Anti-methicillin resistant Staphylococcus aureus activity, synergism with oxacillin and molecular docking studies of metronidazole-triazole hybrids

Article abstract of DOI:10.1016/j.ejmech.2016.03.041

MRSA causes 60-70% of Staphylococcus aureus infection in hospitals and it has developed resistance against the currently available drugs. Interestingly, a series of 35 metronidazole-triazole hybrids on screening against MRSA were found to be active. Compound 22 was found to be effective at 4 μg/mL concentration against nine strains of MRSA. The inhibitory activity was further enhanced upto 1 μg/mL when this compound was used in combination with oxacillin in 1:1 ratio. All the compounds were found to be non-toxic in THP-1 cell line upto a concentration of 50 μM. The time-kill kinetics studies suggested bacteriostatic nature of the compounds. In silico studies show that these compounds interact with Thr600, Ser598, Asn464, His583 and Tyr446 in the active site of PBP2a crystal structure from MRSA.

Full text of DOI:10.1016/j.ejmech.2016.03.041

Accepted Manuscript
Anti-methicillin resistant Staphylococcus aureus activity, synergism with oxacillin and
molecular docking studies of metronidazole-triazole hybrids
Beena Negi, Deepak Kumar, Widuranga Kumbukgolla, Sampath Jayaweera, Prija
Ponnan, Ramandeep Singh, Sakshi Agarwal, Diwan S. Rawat
PII:
S0223-5234(16)30223-9
10.1016/j.ejmech.2016.03.041
EJMECH 8466
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 October 2015
Revised Date: 12 February 2016
Accepted Date: 16 March 2016
Please cite this article as: B. Negi, D. Kumar, W. Kumbukgolla, S. Jayaweera, P. Ponnan, R. Singh,
S. Agarwal, D.S. Rawat, Anti-methicillin resistant Staphylococcus aureus activity, synergism with
oxacillin and molecular docking studies of metronidazole-triazole hybrids, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.041.
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ACCEPTED MANUSCRIPT
Anti-Methicillin Resistant Staphylococcus aureus Activity, Synergism with
Oxacillin and Molecular Docking Studies of Metronidazole-Triazole
Hybrids
Beena Negi,† Deepak Kumar,† Widuranga Kumbukgolla,‡ Sampath Jayaweera,‡ Prija
Ponnan,† Ramandeep Singh,§ Sakshi Agarwal,§ and Diwan S. Rawat*
†
†Department of Chemistry, University of Delhi, Delhi-110007, India.
‡Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Mihinthale, Sri
Lanka
§Translational Health Science and Technology Institute, Vaccine and Infectious Disaease
Research Centre, Gurgaon-122016, India
Graphical abstract
MIC upto 4µg/mL against MRSA
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Anti-Methicillin Resistant Staphylococcus aureus Activity, Synergism with
Oxacillin and Molecular Docking Studies of Metronidazole-Triazole
Hybrids
Beena Negi,† Deepak Kumar,† Widuranga Kumbukgolla,‡ Sampath Jayaweera,‡ Prija
Poonan,† Ramandeep Singh,§ Sakshi Agarwal,§ and Diwan S. Rawat*
†
†Department of Chemistry, University of Delhi, Delhi-110007, India.
‡Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Mihinthale, Sri
Lanka
§Translational Health Science and Technology Institute, Vaccine and Infectious Disaease
Research Centre, Gurgaon-122016, India
Abstract
MRSA causes 60-70% of S. aureus infection in hospitals and it has developed resistance
against the currently available drugs. Interestingly, a series of 35 metronidazole-triazole
hybrids on screening against MRSA were found to be active. Compound 22 was found to be
effective at 4 µg/mL concentration against nine strains of MRSA. The inhibitory activity was
further enhanced upto 1 µg/mL when this compound was used in combination with oxacillin
in 1:1 ratio. All the compounds were found to be non-toxic in THP-1 cell line upto a
concentration of 50 µM. The time-kill kinetics studies suggested bacteriostatic nature of the
compounds. In silico studies show that these compounds interact with Thr600, Ser598,
Asn464, His583 and Tyr446 in the active site of PBP2a crystal structure from MRSA.
Key Words: MRSA, Metronidazole, PBP2a, Oxacillin, Triazole, Methicillin, Oxacillin.
*Corresponding author:
E. Mail: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27667465
Fax No: 91-11-27667501
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1. Introduction
Staphylococcus aureus is the leading cause of infections in the hospitals worldwide.
Staphylococcus aureus strains resistant to methicillin were first found in England, in 1961.1
Since, then the incidences of hospital acquired (HA) and community acquired (CA)
methicillin-resistant Staphylococcus aureus (MRSA) is ever increasing.2-7 The number of
deaths due to MRSA is near to the total number of deaths due to AIDS, tuberculosis and viral
hepatitis all together. Over the years, the semi-synthetic β-lactams such as oxacillin,
cloxacillin, flucloxacillin were antibiotics of choice for the treatment of S. aureus infections.
Besides β-lactam antibiotics, presently other classes of compounds have also been approved
by FDA as anti-MRSA agents such as, peptide antibiotics (vancomycin and daptomycin),
glycylcyclines (tigecycline), and oxazolidinone class of drugs (linezolid).8,9 Bouley et al.,
have recently reported quinazolinones as a promising antibiotic for the treatment of highly
resistant MRSA infections.10 Unfortunately, MRSA has developed resistance to β-lactam
antibiotics (penicillin, methicillin, oxacillin, dicloxacillin and ceftaroline), vancomycin and
glycopeptides.11-13 With the emergence of multidrug resistant MRSA, new anti-MRSA
therapeutic strategies are needed, especially agents that are orally bioavailable. The rapid
evolution of antibiotic resistant bacterial pathogens and the slower pace for the discovery of
novel antibiotics, has a devastating effect on the potential for treatment of bacterial
infections.14-16
.
When methicillin-susceptible S. aureus (MSSA) acquires the methicillin-resistance
gene mecA by horizontal gene transfer mediated by staphylococcal cassette chromosome
(SCC), MRSA is born.17,18 The β-lactam antibiotics act by associating with the penicillin
binding proteins (PBPs), resulting in the inhibition of the cell wall synthesis, leading to
growth inhibition or cell lysis. Resistance to β-lactams emerge if penicillin binding protein 2a
(PBP2a) is over expressed by MRSA. Because of the low binding affinity between PBP2a
(encoded by the gene mecA) and β-lactams, the cell wall synthesis can be renewed. This
protein thus acts as an effective equipment for the MRSA to prevent the attack from β-
lactams. The reason for the resistance is credited to the induction of specific genes coding for
a monofunctional DD-transpeptidase enzyme penicillin binding protein 2a (PBP2a) by β-
lactam exposure.19 These β-lactam antibiotics irreversibly inhibit PBP2 by acylation of active
site serine residue (Ser403),20 in contrast, the PBP2a of MRSA is resistant to β-lactam
acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete
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the cell wall. Recent report highlights the presence of an allosteric site located 60Å distance
from the PBP2a active site and it is capable of binding three ligands including one molecule
of ceftaroline, muramic acid and peptidoglycan.21,22 Such allosteric binding triggers
conformational changes in PBP2a active site, allowing another inhibitor molecule
(ceftaroline) to bind there.20 Although PBP2a have also developed resistance against
ceftaroline in certain strains with mutations (N146K and E150K) observed in allosteric
domain and H351N mutation in region away from allosteric and active sites.
Metronidazole (MTZ) and other imidazole derivatives are known for their
antibacterial activity.23-27 The triazole28 nucleus is a well known pharmacophore for anti-
MRSA activity. In continuation with our earlier work on antibacterial agents, we screened the
MTZ-triazole hybrids against MRSA.29-35 These MTZ-triazole hybrids were found to be
effective against MRSA strains upto 4 µg/mL concentration. We determined that the MTZ-
triazoles can also decrease the minimal inhibitory concentration of oxacillin, many-fold
against MRSA. The synergistic activity shows that MTZ-trizoles can be used in combination
with oxacillin.
2. Chemistry
Click chemistry has been widely used for the construction of various biologically
active molecules due to its ease of synthesis and the possibility of the generation of a library
of compounds through various modifications. The copper catalysed azide-alkyne
cycloaddition reaction results in the formation of 1,4-disubstituted 1,2,3-triazoles, which are
bioisosteres of the amide bond. In the present study, the synthesis of MTZ-triazole hybrids
were accomplished in the sequence of reactions as depicted in scheme 1. Various terminal
alkynes were synthesized using Williamson ether synthesis starting from various substituted
phenols and anilines with propargyl bromide under basic conditions. The hydroxyl group of
MTZ was converted to tosylate, an easily leaving group, by reaction of MTZ (1) with p-
toluenesulfonyl chloride (p-TsCl) in pyridine (scheme 1). The resulting compound (2) on
treatment with sodium azide gave 1-(2-azidoethyl)-2-methyl-5-nitro-1H-imidazole (3).
