Design, synthesis and biological activity of novel substituted 3-benzoic acid derivatives as MtDHFR inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115600

The enzyme dihydrofolate reductase from M. tuberculosis (MtDHFR) has a high unexploited potential to be a target for new drugs against tuberculosis (TB), due to its importance for pathogen survival. Preliminary studies have obtained fragment-like molecule

Full text of DOI:10.1016/j.bmc.2020.115600

Journal Pre-proofs
Design, synthesis and biological activity of novel substituted 3-benzoic acid
derivatives as MtDHFR inhibitors
Thales Kronenberger, Glaucio Monteiro Ferreira, Alfredo Danilo Ferreira de
Souza, Soraya da Silva Santos, Antti Poso, João Augusto Ribeiro, Maurício
Temotheo Tavares, Fernando Rogério Pavan, Gustavo Henrique Goulart
Trossini, Marcio Vinícius Bertacine Dias, Roberto Parise-Filho
PII:
DOI:
Reference:
S0968-0896(20)30430-2
https://doi.org/10.1016/j.bmc.2020.115600
BMC 115600
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 February 2020
2 June 2020
16 June 2020
Please cite this article as: Kronenberger, T., Monteiro Ferreira, G., Danilo Ferreira de Souza, A., da Silva Santos,
S., Poso, A., Augusto Ribeiro, J., Temotheo Tavares, M., Rogério Pavan, F., Henrique Goulart Trossini, G.,
Vinícius Bertacine Dias, M., Parise-Filho, R., Design, synthesis and biological activity of novel substituted 3-
benzoic acid derivatives as MtDHFR inhibitors, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/
10.1016/j.bmc.2020.115600
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1
2
Design, synthesis and biological activity of novel substituted 3-benzoic
acid derivatives as MtDHFR inhibitors
3
4
5
6
7
Thales Kronenbergera,b¥, Glaucio Monteiro Ferreirac¥, Alfredo Danilo Ferreira de Souzad¥, Soraya
da Silva Santose, Antti Posoa,b, João Augusto Ribeirof, Maurício Temotheo Tavaresd, Fernando
Rogério Pavang, Gustavo Henrique Goulart Trossinih, Marcio Vinícius Bertacine Diasf,i, Roberto
Parise-Filhod*
8
a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Department of Oncology and Pneumonology, Internal Medicine VIII, University Hospital
Tübingen, Otfried-Müller-Straße 10, DE 72076, Tübingen, Germany.
b School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio,
Finland.
c Laboratory of Molecular Biology applied to Diagnosis (LBMAD), Department of Pharmacy,
Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
d
Laboratory of Design and Synthesis of Bioactive Substances (LAPESSB), Faculty of
Pharmaceutical Sciences, University of São Paulo, Prof. Lineu Prestes Avenue, 580, Bl.13, São
Paulo, SP, Brazil.
Laboratory of Design and Synthesis of Chemotherapeutics Potentially Active in Neglected
Diseases (LAPEN), Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of
São Paulo, São Paulo, SP, Brazil.
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São
Paulo, SP, Brazil
Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State
e
f
g
University, Araraquara (UNESP Araraquara), São Paulo, Brazil
h Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, Prof.
Lineu Prestes Avenue, 580, Bl.13, São Paulo, SP, Brazil.
i Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK
28
29
30
31
¥The authors contributed equally to this work
*Roberto Parise-Filho. Tel.: +55 11 30913793. E-mail: roberto.parise@usp.br
32 Abstract
33 The enzyme dihydrofolate reductase from M. tuberculosis (MtDHFR) has a high
34 unexploited potential to be a target for new drugs against tuberculosis (TB), due
35 to its importance for pathogen survival. Preliminary studies have obtained
36 fragment-like molecules with low affinity to MtDHFR which can potentially
37 become lead compounds. Taking this into account, the fragment MB872 was
38 used as a prototype for analogue development by bioisosterism/retro-
39 bioisosterism, which resulted in 20 new substituted 3-benzoic acid derivatives.
40 Compounds were active against MtDHFR, with IC50 values ranging from 7 to
41 40 μM, where compound 4e not only had the best inhibitory activity (IC50 = 7 μM),
42 but also was 71-fold more active than the original fragment MB872. The 4e
43 inhibition kinetics indicated an uncompetitive mechanism, which was supported
44 by molecular modeling which suggested that the compounds can access an
1

45 independent backpocket from the substrate and competitive inhibitors. Thus,
46 based on these results, substituted 3-benzoic acid derivatives have strong
47 potential to be developed as novel MtDHFR inhibitors and also anti-TB agents.
48
49 Keywords: bioisosterism, fragment optimization and drug design, MtDHFR,
50 tuberculosis.
51
52 1. Introduction
53
Tuberculosis (TB) is an infectious disease caused by mycobacteria,
54 especially Mycobacterium tuberculosis, and generally affects the lung tissue,
55 inducing extensive lesions [1]. WHO estimates that one-third of the world
56 population is contaminated with M. tuberculosis, highlighting that about 10.4
57 million cases and 1.7 million deaths from TB have occurred in 2016 [2]. HIV/AIDS
58 co-infection is an important risk factor, which increases the rate of disease
59 progression from latent to active illness [3]. In addition, the ever-increasing
60 number of cases of multidrug resistance to the first-choice drugs, such as
61 rifampicin, isoniazid, ethambutol and pyrazinamide (Fig. 1A), is alarming. The
62 phenomenon of resistance occurs, besides other factors, as a consequence of
63 low adherence to long-term treatment [4].
64
Recently, M. tuberculosis resistant strains to bedaquiline (Fig. 1A), one of
65 the last standing approved antituberculosis drugs, were reported [1,5,6]. The rise
66 of new resistant strains emphasizes the urgent need for new therapeutic options
67 against TB. The process of discovery of new therapeutics can include the
68 validation of new potential drug targets and the search of new small molecules
69 with antibiotic activity.
70
In this sense, the folate pathway is an excellent option of validated drug
71 targets, since most of the bacteria cannot uptake folate from the medium and
72 depend on the de novo biosynthesis for survival within the host [7]. The folate
73 biosynthesis in M. tuberculosis is composed of six enzymes that convert GTP to
74 tetrahydrofolate, which is essential for the production of some amino acids and
75 DNA [8].
76
Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) is an
77 essential enzyme in the pathogen folate metabolism. MtDHFR catalyzes the
78 reduction of dihydrofolic acid to tetrahydrofolic acid (Fig. 1B), an essential step
2

79 for the activation of folate [9,10], using NADPH as a cofactor. Mechanistically,
80 upon substrate binding, the nicotinamide moiety from NADPH can engage in the
81 active site and promote catalysis. This movement of the nicotinamide moiety in
82 and out of the active site allows the enzyme to adopt two distinct conformations,
83 an open and a closed state, which has also been described for other DHFR’s,
84 including for human one [11].
85
Antifolates, such as cycloguanil, pyrimethamine, trimethoprim and
86 WR99210 (Fig. 1C) have shown moderate inhibitory activity against MtDHFR
87 [12,13] and recent structural studies hypothesize that this is due to the enzyme
88 flexibility, where the inhibitors would bind to only one of the two conformational
89 states [14,15]. Nevertheless, Wiktor and colleagues demonstrated that synergism
90 treatment using trimethoprim (Fig. 1) and sulfamethoxazole could still greatly
91 reduce TB morbidity and mortality [16]. Although trimethoprim and other DHFR
92 inhibitors have important roles in combating several infections, they are often not
93 used in the clinical treatment of tuberculosis, since monotherapy has little or no
94 effect on the M. tuberculosis growth [14,15].
95
3

96
97
98
99
Fig. 1. A) Drugs in clinical use for tuberculosis treatment. B) The tetrahydrofolate, product from
MtDHFR. C) Compounds with reported activity against MtDHFR.
100
MtDHFR is an essential enzyme for the pathogen and the disruption of
101 folate production impairs DNA synthesis and leads to metabolic stress in the
102 mycobacteria [17–22]. However, despite the obvious druggable potential, there
103 are still no MtDHFR inhibitors available for TB treatment and, consequently, this
104 enzyme rises as a promising drug target. Overall, DHFR inhibitors reported in the
105 literature suffer from low selectivity against the human counterpart leading to
106 side-effects [23], low bacteria cell permeability, and high resistance rate [14,24].
107
Recently, our group identified, from a fragment-based drug design
108 campaign, the compound MB872 (Fig. 1C), which, despite a low affinity for
109 MtDHFR (0.5 mM on binding assays), has an on-target specific activity and a
4

