Design, synthesis, antibacterial activity and docking study of some new trimethoprim derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.10.051

In present study, nineteen novel trimethoprim (TMP) derivatives were designed, synthesized and evaluated for their antibacterial potential. Hydroxy trimethoprim 2 (HTMP) was synthesized by following the demethylation of 4-methoxy group at trimethoxy benzy

Full text of DOI:10.1016/j.bmcl.2016.10.051

Accepted Manuscript
Design, synthesis, antibacterial activity and docking study of some new trime-
thoprim derivatives
Umer Rashid, Waqas Ahmad, Syed Fahad Hassan, Naveeda Akhtar Qureshi,
Basit Niaz, Bakhtiar Muhammad, Sameera Imdad, Muhammad Sajid
PII:
S0960-894X(16)31089-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.051
BMCL 24354
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 June 2016
29 September 2016
17 October 2016
Please cite this article as: Rashid, U., Ahmad, W., Fahad Hassan, S., Akhtar Qureshi, N., Niaz, B., Muhammad, B.,
Imdad, S., Sajid, M., Design, synthesis, antibacterial activity and docking study of some new trimethoprim
derivatives, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.051
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis, antibacterial activity and docking study of some new
trimethoprim derivatives
Umer Rashid a,b,*, Waqas Ahmad a, Syed Fahad Hassan c, Naveeda Akhtar Qureshi d,
Basit Niaz a, Bakhtiar Muhammad a, Sameera Imdad e, Muhammad Sajid e
a Department of Chemistry, Hazara University, Mansehra 21120, Pakistan
b Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Department of Pharmacy, The University of Lahore, Defense Road Campus, Lahore 53700, Pakistan
d Department of Animal Sciences, Quaid-i-Azam University 45320, Islamabad
e Department of Biochemistry, Hazara University, Mansehra 21120, Pakistan
*Corresponding author. E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com
Corresponding Author Tel. +92 (992) 383591-6
.
_____________________________________________________________________________________
1

Abstract
In present study, nineteen novel trimethoprim (TMP) derivatives were designed, synthesized and
evaluated for their antibacterial potential. Hydroxy trimethoprim 2 (HTMP) was synthesized by following
the demethylation of 4-methoxy group at trimethoxy benzyl ring of TMP. Structure-activity relationship
(SAR) studies were explored on HTMP by incorporating various substituents leading to the identification
of some new compounds with improved antibacterial activities. The results revealed that the introduction
of benzyloxy (4a-e) and phenyl ethanone (5a-e) group at 4-position of dimethoxy benzyl ring leads to
overall increase in the antibacterial activity. The most potent antibacterial compound discovered is
benzyloxy derivative 4b with MIC value of 5.0 µM against S. aureus and 4.0 µM against E. coli strains
higher than the standard TMP (22.7 µM against S. aureus and 55.1 µM against E. coli). Substitution at 4-
NH2 group was not tolerated and the resulting Schiff base derivatives 3a-h demonstrated very little or no
antibacterial activity in the tested concentration domain. We further performed exploratory docking
studies on dihydrofolate reductase (DHFR) to rationalize the in vitro biological data and to demonstrate
the mechanism of antibacterial activity. For the ability to cross lipophilic outer membrane, log P was
computed. It was found that the compounds possessing high hydrophobicity have high activity against E.
coli.
Key words: Trimethoprim, dihydrofolate reductase, antibacterial, hydrophobicity, docking
2