Reactions of compound 3 with terminal alkynes in presence of sodium ascorbate and
CuSO4.5H2O in t-BuOH/H2O (1:1) (scheme 1) lead to the formation of desired hybrid
molecules (4-38) in good yield (51-75%). All the compounds were purified over silica gel
column. The structures of the compounds were confirmed by various spectroscopic
techniques. The absence of characteristic IR peaks for C≡C (alkyne) near 2120 cm-1 and for
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azido group of compound 3 near 2100 cm-1 indicates the formation of triazole. The N=O
stretching bands for all the compounds were observed in the region 1502-1599 and 1358-
1
1372 cm-1. In the H NMR spectra, the protons of methyl group attached to imidazole ring
resonate at about 1.7-2.0 ppm as singlet, the four protons of the ethylene (CH2CH2) linker
appeared in the region 4.5-5.2 ppm. The imidazole and triazole ring protons appeared as
singlets in the region 7.7-8.2 ppm.
<Insert Scheme 1 here>
3. Results and Discussion
The agar plate dilution method was conducted using 35 metronidazole-triazole
hybrids against 30 MRSA strains and the MIC of each compound against each MRSA strain
was obtained. Table 1 shows the number of MRSA strains against which the compounds are
effective at a particular concentration. All the compounds were active against one or another
strain of MRSA at 64 µg/mL concentration. Ten compounds (4, 6, 10, 11, 12, 17, 22, 25, 28
and 31) were active at 8 µg/mL. Compound 4, 11, 22, 25 and 28 were active at 4 µg/mL
against 1, 3, 9, 1 and 1 strains of MRSA, respectively. Compound 11 with benzene ring
linked via an ethereal bridge to triazole nucleus exhibited MIC 4 µg/mL against 3 strains of
MRSA. In general, compounds having halogen substituents at the benzene nucleus exhibited
better activity than the other substituents. Compound 22 with 2,4-dichloro substituent in the
phenyl ring was found to be highly active against 9 strains of MRSA at a concentration of 4
µg/mL. Compounds having substituents such as Me, i-Pr, t-Bu, CHO, NO2 at the benzene
ring were found to be less active. Compounds with NH instead of oxygen atom were also less
active than the oxygen linked compounds. Though in these compounds a halogen substituent
at 2-position is well tolerated and the compounds 28 and 31 were active at MIC 8 µg/mL.
Compound 22 showed better activity with MIC 4 µg/mL, than oxacillin against 9
strains, whereas oxacillin is effective upto 64 µg/mL. This most active compound 22 was also
examined for synergy with the reference drug oxacillin. A mixture of compound 22 and
oxacillin was prepared in 1:1 ratio. The concentration of the compound 22 or oxacillin in the
mixture was 32 µg/mL. The agar plate dilution method was conducted using the mixture. The
study showed that both in combination in ratio 1:1 exhibited promising activity. The activity
enhanced upto 1 µg/mL against eleven strains of MRSA. Five strains for which MIC for both
oxacillin and compound 22 were >128 or 128 µg/mL, surprisingly showed MIC 2 or 4 µg/mL
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when used in combination. Thus it can be concluded that these hybrids have a strong ability
to lower the minimal inhibitory concentration (MIC) of a conventional β-lactam, oxacillin.
The synergy between MTZ and oxacillin could be explained by two possible
mechanisms; structural modification or altered synthesis of PBP2a. In this study, we have
shown the possibility of binding MTZ hybrids to PBP2a in a computational model. This kind
of binding can lead to allosteric modification of PBP2A which likely can restore the affinity
of PBP2a to oxacillin.20 Therefore, oxacillin can bind efficiently and inhibit PBP2a which
ultimately reduces the cell wall synthesis In a situation of altered synthesis of PBP2a, MTZ
can act in DNA level. It is well known that MTZ generate reactive intermediates and those
intermediates damage DNA.36,37 Here, the reactive intermediates can affect the activity of
MecA gene altering the synthesis of PBP2a. If PBP2a synthesis reduces, oxacillin can inhibit
the cell wall synthesis more efficiently.
<Insert Table 1 here>
<Insert Table 2 here>
The most active compound 22, at 32 µg/mL concentration was selected for kill
kinetics activity (Table 3). The experiment was done in duplicate and the mean value was
used to draw the kill kinetics curve (Figure 1). The activity was monitored over 24 hour. The
compound 22 inhibited the growth of bacteria entering to the exponential phase resulting
nearly constant bacterial counts. Compound 22 and its combination with oxacillin also
showed bacteriostatic effects, however, with a slight reduction of colony counts after 14 h;
may be due weak cell wall causing the lysis of some bacterial cells. Overall, the time-kill
curves indicate that the compounds are bacteriostatic in nature. Further, all the compounds
were tested for their toxicity using THP-1 cell line. These cell lines may be used to test the
toxicity of the newly synthesized compounds.38,39 The compounds were found to be non-toxic
upto a concentration of 50 µM the highest concentration tested.
<Insert Table 3 here>
<Insert Fig. 1 here>
In the present work, we have attempted to study the interactions of novel MTZ-
triazole hybrids with PBP2a structure from MRSA, targeting the PBP2a active site. For this
purpose molecular docking studies of some compounds (4, 10, 11, 12, 22, 25 and 28) were
performed in the binding pocket of PBP2a crystal structure from MRSA (PDB ID:3ZFZ).
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The results of docking studies and the docked conformations of best scored ligands in the
active site of PBP2a are shown in table 4 and figures 2 to figure 6. These docking results
clearly indicate that the most active compounds exhibited significant binding affinities
towards the PBP2a structures (Glide energy range -55.457 kcalmol-1 to -40.322 kcalmol-1)
and the energy ranges are comparable to that of reference compound oxacillin (Table 4). The
compounds showed hydrogen bond interactions along with π-π interactions in the predicted
binding poses. Compound 25 having lowest binding energy (-55.457 kcalmol-1) and
considerable high Glide XP score (-7.293 kcalmol-1) shows hydrogen bonding interaction
between oxygens of nitro group of the compound and side chain atoms of Thr600 and Ser598
of the protein (Figure 5). Another hydrogen bonding interaction is observed between the
oxygen atom present in the linker between triazole and naphthalene ring of compound 25
with side chain NH of Asn464. Further π-π interaction between aromatic ring of His583 and
Tyr446 of PBP2a and imidazole ring of compound 25 is observed (Figure 5).
<Insert Table 4 here>
Another compound 11, predicted to have low binding energy (-53.285 kcalmol-1) and
high glide score (-6.596 kcalmol-1) and displayed similar H-bonding pattern between oxygens
of nitro group substituent in imidazole ring of the compound and side chain atoms of Thr600
and Ser598 of the protein. Hydrogen bonding interaction is also observed between the oxygen
atom present in the linker between triazole and benzene ring of compound 11 with side chain
NH of Asn464 (Figure 3).
Compound 22 having 2,6-dichloro substitution the benzene ring also show high
docking score (-6.323) and low binding energy (-49.945). In the binding pose nitrogen atom
of the imidazole ring of compound 22 was found to form a hydrogen bond with OH group of
Asn464 side chain (Figure 4). Backbone nitrogen atom of Thr600 is forming two hydrogen
bonds, one with oxygen atom of the nitro substituent of imidazole ring and one of the
nitrogen atoms of the triazole. Further, OH atom in the side chain of Ser598 forms two
hydrogen bonds with the nitrogen atoms of triazole ring of compound 22 (Figure 4).
<Insert Fig. 2 here>
<Insert Fig. 3 here>
<Insert Fig. 4 here>
<Insert Fig. 5 here>
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<Insert Fig. 6 here>
The interaction pattern observed for MTZ-triazole hybrids in the active site of PBP2a
involves amino acids Thr600, Ser598, Asn464, His583 and Tyr446 that play pivotal roles in
the binding of β-lactam antibiotics in the PBP2a active site. Interestingly, none of the MTZ-
triazole hybrids have shown to interact with Ser403 which is involved in acylation of β-
lactam antibiotics. Such binding mode is reminiscent of the suggestions provided by Lim and
Strynadka for designing anti-MRSA drug that could tightly bind non-covalently to PBP2a
binding site and is devoid of β-lactam moiety.40 Hence these observations could be viewed
beneficial for developing antibiotics against broadly resistant MRSA strains.
Different pharmacokinetic (PK) parameters for MTZ-triazole hybrids were calculated.
The most important of these parameters together with its permissible ranges are listed in the
tables S1 and S2. In the present study, most of the test compounds have value for Lipinski’s
rule of 5 violations less than the maximum permissible value of 4,41 indicating that these test
compounds are endowed with drug likeness properties. Lipinski’s rule of 5 is a preliminary
test for the drug-likeness of the compounds that require an ideal orally bioactive compound to
have no more than 5 and 10 hydrogen bond donors (donorHB) and acceptors (accptHB),
respectively, molecular weights (mol_MW) less than 500 amu, and partition coefficients
between octanol and water (QPlog P(oct/wat)) less than 5. Most of the MTZ-triazole hybrids
in the study show no violation of Lipinski’s rule of 5, except for compounds 19 (CH2OPh-4-
NO2), 24 (CH2OPh-2,4,6-t-Bu) and 36 (NHPh-3,5-OMe) that possess Lipinski’s rule of 5
violation of one (Table S1).