110 moderate ligand efficiency (LE=0.26) [25]. MtDHFR:NADPH in complex with
111 MB872 suggests a potential interaction between the carboxylic acid moiety with
112 Arg32 or Arg60, depending on the pose. So, MB872 was used as a ligand
113 prototype for a ligand-based drug design strategy, resulting in a novel series of
114 twenty substituted 3-benzoic acid derivatives active against MtDHFR.
115
116 2. Results and Discussion
117 2.1. Molecular design
118
A screening study regarding the affinity of 1,250 chemical fragments for
119 MtDHFR identified MB872 as an interesting protein ligand with an estimated
120 affinity value of about 0.5 mM. Later structural results [25] led us to design
121 molecules derived from MB872. The proposed modifications address three
122 regions (A, B and C) and a total of 20 compounds were designed and distributed
123 into three distinct series (I, II and III), as shown in Figure 2.
124
A
B
C
O
O
HO
Series III
Series I
Series II
REGION C
REGION B
REGION A
Explore different modes
of interaction with
Arg60 residue
Biososteric replacement
or introduction of
Insertion of HBA
and/or HBD groups
heterocyclic ring
Y
W
R1
R2
O
R1
O
R2
R2
R1 = OMe R2 = m-COOH
R1 = OMe R2 = m-COOMe
R1 = NO2 R2 = m-COOH
R1 = NO2 R2 = m-COOMe
R1 = OH R2 = m-COOH
R1 = H;
R1 = H;
R1 = H;
R1 = OMe; R2 = p-COOH
R1 = OMe; R2 = p-COOMe
R2 = m-COOMe
R2 = p-COOH
R2 = p-COOMe
W = NH; Y = CH2; R2 = m-COOH
W = S; Y = CH2; R2 = m-COOH
W = S; Y = CH2; R2 = m-COOMe
W = CH2; Y = O; R2 = m-COOH
W = CH2; Y = O; R2 = m-COOMe
W/Y = 1,2,3-triazol; R2 = m-COOH
W/Y = 1,2,3-triazol; R2 = m-COOMe
R1 = H;
R2 = m-tetrazol
W = O; Y = CH2-1,2,3-triazol; R2 = m-COOH
W = O; Y = CH2-1,2,3-triazol; R2 = m-COOMe
125
126
Fig. 2. Design of 20 compounds from MB872 divided into three compound series.
127
128
In 2020, Ribeiro et al., reported that region A is near residues, which are
129 potentially acceptors and donors of hydrogen bond (Tyr100, Ser49 and with the
5

130 oxygen of backbone Ile94). Thus, for the moiety A, hydrogen bonding donor and
131 acceptor groups were introduced into the ring, which may have provided a higher
132 possibility of polar interaction with the pocket (Fig. 2). Besides evaluating the
133 possible hydrogen bonds, those groups might have also revealed some
134 electronic/lipophilic effects of the substituents on the ring (Series I).
135
In region B (Fig. 2), we have employed bioisosterism and retro-bioisosterism
136 approaches in order to obtain systematic changes of substituents and thus,
137 generate diversities for structural and biological effects (Series II). Analogues
138 containing a third ring (triazole) in the presence of a spacer group and/or a third
139 ring in this region intended to increase hydrophobic interactions, in the case of
140 the rings, or hydrogen bond interactions, by varying the position of the
141 heteroatom.
142
In region C (Fig. 2), variations in the carboxyl group’s position were
143 performed to evaluate the relevance of ionic interaction with polar residues at the
144 active site. Additionally, also using the bioisosterism, the carboxyl was substituted
145 by a tetrazole, which is an acid bioisostere with a hydrophobic feature.
146 Additionally, esters counterparts of the free carboxylic acids were synthesized to
147 evaluate their role in the biological activity (Series III).
148
149 2.2. Chemistry
150
The prototype MB872 and the final products 4b-i, 7 and 10a-b were obtained
151 in two/three reaction steps, as shown in Scheme 1. The synthesis of the
152 intermediates 3a-j was based on classical alkylations of several phenols and
153 anilines, substituted or not, with the corresponding alkyl halide. For the synthesis
154 of compound 7, the intermediate 3j was previously reduced to the amine
155 derivative 5, which reacted to form a diazonium salt, which underwent
156 replacement by a cyanide ion to obtain the intermediate 6. Lastly, the nitrile
157 underwent a 1,3-dipolar cycloaddition resulting in the tetrazole analogue 7. In
158 order to synthesize the intermediates 9a and 9b, we also performed a 1,3-dipolar
159 cycloaddition reaction. The compounds MB872, 4b-i and 10a-b were synthesized
160 by hydrolysis of the methyl ester, using potassium hydroxide. Overall, we
161 obtained 20 final products, which were characterized by melting point, 1H and 13C
162 NMR.
163
6

Y
W
Y
W
i
ii
R1
W
Y
+
R2
R1
R2
R1
R2
3a
3b
3c
3d
3e
3f
3g
3h
3i
R1= H; R2= m-COOMe; W = O; Y= CH2
R1= MeO; R2= m-COOMe; W = O; Y= CH2
R1= OH; R2= m-COOMe; W = O; Y= CH2
R1= NO2; R2= m-COOMe; W = O; Y= CH2
R1= H; R2= m-COOMe; W = NH; Y= CH2
R1= H; R2= m-COOMe; W = S; Y= CH2
R1= H; R2= m-COOMe; W = CH2; Y= O
R1= H; R2= p-COOMe; W = O; Y= CH2
R1= OMe; R2= p-COOMe; W = O; Y= CH2
2a
4a R1= H; R2= m-COOH; W = O; Y= CH2 (MB872)
4b R1= MeO; R2= m-COOH; W = O; Y= CH2
4c R1= OH; R2= m-COOH; W = O; Y= CH2
4d R1= NO2; R2= m-COOH; W = O; Y= CH2
Y= CH2Br; R2= m-COOMe
Y= OH; R2= m-COOMe
Y= CH2Br; R2= p-COOMe
Y= CH2Br; R2= m-NO2
1a
R1= H; W = OH
2b
2c
2d
1b
1c
1d
1e
1f
R1= MeO; W = OH
1 OH; W = OH
R
R1= NO2; W = OH
R1= H; W = NH2
R1= H; W = SH
4e
R1= H; R2= m-COOH; W = NH; Y= CH2
R1= H; R2= m-COOH; W = S; Y= CH2
R1= H; R2= m-COOH; W = CH2; Y= O
R1= H; R2= p-COOH; W = O; Y= CH2
R1= OMe; R2= p-COOH; W = O; Y= CH2
4f
4g
4h
4i
1g
R1= H; W = CH2Br
3j
R
1 = H; R2= m-NO2; W = O; Y= CH2
iii
iv
v
O
O
O
O
N
NO2
NH2
CN
HN
N
N
3j
5
6
7
N
N
N
N
N
X
vi
X
ii
W
N
COOMe
COOH
8a
8b
9a
9b
10a
10b
W = CCH
W = OCH2CCH
X = Phenyl
X = Phenoxymethyl
X = Phenyl
X = Phenoxymethyl
164
165
166
167
168
Scheme 1. Synthesis of MB872, 4b-i, 7 and 10a-b. Reagents and conditions: (i)
K2CO3/acetone/reflux/16 h; (ii) KOH/THF/HCl/r.t./12 h; (iii) Pd/H2/MeOH/EtOH/r.t./4 h; (iv)
NaNO2/HCl/CuCN/NaCN/r.t./2 h; (v) NaN3/NH4Cl/DMF/reflux/16 h; (vi) ascorbate/CuI/r.t./12
h/methyl 3-azidobenzoate.
169
170 2.3. MtDHFR inhibitory activity and structure-activity relationship (SAR)
171 The MtDHFR inhibitory activity of our compound series was evaluated
172 in vitro by following the fluorescence emitted by MtDHFR substrates when excited
173 at 340 nm (Table 1). The enzymatic activity was measured by consuming the
174 enzyme substrate as the product (NADP+) does not emit fluorescence [27,28]. In
175 this way, trimethoprim IC50 value (91 μM) was determined and used as a positive
176 control for the assay, being in agreement with the literature data (88 μM) [15].
177
Most of the tested compounds were significantly more active against
178 MtDHFR (Table 1) than the initial prototype MB872 and eight of them (3d, 3f, 3i,
179 4c, 4d, 4e, 4i and 9b) showed stronger inhibition profile than trimethoprim. We
180 observed that compounds from series I, which have R1 substituents as hydroxyl
181 in 4c (36 μM ± 0.12) and nitro group in 3d and 4d (35 μM ± 0.23 and 23 μM ±
182 0.12), provided increased inhibitory activities ranging from 13- to 21-fold,
183 respectively, when compared to MB872 (>500 μM). Moreover, 4d (-NO2) had the
184 inhibitory activity increased by about 1.5-fold in comparison to 4c (-OH), which
185 might have been caused by electron density decrease of the aromatic ring, due
186 to the resonance withdrawing effect of the nitro group. Regarding the introduction
187 of methoxyl in R1, an electron donor group (derivatives 3b and 4b presented IC50
188 of 242 μM ± 0.08 and 119 μM ± 4.8, respectively), the compounds did not show
7

