98 (dd,
J
J
J
9.63 (br s, 1H, NH), 10.61 (br s, 1H, NH); 13C-NMR (75 MHz, DMSO-d6): 32.4, 56.7, 109.6,
112.4, 121.1, 121.2, 122.1 (2C), 124.1, 126.5 (2C), 134.3, 141.4, 147.5 (2C), 162.5, 172.0.; LC-
MS: m/z= 324.1 [M+H]+;Anal. Calcd for C17H17N5O2: C, 63.15; H, 5.30; N, 21.66; Found: C,
63.18; H, 5.32; N, 21.63.
460
4.3.6. N'-(2-amino-3-phenylpropanoyl)isonicotinohydrazide (2c)
1
461
462
463
464
465
White solid; Rf = 0.47 (CHCl3/MeOH 4:1); Yield 80%; mp. 171-173 ºC; H NMR (300 MHz,
DMSO-d6):
1H,
δ
3.12 (dd, 1H,
J=16.8, 9.6 Hz, C
HH), 3.64 (dd, 1H,
J
=16.5, 9.6 Hz, CH
H), 4.86 (dd,
J
=10.2, 9.9 Hz, C
H), 6.71 (brs, 2H, NH2), 7.16 (m, 5H, Ar-H), 7.66 (d, 2H, J= 6.0 Hz, Ar-
H), 8.73 (d, 2H, J
= 6.0 Hz, Ar-H), 9.23 (br s, 1H, NH), 9.65 (brs, 1H, NH); 13C-NMR (75 MHz,
DMSO-d6): 38.3, 55.9, 121.1 (2C), 124.8, 126.2 (2C), 128.9 (2C), 137.3, 141.3, 147.4, 148.7,
28