Prediction of oral drug absorption (Percent Human Oral Absorption) was highly
satisfactory for all the test compounds. It is noteworthy that most of highly active test
compounds is predicted to have high percentage (>80%) of Human Oral Absorption as
compared to the reference compounds oxacillin, methicillin and metronidazole (Table S2),
indicating that MTZ-triazole hybrids could be developed as an orally dosed drug for the
treatment of MRSA infection. Studies suggest that the oral bioavailability is influenced by
compound’s flexibility and can be measured by the number of rotatable bonds (<15) and
polar surface area (70 Å2-200 Å2).42 In the present study, all the test compounds have a
number of rotatable bonds (<15) and the polar surface area falls satisfactorily within the
permissible range (Table S2). Moreover, molecules possessing values for Lipinski’s rule of 5
in permissible ranges could be viewed more likely to have good intestinal absorption or
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permeation and is confirmed by the predicted Caco-2 cells permeability (QPPCaco), used as a
model for the gut-blood barrier.43 QPPCaco predictions for all the test compounds showed
very good values compared to the β-lactams (oxacillin and methicillin) (Table S2). Further,
QPlogKhsa which is the prediction for human serum albumin binding, shows that the
predicted values for all the inhibitors lie within the expected range for 95% of known drugs (-
1.5 to 1.5). Also, the QikProp descriptor for brain/blood partition coefficient (QPlogBB)
show satisfactory predictions for all the test compounds and the reference compounds.
Furthermore, QPlogHERG descriptor for the prediction of IC50 value of HERG K+ channel
blockage was predicted for the test compounds. Compounds 4 (CH2Br), 12 (CH2OCH3), and
22 (CH2OPh-2,6-Cl) have been predicted to possess QPlogHERG values in below concern
range (< -5) comparable to reference compounds oxacillin, methicillin and metronidazole
(Table S2).
4. Conclusions
All the synthesized compounds were found to be effective against MRSA. The best
active compound 22 showed good synergism with the reference drug oxacillin. All the
compounds were found to be nontoxic up to 50 ꢀM concentration against THP-1 cell line.
Also, the compounds exhibited good pharmacokinetic properties and follow the Lipinski’s
rule of 5. Thus we believe that these compounds can be considered as a possible lead for the
development of new anti-MRSA agents.
5. Experimental Protocols
5.1. Biological Activity
5.1.1. In vitro anti-MRSA activity
MRSA strains were isolated using axilla and nasal swabs obtained from Medical
Students and Patients at Teaching Hospital Anuradhapura, Sri Lanka. After swabbing axilla
and nasal areas, the swabs were dipped in NaCl enriched nutrient broth. Following overnight
o
incubation at 37 C, those swabs were streaked on Manitol Salt Agar Plates. The colonies
which turned adjacent media into yellow color were selected and the Gram stained smears of
those were observed through light microscopy. The cultures which showed Gram positive
clusters were followed by coagulase test to identify S. aureus. The MIC value of oxacillin
against each S. aureus strain was determined by agar plate dilution method (Andrews et al.)
The dilution series was quality controlled using NCTC 6571 S. aureus organism. A total
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number of 100 strains of MRSA strains were maintained in the culture collection and 30
strains of those were used for experiments.
5.1.1.1. Agar well method: The antibacterial activity of the synthesized compounds was
initially screened using agar well method. The bacteria (MRSA) which were obtained from a
one day old culture were dissolved in sterile water. And the visual density of the organism
was adjusted according to the McFarland-0.5 standard. By soaking a cotton swab in the
microbial solution, and streaking the swab on the surface of the Mueller-Hinton agar, a
microbial lawn was prepared. A well was dug using the cokeborer (16 mm diameter) in the
microbial lawn containing agar plate. The well was filled with the aqueous solution of the
compound (1000 µg/mL) using a pasture pipette. The plates were observed for a clear zone
o
around the wells after overnight (24 h) incubation at 37 C. The diameter of each clear zone
was measured. The compounds which were exhibited a clear zone around the wells, were
further subjected for the determination of MIC against 30 MRSA strains using agar plate
dilution method.
5.1.1.2. Agar plate dilution method: A dilution series was prepared using anti-bacterial
compounds in the concentration range 0.03–128 µg/mL. MRSA was suspended in water
using a cotton swab until the visual density equals to McFarland 0.5 standard. The agar plates
which contain antibacterial compounds were inoculated using MRSA suspension. The plates
o
were incubated 24 h at 37 C and were observed for white colored spots which indicate the
growth of the organism. NCTC 6571 S. aureus was used to quality control the dilution series.
5.2. Bacterial counts for Time-kill curve
Four separate incubation mixtures containing antibacterial compounds (Oxacillin,
MTZ, Compound 22 and the compound 22 with Oxacillin in 1:1 ratio) were prepared in
nutrient broth and the control was set without introducing any compound. After adding equal
amount of MRSA to each incubation mixture including the control, the final volume was
adjusted to 10 mL and the concentration of each antibacterial compound was kept at 32
µg/mL. The initial colony count and subsequent colony counts were obtained using pour
plate method, at each 4 h time intervals, over 24 h period. In conducting pour plate technique
the bacteria was diluted in the range of 101 to 10-7 using nutrient broth. The inoculum (1 mL)
which was obtained from each dilution was mixed with melted nutrient agar, separately and
poured into separate petri-dishes. The plate which contained 30-300 colonies in prepared
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dilution series was used to calculate the colony counts. Same procedure was followed for
each mixture of compounds at each time interval.
5.3. In silico ADMET prediction
The ADME properties of test compounds and reference compounds (oxacillin,
methicillin and metronidazole) are predicted for the pharmaceutically relevant properties to
assess the drug likeness and pharmacokinetic properties. The Qikprop v3.5 (Schrödinger,
Inc., New York, NY, 2012) was used for the evaluation of some important absorption,
distribution, metabolism and elimination (ADME) parameters and its permissible ranges are
listed in the Tables 4 and 5.
All the compounds were prepared in neutralized form for the calculation of
pharmacokinetic properties by Maestro Build module and LigPrep, saved in SD format. In
the present study, the test compounds showed good preliminary test of the druglikeness based
on Lipinski’s rule of 5 showing zero violation of the rule, proving all the test compounds to
be orally active. The descriptor QPPCaco indicating Caco-2 cells permeability, a model used
for the gut-blood barrier, showed good values for all the test compounds. Similarly the values
of descriptor model such as number of rotatable bond (#rotor) and polar surface area (PSA),
used as an indicator of bioavailability for the test compounds lie in expected ranges. Further,
the prediction for human serum albumin binding (QPlogKhsa) and QikProp descriptor for
brain/blood partition coefficient (QPlogBB) and the blood-brain barrier mimic MDCK cell
permeability (QPPMDCK) show satisfactory predictions for all the test compounds (Table
S1).
5.4. Molecular docking studies
The 2D structures of all the compounds were generated by drawing on ChemBioDraw Ultra
12.0 (www.cambridgesoft.com). Ligprep module implemented in Schrödinger was used to
generate energy minimized 3D structures. Partial atomic charges were computed using the
OPLS_2005 force field. The correct Lewis structure, tautomers and ionization states (PH 7.0
+/− 2.0) for each of these ligands were generated and optimized with default settings
(Ligprep 2.5, Schrödinger, LLC, New York, NY, 2012). The results of docking studies using
Glide XP method could be evaluated using Glide energy and GlideScore XP. Glide Energy is
a modified Coulomb-van der Waals interaction energy which is used for comparing binding
affinities of different ligands using Charge-dipole and dipole interactions. GlideScore XP
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includes XP scoring functions, penalities and XP terms such as Van der Waals energy,
Coulomb energy, Lipophilic term, Hydrogen-bonding term, rewards and penalties for various
features, such as buried polar groups, hydrophobic enclosure, correlated hydrogen bonds,
amide twists, penalty for freezing rotatable bonds, pi-pi stacking reward, reward for pi-cation
interactions etc.44 The 3D crystal structure of PBP2a protein from MRSA (PDB ID:3ZFZ;
resolution 2.25 Å), was retrieved from protein data bank (www.rcsb.org). The proteins were
prepared for docking using Protein Preparation Wizard (Maestro 10.0 Schrödinger, LLC,
New York, NY, 2012). Water molecules within 5 Å of the protein structures was considered.
Bond order and formal charges were assigned and hydrogen atoms were added to the crystal
structure. Further to refine the structure OPLS-2005 force field parameter was used to
alleviate steric clashes and the minimization was terminated when RMSD reached maximum
cut-off value of 0.30 Å. The location of co-crystalized ligand Ceftaroline in protein active site
was used to choose the center and size of the receptor grid, which was generated using Glide
5.8 (Schrödinger, LLC, New York, NY, 2012) with default settings for all parameters. The
grid size was chosen sufficiently large to include all active site residues involved in substrate
binding. All ligand conformers were docked to each of the receptor grid files (PfDHFR-TS
wild and mutant structures) using Glide extra precision (XP) mode. Default settings were
used for the refinement and scoring.