189 a significant inhibitory activity when compared to MB872 (>500 μM), although
190 they were modestly active.
191
Compound 4e (Series II), a classical bioisostere of MB872, was the most
192 active against in vitro MtDHFR (IC50 values of 7 ± 0.13 μM) and exhibited better
193 activity than the original prototype (by approx. 71-fold) and trimethoprim (by 13-
194 fold). The bioisostere replacement, exchanging the oxygen atom by nitrogen,
195 provided an extra hydrogen-bond donor feature, which could allow additional
196 stabilizing interactions. Compounds with a free ionizable carboxylic acid group in
197 R2 were active (4c, 4d, and 4i had an IC50 of 36 μM, 23 μM, and 31 μM,
198 respectively), in comparison to esterified versions 3f and 9b, which were less
199 active (76 μM ± 0.01 and 81 μM ± 0.11, respectively), or even inactive (3g and
200 9a). Noteworthy, the insertion of a triazole group in region B led to a complete
201 loss of inhibitory effect (9a and 10a). Moreover, the insertion of the OCH2-1,2,3-
202 triazole group (9b and 10b) was also not beneficial to MtDHFR inhibitory activity.
203
Compounds from series III, containing a methoxyl group in R1 and
204 associated with the para-substitution in R2, had a significant increase in the
205 enzyme inhibition, as observed for compounds 3i and 4i (26 μM ± 0.16 and 31 μM
206 ± 0.10), which are some of the most active derivatives of series III. Therefore,
207 analogues 3i and 4i presented IC50 values 16- and 19-fold lower than the
208 prototype MB872, respectively. Finally, in series III, the bioisostere group
209 tetrazole in meta-substitution (compound 7) was not beneficial, demonstrating
210 low activity against MtDHFR (IC50 = 454 μM).
211
212
Table 1. MtDHFR inhibition activity.
Y
R1
W
R2
Compound
R1
H
W
O
Y
R2
IC50 (µM) ± SDa
>500
LLE
0.98
2.59
0.05
1.31
2.24
1.24
MB872
TMPb
3a
CH2
m-COOH
91 ± 0.16
>500
H
OMe
NO2
H
O
O
O
S
CH2
CH2
CH2
CH2
m-COOMe
m-COOMe
m-COOMe
m-COOMe
3b
242 ±0.08
35 ±0.23
76 ±0.01
3d
3f
8

213
3g
3h
3i
H
H
CH2
O
O
m-COOMe
p-COOMe
p-COOMe
m-COOH
m-COOH
m-COOH
m-COOH
m-COOH
m-COOH
p-COOH
p-COOH
>500
152 ±0.10
26 ±0.16
119 ±4.8
36 ±0.12
23 ±0.13
7 ±0.13
128 ±0.11
230 ±0.09
442 ±0.13
31 ±0.1
454 ±0.15
NDc
0.98
0.56
1.35
1.62
2.40
2.42
3.10
1.01
1.31
1.03
2.20
0.176
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
OMe
OMe
OH
NO2
H
O
4b
4c
4d
4e
4f
O
O
O
NH
S
H
4g
4h
4i
H
CH2
O
H
CH2
CH2
Ome
H
O
7
O
CH2 m-tetrazole
9a
9b
10a
10b
H
1,2,3-triazole m-COOMe
OMe 1,2,3-triazole m-COOMe
81 ±0.11
ND
0.89
H
1,2,3-triazole
m-COOH
OMe 1,2,3-triazole
m-COOH
>500
Approx. 1.04
214
215
216
217
aData are expressed as means and the standard deviation intervals (SD) were obtained from
b
three independent experiments. TMP stands for trimethoprim. cND stands for not determined.
LLE stands for ligand lipophilicity efficiency, which is calculated from the difference between pIC50
values and the LogP.
218
219
The most potent compound (4e) against MtDHFR was selected for further
220 enzyme kinetics studies. The Michaelis constant (Km) and the maximum velocity
221 (Vmax) were determined, in the presence of varying concentrations of inhibitor.
222
The substrate’s Km remained constant (0.22 µM ± 0.04), while Vmax varied (0.34,
223 0.41 and 0.43), when tested using increasing concentrations of compound 4e,
224 which is consistent with uncompetitive inhibition [29,30]. The results suggest that
225 the binding mode explores a distinct (sub-)pocket from the substrate or cofactor
226 binding sites. Srinivasan and colleagues [31] reported ten novel small-molecule
227 inhibitors of E. coli DHFR (EcDHFR), which have shown a unique uncompetitive
228 inhibition mechanism, acting by displacing the DHF substrate. However, the
229 precise structural binding mode suggested based on pocket similarity analyses,
230 would need further validation.
231
9

232 2.4. Uncompetitive DHFR inhibition as a result of compounds accessing a unique
233 backpocket
234
In order to understand at the molecular level the uncompetitive mechanism
235 of the most potent designed inhibitor (4e), comparative molecular dynamics (MD)
236 simulation of co-crystallized substrate (DHF) and known competitive DHFR
237 inhibitors (methotrexate – MTX, pemetrexed – PMX and diaverdine – DIA), as
238 well as docking poses of selected compounds from our series (namely, 4b, 4e
239 and 4h) were performed. Compound 4e was chosen as the most potent of the
240 series, 4b due to the change in the linker region and 4h aiming to discuss the
241 different positioning of the R2 substituent.
242
MtDHFR can shift between two conformations, from the perspective of the
243 loop covering the active site: one open, where it can receive the substrates and
244 the cofactor, and another closed, which is bound to the cofactor. Simulations of
245 the MtDHFR closed conformation in complex with DHF revealed stable hydrogen
246 interactions between the DHF pteridine ring N8 and the Ile5 backbone’s oxygen
247 atom (Fig. 3A) and between the carboxylic acid moiety and the Arg32 and Arg60
248 sidechains (Fig. 3A,B). The original crystal structures of MtDHFR complexed with
249 DHF and MTX showed that the pteridine rings have different conformations [26].
250 The different initial conformations reflect on different stable interactions of the
251 pteridine moiety, such as the MTX 2,4-diamino group addressing Ile5 and Ile94
252 (Fig. 3A,C) oxygen backbone atoms, while the DHF 4-oxopteridine group points
253 away from them (Fig. 3A,B). Interestingly, even though PMX does not directly
254 interact with Ile94 main chain, we observed an intermittent water-mediated
255 hydrogen bond with N7 from the pyrrolopyrimidine ring (40% of the simulation
256 time, data not shown). The orientation of the dihydropyrimidine ring is further
257 stabilized by the hydrogen bond interaction between the sidechain Asp27
258 (Fig. 3A for frequency) and the 2,4-diamino moieties of the MTX and DIA
259 inhibitors (Fig. 3C and E, respectively) and by the 2-amino pyrrolopyrimidine of
260 the PMX (Fig. 3D). On the other hand, the stabilization due to the Asp27
261 interaction, thus occupation of this particular pocket, cannot be observed in our
262 series (Fig. 3A and Fig. 4).
263
10