The infectious diseases are associated with high rates of deaths worldwide. The discovery of antibacterial
chemotherapeutic agents has been considered as one of the most important achievements since the past
fifty years. However, resistance to current clinical antimicrobial drugs has certainly increased the global
health concern. Bacteria have the ability to render all the current anti-infective treatments useless. This
stimulates drug discovery researchers to develop new antimicrobial agents. Since the last decade, a
number of antibacterial drug discovery strategies have emerged.1-3 Among these strategies, structural
manipulation or chemical alteration and exploitation of the biochemical pathways inhibited by known and
previously characterized clinical antimicrobial drugs are the important strategies in drug design.4-5
Trimethoprim (TMP) is a small drug molecule containing 2-aminopyrimidine scaffold (Figure 1). TMP
was initially considered as antimalarial agent, but now considered as highly effective against bacterial
infections, especially in synergistic combination with sulfamethoxazole (Co-trimoxazole). TMP binds to
dihydrofolate reductase (DHFR) and its affinity for bacterial DHFR is 3000 time greater than human
DHFR.6-7 Dihydrofolate reductase (DHFR, EC 1.5.1.3) is a critical enzyme that catalyses the reduction of
dihydrofolate to tetrahydrofolate. DHFR has been the subject of much research as the target of
antimicrobial agents. Methotrexate (MTX), trimethoprim (TMP) and pyrimethamine, having 2-
aminopyrimidine moiety, are the selective inhibitors of DHFR. Increase resistance to TMP has limited its
use. The two rings of TMP allow for the structural changes which may resulting in different lipophilicity
and resistance profiles.8-9
The experience achieved by our research group in the field of computer aided drug design and synthesis of
chemical entities as putative drugs for the treatment of various diseases prompted us to further explore
new compounds as antibacterial drugs.10-13 Recently, we have reported medicinal chemistry approaches to
11
design and synthesize antileishmanial and antibacterial agents.10,
Considering the pharmacological
importance of TMP scaffold, it was planned to design and synthesize a variety of TMP-based potent
antibacterial compounds to diversify the current antimicrobials.
Strong DHFR inhibitors like trimethoprim differs from the folate substrates only in the replacement of 4-
oxo group by 4-NH2 group (Figure 1). Therefore, it is observed that the essential feature of the strong
DHFR inhibitors is 2,4-diaminopyrimidine ring. 2,4-diaminopyrimidine moiety forms hydrogen bond
3

interactions with Asp27, Leu5 and Phe92. While, the trimethoxy benzyl group is embedded in the
hydrophobic pocket. It is evident that trimethoxy benzyl group was not tightly fitted into the active site as
observed with para-aminobenzoate (pABA)-glutamate tail of methotrexate (MTX).14 From these
observations, we envisioned that the development of compounds that can bind tightly into the target may
show the desirable effects on their antibacterial properties. With a view to synthesize potent antibacterial
agents, we designed TMP derivatives by structural changes at both the TMP rings. Compound 2 (HTMP,
Scheme 1) was designed by following the demethylation strategy of methoxy group at para-position
trimethoxy benzyl ring. With an aim to design structural variants of HTMP (2) and to understand
structural requirements for DHFR binding, compounds 3a-h were designed by incorporating benzylidene
group at 4-NH2 . Similarly, compounds 4a-e and 5a-e were designed to enhance the binding affinity
through extra binding interactions with the DHFR.
In the first step, trimethoprim was reacted with 48% HBr to hydrolyse the ether into hydroxyl group.15
1
This hydroxyl group could be further used in nucleophilic substitution reactions. In H NMR spectrum of
HTMP 2, a broad singlet at 8.693 ppm of hydroxyl group indicates the hydrolysis (demethylation) of
methoxy group at 4-position. Two broad singlet signals at 7.89 and 7.44 ppm designates the amino protons
at 2- and 4-position respectively of 2,4-diaminopyrimidine ring. A singlet appears at 6.72 ppm shows the
presence two aromatic protons at dimethoxy benzyl ring. Similarly, a proton singlet signal at 7.68
assignable for an aromatic proton at 2,4-diaminopyrimidine ring. A singlet with six proton integration,
appears at 3.89 ppm, designates the methoxy protons. Another singlet at 3.49 ppm indicates the two
protons of –CH2 (Figure S-1 in Supplementary data). In LC-MS spectrum, a peak present at m/z 277.12
confirms the protonated molecular ion [M+H]+.
For the SAR studies, HTMP (2) was further used to design a set of novel antibacterials. First, we planned
the synthesis of benzylideneamino derivatives to evaluate the effect of modification on 4-amino group.
Eight Schiff base derivatives (3a-h) were synthesized by the reaction of different aryl aldehydes with
HTMP (2) as shown in Scheme 1.16 In 1H NMR spectrum of 3a, disappearance of peak of 4-NH2 protons
of HTMP (2) (at 7.44 ppm) and appearance of a downfield singlet signal at 8.30 ppm designates the imine
proton (–N=CH) and confirms the formation of Schiff base derivative 3a. A multiplet signal appears at
7.16 ppm shows the presence of five aromatic protons of the phenyl ring.
4