5.5. Materials and methods
All of the chemicals used in the synthesis were purchased from Sigma-Aldrich and were used
as such. The progress of the reactions was monitored by using thin layer chromatography (E.
Merck Kieselgel 60 F254). Compounds were purified over silica gel (60-120 mesh) column.
Meting points were recorded on an ERS automated melting point apparatus and are
uncorrected. IR spectra were recorded using Perkin-Elmer and Bruker FT-IR and the values
are expressed as λmax cm-1. Mass spectral data were recorded on a Jeol-AccuTOF JMS-
T100LC and micromass LCT Mass Spectrometer. 1H NMR and 13C NMR spectral data were
recorded on Bruker Spectrospin spectrometer at 300 MHz and 75 MHz and on Jeol ECX
spectrospin instrument at 400 and 100 MHz, respectively using CDCl3 and DMSO-d6 as
solvent and TMS as internal reference. The chemical shift values are recorded on δ scale and
the coupling constants (J) are in Hz.
5.5.1. Typical procedure for the synthesis of metronidazole-triazole conjugates (4-38): To a
vigorously stirred solution of 1-(2-azido-ethyl)-2-methyl-5-nitro-1H-imidazole (3), (2.5
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mmol) and appropriate alkyne (2.5 mmol) in tert-butyl alcohol was added a solution of
CuSO4.5H2O (0.51 mmol) and sodium ascorbate (1.02 mmol) in distilled water (scheme 1).
The amount of tert-butyl alcohol and distilled water was kept 1:1 (v/v). The deep yellow
mixture was stirred vigorously at room temperature and progress of reaction was monitored
by thin layer chromatography. After 2 h, reaction was completed and crude reaction mixture
was extracted with CHCl3 (3 × 10 mL) and dried over anhydrous Na2SO4. Excess of solvent
was removed under vaccum. The crude mixture was purified over SiO2 column using ethyl
acetate/hexane as an eluent.
5.5.2. 4-Bromomethyl-1-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]triazole (4):
o
Yield 52%; mp 108-110 C; IR (KBr, cm-1): 3127, 2947, 1526, 1461, 1429, 1369, 1268,
1
1199, 1117, 1028, 1008; H NMR (300 MHz, DMSO-d6) δ: 1.83 (s, 3H, CH3), 3.73 (s, 2H,
CH2Br), 4.59-4.81 (m, 4H, 2NCH2), 7.95 (s, 1H), 8.03 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) δ: 12.80, 18.91, 24.98, 30.05, 46.25, 48.70, 59.01, 61.26, 96.56, 124.92, 133.25, 138.39,
144.21, 151.21; ESI-MS (m/z) calculated for C9H11BrN6O2: 314.01, found: 315.10 [M + H]+,
317.11 [M + 2]+; Anal. calcd. for C9H11BrN6O2: C, 34.30; H, 3.52; Br, 25.36; N, 26.67; O,
10.15; found: C, 34.33; H, 3.23; Br, 25.55; N, 26.57; O, 10.28.
5.5.3. Propionicacid-1-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]triazol-4-ylmethyl
o
ester (5): Yield 58%; mp 116-118 C; IR (KBr, cm-1): 3118, 3073, 2979, 1732, 1530, 1467,
1
1425, 1372, 1263, 1174, 1143; H NMR (300 MHz, DMSO-d6) δ: 1.02 (t, J = 6 Hz, 3H,
CH2CH3), 1.83 (s, 3H, CH3), 2.32 (q, J = 6 Hz, 2H, CH2CH3), 4.68-4.72 (m, 2H, NCH2),
4.82-4.85 (m, 2H, NCH2), 5.09 (s, 2H, OCH2), 8.01 (s, 1H), 8.06 (s, 1H); ESI-MS (m/z)
calculated for C12H16N6O4: 308.12, found: 309.21 [M + H]+; Anal. calcd. for C12H16N6O4: C,
46.75; H, 5.23; N, 27.26; O, 20.76; found: C, 46.01; H, 5.54; N, 27.57; O, 20.28.
5.5.4.
1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-((tetrahydro-2H-pyran-2-yloxy)
methyl)-1H-1,2,3-triazole (6): Yield 60%; IR (KBr, cm-1): 3423, 2931, 2861, 1526, 1461,
1
1429, 1366, 1267, 1190, 1148, 1018; H NMR (300 MHz, DMSO-d6) δ: 1.30-1.36 (m, 2H),
1.44 (brs, 2H), 1.55-1.58 (m, 2H), 2.39 (s, 3H, CH3), 3.74-3.82 (m, 1H), 4.37-4.40 (m, 2H),
4.46-4.55 (m, 2H), 4.81-4.83 (m, 2H), 4.92-4.96 (m, 2H), 7.79 (s, 1H), 8.04 (s, 1H); ESI-MS
(m/z) calculated for C14H20N6O4: 336.15, found: 337.22 [M + H]+; Anal. calcd. for
C14H20N6O4: C, 49.99; H, 5.99; N, 24.99; O, 19.03; found: C, 50.22; H, 5.73; N, 25.51; O,
19.28.
13

ACCEPTED MANUSCRIPT
5.5.5. 1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(trimethylsilyl)-1H-1,2,3-triazole (7):
Yield 60%; mp 189-191 oC; 1H NMR (300 MHz, DMSO-d6): 0.20 (s, 9H, Si(CH3)3), 1.54 (s,
3H, CH3), 4.47 (t, J = 4.2 Hz, 2H, NCH2), 4.62 (t, J = 4.5 Hz, 2H, NCH2), 7.72 (s, 1H), 7.83
(s, 1H); ESI-MS (m/z) calculated for C11H18N6O2Si: 294.12, found: 295.31 [M + H]+; Anal.
calcd. for C11H18N6O2Si: C, 44.88; H, 6.16; N, 28.55; O, 10.87; Si, 9.54; found: C, 45.01; H,
6.23; N, 28.87; O, 10.68; Si, 9.77.
5.5.6. 1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-propyl-1H-1,2,3-triazole (8): Yield
o
75%; mp 121-122 C; IR (film, cm-1): 3123, 2961, 2910, 2861, 1525, 1461, 1370, 1266,
1
1199, 1148, 1050, 827, 748; H NMR (400 MHz, CDCl3) δ: 0.82 (t, J = 7.63 Hz, 3H,
CH2CH2CH3), 1.49 (sextet, J = 7.63 Hz, 2H, CH2CH2CH3), 1.78 (s, 3H, CH3), 2.49 (t, J =
7.63 Hz, 2H, CH2CH2CH3), 4.63-4.66 (m, 2H, NCH2CH2N), 4.72-4.75 (m, 2H, NCH2CH2N),
7.65 (s, 1H), 8.01 (s, 1H); 13C NMR (100 MHz, CDCl3) δ: 13.30, 13.64, 22.70, 27.39, 46.68,
49.39, 121.55, 133.82, 149.11, 151.42; ESI-MS (m/z) calculated for C11H16N6O2: 264.13,
found: 265.24 [M + H]+.; Anal. calcd. for C11H16N6O2: C, 49.99; H, 6.10; N, 31.80; O, 12.11;
found: C, 50.14; H, 6.33; N, 32.09; O, 12.37.
5.5.7. 1-(1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)cyclohexanol
(9): Yield 68%; mp 151-152 oC; IR (KBr, cm-1): 3394, 2933, 1533, 1459, 1371, 1265, 1197,
1
1146, 1049, 826; H NMR (300 MHz, DMSO-d6): 1.33-1.37 (m, 1H), 1.50-1.59 (m, 3H),
1.61-1.80 (m, 6H), 1.94 (s, 3H, CH3), 2.28 (s, 1H, OH), 4.78 (s, 4H, NCH2CH2N), 7.10 (s,
1H), 8.0 (s, 1H); ESI-MS (m/z) calculated for C14H20N6O3: 320.15, found: 321.21 [M + H]+;
Anal. calcd. for C14H20N6O3: C, 52.49; H, 6.29; N, 26.23; O, 14.98; found: C, 52.76; H, 6.51;
N, 26.48; O, 15.20.
5.5.8. 1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-4-phenyl-1H-[1,2,3]triazole (10): Yield
59%; mp 178-180 oC; IR (KBr, cm-1): 3124, 1527, 1460, 1367, 1263, 1192, 1149, 1082, 825,
1
771; H NMR (300 MHz, DMSO-d6) δ: 1.92 (s, 3H, CH3), 4.77 (brs, 2H, NCH2), 4.88 (brs,
2H, NCH2), 7.31-7.36 (m, 3H, ArH), 7.75-7.77 (m, 2H, ArH), 8.06 (s, 1H), 8.43 (s, 1H); ESI-
MS (m/z) calculated for C14H14N6O2: 298.11, found: 298.30 [M]+; Anal. calcd. for C14H14N6O2:
C, 56.37; H, 4.73; N, 28.17; O, 10.73; found: C, 55.99; H, 4.52; N, 28.42; O, 10.95.