264
265
266
267
268
269
270
271
272
273
Fig. 3. A) Frequency of hydrogen interactions observed along with the production phase of
molecular dynamics simulation. Arginine 32’s hydrogen bond interactions were separated into
two groups: HE, performed by the hydrogen’s NH in the base of the guanidinium group, and HHx
which is one of either HH atoms in the extremity of this group, indistinguishably. Representative
snapshots from the molecular dynamics simulation (1 μs per system), highlighting the residues
involved in interactions B) DHF (PDB ID: 6NND), C) MTX (1DF7), D) PMX (6NNC) and E) DIA
(6NNE). Interactions are represented by dashed lines. DHFR residues are colored according to
the atom types of the interacting amino acid residues (protein’s carbon, light gray; nitrogen, blue;
oxygen, red).
274
275
All known DHFR inhibitors, except DIA (Fig. 3E), share a common hydrogen
276 bond and ionic interaction networks between their carboxylic acid moieties and
277 the Arg32 and Arg60 (Fig. 3A – D). A similar network is also explored by our
278 inhibitors (Fig.3A and Fig. 4), independently from the ring attachment point (-meta
279 or -para), as long as the substituent remains unesterified and, therefore,
280 ionizable. Moreover, hydrophobic (Supplementary Data, Fig. 4A) and π-π
281 interactions (Supplementary Data, Fig. 4B) were suggested by the initial docking
282 pose with our inhibitors and also between the Phe31 and
283 pteridine/pyrrolopyrimidine moieties from known inhibitors, which were not
284 maintained during the simulations.
11

285
286
287
288
289
290
Fig. 4. Representative snapshots from the molecular dynamics simulation, highlighting the
residues involved in interactions: compound 4e (A – 100 ns, B – 1 μs), compound 4b (C, D) and
compound 4h (E, F). DHFR residues are colored according to the atom types of the interacting
amino acid residues (protein’s carbon, light gray; nitrogen, blue; oxygen, red).
291
292
The most active inhibitor from the series (4e, Fig. 4A,B) establishes
293 transient interactions with the Gln28, Ile20 and Ile94 main-chain oxygen atoms.
294 However, the proposed binding mode for 4e cannot be explained by a single
295 conformation, as highlighted by the different positioning of the phenoxy ring upon
296 simulation, seldomly interacting with Phe31. Interestingly, simulations with the
297 least potent inhibitors (4b and 4h) show the inability to stabilize the loop covering
298 the active site, even after 1 μs simulation (Fig. 4D,F). We hypothesize that
299 compounds with an uncompetitive inhibition mechanism would address only one
300 of the two possible DHFR conformations (open and closed) with the most potent
301 ones stabilizing the closed state. However, we do not discard the possibility of
302 the compounds to address other transient pockets or have an effect on higher
303 quarternary structures [11,32]. Finally, compound 4e presented the highest LLE
12

304 values among the tested set, including controls. This suggests that, despite the
305 low potency, compound 4e has the potential to be further optimized into a great
306 lead compound.
307
Interestingly, the EcDHFR inhibitors SAR model describes the presence of
308 two benzene rings, a spacer with a minimum of three degrees of freedom and the
309 presence of polar substituents, which are able to perform hydrogen bond
310 interactions, on the benzene rings [31]. Those chemical features are shared both
311 by our MtDHFR inhibitors and also by stilbenoid, deoxybenzoin and chalcone
312 scaffolds, with a reported uncompetitive mechanism of action [31]. Moreover, in
313 the described uncompetitive inhibition [31], the inhibitor reversibly binds to the
314 ES1 complex (DHFR + NADPH) yielding an inactive ES1I complex. Hence, Vmax
315 in the presence of the inhibitor would be lower than in its absence, without
316 affecting the Km, which we also observe in the 4e inhibition (Supplementary Data,
317 Fig. 1 and Table 1).
318
We therefore suggest that 4e could also present an uncompetitive inhibition
319 mechanism since our inhibitors do not share the most essential interaction
320 features with known substrate competitive DHFR inhibitors (for instance, the
321 interaction with Asp27 and Ile94), but rather occupy a unique pocket near the
322 loop, composed by Arg32, Arg60 and, intermittently, interacts with Phe31.
323
324
325
3. Conclusions
Novel substituted 3-benzoic acid derivatives were designed and
326 synthesized as potential inhibitors against MtDHFR. After having their activities
327 evaluated, compound 4e showed the best inhibitory activity with an IC50 of 7 μM,
328 which is 71-fold more active than the start fragment prototype MB872. Moreover,
329 the kinetics of MtDHFR inhibition revealed that 4e did not compete with DHF or
330 NADPH to inhibit MtDHFR since Km keeps constant, while Vmax changed
331 considerably, indicating an uncompetitive mechanism. Hence, 4e is the first
332 uncompetitive inhibitor reported for DHFR from M. tuberculosis, with a suggested
333 binding mode that partially accesses the binding pocket of known inhibitors, in
334 the active site. In conclusion, these findings provide new insights into substituted
335 3-benzoic acids and 4e analogs can have strong potential for further development
336 into new promising anti-TB compounds.
337
13

338 4.
339
Experimental section
4.1 MtDHFR production
340
DHFR from Mycobacterium tuberculosis was purified according to the
341 protocol described in Ribeiro et al. (2019) [26]. Briefly, a pET28(a)+ vector
342 carrying the folA gene was transformed in BL21(DE3) competent cells. The
343 transformed cells were grown at a temperature of 37 °C until the OD600 reached
344 0.6, when the protein expression was induced by the IPTG (0.2 mM) at 18 ºC
345 during 18 h. After that, cells were harvested by centrifugation and resuspended
346 in a buffer containing 20 mM NaPO4 and 50 mM KCl (Buffer A). Cells were
347 disrupted using a sonicator (Branson Sonifier) in presence of 1 mM PMSF,
348 0.2 mg.mL-1 lysozyme and 1 mM DNAse (Sigma–Aldrich) and cell lysate was
349 clarified by centrigutaion at 13,000 g (at 4 °C for 1 h). The purification was
350 performed using an IMAC column charged with Ni2+ and an imidazole gradient in
351 an Äkta Purifier System using a Buffer B (Buffer A in addition to increasing
352 concentrations of imidazole, until 500 mM) and by gel filtration using a S200
353 chromatography column. The pure protein was eluted using Buffer A,
354 concentrated up to 10 mg/mL and stored at -80 °C until use.
355
356
357
4.1.1 Kinetics assay
Kinetics measurements were carried out Fluorimeter (TECAN Infinite Pro
358 M200) using a standard assay by Meek and coworkers [33], at pH 7.5 (50 mM
359 sodium phosphate). The Michaelis-Menten constant was calculated for the
360 NADPH cofactor and DHF substrate by following a decrease in fluorescence at
361 480 nm, upon excitation at 340 nm, using a range of concentrations (0.1 to
362 100 µM).
363
For IC50 determination and mode of inhibition studies, we used a classic
364 kinetic assay. Therefore, the IC50 determination was carried out in balanced
365 conditions (0.09 μM MtDHFR, 5 μM DHF and 10 μM cofactor NADPH) at pH 7.5
366 (50 mM sodium phosphate buffer) by investigating the effect of, at least, six
367 concentrations (0.01 to 30 µM) of each compound over the catalytic activity of
368 MtDHFR. All compounds were solubilized in DMSO (at the maximum
369 concentration of 5% during the assays). The inhibition percentage was calculated
370 with reaction velocities (Inhibition %=(100-V1/Vc)x100)), where V1 represents the
371 initial velocity in the presence of the putative MtDHFR inhibitor and Vc is the initial
14