The synthesis of target molecules 4a-e starts with the nucleophilic reaction of 4-OH group with substituted
benzyl and acyl bromides in the presence of K2CO3 as base and acetonitrile as solvent (Scheme 2).17 The
synthesis of 5a-e was carried out as depicted in Scheme 3. For the synthesis of 5a-e, α-bromoketones (3-
7) were synthesized using substituted acetophenones, N-bromosuccinimide (NBS) and p-toluenesulfonic
acid (PTSA) under ultrasonic irradiations (Scheme 3).18 Purified α-bromoketones (3-7) were used for the
synthesis of target molecules 5a-e (Scheme 3).19 The reactions were monitored by using thin layer
chromatography (TLC). The synthesized 4a-e and 5a-e were characterized by their melting points (m.p.)
and spectroscopic analysis (1H-NMR, 13C-NMR and LC-MS). In 1H NMR spectra, these compounds were
confirmed by the disappearance of –OH peak of HTMP 2 at 8.69 ppm. Singlet signals appeared at 4.7
ppm assignable to –OCH2 protons for compounds 4a-e.
In vitro antibacterial screening was carried out and TMP was used as a standard drug for comparison with
the studied compounds. The compounds having antibacterial activity were then subjected to determine
Minimum Inhibitory Concentration (MIC).20 Table 1 enlists the in vitro MIC values against gram positive
and negative strains of bacteria. It is evident from Table 1 that some analogues were found to have high
potency than the standard drug trimethoprim (TMP). Demethylation of methoxy group at 4-position of
TMP resulted in comparable activity to TMP. Except compound 3c, all Schiff base derivatives (3a-h)
showed very little or no antibacterial activity in the tested concentration domain and hence these are
considered as poor antimicrobial compounds. 3-hydoxyphenyl containing compound (3c) has shown some
little activity with MIC value of 336 µM (128 µg/ml) against E. coli strain. Optimization studies of the R1-
position revealed that the potency of the compounds with substituted benzyloxy derivatives (4a-e) was
excellent. As can be seen, compound 4b with 4-OCH3 group has shown remarkable potency with MIC
value of 5 µM (2 µg/ml) for S. aureus and 4 µM (1.6 µg/ml) for E. coli strains. Replacement of methoxy
with chlorine and methyl (4c and 4d) resulted in the slight decrease in activity against S. aureus.
Derivatives with phenylethanone group (5a-e) have shown moderate-to-good activity with MIC value.
Compounds with 4-OCH3 and methyl group (5c and 5d) has shown excellent activity with MIC value of
18.7 µM (8 µg/ml) for both S. aureus and E. coli strains. Compound 2d with 4-CH3 group showed good
activity against E. coli strains (MIC 15.5 µM).
5