5.5.9. 1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-4-phenoxymethyl-1H-[1,2,3]triazole (11):
o
Yield 54%; mp 146-148 C; IR (KBr, cm-1): 3128, 2932, 1527, 1466, 1428, 1367, 1252,
1196, 1151, 1049, 884; 1H NMR (300 MHz, DMSO-d6) δ: 1.80 (s, 3H, CH3), 4.71 (t, J = 5.4
14

ACCEPTED MANUSCRIPT
Hz, 2H, NCH2), 4.85 (t, J = 5.4 Hz, 2H, NCH2), 5.12 (s, 2H, OCH2), 6.92-7.01 (m, 3H, ArH),
7.26-7.31 (m, 2H, ArH), 8.04 (s, 1H), 8.11 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 12.74,
46.21, 48.78, 60.77, 114.70, 120.85, 125.34, 129.50, 133.28, 138.37, 143.17, 151.23, 157.86;
ESI-MS (m/z) calculated for C15H16N6O3: 328.12, found: 329.23 [M + H]+; Anal. calcd. for
C15H16N6O3: C, 54.87; H, 4.91; N, 25.60; O, 14.62; found: C, 54.99; H, 5.14; N, 25.82; O,
14.45.
5.5.10. 1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-4-o-tolyloxymethyl-1H-[1,2,3]triazole (12):
o
Yield 60%; mp 134-136 C; IR (KBr, cm-1): 3128, 2924, 2854, 1526, 1461, 1430, 1268,
1243, 1193, 1120, 1052, 1006; 1H NMR (300 MHz, DMSO-d6) δ: 1.74 (s, 3H, CH3), 2.09 (s,
3H, CH3), 4.69-4.71 (m, 2H, NCH2), 4.83-4.85 (m, 2H, NCH2), 5.10 (s, 2H, OCH2), 6.81-
6.86 (m, 1H, ArH), 7.03-7.06 (m, 1H, ArH), 7.11-7.13 (m, 2H, ArH), 8.03 (s, 1H), 8.08 (s,
13
1H); C NMR (100 MHz, DMSO-d6) δ: 12.82, 46.24, 48.73, 57.10, 64.71, 124.85, 133.27,
138.39, 144.14, 151.23; ESI-MS (m/z) calculated for C16H18N6O3: 342.14, found: 343.19 [M +
H]+; Anal. calcd. for C16H18N6O3: C, 56.13; H, 5.30; N, 24.55; O, 14.02; found: C, 56.44; H,
5.67; N, 24.79; O, 14.31.
5.5.11. 1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-4-p-tolyloxymethyl-1H-[1,2,3]triazole (13):
o
Yield 52%; mp 142-143 C; IR (KBr, cm-1): 2924, 2854, 1538, 1509, 1465, 1369, 1266,
1
1235, 1190, 1155, 1047; H NMR (300 MHz, DMSO-d6) δ: 1.78 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 4.70 (brs, 2H, NCH2), 4.82 (brs, 2H, NCH2), 5.05 (s, 2H, OCH2), 6.85 (d, J = 8 Hz,
13
2H, ArH), 7.05 (d, J = 8 Hz, 2H, ArH), 8.02 (s, 1H), 8.07 (s, 1H); C NMR (75.5 MHz,
DMSO-d6): 12.73, 20.05, 48.73, 60.87, 114.56, 125.23, 129.49, 129.78, 133.23, 138.34,
143.26, 151.19, 155.74; ESI-MS (m/z) calculated for C16H18N6O3: 342.14, found: 343.22 [M +
H]+; Anal. calcd. for C16H18N6O3: C, 56.13; H, 5.30; N, 24.55; O, 14.02; found: C, 56.47; H,
5.61; N, 24.69; O, 14.24.
5.5.12. 4-((4-iso-Propylphenoxy)methyl)-1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-
o
1,2,3-triazole (14): Yield 62%; mp 147-149 C; IR (KBr, cm-1): 3131, 2958, 2924, 1747,
1700, 1650, 1611, 1511, 1430, 1364, 1264, 1241, 1191, 1147; 1H NMR (300 MHz, CDCl3) δ:
1.12 (d, J = 6.87 Hz, 6H, CH(CH3)2), 1.74 (s, 3H, CH3), 2.78 (septet, J = 6.87 Hz, 1H,
CH(CH3)2), 4.65-4.68 (m, 2H, NCH2CH2NH2), 4.79-4.82 (m, 2H, NCH2CH2NH2), 5.04 (s,
2H, OCH2), 6.86 (d, J = 8.39 Hz, 2H, ArH), 7.10 (d, J = 8.39 Hz, 2H, ArH), 8.01 (s, 1H),
13
8.06 (s, 1H); C NMR (100 MHz, DMSO-d6) δ: 12.73, 24.11, 32.60, 36.21, 48.77, 60.88,
114.53, 125.26, 127.14, 133.27, 138.37, 140.73, 143.34, 151.23, 155.96; ESI-MS (m/z)
15

ACCEPTED MANUSCRIPT
calculated for C18H22N6O3: 370.17, found: 371.24 [M + H]+; Anal. calcd. for C18H22N6O3: C,
58.37; H, 5.99; N, 22.69; O, 12.96; found: C, 58.68; H, 6.22; N, 22.84; O, 13.09.
5.5.13. 4-((4-tert-Butylphenoxy)methyl)-1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-
o
1,2,3-triazole (15): Yield 64%; mp 141-142 C; IR (film, cm-1): 3135, 3108, 2962, 2868,
1608, 1527, 1513, 1466, 1429, 1363, 1260, 1244, 1188, 1148; 1H NMR (300 MHz, CDCl3) δ:
1.20 (s, 9H, (CH3)3), 1.73 (s, 3H, CH3), 4.66-4.68 (m, 2H, NCH2CH2NH2), 4.79-4.82 (m, 2H,
NCH2CH2NH2), 5.05 (s, 2H, OCH2), 6.86 (d, J = 8.39 Hz, 2H, ArH), 7.24 (d, J = 8.39 Hz,
2H, ArH), 8.01 (s, 1H), 8.06 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 12.73, 31.30, 33.76,
46.23, 48.77, 60.84, 114.18, 125.24, 126.06, 133.27, 142.97, 143.35, 155.60; ESI-MS (m/z)
calculated for C19H24N6O3: 384.19, found: 385.26 [M + H]+; Anal. calcd. for C19H24N6O3: C,
59.36; H, 6.29; N, 21.86; O, 12.49; found: C, 59.22; H, 6.51; N, 22.01; O, 12.86.
5.5.14. 2-{1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]triazol-4-ylmethoxy}-benzal-
o
dehyde (16): Yield 60%; mp 180-182 C; IR (KBr, cm-1): 3141, 3031, 2870, 1679, 1599,
1
1534, 1475, 1370, 1261, 1241, 1187, 1049, 1004; H NMR (300 MHz, DMSO-d6) δ: 1.78
(s, 3H, CH3), 4.72 (brs, 2H, NCH2), 4.87 (brs, 2H, NCH2), 5.33 (s, 2H, OCH2), 7.08-7.13
(m, 1H, ArH), 7.38-7.41 (m, 1H, ArH), 7.65-7.72 (m, 2H, ArH), 8.04 (s, 1H), 8.19 (s, 1H),
13
10.33 (s, 1H, CHO); C NMR (100 MHz, DMSO-d6) δ: 12.68, 46.23, 48.89, 61.85, 114.06,
121.12, 124.43, 125.56, 127.60, 133.26, 136.36, 138.36, 142.69, 151.17, 160.25, 189.10; ESI-
MS (m/z) calculated for C16H16N6O4: 356.12, found: 356.17 [M]+; Anal. calcd. for
C16H16N6O4: C, 53.93; H, 4.53; N, 23.58; O, 17.96; found: C, 54.11; H, 4.75; N, 23.78; O,
18.25.
5.5.15.
4-{1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]triazol-4-ylmethoxy}-
o
benzaldehyde (17): Yield 60%; mp 170-172 C; IR (KBr, cm-1): 2924, 2854, 1683, 1601,
1
1577, 1524, 1454, 1370, 1261, 1221, 1172, 826; H NMR (300 MHz, DMSO-d6) δ: 1.77 (s,
3H, CH3), 4.68-4.71 (m, 2H, NCH2CH2NH2), 4.83-4.86 (NCH2CH2NH2), 5.25 (s, 2H,
OCH2), 7.18 (d, J = 8.39 Hz, 2H, ArH), 7.85 (d, J = 8.39 Hz, 2H, ArH), 8.03 (s, 1H), 8.14 (s,
1H), 9.86 (s, 1H, CHO); 13C NMR (75.5 MHz, DMSO-d6): 12.68, 46.10, 48.77, 61.19,
115.19, 125.61, 129.82, 131.73, 133.16, 138.32, 142.41, 151.15, 162.73, 191.31; ESI-MS
(m/z) calculated for C16H16N6O4: 356.12, found: 357.18 [M + H]+; Anal. calcd. for
C16H16N6O4: C, 53.93; H, 4.53; N, 23.58; O, 17.96; found: C, 54.06; H, 4.62; N, 23.85; O,
18.17.