372 velocity of the control (5% DMSO v/v). The IC50 values were calculated using
373 least-squares non-linear regression, available in the GraphPad Prism
374 (GraphPad® software, version 8.1, La Jolla, California, USA). The mechanism
375 using the same reaction conditions as above with the most potent derivative (4e)
376 was investigated by determining its effect over the apparent Km and Vmax values
377 for DHF and NADPH. Apparent Km and Vmax values in the presence of the inhibitor
378 were obtained by non-linear regression to the Michaelis-Menten equation and
379 compared to each other (F test of the sim of the extra squares, values considered
380 significantly different when p < 0.05), as available in the GraphPad Prism
381 (GraphPad® software, version 8.1, GraphPad software, version 8.1, La Jolla,
382 California, USA, www.graphpad.com).
383
384 4.2 Chemistry
385
All reagents were purchased from Synth, Merck and Sigma Aldrich and
386 were used directly without any purification. Thin-layer chromatography (TLC) was
387 performed using silica GF254 gel-coated aluminum plates with a fluorescent
388 indicator (Merck). The TLCs were revealed by UV light irradiation at 254 nm
389 and/or staining solution. Chromatographic purification was performed on silica
390 gel columns (SiGel 60, 220 – 440 mesh, Sigma-Aldrich). The purity of the
391 synthesized compounds was assessed on a Shimadzu Prominence HPLC
392 system using a C18 column (5 µm, 150 x 4.6 mm, Gemini), with ultraviolet
393 detection set at 254 nm. The mobile phase employed was deionized water
394 (Solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (Solvent B) in
395 gradient elution. The overall run time was 20 minutes and the injection volume
1
13
396 was 20 μL. For structural characterization, H NMR and C NMR spectra were
397 recorded on a Bruker Advanced DPX-300 NMR spectrometer (300 MHz and 75
398 MHz, respectively) and the samples were prepared in CDCl3, CD3OD, D2O or
399 DMSO-d6. Chemical shifts are given in parts per million (ppm), with
400 tetramethylsilane as an internal standard. The following multiplicity abbreviations
401 are used: singlet (s), doublet (d), broad singlet (br s), triplet (t) and multiplet (m).
402 Coupling constants (J values) are given in Hertz (Hz). All NMR spectra for each
403 compound can be found in the Supplementary Data.
404
405
4.2.1 Procedure for the synthesis of compounds 3a-i
15

406
Methyl 4-(bromomethyl)benzoate or methyl 3-(bromomethyl)benzoate
407 (1 mmol) was dissolved in acetone (5 mL/mmol) and then potassium carbonate
408 (3 eq., 3 mmol) was added. The compound to be alkylated (1a-g) (1.2 eq.) was
409 added to the mixture and the reaction was kept under reflux for 16 h. The solvent
410 was then removed under reduced pressure. The residue was solubilized in ethyl
411 acetate and then washed with deionized water (3 x 10 mL). The organic layer
412 was dried (Mg2SO4), filtered, and concentrated under reduced pressure. The
413 products were purified by flash column chromatography (EtOAc/hexane in the
414 proportion of 7:3) [34].
415
416 Methyl 3-phenoxymethyl benzoate (3a)
417 Yellow liquid; yield: 74%. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.11 (s, 1H, H2),
418 8.00 (d, J = 7.7 Hz, 1H, H6), 7.64 (d, J = 7.7 Hz, 1H, H4), 7.45 (t, J = 7.7 Hz, 1H,
419 H5), 7.29 (t, J = 8.0 Hz, 2H, H5’-H3’), 6.98 (m, 3H, H2’-H4’-H6’) 5.10 (s, 2H, H7),
420 3.92 (s, 3H, H9). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.9 (C8), 158.6 (C1’),
421 137.6 (C3), 131.8 (C4), 130.5 (C6), 129.5 (C5’-C3’), 129.1 (C1), 128.7 (C2), 128.5
422 (C5),121.2 (C4’), 114.9 (C6’-C2’), 69.4 (C7), 52.1 (C9).
423
424 Methyl 3-((4-methoxyphenoxy)methyl)benzoate (3b)
425 White solid; yield: 70%. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.09 (s,1H, H2), 7.99
426 (d, J = 7.6 Hz, 1H, H6), 7.62 (d, J = 7.3 Hz, 1H, H4), 7.44 (t, J = 7.6 Hz, 1H, H5),
427 6.90 (d, J = 8.6 Hz, 2H, H5’-H3’) 6.83 (d, J = 8.9 Hz, 2H, H6’-H2’), 5.04 (s, 2H,
13
428 H7), 3.92 (s, 3H, H9), 3.76 (s, 3H, H10). C NMR (75 MHz, CDCl3) δ (ppm):
429 166.9 (C8), 154.2 (C4’), 152.7 (C1’), 137.8 (C3), 131.8 (C4), 130.5 (C6), 129.1
430 (C1), 128.6 (C2),128.5 (C5), 115.9 (C6’-C2’), 114.7 (C5’-C3’), 70.2 (C7), 55.7
431 (C10), 52.1 (C9).
432
433 Methyl 3-((4-nitrophenoxy)methyl)benzoate (3d)
434 Yellow solid; yield: 74%. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.21 (d, J = 9.1 Hz,
435 2H, H5’-H3’), 8.11 (s, 1H, H2), 8.04 (d, J = 7.7 Hz, 1H, H6), 7.63 (d, J = 7.5 Hz,
436 1H, H4), 7.49 (t, J = 7.7 Hz, 1H, H5) 7.04 (d, J = 9.1 Hz, 2H, H6’-H2’), 5.20 (s,
437 2H, H7), 3.94 (s, 3H, H9). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.6 (C8), 163.4
438 (C1’), 141.9 (C4’), 136.0 (C3), 131.8 (C1), 130.8 (C4), 129.6 (C6), 128.9 (C2),
439 128.3 (C5), 128.6 (C5’) 126.0 (C3’), 114.9 (C6’-C2’), 70.1 (C7), 52.2 (C9).
16

440
441 3-((Phenylthio)methyl)benzoate (3f)
442 Yellow liquid; yield: 77%. 1H NMR (300 MHz, (CDCl3): δ = 7.96 (s, 1H, H2), 7.90
443 (d, J = 7.7 Hz, 1H, H6), 7.45 (d, J = 7.6 Hz, 1H, H4), 7.38 – 7.14 (m, 5H), 4.13 (s,
444 2H, H7) 3.89 (s, 3H, H9). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.8 (C8), 138.1
445 (C1’), 135.7 (C3), 133.3 (C4), 130.4 (C6), 130.3 (C3’-C5’), 130.0 (C1), 128.9 (C2),
446 128.5 (C5), 128.8 (C4’), 126.7 (C6’-C2’), 52.1 (C9), 38.9 (C7).
447
448 Methyl 3-(benzyloxy)benzoate (3g)
1
449 White solid; yield: 73%. H NMR (300 MHz, CDCl3) δ (ppm): 7.66 (s, 1H, H2),
450 7.64 (d, J = 6.7 Hz, 1H, H6), 7.47 – 7.29 (m, 6H), 7.19 – 7.14 (m, 2H), 5.10 (s,
451 2H, H7), 3.91 (s, 3H, H9). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.9 (C8), 158.8
452 (C3), 136.6 (C1’), 131.5 (C1), 129.4 (C5), 128.6 (C5’-C3’), 128.1 (C1), 127.5 (C6’-
453 C2’), 122.3 (C4), 120.3 (C6), 115.2 (C2), 70.2 (C7), 52.1 (C9).
454
455 Methyl 4-(benzyloxy)benzoate (3h)
456 Brown solid; yield: 92%. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.05 (d, J = 8.2 Hz,
457 2H, H6-H2), 7.50 (d, J = 8.0 Hz, 2H, H5-H3), 7.29 (t, J = 6.0 Hz, 2H, H5’-H3’),
458 6.95-6.99 (m, 3H, H6’-H2’-H4’), 5.13 (s, 2H, H7), 3.92 (s, 3H, H9). 13C NMR (75
459 MHz, CDCl3) δ (ppm): 166.9 (C8), 158.5 (C1’), 142.4 (C4), 129.9 (C6-C2), 129.6
460 (C5-C3), 127.0 (C5’-C3’), 121.3 (C4’), 114.9 (C6’-C2’), 69.3 (C7), 52.1 (C9).
461
462 Methyl 4-((4-methoxyphenoxy)methyl)benzoate (3i)
463 Brown solid; yield: 84%. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.04 (d, J = 8.2 Hz,
464 2H, H6-H2), 7.49 (d, J = 8.2 Hz, 2H, H5-H3), 6.90 (d, J = 9.2 Hz, 2H, H5’-H3’),
465 6.83 (d, J = 9.2 Hz, 2H, H6’-H2’), 5.08 (s, 2H, H7), 3.92 (s, 3H, H9), 3.76 (s, 3H,
466 H10). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.9 (C8), 154.2 (C4’), 152.7 (C1’),
467 142.6 (C4), 129.8 (C5-C3), 127.0 (C6-C2), 115.9 (C6’-C2’), 114.7 (C5’-C3’), 70.1
468 (C7), 55.7 (C10), 52.1 (C9).
469
470
471 4.2.2.1 Synthesis of reagent methyl 3-aminobenzoate
472 Methyl 3-amino benzoate was dissolved in water, then the medium was
4.2.2 Synthesis of triazoles
473 acidified (at 0 °C) using 5% of HCl solution. Afterward, a solution of NaNO2
17