We have analyzed the variations in the in vitro antibacterial results of both the strains. We were in opinion
that these variations may be due to: 1) ability to enter the bacterial cytoplasm and even to cross nuclear
envelope; 2) Interaction/fitting of the compounds into the active site. High value of log P has a direct
effect with the potency of compounds against S. aureus and E. coli. Log P was predicted by ChemAxon
software package (Marvin v15.4.6, 2015).21 It is clear from the Table 1 that the compounds with high log
P value were found more active towards Gram negative strains than Gram positive strains. The reason is
the presence of more lipophilic outer membrane in Gram negative bacteria (E. coli) which act as an
efficient permeability barrier. This makes the results very promising against E. coli. The in vitro results in
Table 1 revealed that derivatives with high Log P values are found to be more potent against E. coli than
standard drug and gram positive strain.
Interactions of the compounds with important residues were determined by docking studies.
Computational docking studies were carried out using GOLD (Genetic Optimization for Ligand Docking)
suit v5.4.1 to understand the types of interactions and binding orientations.22-23 Based on the mechanism of
action of TMP, DHFR was chosen as a bacterial target to deduce the interactions and binding mode of
synthesized derivatives. X-ray structure of DHFR from S. aureus was retrieved from PDB (PDB ID
2W9S) with trimethoprim as co-crystallized ligand. The ability of the docking algorithm was validated to
reproduce the co-crystallized pose of TMP in the 2W9S pocket. This yielded a good agreement between
the docked and crystal structures as it further confirmed the interaction region of the ligand and a
favorable interaction with DHFR. The binding mode of hydroxy trimethoprim (2, HTMP) resembles that
of TMP. 2,4-diaminopyrimidine moiety forms hydrogen bond interactions with Asp27, Ile5 and Phe92.
Ser49 forms hydrogen bond with hydroxyl group (Figure 2a-b). The GOLD fitness score of TMP (62.34)
compared to HTMP (62.67) is in good agreement with bioactivity data.
We have found the different binding mode of these Schiff base derivatives (3a-h) due to their different
orientation in the binding site. This orientation could explain the very little or no antibacterial activity of
the Schiff base derivatives (Table 1). 2D binding orientations of 4-hydroxy (3b) and 3-hydroxy phenyl
(3c). These poses are generated by Discovery Studio Visualizer.24 Compound 3c with 4-hydroxy group
oriented itself in such a way that it forms hydrogen bonds with Ala7 and Asn18. While, compound 3c
forms hydrogen bond interactions with Ala7, Asp27 and Ser49 (Figure S-2 in Supplementary data).
6

These interactions along with high logP value may be reason of high activity of 3c against E. coli strain.
The good activity of 3c compared to 3b was further confirmed by Gold fitness score. The calculated
GOLD fitness score for inhibitor 3b is 41.05 and for 3c is 45.42.
The designed compounds 4a-e showed excellent activity and this was confirmed by the mode of
orientation of these compounds and their Gold fitness score. The docking results showed that these
compounds fit well in the binding cavity of DHFR. Compound 4b is considered as the best compound into
the binding site of 2W9S with the highest GOLD docking score of 79.89. It can be seen from the 2D
diagram from Discovery Studio Visualizer that compound 4b with exhibited four conventional hydrogen
bonds with Ile5 (1.84 Å), Asp27 (1.49 Å), Phe92 (2.99 Å). Another hydrogen bond at a distance of 2.10 Å
with Gln95 and 4-OCH3 stabilizes the ligand-enzyme complex (Figure 3a). By replacing methoxy group
(4b) with chlorine (4c), antibacterial activity decreased. Interaction pattern of compound 4c is shown in
Figure 3b. It can be seen that chlorophenyl moiety is located in the cavity space surrounded by Gly15,
Gln19, Lys45, Gln95 and Thr121. The computed Gold fitness score for 4c is 70.81.
Visual inspection of the docked poses of compounds 5a-e showed that these compounds are also well
stabilized in the cavity. Compound 5b is properly fitted into the binding site of 2W9S with the GOLD
docking score of 70.04. It can be seen from the 2D diagram from Discovery Studio Visualizer that
compound 5b exhibited six conventional hydrogen bonds with Asp27 (3.11 Å), Thr46 (3.78 Å), Phe92
(2.34 Å), Gln95 (2.25 Å) and with Lys45 (2.65 Å). On the other hand compounds with chloro and nitro
groups showed less affinity and Gold fitness score. The methyl substituted analogue (5d) showed four
conventional hydrogen bonds. 4-NH2 group forms HB interactions with Ile5 and Phe92 at the distance of
3.72 Å and 2.95 Å respectively. Carbonyl oxygen forms HB interactions with Tyr16 and Gln19 at the
distance of 1.90 Å and 3.21 Å respectively (Figure S-3 in Supplementary data).
In conclusion, by using rational design approach, we have designed and synthesized some novel
trimethoprim derivatives. Two strategies were adopted to understand structural requirements for DHFR
binding. In the first strategy, we designed compounds by incorporating substituents at 4-NH2 of 2,4-
diaminopyrimidine ring. In the second strategy, trimethoxy benzyl ring was used for the SAR study and
some benzyloxy and phenylethanone derivatives were synthesized. SAR studies have shown that
7