16

ACCEPTED MANUSCRIPT
5.5.16. 4-(2-Chloro-phenoxymethyl)-1-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]
o
triazole (18): Yield 63%; mp 140-142 C; IR (KBr, cm-1): 3133, 2924, 1525, 1501, 1460,
1430, 1366, 1264, 1195, 1037; 1H NMR (300 MHz, CDCl3) δ: 1.98 (s, 3H, CH3), 4.79 (s, 4H,
NCH2CH2N), 5.15 (s, 2H, OCH2), 6.66-6.69 (m, 1H, ArH), 6.74-6.75 (m, 1H, ArH), 7.01-
7.04 (d, J = 8.4 Hz, 1H, ArH), 7.33 (s, 1H), 8.00 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
12.73, 18.44, 19.64, 46.21, 48.76, 60.83, 111.67, 116.11, 125.20, 128.33, 130.15, 133.28,
137.29, 138.37, 143.38, 151.22, 155.98; ESI-MS (m/z) calculated for C15H15ClN6O3: 362.08,
found: 363.12 [M + H]+, 365.35 [M + 2]+; Anal. calcd. for C15H15ClN6O3: C, 49.66; H, 4.17;
Cl, 9.77; N, 23.17; O, 13.23; found: C, 49.59; H, 4.33; Cl, 10.00; N, 23.38; O, 13.54.
5.5.17.
1-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-4-(4-nitro-phenoxymethyl)-1H-
o
[1,2,3]triazole (19): Yield 54%; mp 196-198 C; IR (KBr, cm-1): 3127, 2916, 1593, 1502,
1
1457, 1361, 1259, 1186, 1044, 1106; H NMR (300 MHz, CDCl3) δ: 1.99 (s, 3H, CH3), 4.82
(brs, 4H, NCH2CH2N), 5.26 (s, 2H, OCH2), 7.04 (d, J = 9 Hz, 2H, ArH), 7.36 (s, 1H), 8.00 (s,
1H), 8.21 (d, J = 9 Hz, 2H, ArH); ESI-MS (m/z) calculated for C15H15N7O5: 373.11, found:
373.15 [M]+; Anal. calcd. for C15H15N7O5: C, 48.26; H, 4.05; N, 26.26; O, 21.43; found: C,
48.42; H, 4.25; N, 26.47; O, 21.60.
5.5.18. 4-((2,3-Dimethylphenoxy)methyl)-1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-
o
1,2,3-triazole (20): Yield 58%; mp 144-146 C; IR (film, cm-1): 3128, 2923, 1703, 1656,
1584, 1525, 1461, 1429, 1372, 1269, 1251, 1198, 1093; 1H NMR (300 MHz, CDCl3) δ: 1.95
(s, 3H, CH3), 2.11 (s, 3H, CH3), 2.30 (s, 3H, CH3), 4.81 (s, 4H, NCH2CH2N), 5.16 (s, 2H,
OCH2), 6.76-6.82 (m, 2H, ArH), 7.02-7.07 (m, 1H, ArH), 7.30 (s, 1H), 8.0 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 11.46, 12.66, 19.73, 46.31, 48.82, 61.39, 109.63, 122.38, 124.34,
125.13, 125.85, 133.28, 137.34, 138.37, 143.61, 151.20, 155.80; ESI-MS (m/z) calculated for
C17H20N6O3: 356.15, found: 357.20 [M + H]+; Anal. calcd. for C17H20N6O3: C, 57.29; H, 5.66;
N, 23.58; O, 13.47; found: C, 57.46; H, 5.80; N, 23.72; O, 13.66.
5.5.19. 4-((2,4-Dichlorophenoxy)methyl)-1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-
o
1,2,3-triazole (21): Yield 67%; mp 120-121 C; IR (film, cm-1): 3133, 2941, 1695, 1529,
1
1472, 1366, 1260, 1191, 1146, 1056, 997, 809; H NMR (300 MHz, DMSO-d6) δ: 1.72 (s,
3H, CH3), 4.65-4.68 (m, 2H, NCH2CH2NH2), 4.81-4.83 (t, 2H, NCH2CH2NH2), 5.20 (s, 2H,
13
OCH2), 7.28-7.35 (m, 2H, ArH), 7.53-7.54 (m, 1H, ArH), 8.00 (s, 1H), 8.10 (s, 1H); C
NMR (100 MHz, DMSO-d6) δ: 12.69, 46.27, 48.87, 62.04, 115.49, 122.48, 124.79, 125.74,
128.01, 129.34, 133.27, 138.36, 142.34, 151.17, 152.34; ESI-MS (m/z) calculated for
17

ACCEPTED MANUSCRIPT
C15H14Cl2N6O3: 396.05, found: 397.11 [M + H]+, 399.18 [M + 2]+, 401.20 [M + 4]+; Anal.
calcd. for C15H14Cl2N6O3: C, 45.36; H, 3.55; Cl, 17.85; N, 21.16; O, 12.08; found: C, 45.55;
H, 3.71; Cl, 17.93; N, 21.30; O, 12.29.
5.5.20. 4-((2,6-Dichlorophenoxy)methyl)-1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-
o
1,2,3-triazole (22): Yield 68%; mp 170-171 C; IR (film, cm-1): 3211, 2957, 1696, 1530,
1
1459, 1365, 1258, 1191, 1146, 1044, 968, 755; H NMR (300 MHz, DMSO-d6) δ: 1.93 (s,
3H, CH3), 4.79 (t, J = 5.7 Hz, 2H, NCH2), 4.94 (t, J = 5.7 Hz, 2H, NCH2), 5.15 (s, 2H,
OCH2), 7.28 (d, J = 8.1 Hz, 1H, ArH), 7.56 (d, J = 8.1 Hz, 2H, ArH), 8.12 (s, 1H), 8.29 (s,
13
1H); C NMR (100 MHz, DMSO-d6) δ: 12.92, 46.16, 48.76, 65.94, 126.07, 126.32, 128.76,
129.28, 133.27, 138.40, 142.39, 150.17, 151.28; ESI-MS (m/z) calculated for C15H14Cl2N6O3:
396.05, found: 397.15 [M + H]+, 399.18 [M + 2]+, 401.18 [M + 4]+; Anal. calcd. For
C15H14Cl2N6O3: C, 45.36; H, 3.55; Cl, 17.85; N, 21.16; O, 12.08; found: C, 45.49; H, 3.67; Cl,
17.91; N, 21.52; O, 12.16.
5.5.21.
4-(Benzyloxymethyl)-1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazole
(23): Yield 58%; mp 104-106 oC; IR (film, cm-1): 3189, 2922, 1715, 1530, 1464, 1364, 1263,
1
1189, 1145, 1049, 823; H NMR (300 MHz, DMSO-d6) δ: 1.88 (s, 3H, CH3), 4.48-4.56 (m,
4H, 2NCH2), 4.75-4.87 (m, 4H, 2OCH2), 7.36 (brs, 5H, ArH), 8.06 (s, 2H); ESI-MS (m/z)
calculated for C16H18N6O3: 342.14, found: 343.19 [M + H]+; Anal. calcd. for C16H18N6O3: C,
56.13; H, 5.30; N, 24.55; O, 14.02; found: C, 56.44; H, 5.52; N, 24.70; O, 14.28.
5.5.22. 1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-((2,4,6-tri-tert-butylphenoxy)methyl)-
1H-1,2,3-triazole (24): Yield 77%; mp 199-200 oC; IR (film, cm-1): 3125, 2960, 1718, 1531,
1
1468, 1364, 1214, 1176, 1052, 746; H NMR (300 MHz, DMSO-d6) δ: 1.29 (s, 9H, (CH3)3),
1.38 (s, 18H, 2(CH3)3), 1.83 (s, 3H, CH3), 4.75 (s, 4H, 2NCH2), 4.91 (s, 2H, OCH2), 7.25 (s,
2H, ArH), 8.07 (s, 1H), 8.13 (s, 1H); ESI-MS (m/z) calculated for C27H40N6O3: 496.31, found:
497.38 [M + H]+; Anal. calcd. for C27H40N6O3: C, 65.30; H, 8.12; N, 16.92; O, 9.66; found:
C, 65.48; H, 8.66; N, 17.12; O, 9.81.
5.5.23. 1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-((naphthalen-1-yloxy)methyl)-1H-
o
1,2,3-triazole (25): Yield 60%; mp 168-169 C; IR (film, cm-1): 3127, 2916, 1577, 1524,
1
1459, 1429, 1371, 1267, 1238, 1198, 1096, 1052; H NMR (300 MHz, DMSO-d6) δ: 1.87 (s,
3H, CH3), 4.80-4.81 (m, 2H, NCH2), 4.94 (s, 2H, NCH2), 5.37 (s, 2H, OCH2), 7.19 (d, J = 7.2
Hz, 1H, ArH), 7.48-7.56 (m, 4H), 7.93 (d, J = 6.9 Hz, 1H), 8.12-8.15 (m, 2H), 8.27 (s, 1H);
18

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ESI-MS (m/z) calculated for C19H18N6O3: 378.14, found: 379.19 [M + H]+; Anal. calcd. for
C19H18N6O3: C, 60.31; H, 4.79; N, 22.21; O, 12.69; found: C, 60.52; H, 4.96; N, 22.55; O, 12.83.