474 (2.5 eq) was added dropwise and the mixture was kept under stirring. After 30
475 min, NaN3 (1.6 eq.) was added slowly. At the end of the bubbles, the mixture
476 reaction was extracted with EtOAc. The organic layer was dried over with
477 Na2SO4, filtered, and concentrated under reduced pressure [35].
478
479 Methyl 3-aminobenzoate
480 Colorless liquid; yield: 95%. 1H NMR (300 MHz, CDCl3) δ (ppm): 7.85 – 7.76 (m,
481 1H, H6), 7.74 – 7.61 (m, 1H, H2), 7.42 (t, J = 7.9 Hz, 1H, H5), 7.21 – 7.18 (m, 1H,
482 H4), 3.93 (s, 3H, H8). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.2 (C7), 140.6 (C3),
483 131.9 (C5), 129.8 (C1), 126.0 (C4), 123.4 (C6), 120.0 (C2), 52.3 (C8).
484
485 4.2.2.2 Synthesis of reagent (prop-2-yn-1-yloxy)benzene
486
Propargyl bromide (80% in toluene) (1 mmol) was dissolved in acetone
487 (5 mL/mmol) and then K2CO3 (3 eq, 3 mmol) was added. Phenol (1.2 eq.) was
488 added under stirring and the reaction was maintained at reflux for 16 hours. In
489 the end, the solvent was removed under reduced pressure and the mixture was
490 solubilized in EtOAc and then it was washed with deionized water (3 x 10 mL).
491 The organic layer was dried (Na2SO4), filtered, and concentrated in vacuum. The
492 residue was purified by flash column chromatography (EtOAc/hexane) [34].
493
494 (prop-2-yn-1-yloxy) benzene
495 Yellow liquid; yield: 91%. 1H NMR (300 MHz, CDCl3) δ (ppm): 7.33 – 7.27 (m, 2H,
496 H3-H6), 7.01 – 7.61 (m, 3H, H2-H5-H4), 4.68 (d, J = 2.4 Hz, 2H, H7), 2.50 (t, J =
497 2.4 Hz, 1H, H9) . 13C 1H NMR (75 MHz, CDCl3) δ (ppm): 157.6 (C1), 129.5 (C3-
498 C6), 121.6 (C4), 114.9 (C2-C5), 78.7 (C8), 75.4 (C9), 55.8 (C7).
499
500 4.2.2.3 Cycloaddition – Synthesis of compounds 9a-b
501
In the mixture of tetrahydrofuran/H2O or t-BuOH/H2O were added CuI
502 (1.1 eq.) and sodium ascorbate (0.5 eq.). The reaction was kept under stirring
503 and light protection for 12 hours. The mixture was filtered through Celite® 545,
504 then the solvent was evaporated under reduced pressure and the residue was
505 dissolved in ethyl acetate and washed with and water (1:1). Finally, the products
506 were purified using flash chromatography column (EtOAc/hexane) to give the
507 compounds 9a-b [36].
18

508
509 Methyl 3-(4-phenyl-1H-1,2,3-triazol-1-yl)benzoate (9a)
1
510 Yellow solid; yield: 30%. H NMR (300 MHz, CDCl3) δ (ppm): 8.40 (s, 1H, H8),
511 8.27 (s, 1H, H2), 8.12 (d, J = 6.8 Hz, 1H, H6), 8.09 (d, J = 7.0 Hz, 1H, H4), 7.91
512 (d, J = 7.1 Hz, 2H, H6’-H3’), 7.63 (t, J = 7.9 Hz, 1H, H5) 7.46 (t, J = 7.3 Hz, 2H,
513 H5’-H3’), 7.37 (t, J = 7.3 Hz, 1H, H4’) 3.98 (s, 3H, H9). 13C NMR (75 MHz, CDCl3)
514 δ (ppm): 165.8 (C9), 148.7 (C7), 137.2 (C3), 131.9 (C1), 130.1 (C1’), 129.6 (C5),
515 128.9 (C3’-C5’), 128.6 (C4’), 125.9 (C2’-C6’),124.7 (C4), 121.0 (C8), 117.5 (C2),
516 52.6 (C10).
517
518 Methyl 3-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)benzoate (9b)
1
519 Yellow solid; yield: 80%. H NMR (300 MHz, CDCl3) δ (ppm): 8.35 (s, 1H, H9),
520 8.13 – 8.10 (m, 2H, H2-H6), 8.02 (d, J = 8.1 Hz, 1H, H4), 8.09 (d, J = 7.0 Hz, 1H,
521 H4), 7.61 (t, J = 8.0 Hz, 1H, H5), 7.31 (t, J = 7.9 Hz, 2H, H5’-H3’), 7.04 – 6.96 (m,
522 3H, H6’-H2’-H4’), 5.31 (s, 2H, H7), 3.96 (s, 3H, H11). 13C NMR (75 MHz, CDCl3)
523 δ (ppm): 165.7 (C10), 158.2 (C1), 145.4 (C8), 137.2 (C3), 132.0 (C1), 130.0 (C5),
524 129.6 (C3’-C5’), 129.7 (C6) 124.8 (C4), 121.4 (C2), 121.2 (C4’), 120.8 (C9), 114.8
525 (C2’-C6’), 62.0 (C7), 52.5 (C11).
526
527 4.2.2.4 Hydrolysis of esters – Synthesis of compounds MB872, 4b-I and 10a-b
528
Methyl esters (1 eq, 1 mmol) were dissolved in THF and water
529 (2 mL/mmol), after a KOH solution 2 mol/L was added (3 eq, 3 mmol, 1.5 mL
530 solution). The mixture was kept under stirring and room temperature until TLC
531 indicated that no starting material remained. THF was removed under reduced
532 pressure and the mixture was acidified with a solution of HCl (5% v/v) until pH 2.0.
533 The precipitate was filtered and collected. Lastly, the products were purified by
534 flash chromatography column using DCM/MeOH (9:1, respectively), as the eluent
535 system [34].
536
537 3-Phenoxymethyl benzoic acid (MB872)
538 White solid; yield: 84%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.04 (s, 1H, H2),
539 7.92 (d, J = 7.7 Hz, 1H, H6), 7.70 (d, J = 7.6 Hz, 1H, H4), 7.53 (t, J = 7.7 Hz, 1H,
540 H5), 7.31 (t, J = 7.9 Hz, 2H, H5’-H3’), 7.03 (d, J = 8.0 Hz, 2H, H2’-H6’) 6.95 (t, J
541 = 7.3 Hz, 1H, H4’), 5.18 (s, 2H, H7). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 167.1
19