benzyloxy derivatives leads to excellent in vitro antibacterial against S. aureus and E. coli strains. Among
the derivatives synthesized, 4a-d exhibited the most potent activity. Based on the mechanism of action of
TMP, a comparative docking study on DHFR as target proved useful to explain to rationalize the in vitro
biological data and mechanism of antibacterial activity. Therefore, it is concluded that docking
investigations and in vitro activity revealed a successful drug design.
Acknowledgments
We are thankful to The Cambridge Crystallographic Data Centre (CCDC) for providing license of
GOLD software to COMSATS Institute of Information Technology. We are also extend our sincere
thanks to Islamabad Pharmaceutical Products (IPP), Islamabad for providing sample of TMP.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Silver, L. L. Clin. Microbiol. Rev. 2011, 24, 71–109.
Payne, D.J.; Gwynn, M.N.; Holmes, D.J.; Pompliano, D.L. Nat. Rev. Drug Discov. 2007, 6, 29.
Gwynn, M.N.; Portnoy, A.; Rittenhouse, S.F.; Payne, D.J. Ann. N.Y. Acad. Sci. 2010, 1213, 5.
Chopra, I.; Hodgson, J.; Metcalf, B.; Poste, G. Antimicrob. Agents Chemother. 1997, 41, 497.
Knowles, D.J.C. Trends Microbiol. 1997, 5, 379.
Tawari, N.R.; Bag, S.; Degani, M.S. Curr. Pharm. Des. 2011, 17, 712.
Rao, A.S.; Tapale, S.R. Intern. J Pharm. Sci. Res. 2013, 4, 2535.
Sharma, M.; Chauhan, P.M. Future Med. Chem. 2012, 4, 1335.
Banerjee, D.; Kuckuka, P.M.; Capiauxa, G.; Alpdogana, T. B.; Gorlickc, R.; Bertino, J.R.
Biochimica et Biophysica Acta, 2002, 1587, 164.
10. Rashid, U.; Sultana, R.; Shaheen, N.; Hassan, S.F.; Yaqoob, F.; Ahmad, M.J.; Iftikhar, F.;
Sultana, N.; Asghar, S.; Yasinzai, M.; Ansari, F.L.; Qureshi, N.A. Eur. J. Med. Chem. 2016, 115,
230.
11. Ahmad, M.J.; Hassan, S. F.; Nisa, R.U.; Ayub, K.; Nadeem, M.S.; Nazir, S.; Ansari, F.L.;
Qureshi, N.A.; Rashid, U. Med. Chem. Res. (in press) 2016, DOI. 10.1007/s00044-016-1613-z
12. Rashid, U.; Rahim, F.; Taha, M.; Arshad, M.; Ullah, H.; Mahmood, T.; Ali. M. Bioorg. Chem.
2016, 66, 111.
8