5.5.24. N-((1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl) aniline
(26): Yield 62%; mp 137-138 oC; IR (KBr, cm-1): 3132, 2958, 2928, 2869, 1611, 1583, 1525,
1
1510, 1461, 1431, 1371, 1268, 1241, 1198, 1176, 1150, 1116, 1051, 1006, 884, 862; H
NMR (300 MHz, DMSO-d6) δ: 1.79 (s, 3H, CH3), 3.30 (s, 1H, NH), 4.70-4.71 (m, 2H,
NCH2), 4.82-4.83 (m, 2H, NCH2), 5.07 (s, 2H, CH2NH), 6.90 (d, J = 7.8 Hz, 3H), 7.07 (d, J =
13
7.8 Hz, 2H, ArH), 8.03 (s, 1H), 8.08 (s, 1H); C NMR (100 MHz, DMSO-d6) δ: 12.74,
13.59, 24.34, 36.39, 46.21, 48.78, 60.86, 114.48, 125.27, 129.22, 133.27, 134.40, 138.37,
143.33, 151.21, 155.97; ESI-MS (m/z) calculated for C15H17N7O2: 327.14, found: 327.18
[M]+; Anal. calcd. for C15H17N7O2: C, 55.04; H, 5.23; N, 29.95; O, 9.78; found: C, 55.31; H,
5.42; N, 30.15; O, 10.01.
5.5.25.
2-Methyl-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (27): Yield 58%; mp 151-152 oC; IR (nujol, cm-1): 3111, 3074, 2924, 2855,
1
1604, 1523, 1459, 1367, 1314, 1258, 1188, 1050, 742; H NMR (300 MHz, DMSO-d6) δ:
1.72 (s, 3H, CH3), 2.05 (s, 3H, CH3), 4.30 (d, J = 5.7 Hz, 2H, CH2NH), 4.60-4.65 (m, 2H,
NCH2), 4.75-4.76 (m, 2H, NCH2), 5.40 (t, J = 5.7 Hz, 1H, CH2NH), 6.44-6.47 (m, 2H, ArH),
6.91-6.93 (m, 2H, ArH), 7.76 (s, 1H), 7.99 (s, 1H); ESI-MS (m/z) calculated for C16H19N7O2:
341.16, found: 341.13 [M]+; Anal. calcd. for C16H19N7O2: C, 56.29; H, 5.61; N, 28.72; O, 9.37;
found: C, 56.52; H, 5.90; N, 28.88; O, 9.60.
5.5.26.
2-Fluoro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (28): Yield 65%; mp 110-111 oC; IR (KBr, cm-1): 3368, 3106, 2858, 1623,
1
1527, 1461, 1365, 1332, 1294, 1265, 1191, 1153, 1115, 1051, 823; H NMR (300 MHz,
DMSO-d6) δ: 1.72 (s, 3H, CH3), 4.33 (d, J = 6 Hz, 2H, CH2NH), 4.65 (t, J = 5.1 Hz, 2H,
NCH2), 4.78 (t, J = 5.1 Hz, 2H, NCH2), 5.97 (t, J = 6 Hz, 1H, NH), 6.53-6.55 (m, 1H, ArH),
6.64 (t, J = 8.1 Hz, 1H, ArH), 6.91 (t, J = 7.8 Hz, 1H, ArH), 6.99 (dd, J = 7.8, 4.2 Hz, 1H,
ArH), 7.83 (s, 1H), 8.01 (s, 1H); ESI-MS (m/z) calculated for C15H16FN7O2: 345.13, found:
345.09 [M]+; Anal. calcd. for C15H16FN7O2: C, 52.17; H, 4.67; F, 5.50; N, 28.39; O, 9.27;
found: C, 52.29; H, 4.80; F, 5.72; N, 28.54; O, 9.41.
5.5.27.
3-Fluoro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (29): Yield 55%; mp 139-140 oC; IR (film, cm-1): 3384, 3121, 3021, 1617,
19

ACCEPTED MANUSCRIPT
1
1526, 1464, 1363, 1261, 1187, 1147, 1046, 925, 823, 752; H NMR (400 MHz, CDCl3) δ:
1.94 (s, 3H, CH3), 4.33 (brs, 1H, NH), 4.40 (s, 2H, CH2NH), 4.77 (s, 4H, NCH2CH2N), 6.27-
6.31 (m, 1H, ArH), 6.37 (dd, J = 8.0, 1.5 Hz, 1H, ArH), 6.40-6.45 (m, 1H, ArH), 7.07-7.13
(m, 1H, ArH), 7.15 (s, 1H), 7.97 (s, 1H); ESI-MS (m/z) calculated for C15H16FN7O2: 345.13,
found: 346.19 [M + H]+; Anal. calcd. for C15H16FN7O2: C, 52.17; H, 4.67; F, 5.50; N, 28.39;
O, 9.27; found: C, 52.33; H, 4.82; F, 5.73; N, 28.76; O, 9.43.
5.5.28.
4-Fluoro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (30): Yield 57%; mp 138-139 oC; IR (KBr, cm-1): 3375, 3160, 3113, 3033,
1
2838, 1612, 1558, 1528, 1510, 1460, 1363, 1262, 1220, 1191, 1155, 1114, 1051; H NMR
(400 MHz, CDCl3) δ: 1.92 (s, 3H, CH3), 4.11 (brs, 1H, NH), 4.37 (s, 2H, CH2NH), 4.76 (s,
4H, NCH2CH2N), 6.53-6.56 (m, 2H, ArH), 6.86-6.90 (m, 2H, ArH), 7.13(s, 1H), 7.97 (s, 1H);
ESI-MS (m/z) calculated for C15H16FN7O2: 345.13, found: 346.20 [M + H]+; Anal. calcd. for
C15H16FN7O2: C, 52.17; H, 4.67; F, 5.50; N, 28.39; O, 9.27; found: C, 52.30; H, 4.59; F, 5.72;
N, 28.68; O, 9.50.
5.5.29.
2-Chloro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (31): Yield 59%; mp 146-142 oC; IR (nujol, cm-1): 3380, 3110, 3033, 2924,
2854, 1595, 1560, 1530, 1515, 1460, 1424, 1367, 1320, 1262, 1190, 1153, 1092, 1052, 1037;
1H NMR (300 MHz, DMSO-d6) δ: 1.74 (s, 3H, CH3), 4.39 (d, J = 6 Hz, 2H, CH2NH), 4.65 (t,
J = 5.1 Hz, 2H, NCH2), 4.78 (t, J = 5.1 Hz, 2H, NCH2), 5.88 (t, J = 6 Hz, 1H, NH), 6.55-6.65
(m, 2H, ArH), 7.05-7.10 (m, 1H, ArH), 7.23 (d, J = 7.8 Hz, 1H, ArH), 7.83 (s, 1H), 8.01 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 12.74, 38.28, 46.26, 48.70, 111.55, 116.81, 117.95,
123.60, 127.92, 128.93, 133.22, 138.33, 143.55, 145.96, 151.20; ESI-MS (m/z) calculated for
C15H16ClN7O2: 361.10, found: 361.14 [M]+, 363.12 [M + 2]+; Anal. calcd. for C15H16ClN7O2:
C, 49.80; H, 4.46; Cl, 9.80; N, 27.10; O, 8.84; found: C, 50.13; H, 4.69; Cl, 9.99; N, 27.27;
O, 9.11.
5.5.30.
3-Chloro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (32): Yield 60%; mp 101-102 oC; IR (KBr, cm-1): 3382, 3112, 3075, 2851,
1
1599, 1528, 1461, 1424, 1367, 1323, 1262, 1190, 1154, 1086, 1051, 986; H NMR (300
MHz, DMSO-d6) δ: 1.76 (s, 3H, CH3), 4.27 (d, J = 5.4 Hz, 2H, CH2NH), 4.67 (d, J = 3.3 Hz,
2H, NCH2), 4.79 (s, 2H, NCH2), 6.45-6.60 (m, 3H, ArH), 7.02-7.07 (m, 1H, ArH), 7.86 (s,
1H), 8.02 (s, 1H); ESI-MS (m/z) calculated for C15H16ClN7O2: 361.10, found: 362.14 [M +
20

ACCEPTED MANUSCRIPT
H]+, 364.16 [M + 2]+; Anal. calcd. for C15H16ClN7O2: C, 49.80; H, 4.46; Cl, 9.80; N, 27.10;
O, 8.84; found: C, 50.07; H, 4.69; Cl, 9.95; N, 27.31; O, 8.98.
5.5.31.
4-Bromo-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methyl) aniline (33): Yield 62%; mp 145-146 oC; IR (film, cm-1): 3402, 3134, 3012, 1594,
1
1527, 1465, 1428, 1362, 1260, 1188, 1145, 1048, 818, 752; H NMR (300 MHz, CDCl3) δ:
1.93 (s, 3H, CH3), 4.23 (brs, 1H, NH), 4.38 (d, J = 5.1 Hz, 2H, CH2NH), 4.76 (s, 4H,
NCH2CH2N), 6.49 (d, J = 8.8 Hz, 2H, ArH), 7.12 (s, 1H), 7.25 (dd, J = 8, 1.4 Hz, 2H, ArH),
7.97 (s, 1H); ESI-MS (m/z) calculated for C15H16BrN7O2: 405.05, found: 406.13 [M + H]+,
408.17 [M + 2]+; Anal. calcd. for C15H16BrN7O2: C, 44.35; H, 3.97; Br, 19.67; N, 24.14; O,
7.88; found: C, 44.66; H, 4.14; Br, 19.55; N, 24.66; O, 8.05.