542 (C8), 158.2 (C1’), 137.7 (C3), 131.8 (C1), 131.0 (C4), 129.4 (C5’-C3’), 128.7 (C6),
543 128.6 (C2), 128.2 (C5), 120.8 (C4’), 114.8 (C6’-C2’), 68.5 (C7).
544
545 3-((4-Methoxyphenoxy)methyl)benzoic acid (4b)
546 White solid; yield: 77%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.02 (s, 1H, H2),
547 7.90 (d, J = 7.7 Hz, 1H, H6), 7.67 (d, J = 7.6 Hz, 1H, H4), 7.51 (t, J = 7.6 Hz, 1H,
548 H5), 6.96 (d, J = 9.1 Hz, 2H, H5’-H3’), 6.86 (d, J = 9.1 Hz, 2H, H6’-H2’), 5.12 (s,
549 2H, H7), 3.69 (s, 3H, H10). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 167.2 (C8),
550 153.6 (C4’), 152.1 (C1’), 137.9 (C3), 131.7 (C1), 131.0 (C4), 128.6 (C6), 128.5
551 (C2), 128.1 (C5), 115.8 (C6’-C2’), 114.6 (C5’-C3’), 69.1 (C7), 55.3 (C10).
552
553 3-((4-Hydroxyphenoxy)methyl)benzoic acid (4c)
554 Yellow solid; yield: 30%. 1H NMR (300 MHz, Methanol-d4) δ (ppm): 8.10 (s, 1H,
555 H2), 7.98 (d, J = 7.6 Hz, 1H, H6), 7.67 (d, J = 7.5 Hz, 1H, H4), 7.49 (t, J = 7.7 Hz,
556 1H, H5), 6.86 (d, J = 8.9 Hz, 2H, H6’-H2’), 6.73 (d, J = 8.9 Hz, 2H, H5’-H3’), 5.06
13
557 (s, 2H, H7). C NMR (75 MHz, Methanol-d4) δ (ppm): 169.9 (C8), 153.4 (C4’),
558 152.7 (C1’), 139.7 (C3), 132.42 (C1), 132.9 (C4), 130.0 (C6), 129.7 (C2), 129.6
559 (C5), 117.2 (C6’-C2’), 116.9 (C5’-C3’), 71.4 (C7).
560
561 3-((4-Nitrophenoxy)methyl)benzoic acid (4d)
562 Yellow solid; yield: 70%. 1H NMR (300 MHz, D2O) δ (ppm): 7.99 (d, J = 9.1 Hz,
563 2H, H5’-H3’), 7.89 (s, 1H, H2), 7.81 (d, J = 7.3 Hz, 1H, H6), 7.45 (t, J = 6.5 Hz,
564 1H, H5), 7.39 (d, J = 7.5 Hz, 1H, H4), 6.94 (d, J = 9.1 Hz, 2H, H6’-H2’), 5.10 (s,
565 2H, H7). 13C NMR (75 MHz, D2O) δ (ppm): 174.9 (C8), 163.3 (C1’), 141.1 (C4’),
566 136.9 (C3), 135.7 (C1), 130.3 (C4), 128.9 (C6), 128.7 (C2), 128.3 (C5), 126.7
567 (C5’-C3’), 115.1 (C6’-C2’), 70.4 (C7).
568
569 3-((Phenylamino)methyl)benzoic acid (4e)
570 Yellow solid; yield: 64%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 7.93 (s, 1H, H2),
571 7.83 (d, J = 6.2 Hz, 1H, H6), 7.41 – 7.38 (m, 2H, H4-H5), 7.06 (t, J = 7.4 Hz, 2H,
572 H5’-H3’), 6.65 (d, J = 7.9 Hz 2H, H6’-H2’), 6.58 (t, J = 7.1 Hz, 1H, H4’), 6.60 (d, J
13
573 = 7.7 Hz, 2H, H6’-H2’), 4.72 (s, 2H, H7), 4.54 (s, 1H, NH). C NMR (75 MHz,
574 DMSO-d6) δ (ppm): 166.5 (C8), 148.1 (C1’), 139.1 (C3), 130.2 (C4), 128.9 (C3’-
20

575 C5’),128.3 (C6), 127.8 (C1), 127.5 (C2), 128.5 (C5), 116.3 (C4’), 112.5 (C6’-C2’),
576 48.6 (C7).
577
578 3-((phenylthio)methyl)benzoic acid (4f)
579 White solid; yield: 70%. 1H NMR (300 MHz, DMSO-d6): δ = 7.95 (s, 1H, H2), 7.81
580 (d, J = 7.7 Hz, 1H, H6), 7.58 (d, J = 7.6 Hz, 1H, H4), 7.41 (t, J = 7.7 Hz, 1H H5),
581 7.34 (d, J = 7.1 Hz, 2H, H6’-H2’), 7.29 (t, J = 7.5 Hz, 2H, H5’-H3’), 7.18 (t, J = 7.0
582 Hz, 1H, H4’), 4.32 (s, 2H, H7). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 167.0 (C8),
583 147.9 (C1’), 140.1 (C3), 131.9 (C4), 130.6 (C6), 129.3 (C3’-C5’),128.8 (C1), 128.7
584 (C2), 128.5 (C5), 117.8 (C4’), 113.0 (C6’-C2’), 48.0 (C7), 52.1 (C9).
585
586 3-benzyloxy benzoate (4g)
587 Yellow solid; yield: 80%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 13.0 (s, 1H, H9),
588 7.60 – 7.29 (m, 9H), 5.21 (s, 2H, H7). 13C NMR (75 MHz, CDCl3) δ (ppm): 167.0
589 (C8), 158.3 (C3), 136.8 (C1’), 132.2 (C1), 129.7 (C5), 128.4 (C5’-C3’), 127.8 (C1),
590 127.6 (C6’-C2’), 121.8 (C4), 119.7 (C6), 114.9 (C2), 69.3 (C7).
591
592 4-Phenoxymethyl benzoic acid (4h)
1
593 White solid; yield: 68%. H NMR (300 MHz, DMSO-d6) δ (ppm): 12.93 (s, 1H,
594 H9), 7.97 (d, J = 7.7 Hz, 2H, H6-H2), 7.57 (d, J = 7.7 Hz, 2H, H5-H3), 7.30 (t, J =
595 7.9 Hz, 1H, H5’-H3’), 7.02 (d, J = 7.9 Hz 2H, H6’-H2’), 6.95 (t, J = 7.3 Hz, 1H,
596 H4’), 5.19 (s, 2H, H7). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 167.1 (C8), 158.1
597 (C1’), 142.2 (C4), 130.1 (C6-C2), 129.5 (C5-C3), 127.3 (C5’-C3’), 120.9 (C4’),
598 114.8 (C6’-C2’), 68.5 (C7).
599
600 4-((4-Methoxyphenoxy)methyl)benzoic acid (4i)
601 White solid; yield: 83%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 7.96 (d, J = 8.0
602 Hz, 2H, H6-H2), 7.54 (d, J = 8.0 Hz, 2H, H5-H3), 6.95 (d, J = 9.0 Hz, 2H, H5’-
13
603 H3’), 6.86 (d, J = 9.0 Hz, 2H, H6’-H2’), 5.13 (s, 2H, H7), 3.69 (s, 3H, H10). C
604 NMR (75 MHz, DMSO-d6) δ (ppm): 167.1 (C8), 153.6 (C4’), 152.1 (C1’), 142.4
605 (C4), 130.2 (C5-C3), 127.2 (C6-C2), 115.8 (C6’-C2’), 114.6 (C5’-C3’), 69.1 (C7),
606 55.3 (C10).
607
608 3-(4-Phenyl-1H-1,2,3-triazol-1-yl)benzoic acid (10a)
21

609 Yellow solid; yield: 90%. 1H NMR (300 MHz, Methanol-d4) δ (ppm): 8.80 (s, 1H,
610 H8), 8.35 (s, 1H, H2) 7.99 (d, J = 7.7 Hz, 1H, H6), 7.91 (d, J = 7.1 Hz, 1H, H4),
611 7.83 (d, J = 8.1 Hz, 2H, H6’-H3’), 7.52 (t, J = 7.9 Hz, 1H, H5), 7.36 (t, J = 7.6 Hz,
612 2H, H5’-H3’), 7.27 (t, J = 7.3 Hz, 1H, H4’). 13C NMR (75 MHz, Methanol-d4) δ
613 (ppm): 172.3 (C9), 149.7 (C7), 140.0 (C3), 138.2 (C1), 131.5 (C1’), 130.8 (C5),
614 130.6 (C4’), 130.0 (C3’-C5’), 129.6 (C4), 126.8 (C2’-C6’), 123.6 (C8), 122.2 (C2).
615
616 3-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl) benzoic acid (10b)
617 Yellow solid; yield: 98%. 1H NMR (300 MHz, Methanol-d4) δ (ppm): 8.63 (s, 1H,
618 H9), 8.40 (s, 1H, H2), 8.10 (d, J = 7.6 Hz, 1H, H6), 7.95 (d, J = 8.0 Hz, 1H, H4),
619 7.61 (t, J = 7.9 Hz, 1H, H5), 7.31 (t, J = 7.4 Hz, 2H, H5’-H3’), 7.06 (d, J = 8.0 Hz,
620 2H, H6’-H2’), 6.97 (t, J = 7.3 Hz, 1H, H4’), 5.27 (s, 2H, H7). 13C NMR (75 MHz,
621 Methanol-d4) δ (ppm): 172.5 (C10), 159.8 (C1), 146.1 (C8), 140.1 (C3), 138.1
622 (C1), 130.8 (C5), 130.6 (C3’-C5’), 123.7 (C2), 123.6 (C4’), 122.8 (C9), 116.0 (C2’-
623 C6’), 62.4 (C7).
624
625 4.2.3 Synthesis of tetrazoles (7)
626
627
628
629
630
631
632
633
Methyl 3-(4-phenyl-1H-1,2,3-triazol-1-yl)benzoate was dissolved in DMF
(4 mL/mmol) and then NaN3 (1.2 eq.) and NH4Cl (1.2 eq.) were added. The
mixture was maintained under stirring and reflux for 16 hours. Afterward, water
was added and the mixture was washed with EtOAc. Lastly, the pH of the
aqueous phase was adjusted to 1.00 with the addition of 5% (v/v) HCl solution
and the mixture was kept under stirring for 30 minutes. The precipitate was
filtered and washed with water [37].
634 5-(3-(phenoxymethyl)phenyl)-1H-tetrazole (7)
1
635 Yellow solid; yield: 5%. H NMR (300 MHz, Methanol-d4) δ (ppm): 8.03 (s, 1H,
636 H2), 7.87 (d, J = 7.5 Hz, 1H, H6), 7.56 (d, J = 7.7 Hz, 1H, H4), 7.49 (t, J = 7.6 Hz,
637 1H, H5), 7.18 (t, J = 7.9 Hz, 2H, H5’-H3’), 6.62 (d, J = 8.0 Hz, 2H, H2’-H6’) 6.84
638 (t, J = 7.3 Hz, 1H, H4’), 5.07 (s, 2H, H7).13C NMR (75 MHz, Methanol-d4) δ (ppm):
639 160.1 (C1’), 157.5 (C8), 140.6 (C3), 131.0 (C4), 130.7 (C6), 130.6 (C5’-C3’),
640 127.6 (C5), 127.1 (C4), 125.7 (C1), 122.2 (C4’), 116.0 (C6’-C2’), 70.2 (C7).
641
642 4.3 Molecular modeling and molecular dynamics simulation
22