13. Rashid, U.; Batool, I.; Wadood, A.; Khan, A.; Qasmi, Z.U.; Chaudary, M.I.; Ansari, F.L. J. Mol.
Graphics Modell. 2013, 43, 47.
14. Heaslet, H.; Harris, M.; Fahnoe, K.; Sarver, R.; Putz, H.; Chang, J. Subramanyam, C. Barreiro,
G. Miller, J.R. Proteins 2009, 76, 706.
15. Ando, T.; Tsukiji, S.; Tanaka, T.; Nagamune, T. Chem. Commun. (Camb). 2007, 21, 4995.
General procedure for synthesis of compound 2. 20.0 g of trimethoprim (68.88 mmol) was
dissolved in 40 % HBr at room temperature. The mixture was then stirred at 100 ºC. After
completion of reaction (TLC), the mixture was quenched addition of 50 % NaOH aq. (40-50
mL). After cooling to room temperature, it was placed at 4 ºC overnight to form crystals. The
crystals were filtered and washed with ice-cold water (ca. 100 mL). After the crystals were
dissolved in boiling water, the solution was neutralized to ca. pH 7 with 28% aqueous ammonia
and placed at 4 ºC overnight for recrystallization. White crystals were filtered, washed with ice-
cold water (ca. 100 mL), affording compound 2.
White crystals. Yield 70%. Rf: 0.47 (n-hexane/ethyl acetate 1:1). 1H NMR (300 MHz, DMSO-
d6): δ 8.69 (brs, 1H, OH), 7.89 (brs, 2H, NH2), 7.68 (s, 1H, Ar-H), 7.44 (brs, 2H, NH2), 6.73 (s,
2H, Ar-H), 3.89 (s, 6H, O-CH3), 3.49 (s, 2H, CH2). 13C-NMR (75 MHz, DMSO- d6): δ 162.0,
161.1, 160.1, 152.3, 130.9, 129.8, 106.8, 105.1, 56.2, 37.3. LC-MS for C13H16N4O3 (m/z): Found
277.12 [M+H].
16. General procedure for synthesis of Schiff base derivatives of hydroxyl trimethoprim
(HTMP, 3a-h). In a pyrex tube containing 5 ml ethanol, add appropriate aromatic aldehydes (4
mmol) and compound 2 (4 mmol). Then one drop of sulphuric acid as catalyst was added. The
o
reaction mixture was then irradiated under ultrasonic bath at 40 C for 3-4 hr. The reaction was
monitored by TLC. After the completion of reaction, cool the reaction mixture and evaporated
the solvent and the resulting crude product was recrystallized from ethanol.
17. General procedure for synthesis of benzyloxy derivatives of HTMP (4a-e): To a solution of
hydroxyl TMP (HTMP, 2) (4 mmol) in acetonitrile (25 mL) in a Pyrex tube was added potassium
carbonate (8 mmol) and substituted benzyl halides (4 mmol). The mixture was stirred at 40-45 ºC
for 30-45 minutes in ultrasonic bath. Reaction mixture was then filtered, diluted with ethyl
9

acetate. Organic layer was washed with 3 x 10 ml of NaHCO3 and then with brine (20 ml). The
crude residue after drying over anhydrous MgSO4, purified with silica gel column
chromatography to afford the corresponding benzyloxy derivatives.
18. Guan, X.Y.; Misba, Z.A.; Huang, K.-W. Arab. J. Chem. 2015, 8, 892.
General procedure of the synthesis of α–Bromination of acetophenones (3-7): To a solution
of hydroxyl TMP (HTMP, 2) (4 mmol) in acetonitrile (25 mL) in a Pyrex tube was added
potassium carbonate (8 mmol) and substituted benzyl halides (4 mmol). The mixture was stirred
at 40-45 ºC for 30-45 minutes in ultrasonic bath. Reaction mixture was then filtered, diluted with
ethyl acetate. Organic layer was washed with 3 x 10 ml of NaHCO3 and then with brine (20 ml).
The crude residue after drying over anhydrous MgSO4, purified with silica gel column
chromatography to afford the corresponding benzyloxy derivatives.
19. General procedure for synthesis of phenyl ethanone derivatives of HTMP (5a-e): To a
solution of hydroxyl TMP (HTMP, 2) (4 mmol) in acetonitrile (25 mL) in a Pyrex tube was
added potassium carbonate (8 mmol) and α–bromoketones (3-7, 4 mmol). The mixture was
stirred at 40-45 ºC for 30-45 minutes in ultrasonic bath. Reaction mixture was then filtered,
diluted with ethyl acetate. Organic layer was washed with 3 x 10 ml of NaHCO3 and then with
brine (20 ml). The crude residue after drying over anhydrous MgSO4, purified with silica gel
column chromatography to afford the corresponding phenyl ethanone derivatives.
20. Antibacterial Screening: Trimethoprim derivatives were tested for their antibacterial activity.
Antibacterial assay was used to find the effectiveness of newly synthesized compounds for
inhibition of bacterial growth. The microorganisms Escherichia coli and Staphylococcus aureus
were used for this study. This antibacterial activity was checked by agar well diffusion method.
The compounds having antibacterial activity were then subjected to determine Minimum
Inhibitory Concentration (MIC). MIC is the concentration of test compound at which the
minimum or least inhibitory zone was detected. MIC was checked at lower concentrations of
active test compounds (0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 μg/mL). All the tests were
performed in triplicate. MIC was expressed in µM against each concentration (Table 1, shown in
10