5.5.32. 2,3-Dichloro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-
yl) methyl)aniline (34): Yield 62%; mp 148-149 oC; IR (KBr, cm-1): 3363, 3108, 2857, 1584,
1
1529, 1502, 1460, 1424, 1366, 1321, 1261, 1221, 1189, 1151, 1181, 1041, 899; H NMR
(300 MHz, DMSO-d6) δ: 1.74 (s, 3H, CH3), 4.42 (d, J = 5.7 Hz, 2H, CH2NH), 4.65 (t, J = 4.5
Hz, 2H, NCH2), 4.78 (t, J = 4.5 Hz, 2H, NCH2), 6.29 (t, J = 5.7 Hz, 1H, CH2NH), 6.59 (d, J =
8.4 Hz, 1H, ArH), 6.79 (dd, J = 8.1, 1.2 Hz, 1H, ArH), 7.05-7.11 (m, 1H, ArH), 7.82 (s, 1H),
13
8.01 (s, 1H); C NMR (100 MHz, DMSO-d6) δ: 12.76, 38.40, 46.25, 48.71, 109.84, 115.61,
116.95, 123.64, 128.29, 131.61, 133.22, 138.32, 145.33, 145.63, 151.16; ESI-MS (m/z)
calculated for C15H15Cl2N7O2: 395.06, found: 396.18 [M + H]+, 398.17 [M + 2]+, 400.17 [M +
4]+; Anal. calcd. for C15H15Cl2N7O2: C, 45.47; H, 3.82; Cl, 17.90; N, 24.74; O, 8.08; found:
C, 45.71; H, 3.93; Cl, 18.16; N, 24.98; O, 8.21.
5.5.33. 2,4,5-Trichloro-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-
o
4-yl) methyl)aniline (35): Yield 74%; mp 169-170 C; IR (film, cm-1): 3378 3113, 1581,
1
1528, 1463, 1428, 1363, 1264, 1191, 1149, 1047, 829, 754; H NMR (400 MHz, CDCl3) δ:
1.98 (s, 3H, CH3), 4.45 (d, J = 5.1 Hz, 2H, CH2NH), 4.79 (s, 4H, NCH2CH2N), 4.84 (t, J =
5.9 Hz, 1H, CH2NH), 6.71 (s, 1H, ArH), 7.18 (s, 1H), 7.35 (s, 1H, ArH), 7.98 (s, 1H); ESI-
MS (m/z) calculated for C15H14Cl3N7O2: 429.02, found: 430.08 [M + H]+, 432.11 [M + 2]+,
434.09 [M + 4]+, 436.21 [M + 6]+; Anal. calcd. for C15H14Cl3N7O2: C, 41.83; H, 3.28; Cl,
24.70; N, 22.77; O, 7.43; found: C, 42.11; H, 3.43; Cl, 24.69; N, 22.80; O, 7.58.
5.5.34. 3,5-Dimethoxy-N-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-
o
4-yl) methyl)aniline (36): Yield 69%; mp 136-138 C; IR (film, cm-1): 3397, 3131, 2956,
21

ACCEPTED MANUSCRIPT
1605, 1528, 1465, 1364, 1262, 1197, 1151, 1055, 930, 817, 753; 1H NMR (400 MHz, CDCl3)
δ: 1.95 (s, 3H, CH3), 3.73 (s, 6H, 2OCH3), 4.19 (brs, 1H, NH), 4.39 (s, 2H, CH2NH), 4.76 (s,
4H, NCH2CH2N), 5.80 (d, J = 2 Hz, 2H, ArH), 5.90-5.91 (m, 1H, ArH), 7.16 (s, 1H), 7.98 (s,
1H); ESI-MS (m/z) calculated for C17H21N7O4: 387.16, found: 388.21 [M + H]+; Anal. calcd.
for C17H21N7O4: C, 52.71; H, 5.46; N, 25.31; O, 16.52; found: C, 52.87; H, 5.71; N, 25.52; O,
16.33.
5.5.35.
N-((1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)
o
pyridine-2 amine (37): Yield 51%; mp 154-155 C; IR (film, cm-1): 3019, 1603, 1526, 1469,
1
1365, 1262, 1214, 1148, 1046, 745; H NMR (300 MHz, DMSO-d6) δ: 1.75 (s, 3H, CH3),
4.41 (d, J = 5.1 Hz, 2H, NHCH2), 4.64 (brs, 2H, NCH2), 4.75 (brs, 2H, NCH2), 6.45 (d, J =
6.9 Hz, 2H, ArH), 6.92 (brs, 1H, NH), 7.31-7.36 (m, 1H, ArH), 7.79 (s, 1H), 7.94 (s, 1H), 8.0
(s, 1H); ESI-MS (m/z) calculated for C14H16N8O2: 328.14, found: 328.19 [M]+; Anal. calcd. for
C14H16N8O2: C, 51.21; H, 4.91; N, 34.13; O, 9.75; found: C, 51.40; H, 5.22; N, 34.46; O,
10.03.
5.5.36. N-((1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-2-
o
phenylethanamine (38): Yield 52%; mp 79-80 C; IR (film, cm-1): 3383, 3132, 3026, 1654,
1
1608, 1533, 1466, 1430, 1366, 1263, 1191, 1147, 1046, 825, 753; H NMR (300 MHz,
DMSO-d6) δ: 1.86 (s, 3H, CH3), 2.76 (brs, 3H), 3.86 (s, 4H, CH2NHCH2), 4.70-4.72 (m, 2H,
NCH2), 4.82 (s, 2H, NCH2), 7.19-7.22 (m, 3H, ArH), 7.26-7.29 (m, 2H, ArH), 7.87 (s, 1H),
8.04 (s, 1H); ESI-MS (m/z) calculated for C17H21N7O2: 355.17, found: 355.15 [M]+; Anal.
calcd. for C17H21N7O2: C, 57.45; H, 5.96; N, 27.59; O, 9.00; found: C, 57.68; H, 6.13; N,
27.77; O, 9.25.
Acknowledgements
D.S.R. thanks SERB-Department of Science and Technology [EMR/2014/001127] New
Delhi, India for financial support. D.K. and Beena are thankful to CSIR for the award of
junior and senior research fellowship. Authors are also thankful to CIF-USIC, University of
Delhi, Delhi for spectral data.
References
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Table 1: In Vitro Anti-MRSA Activity of MTZ-Triazole Hybrids
Compound
R
MIC (µg/mL)
>128
13
18
14
6
128
-
64
1
32
3
7
2
-
16
10
-
8
2
-
4
1
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
9
-
-
1
-
-
1
-
-
-
-
-
-
-
-
CH2Br
4
CH2OCOEt
-
5
5
CH2OTHP
-
1
10
-
3
-
6
TMS
-
24
24
24
1
7
CH2CH2CH3
6
-
-
-
-
8
Cyclohexan-1-ol
Ph
6
-
-
-
-
9
12
4
-
0
1
4
8
-
9
1
4
-
8
3
3
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CH2OPh
-
18
2
CH2OCH3
16
9
-
CH2OPh-4-CH3
CH2OPh-4-iPr
CH2OPh-4-t-Bu
CH2OPh-2-CHO
CH2O-4-CHOPh
CH2OPh-2-Cl
CH2OPh-4-NO2
CH2OPh-2,3-Me
CH2OPh-2,4-Cl
CH2OPh-2,6-Cl
CH2OCH2Ph
CH2OPh-2,4,6-t-Bu
CH2O-1-napthyl
CH2NHPh
-
13
24
24
24
7
6
-
-
-
6
-
-
-
-
6
-
-
-
-
14
6
-
3
-
5
-
1
-
-
24
24
24
24
5
6
-
-
-
-
6
-
-
-
-
6
-
-
-
-
7
2
-
5
-
1
-
1
-
6
24
24
1
6
-
-
-
-
12
6
-
3
-
8
-
5
-
-
24
24
8
CH2NHPh-2-CH3
CH2NHPh-2-F
CH2NHPh-3-F
NHPh-4-F
6
-
-
-
-
5
-
0
0
-
12
1
-
4
-
15
7
-
14
23
6
-
-
CH2NHPh-2-Cl
CH2NHPh-3-ClPh
CH2NHPh-4-Br
CH2NHPh-2,3-Cl
CH2NH-2,4,5-ClPh
NHPh-3,5-OMe
7
1
-
6
1
5
-
5
-
5
-
15
7
14
15
14
24
14
1
-
2
1
-
-
15
6
-
-
-
-
15
-
0
1
-
28

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2-Aminopyridyl
NHCH2CH2Ph
6
-
24
14
4
-
-
-
-
-
-
-
-
37
15
20
-
0
4
1
1
38
Oxacillin
1
29