643
The PDB structure of MtDHFR co-crystallized with dihydrofolate substrate
644 (DHF, PDB ID: 6NND) and the antifolates methotrexate (MTX, 1DF7), with
645 pemetrexed (PMX, 6NNC) and diaverdine (DIA, 6NNE) were selected for
646 simulation, as well as the structure with only NADPH (1DG8). A single chain as
647 a monomer was selected for each of the structures for molecular modelling,
648 based on the number of Ramachandran outliers. The Small-Molecule Drug
649 Discovery Suite (v2019.3, Schrödinger, LLC, New York, NY, 2019) was used for
650 all calculations. Protein structures were prepared by adding hydrogen atoms and
651 fixing missing sidechains using the Protein Preparation Wizard (PrepWiz) [38],
652 missing loops were generated using Prime and sulfate/crystallization buffer
653 molecules, such as glycerol (GOL) were removed. Asp27 ionization state as well
654 as the ligand N3, and equivalent in other ligands, were assigned according to the
655 experimental results from Wan et al., 2014, where the folate N3 atom established
656 an ionic interaction with the negatively charged Asp27 [39].
657
Molecular docking of chosen ligands from our series (4b, 4e and 4h) was
658 performed in a grid encompassing residues around 20 Å from the centroid of the
659 co-crystallized ligand, using the default settings of the Glide program (Glide v7.7,
660 Maestro v2019.3) in extra-precision mode, with at least five poses selected for
661 further visual inspection [40]. Amino acid residues were considered rigid and both
662 structural water molecules and cofactor NAD+ were maintained in the active site
663 during the calculation.
664
Protein complexes with the original co-crystallized ligands and with the
665 docked ligands were submitted for molecular dynamics simulations (MD) in order
666 to evaluate both the ligand stability and the effects of ligand-binding upon the
667 protein (the full protocol was previously described [41]. MD simulations were
668 carried out using Desmond [42], with the OPLS3e force-field [43]. The simulated
669 system encompassed the protein-ligand complexes, a predefined water model
670 (TIP3P [44]) as a solvent and counterions (Na+ or Cl- adjusted to neutralize the
671 overall system charge). The system was treated in a cubic box with periodic
672 boundary conditions specifying the shape and the size of the box as 13 Å
673 distance from the box edges to any atom of the protein. We used a time step of
674 1 fs, the short-range coulombic interactions were treated using a cut-off value of
675 9.0 Å using the short-range method, while the smooth Particle Mesh Ewald
676 method (PME) handled long-range coulombic interactions [45].
23

677
Initially, the relaxation of the system was performed using Steepest Descent
678 and the limited-memory Broyden-Fletcher-Goldfarb-Shanno algorithms in a
679 hybrid manner. The simulation was performed under the NPT ensemble for 5 ns
680 implementing the Berendsen thermostat and barostat methods. A constant
681 temperature of 310 K was maintained throughout the simulation using the Nose-
682 Hoover thermostat algorithm and Martyna-Tobias-Klein Barostat algorithm to
683 maintain 1 atm of pressure, respectively.
684
After minimization and relaxation of the system, we proceeded with a single
685 production step of at least 1 μs, with sampling every 500 ps. The representative
686 structure was selected by inspecting changes in the Root-mean-square deviation
687 (RMSD, Supplementary Data, where Figure 2 represents the variation of the
688 RMSD values along with the simulation and the changes in the RMSF values,
689 Root-mean-square fluctuation, by residue for the protein backbone, on Figure 3).
690 Interactions and distances were determined using the Simulation Event Analysis
691 pipeline implemented in Maestro (Maestro v2019.3). The current geometric
692 criteria for protein-ligand hydrogen bond is a distance of 2.5 Å between the donor
693 and acceptor atoms (D — H···A); a donor angle of ≥120° between the donor-
694 hydrogen-acceptor atoms (D — H···A); and an acceptor angle of ≥90° between
695 the hydrogen-acceptor-bonded atom atoms (H···A — X). Similarly, protein-water
696 or water-ligand hydrogen bond had a distance of 2.8 Å between the donor and
697 acceptor atoms (D—H···A); a donor angle of ≥110° between the donor-hydrogen-
698 acceptor atoms (D—H···A); and an acceptor angle of ≥90° between the
699 hydrogen-acceptor-bonded atom atoms (H···A—X). Non-specific hydrophobic
700 interactions are defined by hydrophobic sidechain within 3.6 Å of a ligand's
701 aromatic or aliphatic carbons and π-π interactions required two aromatic groups
702 stacked face-to-face or face-to-edge, within 4.5 Å of distance. Trajectories and
703 interaction data are available on Zenodo repository (under the code:
704 10.5281/zenodo.3663046).
705
706 Conflict of interest
707 Authors declare no conflict of interest
708
709 Acknowledgments
24

710 The authors are grateful to CAPES, CNPq (grant number 153232/2018-8,
711 436791/2018-8 and 310232/2017-1) and FAPESP (grant numbers 2013/15906-
712 5, 2013/18160-4, 2017/00689-0 and 2017/25543-8) for financial support. The
713 authors wish to acknowledge the CSC – IT Center for Science, Finland, for the
714 generous computational resources.
715
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UNIVERSITY OF SAO PAULO
Faculty of Pharmaceutical Sciences
Department of Pharmacy
February 20th, 2020
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Professor F. Romesberg
The Scripps Research Institute
Dear Editor,
We wish to confirm that there are no known conflicts of interest associated with this
publication and there has been no significant financial support for this work that could have
influenced its outcome. We confirm that the manuscript has been read and approved by all named
authors and that there are no other persons who satisfied the criteria for authorship but are not
listed. We further confirm that the order of authors listed in the manuscript has been approved by
all of us. We confirm that we have given due consideration to the protection of intellectual property
associated with this work and that there are no impediments to publication, including the timing
of publication, with respect to intellectual property. In so doing we confirm that we have followed
the regulations of our institutions concerning intellectual property. We understand that the
Corresponding Author is the sole contact for the Editorial process (including Editorial Manager
and direct communications with the office). He/she is responsible for communicating with the
other authors about progress, submissions of revisions and final approval of proofs. We confirm
that we have provided a current, correct email address which is accessible by the Corresponding
Author and which has been configured to accept email from (roberto.parise@usp.br). All authors
have approved the manuscript and agree with its submission.
Sincerely yours,
Roberto Parise Filho, Ph.D. (on behalf of the authors)
Assistant Professor
Laboratory of Design and Synthesis of Bioactive Substances (LAPESSB)
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