parenthesis in µg/mL). The reported MIC value are an average of at least three individual
measurements.
21. www.chemaxon.com
22. Verdonk, M.L.J.; Cole, C.; Hartshorn, M.J.; Murray, C.W.; Taylor, R.D. Proteins 2003, 52, 609.
23. Computational studies: Log P was predicted by ChemAxon software package (Marvin v15.4.6,
2015). Docking experiment was carried using GOLD docking program 5.4.1. GOLD uses the
Genetic algorithm (GA). This method allows a partial flexibility of protein and full flexibility of
ligand. X-ray structure of DHFR from S. aureus was retrieved from PDB (PDB ID 2W9S) with
TMP as co-crystallized ligand. As a first step, the ability of the docking algorithm was validated
to reproduce the co-crystallized pose of TMP in the 2W9S pocket. This yielded a good agreement
between the docked and crystal structures as it further confirmed the interaction region of the
ligand. Gold Score was selected as the criteria for the selection of compounds because they serve
as fitness function for the orientation, and estimates of binding affinity. Binding site was defined
as the residues within 6 Å from the ligand. No water was present in any binding site. The default
docking protocol was applied (1.09 auto settings, 10 GA) and the best pose saved. Each
experiment was then repeated 10 times. The view of the docking results and analysis of their
surface with graphical representations were done using Discovery Studio Visualizer.
24. Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 4.5, San Diego:
Dassault Systèmes, 2015
11

Figure Legends
Figure 1: Comparison of the structures of the inhibitors and substrates of DHFR. Inhibitors differs from
the folate substrates only in the replacement of 4-oxo group (blue circle) by 4-NH2 group (pink circle). a)
Structures of two inhibitors of DHFR; Trimethoprim and Methotrexate; b) Structures of the two substrates
of DHFR; Folic acid and dihydrofolic acid.
Figure 2: 2D interaction plot of: a) TMP (1) and HTMP (2) generated by Discovery Studio Visualizer
showing interaction of with key amino acid residues. Conventional hydrogen bonding is shown by green
color dotted lines.
Figure 3: 2D binding interaction plot of the most active compounds generated by Discovery Studio
Visualizer s: a) Compound 4b; b) Compound 4c.
12

Table 1: MIC values (µM) of trimethoprim derivatives
R
NH
OCH3
N
R1
O
H2N
N
OCH3
MIC (µM)
(µg/ml)
Compounds
R
R1
MW
Log P
S. aureus
E. coli
H
H
H
H
H
H
H
276.1 1.14
364.1 3.84
380.1 3.72
380.1 3.72
399.1 1.16
378.1 4.47
24.1 (6.6)a
NI
672 (256)
672 (256)
NI b
48.2 (13.3)
NI
672 (256)
336 (128)
NI
2
=CH-C6H5
3a
3b
3c
3d
3e
=CH-(4-OH-C6H4)
=CH-(3-OH-C6H4)
=CH-(3-NO2-C6H4)
=CH-(4-CH3-C6H4)
NI
NI
535.3
(213.3)
NI
NI
18.2 (6.6)
5 (2)
20 (8)
10 (4)
32.3 (13.3)
81.2 (32)
535.3
(213.3)
NI
=CH-(4-Cl-C6H4)
H
398.1 4.52
3f
=CH-(4-NMe2-C6H4)
=CH-(4-OCH3-C6H4)
H
H
407.2 4.27
394.2 3.75
366.1 3.01
396.2 2.85
399.1 3.61
380.2 3.52
411.1 2.14
394.1 2.52
3g
3h
4a
4b
4c
4d
4e
5a
NI
H
H
H
H
H
H
-CH2-C6H5
18.2 (6.6)
4.0 (1.6)
8.3 (3.3)
5 (2)
39 (16)
81.2 (32)
-CH2-(4-OCH3-C6H4)
-CH2-(4-Cl-C6H4)
-CH2-(4-CH3-C6H4)
-CH2-(4-NO2-C6H4)
-CH2-CO-C6H5
-CH2-CO-(4-OCH3-
C6H4)
H
428.1 2.36
18.7 (8)
18.7 (8)
5b
H
H
H
-CH2-CO-(4-Cl-C6H4)
408.2 3.12
78.4 (32)
37.7 (16)
72.9 (32)
22.7 (6.6)
78.4 (32)
15.5 (6.6)
72.9 (32)
55.1 (16)
5c
5d
5e
-CH2-CO-(4-CH3-C6H4) 424.2 3.03
-CH2-CO-(4-NO2-C6H4) 439.1 3.04
290.3 1.28
Trimethoprim
13

a The reported MIC values in µg/ml (shown in parenthesis) are an average of at least three individual measurements.
b NI=No inhibition shown in the tested concentration domain
FIGURES
Figure 1: Comparison of the structures of the inhibitors and substrates of DHFR. Inhibitors differs from
the folate substrates only in the replacement of 4-oxo group (blue circle) by 4-NH2 group (pink circle). a)
Structures of two inhibitors of DHFR; Trimethoprim and Methotrexate; b) Structures of the two substrates
of DHFR; Folic acid and dihydrofolic acid.
14

Figure 2: 2D interaction plot of: a) TMP (1) and HTMP (2) generated by Discovery Studio Visualizer
showing interaction of with key amino acid residues. Conventional hydrogen bonding is shown by green
color dotted lines.
15

Figure 3: 2D binding interaction plot of the most active compounds generated by Discovery Studio
Visualizer s: a) Compound 4b; b) Compound 4c.
16

Schemes
R1
NH2
NH2
N
N
OCH3
OCH3
OCH3
OH
N
b
OCH3
OH
a
N
N
H2N
N
H2N
N
H2N
OCH3
OCH3
OCH3
3a-h
1
2
Scheme 1: Synthesis of Schiff bases 3a-h. Reagents and conditions: a) 48% HBr b) Substituted aromatic aldehydes,
ethanol, H2SO4, ultrasonic bath, 40 oC, 3-4 hr.
17

NH2
N
NH2
N
OCH3
O
OCH3
OH
a
N
N
H2N
H2N
OCH3
R1
OCH3
4a-e
2
Scheme 2: Synthesis of compounds 4a-e. Reagents and conditions: a) Substituted benzyl bromides, K2CO3, ACN,
ultrasonic bath, 40-45 oC, 30-45 min.
18

O
O
a
Br
CH3
R
R
3-7
NH2
NH2
N
OCH3
O
OCH3
OH
b
N
N
R2
H2N
N
H2N
OCH3
O
OCH3
5a-e
2
Scheme 3: Synthesis of compounds 5a-e. Reagents and conditions: a) NBS, PTSA CH2Cl2, ultrasonic bath, 40-45 oC,
40 min; (b) α-bromoketones (3-7), K2CO3, ACN, ultrasonic bath, 40-45 oC, 30-45 min.
19

Design, synthesis, antibacterial activity and docking study of some new
trimethoprim derivatives
Umer Rashid*, Waqas Ahmad, Syed Fahad Hassan, Naveeda Akhtar Qureshi, Basit Niaz,
Bakhtiar Muhammad, Sameera Imdad, Muhammad Sajid
*Corresponding author. E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com
Corresponding Author Tel. +92 (992) 383591-6
Graphical Abstract
20